Escolar Documentos
Profissional Documentos
Cultura Documentos
May 2014
www.nature.com/ejhg
ABSTRACTS
EMPAG
EUROPEAN MEETING ON
PSYCHOSOCIAL ASPECTS OF GENETICS
Abstracts
European Society
of Human Genetics
Karin Knob
Andrea Robinson
c/o Vienna Medical Academy
Alser Strasse 4
1090 Vienna
Austria
T: 0043 1 405 13 83 20 or 35
F: 0043 1 407 82 74
E: office@eshg.org
www.eshg.org
President
Han Brunner, NL
Chair
Brunhilde Wirth, DE
Vice-President
Stanislas Lyonnet, FR
Members
Antonio Amoroso, Local Host, Turin, IT
Jeffrey Barrett, Cambridge, UK
Alexis Brice, Paris, FR
Paul de Bakker, Utrecht, NL
Martina Cornel, Amsterdam, NL
Helene Dollfus, Strasbourg, FR
David Fitzpatrick, Edinburgh, UK
Francesca Forzano, Genova, IT
Maurizio Genuardi, Florence, IT
Daniel Grinberg, Barcelona, ES
Gunnar Houge, Bergen, NO
Erik Iwarsson, Stockholm, SE
Xavier Jeunemaitre, Paris, FR
Lidia Larizza, Milan, IT
Jose C. Machado, Porto, PT
Giovanni Neri, Rome, IT
William Newman, Manchester, UK
Minna Nystrm, Helsinki, FI
Francesc Palau, Valencia, ES
Anita Rauch, Zurich, CH
Samuli Ripatti, Helsinki, FI
Peter N. Robinson, Berlin, DE
Marco Seri, Bologna, IT
Joris Veltman, Nijmegen, NL
Joris Vermeesch, Leuven, BE
Karin Writzl, Ljubljana, SI
President-Elect
Helena Kriinen, FI
Secretary-General
Gunnar Houge, NO
Deputy-Secretary-General
Karin Writzl, SI
Treasurer
Andrew Read, UK
Executive Officer
Jerome del Picchia, AT
Board Members
Yasemin Alanay, TR
Martijn Breuning, NL
Pascal Borry, BE
Nina Canki-Klain, CR
Ana Carri, ES
Domenico Coviello, IT
Koen Devriendt, BE
Munis Dundar, TR
Peter Kroisel, AT
Dorit Lev, IL
Milan Macek Jr., CZ
Liaison Members
Julie McGaughran, AU
Bela Melegh, HU
Will Newman, UK
Markus Nthen, DE
Markus Perola, FI
Borut Peterlin, SI
Trine E Prescott, NO
Hans Scheffer, NL
Jrg Schmidtke, DE
Heather Skirton, UK
Martina Cornel, NL
Ros Hastings, UK
Thomas Liehr, DE
Milan Macek Jr., CZ
Tayfun Ozcelik, TR
Heather Skirton, UK
GertJan B. van Ommen, NL
Brunhilde Wirth, DE
Further information on structure and organisation can be found on the website www.eshg.org
Posters
51
370
Indices
517
RAS proteins are small monomeric GTPases that function as molecular switches controlling a major intracellular signaling network that, depending on the
cellular context, guides diverse biological functions, including proliferation, cell
fate determination, survival, migration, differentiation, and senescence. RAS
signaling is switched on upon stimulation by numerous cytokines, hormones,
and growth factors, and mediates the appropriate cell response to external stimuli through the regulation of transcription, cytoskeletal rearrangement, and
metabolism. Within this network, signal flow through the RAF-MEK-ERK pathway, the first identified mitogen-associated protein kinase (MAPK) cascade,
mediates early and late developmental processes, including determination of
morphology, organogenesis, synaptic plasticity, and growth.
Signaling through the RAS-MAPK cascade is tightly controlled, and its enhanced
activation has been known for decades to represent a major event in oncogenesis. Activating somatic RAS gene mutations occur in approximately 30% of
human cancers, and upregulation of this signaling pathway can also result from
enhanced function of upstream signal transducers or RAS effectors, and inefficient function of feedback mechanisms.
Unexpectedly, discoveries derived from a massive disease gene hunting effort
have recently established a novel scenario in which the upregulation of this
signaling cascade underlies a group of clinically related developmental disorders, the RASopathies, characterized by facial dysmorphism, cardiac defects,
reduced postnatal growth, variable cognitive deficits, ectodermal and musculoskeletal anomalies, and increased risk for certain malignancies. These disorders are caused by mutations in genes encoding RAS proteins, regulators
of RAS function, modulators of RAS interaction with effectors or downstream
signal transducers. Based on the relatively high prevalence of some of these
disorders (i.e., Noonan syndrome and neurofibromatosis type 1), the dysregulation of this signaling pathway represents one of the most common events affecting developmental processes. These discoveries have also provided us with
unpredicted molecular mechanisms converging toward the dysregulation of
this signaling network.
PL1.2
Evolution of the HD gene
E. Cattaneo;
Department of Bioscience, University of Milan, Milan, Italy.
The Hdh gene arose with no CAGs in Dictyostelium discoideum (Dd), around 800
million-year ago before the protostome-deuterostome divergence (Zuccato,
Physiol Rev 2010). The CAG then has appeared in and is unique to the deuterostome branch. Two CAGs are found in Hdh in sea urchin (Strongylocentrotus
purpuratus, Sp), the first specie to carry a primitive nervous system, and two
CAGs are present in amphioxus (Branchiostoma floridae, Bf), the first specie to
exhibit a rudimental hollow nerve tube and cephalization. Four CAG are found
in Hdh from the more evolved fishes, amphibians, and birds. The CAG further
expands in mammals and reaches its maximum length in human. A study of 278
normal subjects revealed an increase in grey matter with increasing length of
the CAG repeat (Muhlau, PlosOne 2012), indicating that CAG size could influence normal brain structure. Our hypothesis is that the progressive increase
in CAG length in the Hdh gene observed during evolution may be implicated in
the evolutionary changes that have occurred in the developing and adult nervous system throughout vertebrate phylogeny, with a possible role for the CAG
in newly emerging cognitive functions in the mammalian brain. We have now
collected the Hdh gene from new species both in the protostome and deuterostome branch. In addition to further analyze the CAG tract during mammalian
evolution we have collected genomic DNA and amplified the CAG tract from
non-anthropomorphic and anthropomorphic primates. Our reconstruction of
htt phylogeny supports further that the CAG tract expands during deuterostome evolution and seems to correlate with the appearance and/or evolution of
progressively more complex nervous systems.
PL1.3
Genetic engineering of hematopoietic stem cells for the treatment of
inherited diseases
L. Naldini;
Milan, Italy.
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PL2.1
Early-Onset Stroke and Vasculopathy Associated with Mutations in ADA2
I. Aksentijevich1, Q. Zhou1, A. K. Ombrello1, D. Yang2, A. V. Zavialov3, R. Sood1, M. Boehm2, D.
L. Kastner1;
1
NHGRI/NIH, Bethesda, MD, United States, 2NHLBI/NIH, Bethesda, MD, United States,
3
University of Turku, Turku, Finland.
We initially observed 3 unrelated patients with fevers, livedo reticularis, vasculopathy, and early-onset recurrent ischemic strokes. We performed exome
sequencing on affected patients and their unaffected parents that pointed to
recessively inherited predicted-deleterious mutations in CECR1, encoding
adenosine deaminase 2 (ADA2). All mutations are either novel or present at
low frequency (<0.001) in several large databases, consistent with the recessive inheritance. Candidate gene screening was done in additional 6 patients.
Nine patients shared 4/9 missense mutations in CECR1 along with conserved
haplotypes. Computer modeling based on the crystal structure of the human
ADA2 suggests that CECR1 mutations either disrupt protein stability or impair
enzyme activity. All patients had at least a 10-fold reduction in serum and plasma concentrations of ADA2, and reduced ADA2-specific adenosine deaminase
activity. ADA2 is expressed predominantly in myeloid cells and is a secreted
protein. Animal models suggest that ADA2 is the prototype for a family of growth factors (ADGFs). Although there is no murine homolog of CECR1, there are
2 zebrafish homologs, Cecr1a and Cecr1b. Knockdown of a zebrafish ADA2
homolog caused intracranial hemorrhages and neutropenia, phenotypes that
were rescued by wild type but not mutant human CECR1. Skin, liver, and brain
biopsies from patients demonstrated vasculopathic changes characterized by
compromised endothelial integrity, endothelial cellular activation, and inflammation although ADA2 is not expressed in the endothelial cells. Our data suggest that loss-of-function mutations in CECR1 are associated with a spectrum
of vascular and inflammatory phenotypes ranging from early-onset recurrent
stroke to systemic vasculopathy and/or vasculitis.
PL2.2
Disrupted auto-regulation of SNRPB causes cerebro-costo-mandibular
syndrome
We sequenced hundreds of single-cell transcriptomes to decipher the cell-tocell transcriptional variation. Starting from a homogeneous cell population of
human female primary fibroblasts from one individual, we used the C1 SingleCell-Auto-Prep-System to capture individual cells and to generate pre-amplified
cDNA for next-generation sequencing. On average, 9322 genes per single-cell
(RPKM >0.3) were detected, representing ~50% of the total genes detected by
the bulk sample containing millions of cells. We noted a wide spectrum of transcriptional heterogeneity. Correlation analysis between single-cells showed
an average coefficient of 62% (0.3-0.9). A large number of detected genes are
cell-specific with gene mRNA levels variable between single-cells. Moreover, we
identified cell-specific novel exons, multitude of alternative spliced isoforms
and 3UTR isoforms due to alternative polyadenylation. Hence, 1% of exons
showed difference for exon inclusion between single-cells and 10% of 3UTR
contained alternative transcript termination sites.
To further assess the differential allelic expression at the single-cell level, WGS
has been performed on this sample. Our data revealed that both alleles are actively transcribed for most of the detected genes. Interestingly, we observed a
skewed monoallelic distribution in single-cells in a given snapshot of time. Indeed, for most of the genes, we detected one transcribed allele at the time in an
individual cell. For a specific gene, rare are the individual cells expressing both
alleles simultaneously. The results were validated in fibroblasts from a second
individual. We will also provide a comprehensive survey of imprinted genes, X
inactivation and escape.
S.E.A and E.T.D. laboratories contributed equally.
PL2.5
Chromosome X-wide association analysis discovers new loci for complex
traits including a height locus not dosage compensated between men
and women
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In 2013, the American College of Medical Genetics and Genomics (ACMG) issued Recommendations for incidental findings in clinical exome and genome
sequencing. The Recommendations were written by a Working Group of 14 medical geneticists, laboratory geneticists, genetic counselors and ethicists who
met regularly for 14 months to draft them. The draft Recommendations were
presented for commentary in a public forum at the 2012 Annual Meeting of the
ACMG, reviewed by 15 additional experts, and reviewed several times by the
Board of the ACMG before being issued in March, 2013. The Recommendations
M. Kriek;
Clinical Geneticist, Department of Clinical Genetics, Department of Human Genetics, Leiden
University Medical Center, Leiden, Netherlands.
In 2013 the American College of Medical Genetics and Genomics (ACMG) published a list of 56 genes that are proposed specific targets of a search for pathogenic variants in both children and adults (Green et al.). These genes are associated with diseases with an indication for immediate medical intervention of
the patient or of a family member. The publication asserts that, in principle, the
necessary patient data are available following a NGS procedure and therefore
cannot be ignored. For purposes of clarity, the vast majority of these variants
are removed during the various filtering stages of the normal trio analysis process and that identification of these variants will require additional actions.
During the American Society of Human Genetics meeting 2013, it was argued
that consensus on a basic list of clearly pathogenic variants in the relevant genes is essential if this form of diagnosis is to be implemented in genetic diagnostics. A disadvantage of the approach as proposed by the ACMG (analysis of the
entire gene, rather than focusing on known pathogenic gene variants) is that
variants will be detected that have not been previously reported and/or are
of undefined pathogenicity. This could result in considerable distress for the
patient and family. To avoid this, only selected variants in the genes nominated
by the ACMG will be analysed.
Another point of discussion is whether conditions that manifest later in life
should be investigated in children. While the ACMG is in favour, Ploem et al. are
of a different opinion: When it comes to the unsolicited findings of NGS diagnostics in a young child, the interests of the child prevail over any wishes of the
parents (not) to receive certain information. (NTVG 2014;158:A6757). This is
consistent with ESHG recommendations (EJHG 2013;21:580-4)).
In case of testing minors, guidelines need to be established as to what unsolicited
information should be disclosed in order to balance the autonomy and interests of
the child and the parental rights and needs (not) to receive information that may
be in the interest of their (future) family
A solution to these conflicting interests is as follows: where the index patient
is a minor, a genetic predisposition for late onset diseases for which immediate medical treatment is indicated will only be sought in parents. This is made
possible by the setup of trio analysis in which variant lists are available for each
parent.
Genetic variants that result in clinical manifestations that in require direct intervention during childhood will be analysed in the index patient, even when
a minor.
PL3.4
Debate: Whole-genome sequencing in health care: proceed with caution
and avoid incidental findings
M. C. Cornel;
VU University Medical Center, Amsterdam, Netherlands.
Back to index
M. Capecchi;
Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, UT,
United States.
Gene targeting provides the means for modifying any gene in any desired manner in an intact, living animal, the mouse. This technology permits the evaluation of the function of genes in mammals. Because nearly all biological phenomena are mediated or influenced by the activity of genes, this methodology
permits the analysis of the most complex biological processes such as development, learning, normal and aberrant behavior, cancer, immunology and a multitude of congenital human diseases. In the past, gene targeting has been used
primarily to generate knockout mice, that is mice in which the chosen gene has
been disrupted in the germline, such that every cell in the mouse carries the
mutations. This methodology permits evaluation of the function of that gene in
the intact mouse. However, many, if not most, genes have multiple functions. If
one of those functions is critical to the survival of the mouse, then subsequent
functions of that gene in the mouse cannot be evaluated by the above means.
This problem can be circumvented by generating conditional mutations, that
restrict the effects of the mutation temporally, spatially (to defined sets of cells
or tissue), or both. In mice, conditional mutagenesis is achieved by combining
gene targeting, which is achieved through homologous recombination, with a
site-specific recombination system, such as Cre/loxP or Flp/FRT. Cre and Flp
are recombinases that mediate recombination between specific, short DNA sequences, loxP and FRT, respectively. Gene targeting is used to flank your chosen
gene with either loxP or FRT sites in the mouse germline. By restricting the
production of the Cre or Flp recombinase to either a specific set of cells, a chosen temporal period, or both, within the developing or adult mouse, the gene
is only excised from the genome of the mouse, in those selected cells, during
that chosen temporal period, or both. I will describe a number of applications
of gene targeting and conditional mutagenesis derived in our laboratory that
address interesting biological questions from modeling human cancer to neuropsychiatric disorder in the mouse.
PL5.1
Signatures of Mutational Processes in Human Cancer
M. Stratton;
Wellcome Trust Sanger Institute, Hinxton, United Kingdom.
All cancers are caused by somatic mutations. However, the processes underlying the genesis of somatic mutations in human cancer are remarkably poorly
understood. Recent large-scale cancer genome sequencing initiatives have provided us with new insights into these mutational processes through the mutational signatures they leave on the cancer genome. In this talk I will review the
mutational signatures found across cancer and consider the underlying mutational processes that have been operative.
ABSTRACTS SYMPOSIA
SYMPOSIA
S01.1
From rare disease to management of common disorders
M. Summar;
Washington, DC, United States.
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K. Kutsche;
Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg,
Germany.
Pontocerebellar hypoplasias (PCH) constitute a group of autosomal recessively inherited neurodegenerative disorders with prenatal onset. Classification based on clinical, neuroradiological and neuropathological characteristics divided PCH into eight subtypes; the underlying genetic cause has
been identified in six PCH types (PCH1, PCH2, and PCH4-6 and 8). PCH2
represents the most frequent form of PCHs and typical clinical features are
respiratory and feeding difficulties at birth, dyskinesia and chorea, severe
impairment of cognitive and motor development, progressive microcephaly, and frequent death in late infancy or early childhood. MRI imaging in
PCH2 shows brainstem and cerebellar hypoplasia, with the cerebellar hemispheres more affected than the vermis. In patients with PCH2, 4 and 5,
biallelic mutations in genes encoding three subunits of the heterotetrameric
transfer RNA (tRNA) splicing endonuclease complex, TSEN54, TSEN2, and
TSEN34 have been identified. PCH shows significant overlapping features
with microcephaly with pontine and cerebellar hypoplasia (MICPCH), generally associated with loss-of-functionCASK mutations. The classical MICPCH
phenotype can be found in females who typically have moderate to severe
intellectual disability and progressive microcephaly with pontine and cerebellar hypoplasia. Possible findings are ophthalmologic anomalies and sensorineural hearing loss. Males with a CASK mutation have also been identified. The phenotypic spectrum ranges from severe intellectual disability
and MICPCH, or early-infantile epileptic encephalopathy to mild X-linked
intellectual disability (XLID) with or without nystagmus. I will present an
overview on clinical and genetic data of patients with PCH and MICPCH and
how to discriminate the two conditions. My focus will be on the different
CASK mutations in females and males and their associated phenotypes to
help understanding genotype-phenotype relationships.
S03.2
The neurobiology of lissencephal
A. Wynshaw-Boris;
Cleveland, OH, United States.
S03.3
Neuronal migration defects associated with mutations in tubulins and
MTrelated proteins
L. Broix, K. Poirier, Y. Saillour, N. Bahi-Buisson, J. Chelly;
Cochin Institute, INSERM Unit 1016, CNRS UMR 8104, ParisDescartes University,
Paris, France.
Neuronal migration disorders such as malformations of cortical development are frequent causes of epilepsy and intellectual disability. Disrupted
biological and cellular processes such as neuronal progenitors proliferation,
neuronal migration, and cortical organization, are traditionally used as a
basis for the classification of MCD. Etiologies of these disorders are heterogeneous and genetic studies in humans and mice have identified a spectrum
of mutations in genes involved in an array of crucial processes that often
disrupt the development of the cerebral cortex. Aside from genes such as
ARX, GPR56 and WDR62, the importance of genes encoding cytoskeletal
proteins has become evident. For example, mutations in DCX and LIS1, both
of which encode proteins involved in MT homeostasis, are associated with a
large spectrum of neuronal migration disorders. Moreover, MCD associated
with mutations in - or -tubulin-encoding genes such as TUBA1A, TUBB2B,
TUBB3 and TUBB5, have been also described. These tubulin-related cortical dysgenesis are thought to involve a combination of abnormal neuronal
proliferation, migration and differentiation. More recently, we reported the
association between mutations in TUBG1, DYNC1H1, KIF2A and KIF5C, and
diverse forms of malformations of cortical development with or without
microcephaly. We further showed that the mutations in these MTs-related
proteins: KIF5C, KIF2A and DYNC1H1 affect ATP hydrolysis, productive protein folding and microtubule binding, respectively. In addition, we showed
by in utero electroporation that in vivo downexpression by shRNA of mouse,
Tubb3 and Tubg1, as well as expression of Kif2a mutants, interferes with
proper neuronal polarization and migration, morphogenesis and intermediate progenitor proliferation.
Altogether, these findings together with literature data support the hypothesis that proliferation and migration are genetically and functionally interdependent. Finally, they reinforce the importance of centrosomal and
ABSTRACTS SYMPOSIA
MT-related proteins in cortical development and strongly suggest that MTdependent mitotic and post-mitotic processes are major contributors to the
pathogenesis of MCD.
S04.1
Disease, networks and epistasis
C. Webber;
Neurological Disease Genomics, MRC Functional Genomics Unit, Department of
Physiology, Anatomy & Genetics, Oxford University, Oxford, United Kingdom.
I will give an overview of our recent work in identifying the pathways and
processes underlying complex disorders, illustrating how different functional genomics resources can each provide novel biological insights into the
same phenotype-influencing gene network. The topologies of the identified
networks can identify pathway loading (both additive and epistatic) along
with the direction in which the pathway is perturbed, thereby inviting drug
repurposing. I will also illustrate some of the novel integrative approaches
that weve been applying in GWA/exome studies and used to determine the
significance of a functionally-clustered genome.
S04.2
Understanding molecular mechanisms of human disease mutations
and coding variants through 3D protein networks
H. Yu;
Ithaca, NY, United States.
To better understand the molecular mechanisms and genetic basis of human disease, we combined the massive scale of network systems biology
with the supreme resolution of traditional structural biology to generate
the first comprehensive atomic-resolution 3D interactome-network comprising 3,398 interactions between 2,890 proteins with structurally-defined
interface residues for each interaction. We found that disease mutations are
significantly enriched both among interface residues and other non-interface ones within the same domains, contradicting the previous assumption
that only a few interface residues are mutation hot spots for disease. We
further classified 94,476 disease-associated mutations according to their inheritance modes and found that the widely-accepted guilt-by-association
principle does not apply to dominant mutations. Furthermore, recessive
truncating mutations on the same interface are much more likely to cause
the same disease, even if they are close to the N-terminus of the protein,
indicating that a significant fraction of truncating mutations can generate
functional protein products.
S04.3
From protein networks to disease mechanisms
R. Sharan;
Tel Aviv University, Department of Computer Science, Tel Aviv, Israel.
S05.2
24 chromosome copy number analysis for preimplantation genetic
screening
A. H. Handyside;
Illumina, Cambridge, United Kingdom.
S05.3
Preimplantation genetic diagnosis
Back to index
T. Voet;
Leuven, Belgium.
D. R. M. Timmermans1,2;
1
Department of Public and Occupational Health, EMGO Institute, VU University
Medical Center, Amsterdam, Netherlands, 2National Institute for Public Health and the
Environment, Bilthoven, Netherlands.
Risk communication is an essential component of genetic counseling . Genetic testing and providing information about genetic predisposition may
enable early disease detection, targeted surveillance, and may lead to effective prevention strategies and behavioral change. However, the impact of genetic information on peoples perceptions may be limited. Most people are
unfamiliar with probabilistic thinking and find it hard to understand risks.
Moreover, risks are experienced differently depending on the characteristics
of the risk. A risk is more than a number. It is not only the probability, the
quantification of the uncertainty, but also the nature of the risk, the severity
of the consequences and the degree of control what matters. People process
and evaluate information about potential risks in two different ways: analytical-rational if possible, but always intuitive-affective. The characteristics
of the risks as well as the way risk information is processed impact peoples
understanding and perception of risks. This perception may be in discordance with the way experts perceive genetic risks. In order to enable people
to make informed decisions, information about (genetic) risks should not
only be adequate but should also be in line with peoples perceptions or
mental model. Discrepancies with peoples mental model of genetic risks as
well the abstractness of the risk information may hamper peoples informed
decision making. In this presentation I will discuss the factors affecting the
perception of health risks and genetic risks in particular, the way people
understand the risks communicated to them and what this means for risk
communication
S06.2
Risk perception: what could be at stake in multiple genetic testing?
C. M. Julian-Reynier;
Institut Paoli-Calmettes, UMR912 Inserm, Marseille, France.
First the concept of risk perception will be introduced, highlighting its interest for clinical practice or various research fields. We will focus on associated
factors and in particular on the potential role of emotions in risk perception.
Then the evidence for the relevance of previous findings will be reviewed in
the context of genetic risk assessment and genetic test result disclosure such
as investigated in clinical genetics/genetic counseling. Different application
fields such as cancer genetics will be selected as illustrations of different
situational contexts. Finally the way these previous experiences and body of
knowledge could help to anticipate the potential consequences of multiple
risk information disclosure and to document specific recommendations in
the context of the process of multiple genetic testing or next generation sequencing will be discussed.
S06.3
Risk Communication Methods for Helping Patients Understand the
Risks and Benefits of Genetic Testing
A. Fagerlin;
University of Michigan, Ann Arbor VA Center for Clinical Management Research, Ann
Arbor, MI, United States.
ABSTRACTS SYMPOSIA
S07.1
Gene therapy of human genetic diseases with AAV vectors
A. Auricchio;
Telethon Institute of Genetics and Medicine (TIGEM), Napoli, Italy.
M. De Luca;
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche, Modena, Italy.
Adult stem cells are cells with a high capacity for self-renewal that can produce terminally differentiated progeny. Stem cells generate an intermediate
population of committed progenitors, often referred to as transit amplifying
(TA) cells, that terminally differentiate after a limited number of cell divisions. Human keratinocyte stem cells are clonogenic and are known as holoclones. Human corneal stem cells are segregated in the limbus while limbalderived TA cells form the corneal epithelium. Self-renewal, proliferation and
differentiation of limbal stem cells are regulated by the Np63 (, and ),
C/EBP and Bmi1 transcription factors. Cultivated limbal stem cells generate sheets of corneal epithelium suitable for clinical application. We report
long-term clinical results obtained in an homogeneous group of 154 patients
presenting with corneal opacification and visual loss due to chemical and
thermal burn-dependent limbal stem cell deficiency. The corneal epithelium and the visual acuity of these patients have been restored by grafts of
autologous cultured limbal keratinocytes. In post hoc analyses, success was
associated with the percentage of p63-bright holoclone-forming stem cells
in culture. Graft failure was also associated with the type of initial ocular
damage and postoperative complications. Mutations in genes encoding the
basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder.
Epidermal stem cells transduced with a retroviral vector expressing the 3
cDNA can generate genetically corrected cultured epidermal grafts able to
permanently restore the skin of patients affected by LAM5-3-deficient JEB.
The implication of these results for the gene therapy of different genetic skin
diseases will be discussed.
S07.3
Therapeutic targeting of Phosphatidylinositol-3-kinase/AKT/mTOR
signalling in segmental overgrowth disorders
R. Semple;
University of Cambridge, Metabolic Research Laboratories, Addenbrookes Hospital,
Cambridge, United Kingdom.
S08.2
Insights into European genetic history at fine geographic scales using
haplotype-based approaches
S. Myers;
Dept of Statistics, Oxford University, United Kingdom
Wellcome Trust Centre for Human Genetics, Oxford University, United Kingdom
Modern genetic datasets are revealing features of our genetic history, and
how this has shaped our genomes, in unprecedented detail. Across Europe,
multiple invasions and migrations have taken place over the past several
millennia, but the possible genetic effects of these and of subsequent regional isolation remain uncharacterised, partly due to the extremely subt-
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Hypertrophic cardiomyopathy was one of the first monogenic cardiovascular disorders to be understood at the molecular level. Twenty years after
the discovery of the first disease gene, HCM is still seen principally to be a
disease of the sarcomere. At the biophysical level, the contractile protein
mutations that cause HCM are seen to be activating in that they enhance
Ca(2+) sensitivity, maximal force production, and ATPase activity. These
defects then entrain secondary disturbances of energy deficiency and altered Ca(2+) handling that appear to be major common paths leading to the
phenotype of hypertrophy and risk of sudden cardiac death. Importantly,
these functional consequences of HCM mutations may lend themselves to
specifically targeted approaches to disease modifying therapy for HCM, and
phase I/II clinical trials have been completed or are underway.
In contrast, dilated cardiomyopathy is caused by mutations in genes encoding many different classes of proteins with very diverse roles in cardiomyocyte function, e.g. ranging from the nuclear envelope through to the
contractile and force transduction apparatus. This indicates that the DCM
phenotype is the end result of disparate pathways that lead to myocyte loss
and fibrous replacement, suggesting that finding broadly applicable approaches to disease-modifying therapy may be difficult. In the subset of DCM
caused by sarcomeric mutations the picture is clearer, and may lead to approaches to therapy, as DCM alleles result in functional changes that are the
ABSTRACTS SYMPOSIA
opposite of the changes seen with HCM alleles: depressed motor function
and reduced Ca(2+) sensitivity.
S09.2
Mendelian Randomization
M. V. Holmes;
University of Pennsylvania, Philadelphia, PA, United States.
P. Charron1,2,3;
1
Depart. of genetics, Piti-Salptrire hospital, AP-HP, Paris, France, 2INSERM UMR1166,
Paris, France, 3University UPMC Paris 6, Paris, France.
E. Cuppen1, W. Kloosterman2;
1
Hubrecht Institute and Center for Molecular Medicine UMC Utrecht, Utrecht,
Netherlands, 2Center for Molecular Medicine UMC Utrecht, Utrecht, Netherlands.
De novo genomic rearrangements are a common cause of congenital abnormalities and mental retardation. However, in the majority of patients a
molecular mechanism linking rearranged chromosomes to disease phenotype is lacking. This is particularly true for ultra-complex genomic rearrangements, such as those resulting from catastrophic chromosome shattering
termed chromothripsis. We developed a family-based approach for dissecting the functional consequences of (complex) genomic rearrangements in
patients with congenital disease. This approach combines gene-expression
and chromatin profiling with functional studies in patient-derived cell lines
and model organisms. We highlight two cases where family-based analysis
and functional follow-up studies were instrumental to reveal the drivers of
the disease phenotype. The described methodology is key for understanding disease in a large and broad category of patients with chromosomal
aberrations of unclassified significance and thereby improves diagnosis and
clinical decision-making.
10
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S10.2
Kataegis: a mutation signature identified through whole-genome
sequencing of human cancers
Shortness of stature is one of the most common pediatric concerns and has
an incidence of 3 % in the general population. In the majority of patients
with idiopathic growth deficit the etiology remains elusive in the absence of
morphological details. This unknown etiology prevents a sufficient medical
care in most cases.
As it has been proposed that the growth fundamentally regulated by genetic factors, GWAS found significant evidence for both single nucleotide and
copy number polymorphisms associated with height variation in the general
population. However, these associations explain only a small fraction of the
overall variability of human height.
Based on the early identification of SHOX gene deletions as a common cause
ABSTRACTS SYMPOSIA
of idiopathic and syndromic (Leri-Weil syndrome) short stature as well as
copy number variation (CNV) as a common cause of intellectual disability,
the hypothesis of a rare variant - frequent disease hypothesis seemed to be
feasible for short stature. To address this hypothesis we thoroughly build a
study group of more than 400 families with idiopathic short stature and conducted SNP array analysis to demonstrate the presence of CNVs as a common
underlying cause of short stature. Molecular karyotyping was performed and
CNVs of a minimum size of 50kb scored and compared to healthy controls.
Based on this technique we found a significant odds ratio for aberrations
above 100 kb only. Due to the number of potential disease causing CNVs a
gene-centric analysis comparing known CNVs, gene functions, tissue expression and murine knock-out phenotypes was neccessary. We confirmed that
10 % of the patients had de novo and inherited CNVs in agreement to the
segregation of the short stature phenotype in the families. These CNV regions
include known microdeletion/duplication loci expanding the phenotypical
spectrum of these entities. The pathogenicity of novel loci was substantiated
by comparison to available information, especially the overlap with loci of
genome wide association for short stature. Our data showed a clear connection between the prenatal onset of short stature as well as the severity of
the growth deficit with the likelihood of the identification of causal CNVs.
Thus, we confirmed CNVs as a main cause of idiopathic short stature. Further
improvement of the array technology as well as the application of CNV identification based on next generation sequencing will lead to a more elaborate
and detailed view on even smaller CNVs. Application of these methods can
help to illuminate the complex heterogeneity of short stature.
S12.1
The Epigenetic Basis of Common Human Disease
A. P. Feinberg;
Johns Hopkins University, Baltimore, MD, United States.
Although epigenetic changes in the cancer genome have been known for 3
decades, the role of epigenetics in common human disease generally and
its relationship to genetic variation has only recently begun to be explored.
We have been developing whole-genome approaches to epigenetic analysis
of human disease. Surprising results have been the discovery of CpG island
shores and large hypomethylated blocks corresponding to nuclear lamina
/ heterochromatin lysine-modification regions (LOCKs), and accounting for
the vast majority of epigenetic changes in cancer. We have also been contributing to a new field of epigenetic epidemiology that integrates genetic, epigenetic, and environmental factors, and we are applying these approaches
to the study of autoimmune and neuropsychiatric disease. One of the most
exciting developments in this recent work is the idea that epigenetic plasticity under genetic control may itself confer a survival advantage in evolution, and may also be important in normal tissue differentiation and response
to the environment. In support of this idea, we have identified variably methylated regions (VMRs) in normal development. These same VMRs appear
to be targets for disrupted methylation in cancer, pointing to a unifying model of cancer in which epigenetic dysregulation allows rapid selection for
tumor cell survival at the expense of the host. We have also identified genetic variants that increase epigenetic plasticity in patients with autoimmune
disease. Thus, will need to integrate genetic and epigenetic analysis in common disease risk assessment, prevention, and treatment, and also consider
the role of epigenetic plasticity itself in disease pathogenesis.
S12.2
Intergenerational epigenetic programming in a mouse model of
undernutrition
A. C. Ferguson-Smith;
University of Cambridge, Department of Genetics, Cambridge, United Kingdom.
Environmental factors during early life are critical for the later metabolic
health of the individual and of future progeny. In a mouse model of maternal
caloric restriction during pregnancy the metabolic physiology of offspring
over two generations is affected, including via paternal transmission to the
second generation. Here we explore whether the paternal experience of in
utero undernutrition, with an impact on the health of his offspring, is an
epigenetically inherited memory transmitted via his sperm methylome.
S12.3
Cancer Genetics and Epigenetics: Two Sides of the Same Coin?
P. A. Jones;
Chief Scientific Officer, Van Andel Research Institute, Grand Rapids, MI, United States.
Back to index
Noninvasive prenatal testing (NIPT) for Down syndrome (DS) using massively
parallel sequencing of maternal plasma DNA facilitates early detection of affected fetuses. If NIPT is performed at ~ 12 weeks of gestation it creates a potential 28-week window of opportunity in which to treat the fetus by orally administering small molecules to the mother. Our laboratory is using a 4 phase translational approach that involves human biomaterials (amniocytes and amniotic
fluid), a mouse model of DS, and living human fetuses. In phase 1 we compared
the transcriptome of fetuses with and without DS by analysis of cell-free RNA
in amniotic fluid and showed that oxidative stress was a significant difference
in the affected fetuses. In phase 2 we uploaded differentially-regulated genes
into the Connectivity Map to identify candidate FDA-approved therapeutic
molecules. In phase 3 drugs that had high efficacy in negating oxidative stress
and showed low toxicity were selected for further studies in an animal model.
We use the Ts1Cje mouse model of DS because affected males are fertile, yet
11
ABSTRACTS SYMPOSIA
cognition is significantly impaired. We can therefore use wild type females for
the treatment experiments, ensuring both a normal intrauterine environment
and normal postnatal nurturing behavior. Treated and untreated affected pups
and littermate controls are then evaluated using a variety of brain studies that
include analyses of gene expression, histology, cellular proliferation and migration, and neurobehavior. In parallel, in phase 4 we are preparing for a human
clinical trial by analyzing fetal brain growth using quantitative fetal magnetic
resonance imaging (MRIs). To date, we have identified significant phenotypic
differences in Ts1Cje embryos and neonates as endpoints to evaluate therapy. We have also shown encouraging, statistically-significant improvement in
some neurobehavioral tests in adult mice. These results suggest that prenatal
treatment in DS is an achievable goal.
S13.3
Clinical and social implications of NIPT
K. E. Ormond;
Stanford University School of Medicine, Stanford, CA, United States.
J. OHalloran1, J. Tyson2;
1
Newcastle upon Tyne, United Kingdom, 2QuantuMDx, Newcastle upon Tyne, United
Kingdom.
N. Michalski1,2,3, C. Petit1,2,3,4,5;
1
Unit de Gntique et Physiologie de lAudition, Institut Pasteur, Paris, France, 2UMRS
1120, Institut National de la Sant et de la Recherche Mdicale (INSERM), Paris, France,
3
Sorbonne Universits, UPMC Univ Paris 06, Paris, France, 4Syndrome de Usher et autres
Atteintes Rtino-Cochlaires, Institut de la vision, Paris, France, 5Collge de France, Paris,
France.
The biochemical study of the components and associated molecular networks of the auditory sensory cells, the hair cells, are strongly impeded by
the small number of these cells. In contrast, human and mouse genetic approaches have proven to be powerful to identify such components.
The presentation will focus on the hair bundle of the hair cells that operates
the mechano-electrical transduction (MET). This sensory antenna is comprised of several rows of rigid microvilli, known as stereocilia of a few femtoliters in volume, that are organised in a staircase pattern. The MET process
relies not only on the MET machinery but also on the proper architectural
and biophysical properties of the hair bundle. In addition, any defect that
affects the mechanical elements involved in sound-induced oscillation of the
12
Back to index
hair bundle or its ionic environment necessary to drive hair cell depolarization, also perturbs MET. For each of these contributors to the MET, several
key components have been identified by studying inherited deafness forms;
they are used as entry points to decipher the involved molecular networks.
The presentation will illustrate how the genetic approach led to the discovery of molecules controlling the development, maturation and maintenance
of the correct structure of the hair bundle and the MET process. It will illustrate how it enlightens our understanding of the way these proteins form
molecular complexes in vivo and how these complexes work and cooperate
together. A special emphasis will be put on the networks composed of proteins encoded by the genes responsible for the Usher syndrome, the most frequent cause of hereditary sensorineural deafness associated with retinitis
pigmentosa. Usher genes are critically involved in hair bundle development
and the MET machinery as well as the functioning of the photoreceptor
cells. Recent results based on mouse and human genetics, which provide
evidence for a molecular maturation of the auditory MET machinery, will
be discussed.
S15.2
Genes and cellular pathway of Fanconis anemia
J. Surralls;
Group of Genome Instability and DNA Repair, Department of Genetics and Microbiology,
Universitat Autnoma de Barcelona (UAB) and Center for Biomedical Network Research
on Rare Diseases (CIBERER), Barcelona, Spain.
Fanconi anemia (FA) is a rare genetic disease characterized by bone marrow failure, malformations, chromosome fragility, hypersensitivity to DNA
interstrand cross-linking (ICL) chemotherapy and a high predisposition to
cancer, including leukemia and solid tumors such as head-and-neck and
gynecological carcinomas. The only cure of the hematological disease, which
is the major cause of early death, is bone marrow transplantation using an
HLA compatible donor. Those families with unavailable donor rely on preimplantation genetic diagnosis with selection of an HLA related embryo and
in the future implementation of advanced gene and cell therapies. All these
novel therapeutic applications to human health are further complicated by
the fact that at least 16 genes, from FANCA to FANCQ, are involved in this
disease and their products interact in a complex genome stability and tumor suppression network. Notably, 4 out of 16 FA genes (FANCD1/BRCA2,
FANCN/PALB2, FANCJ/BRIP1 and FANCO/Rad51C) are breast cancer susceptibility genes in otherwise unaffected mutation carriers. Therefore, the
discovery of novel Fanconi anemia genes is important not only for the affected patients but also for the general population. In this context, I will present data on the discovery of a novel Fanconi anemia gene by whole exome
sequencing. Interestingly, this gene (ERCC4 o FANCQ) is involved not only in
FA but also in xeroderma pigmentosum, a melanoma susceptibility disorder
with defective nucleotide excision repair (NER), and in XFE-type progeria.
Our genetic and biochemical results indicate that depending on the balance
between ICL repair and NER, mutations in the same gene lead to clinically
three distinct disorders.
S15.3
Analysis of signalling pathways in Tbx1 mutants identifies a novel
mechanism in coronary artery Morphogenesis
ABSTRACTS SYMPOSIA
CXCR4 pathway in some of the neurological deficits observed in chromosomally engineered mouse models of 22q11DS, suggesting multiple, independent roles for this pathway in 22q11DS.
S16.1
SINEUPs: a new functional class of antisense non-coding RNAs that
activate translation
Back to index
S17.1
Cancer genetic heterogeneity: implications for therapy
responsiveness and acquisition of therapy resistance
A. Bardelli;
Candiolo, Italy.
S. Gustincich;
Trieste, Italy.
S16.2
Molecular function of the repetitive (epi)genome in normal
physiology and in disease
D. Gabellini;
Dulbecco Telethon Institute and San Raffaele Scientific Institute, Milan, Italy.
Only about 1% of the genome encodes for the ~20000 human proteins,
which are similar in number and largely orthologous to those found in organisms of significant lower complexity. On the contrary, the proportion of non
protein-coding DNA has increased with developmental complexity reaching
98.5% in humans. Interestingly, up to two thirds of the human genome is
composed of non protein-coding repetitive sequences. Furthermore, a significant portion of the epigenetic modifications is present in these regions and
DNA repeats are dynamically transcribed in different cells and developmental stages producing a vast pool of non protein-coding RNA (ncRNA) molecules. Thus, ncRNAs produced by DNA repeats may hold the key to understanding the regulatory complexity inherent in advanced biological networks.
Long ncRNAs (lncRNAs) represent the most numerous and functionally diverse class of RNA produced by mammalian cells. Despite the growing interest on lncRNAs, they still remain poorly explored in terms of biological relevance, cellular function, mechanism of action and involvement in disease.
We have recently contributed to this field through the identification of the
first activating lncRNA involved in a human genetic disease: facioscapulohumeral muscular dystrophy (FSHD).
FSHD is one of the most important genetic diseases affecting the skeletal muscle. It is an autosomal dominant disorder with a strong epigenetic
component. Unlike the majority of genetic diseases, FSHD is not caused by
mutation in a protein-coding gene. Instead, the disease is associated with a
reduced copy number of the D4Z4 macrosatellite repeat mapping to 4q35.
Despite years of intensive research, the molecular pathogenesis of FSHD
remains largely unknown. We recently identified DBE-T, a chromatin-associated lncRNA produced preferentially in FSHD patients. DBE-T mediates a
Polycomb to Trithorax epigenetic switch at the FSHD locus, driving chromatin remodeling and transcription of FSHD candidate genes.
Here, I will discuss our recent results regarding the regulation of DBE-T expression, the mechanism responsible for DBE-T tethering to chromatin and
how this lncRNA regulates the epigenetic status of the FSHD locus.
S16.3
The SMN complex: RNA processing and motor neuron disease
L. Pellizoni1,2;
1
Department of Pathology and Cell Biology, Columbia University, New York, NY, United
States, 2Center for Motor Neuron Biology and Disease, Columbia University, New York,
NY, United States.
At the post-transcriptional level, expression of protein-coding genes is controlled by a series of RNA regulatory events including nuclear processing of
primary transcripts, transport of mature mRNAs to specific cellular compartments, translation and ultimately, turnover. These processes are orchestrated through the dynamic association of mRNAs with RNA binding proteins
and ribonucleoprotein (RNP) complexes. Accurate formation of RNPs in vivo
is fundamentally important to cellular development and function, and its impairment often leads to human disease. The survival motor neuron (SMN)
protein is key to this biological paradigm: SMN is essential for the biogenesis
of various RNPs that function in mRNA processing, and genetic mutations
leading to ubiquitous SMN deficiency cause the neurodegenerative disease
spinal muscular atrophy (SMA). I will discuss the expanding role of SMN in
the regulation of gene expression through its multiple functions in RNP assembly and advances in our understanding of how disruption of SMN-dependent RNA processing pathways can cause motor neuron disease.
S17.2
Non-cell autonomous interactions promote sub-clonal heterogeneity
Cancers arise and progress due to the underlying Darwinian somatic evolution. It has been commonly accepted that biological and clinical behaviors of individual tumors reflect properties of the most abundant cells that
have achieved clonal dominance by virtue of carrying the most advanced
complement of oncogenic driver mutations. However, the recent influx of
data from tumor genome sequencing has revealed a remarkable degree of
genetic divergence within tumors, including sub-clonal differences in mutational status of key driver genes. This clonal heterogeneity begs obvious
questions: i) what are the mechanisms that enable co-existence of genetically distinct populations, and ii) what are the biological consequences of
this co-existence? Microenvironmentally constrained tumors represent a
particularly interesting scenario: overcoming the bottleneck requires changes in the tumor environment induced by secreted factors, but it is not clear
a priori whether the expression of a secreted factor capable of driving tumor
outgrowth can provide an autonomous fitness advantage. To address this
scenario, we have developed a mouse xenograft model of microenvironmentally-constrained tumors. Using this model, we have interrogated the impact
of sub-clonal expression of secreted factors in contexts of either competition
against the parental clone or that of polyclonal tumors. We found that tumor
progression can be driven non-cell autonomously by a sub-population of
cells that is unable to achieve clonal dominance. This non-cell autonomous
driving of tumor growth is predicted to stabilize sub-clonal heterogeneity
within tumors throughout clinically relevant time frames. This co-existence
of biologically distinct sub-populations enables inter-clonal interactions
that can affect clinically important phenotypes
S17.3
Circulating tumor cells: Detection, biology and clinical implications
K. Pantel;
Institute of Tumor Biology, University Cancer Center Hamburg, University Medical
Center Hamburg Eppendorf, Hamburg, Germany.
13
ABSTRACTS SYMPOSIA
Back to index
S18.1
Glyco-lipophobia: association with disorders of glycolipid and
glycosylphosphatidylinositol anchor synthesis
S19.2
Using transcriptome sequencing to understand mechanisms of
disease
Thankfully, DNA and protein sequencing technologies revolutionized genetics and proteomics. Sadly, the two other macromolecules, carbohydrates
and lipids, missed that technical revolution creating a malady called Glycolipophobia. Perhaps its development was predictable based on their molecular complexity, template-independent biosynthesis, and structural analysis without having defined functions. Fortunately, this condition is treatable,
and given the discovery of over 100 genetic disorders in these pathways,
treatment should not be delayed.
Glycosylation related genes comprise 1-2% of the human genome and
those genes often fall into a series of distinct (sometimes overlapping) pathways. Each pathway generates sugar chains (glycans) for typical clients.
The best-known pathway is N-glycosylation found in secreted proteins,
membrane-bound receptors, and signaling molecules. Some glycosylation
disorders involve addition of glycans to lipids, not proteins. A few glycolipids are biosynthetic precursors of other pathways, but two define distinct
pathways: glycosylphosphatidylinolsitol (GPI) anchors and glycosphingolipids (GSL). These molecules are often located in lipid rafts. Fifteen distinct
disorders disrupt these pathways. All cells contain GPI and GSL, and affected
patients display a range of mostly severe phenotypes that can include intellectual disability, seizures, ophthalmologic abnormalities, heart defects, and
often plasma hyper- or hypo-phosphatasia. Simple biochemical indicators
can help identify likely candidate genes from exome sequencing, and then
also confirm those candidates. Cellular assays based on the established pathways can help design potential therapeutic screens.
Understanding the biosynthetic pathways not only suggests, and then confirms, candidate genes, but it creates avenues for therapy, based on the restoration of functional readouts. The powerful partnership of biochemistry
and genetics can help eradicate or reduce Glyco-lipophobia. Patients and families have a vested interest in that victory: they need us to collaborate and
cooperate, regardless of the target molecules and genes.
Supported by The Rocket Fund and R01DK55615
H. H. Freeze;
Sanford-Burnham Medical Research Institute, La Jolla, CA, United States.
S18.2
Disorders of phospholipids, sphingolipids and fatty acids biosynthesis
F. Mochel;
Paris, France.
T. Lappalainen;
New York Genome Center, New York, NY, United States.
S19.3
High resolution genetic analysis to detect variants associated with
quantitative traits and diseases in the founder Sardinian population
F. Cucca;
Sassari, Italy.
S18.3
Update on lipidomic approaches in disorders affecting complex lipids
metabolism: the example of cardiolipin
F. M. Vaz;
Academic Medical Center, Laboratory Genetic Metabolic Disease, Amsterdam,
Netherlands.
14
other modulators, including FLI1, whose haploinsufficiency due to 11q23ter deletions determines TCPT or JBS. In case of mutations of GATA1 or
GFI1B, another two hematopoietic transcription factors, thrombocytopenia
associates with red cells defects and reduction of alpha-granules. Absence
of alpha-granules resulting typical gray appearance of platelets is a pathognomic feature of GPS. NBEAL2, the causative gene in this form, is likely to
function in vesicle trafficking and generation of platelet granules.
The largest group of ITs could be related to defects of proplatelet formation,
a process involving dynamic reorganization of the cytoskeleton, signaling
pathways or apoptosis. Regarding the first aspect, mutations in genes encoding for components of the cytoskeleton (MYH9, ACTN1, TUBB1, WAS or
FLNA) are likely to interfere with the correct proplatelet extension and platelet release. Of note, FLNA interacts with the C-terminus of GPIbalpha, one
subunits of the von Willebrand factor receptor (GPIb/IX/V). Alterations of
GPIb/IX/V cause BSS characterized by mild thrombocytopenia associated
with reduction of platelet aggregation. Finally, defects of cytochrome c
(CYCS) could be associated with increased apoptosis and premature release
of platelets into bone marrow instead of blood stream.
This heterogeneity makes the molecular diagnostic testing a complex and
time-consuming process. Next generation sequencing strategies will have
a strong impact in the diagnosis of the known forms and cloning novel IT
genes.
Table 1. Features of inherited thrombocytopenias classified according to
possible defective processes of megakaryopoisis and platelet production.
ES1.1
Genetics of familial forms of thrombocytopenia
A. Savoia1,2;
1
Department of Medical Sciences, University of Trieste, Trieste, Italy, 2IRCCS Burlo
Garofolo, Trieste, Italy.
Inherited thrombocytopenias (ITs) are a heterogeneous group of rare diseases characterized by bleeding risk sometimes associated with platelet
dysfunction or other clinical features. More than twenty different forms
have been characterized (Table 1). They account for almost 50% of the IT
patients, suggesting that many forms are still unrecognizable.
The IT genes play a variety of roles in the complex process of megakaryopoisis and platelet production though their function is often unclear. In
the rare forms of CAMT, CTRUS and TAR, thrombocytopenia is severe because of absence or reduction of megakaryocytes (MKs). Whereas in TAR,
the platelet count tends to rise during life, in CAMT and CTRUS the disease
progresses to bone marrow failure. Loss of function of MPL, the receptor
of thrombopoietin, prevents production of Mks in the bone marrow of the
CAMT patients. Less certain is instead the role of HOXA11 and RBM8A in
CTRUS and TAR, respectively.
In another group of ITs there is a defect of MK maturation. In two of these
forms, ANKRD26RD and PFD/AML, patients are also at risk of developing
leukemias. Whereas the role of ANKRD26 remains obscure, RUNX1 is a master transcription factor regulating hematopoiesis. RUNX1 binds several
Disease
Abbreviation
OMIN
entry
CAMT
604498
TAR
274000
Congenital amegakaryocytic
thrombocytopenia
Amegakaryocytic
thrombocytopenia with radioulnar synostosis
CTRUS
FPD/AML
THC2
JBS
Normal
AR
RBM8A (1q21.1)
Normal
AD
ANKRD26 (10p2)
XL
GATA1 (Xp11)
AD
601399
AD
188025
600588
147791
AD
300367
GATA1RD
314050
Bernard-Soulier
syndrome
Biallelic
Monoallelic
AR
Large
Giant
GFI1B (9q34.13)
Large
MYH9RD
155100
153640,
153650,
605249
AD
MYH9 (22q12-13)
FLNARD
nd
XL
FLNA (Xq28)
Small/giant
ACTN1RD
TUBB1RD
WAS
615193
613112
301000
XLT
313900
231200
BSS
nd
VWDP
177820
CYCS-related thrombocytopenia
THC4
612004
ITGA2/ITGB3-related
thrombocytopenia
Large
210250
STSL
X-linked thrombocytopenia
Large
AD
Wiskott-Aldrich syndrome
Normal
nd
GFI1B
TUBB1-related
macrothrombocytopenia
Large deletion
(11q23-ter)
Normal
Large
GFI1B thrombocytopenia
FLNA-related thrombocytopenia
RUNX1 (21q22)
Normal
References
NBEAL2 (3p21.1)
139090
ACTN1-related thrombocytopenia
HOXA11 (7p1514)
Other features
AR
GPS
MYH9-related disease
Platelet size
MPL (1p34)
605432
188000
TCPT
Gene
Inheritance (chromosome
localization)
AR
nd
187800
AD
ACTN1 (1q24.1)
Large
AD
TUBB1 (6p21.3)
WAS (Xp11)
Small
AR
GP1BA (17p13),
GPIBB (22q11), GP9
(3q21)
Giant
XL
AD
AD
AD
AD
Back to index
Giant
Large
GPIBA (17p13)
Possiblly giant
CYCS (7p15.3)
Normal
ITGA2B (5q23-q31),
ITGB3 (17q21.32)
Large
Bottega et al.
Haematologica. 98:868,
2013
Kunishima et al. Am J
Hum Genet 92:431, 2013
May be associated with
Berrou et al. Arterioscler
periventricular nodular
Thromb Vasc Biol
heterotopia (MIM 300049)
33:e11-8, 2013
Kunishima et al. Blood
None
113:458, 2009
Severe immunodeficiency.
Mahlaoui et al. Blood
Platelets have reduced life span 121:1510, 2013
Albert et al. Bood
No or mild immunodeficiency
115:3231, 2010
Savoia et al.
Haematologica 96:417,
2011
None
Noris et al.
Haematologica 97:82,
2012
Platelet count goes down
Balduini et al. Hum Genet
under stress
131:1821, 2012
Platelet anisotropy, possible
Balduini et al. Hum Genet
defects of platelet function
131:1821, 2012
Morison et al. Nat Genet
None
40:387, 2008
Anisocytosis
15
Until the end of the last century, a few forms of inherited thrombocytopenia (IT) were known and these disorders were considered exceedingly rare.
Spontaneous bleeding was considered as a feature common to all patients
and as the main clinical consequence of these disorders. Recently, many new
forms of ITs have been identified and our view of these disorders has considerably changed. We realized that ITs are less rare than previously thought
and that most patients have mild or no spontaneous bleedings. In many
cases thrombocytopenia is discovered incidentally during adult life and its
genetic origin is missed, exposing patients to the risk of misdiagnosis with
immune thrombocytopenia. To avoid this mistake, it is therefore essential
that ITs are considered in the differential diagnosis of any thrombocytopenia of unknown origin both in children and adults. Family history, to search
for other affected family members, and physical examination, to identify the
defects that typically associate with thrombocytopenia in syndromic forms,
are useful tools in this respect, although negative results do not exclude the
genetic origin of platelet deficiency. Microscope evaluation of blood films is
another key element for differential diagnosis, as morphological anomalies
of platelets (increased or reduced size, defects of granules or vacuolization),
leukocytes (Dhle-like bodies in neutrophils) or red cells (anisocytosis) are
observable in many ITs.
Once an IT is suspected, a diagnostic algorithm can guide further investigation toward the specific patients disease. It is likely that next generation
sequencing techniques will simplify this process in the near future.
A definite diagnosis is essential for proper patient management. In some
forms of IT, the major risk for affected subjects does not derive from bleedings, but from the increased propensity to develop additional disorders, as
hematological malignancies or kidney failure. In syndromic ITs, associated
extra-hematological defects often have a much higher impact on patients
quality of life than the reduced platelet count. The identification of genotype-phenotype correlations for same disorders made feasible to identify
and quantify the risks affecting each patient and to design personalized
follow-up protocols.
A definite diagnosis is also required to personalize treatment, as platelet
transfusions are no longer the only remedy for ITs (Table 1).
Hematopoietic stem cell transplantation can cure ITs and this treatment is
the best option for a few disorders that invariably lead to death during childhood. The thrombopoietin mimetic eltrombopag is effective in increasing
platelet count in the IT induced by MYH9 mutations, the most frequent form
of IT, and this drug has been successfully used instead of platelet transfusions to prepare patients for elective surgery. Splenectomy has no role in
ITs, except for patients with WAS mutations, whose platelet counts increase
after spleen removal. Finally, effective treatments are available also for some
extra hematological defects of syndromic ITs.
Table 1. Treatments for inherited thrombocytopenias
Indications
Comments
All ITs. To stop bleedings when
Use HLA-matched donors to reduce
local measures failed or are
the risk of alloimmunization
not possible
Successfully used also in a few
Hemopoietic
- WiskottAldrich syndrome
patients with biallelic Bernard
Stem Cell
- Congenital amegakaryocytic
Soulier syndrome and life threatening
Transplantation thrombocytopenia
hemorrhages
Used instead of platelet transfusions
for preparing patients to surgery.
Eltrombopag
- MYH9-related disease
Efficacy in other conditions to be
tested
- WiskottAldrich syndrome It increases platelet count but also the
Splenectomy
- X-linked thrombocytopenia risk of infections
Experimental. A test dose is
- All mild forms of IT. To stop
recommended to identify patients who
Desmopressin bleedings and to prepare
might benefit from this treatment for
patients to surgery
future bleedings-surgery
All mild form of IT. To stop
Antifibrinolytic
Experimental (anecdotal evidence of
or prevent bleedings, and to
agents
efficacy)
prepare patients to surgery
All inherited
Recombinant
thrombocytopenias. To stop
Experimental (anecdotal evidence of
factor VIIa
bleedings when all other
efficacy). Risk of thrombosis
treatments failed
Platelet
transfusions
16
Back to index
ES2.1
Using prediction scores in cardiovascular medicine
S. Ripatti1,2,3;
1
Hjelt Institute, University of Helsinki, Helsinki, Finland, 2Institute for Molecular
Medicine Finland (FIMM), Helsinki, Finland, 3Wellcome Trust Sanger Institute, Hinxton,
United Kingdom.
A. Hoischen;
Department of Human Genetics, Radboud Institute for Molecular Life Sciences, Radboud
university medical center, Nijmegen, Netherlands, Nijmegen, Netherlands.
Over the past decade the next generation sequencing has led to a revolution in human genetics and in disease gene identification in particular. This
revolution was driven by new technologies that allowed new applications.
But whats next?
In this educational session I will address the latest technological developments, for example: What to do with more and less expensive sequencing
data? What do longer sequencing reads offer? Can we detect mosaicism reliably? I will also show new applications, for example the benefits of whole
genome sequencing, new developments for exome sequencing and also new
methods for highly multiplexed targeted re-sequencing.
ES3.2
Single cell genome and transcriptome sequencing
J. Lundeberg;
SciLifeLab, KTH- Royal Institute of Technology, Stockholm, Sweden.
Cells in a particular tissue are not identical. Instead, cells that are identical from a genomic point of view may have considerable variation in gene
expression profile and protein levels, giving rise to a heterogeneous collection of cells with different behavior and appearance. Even the genomic
DNA sequence may vary slightly between neighboring normal cells within
a tissue albeit the differences are can be significant in adjacent tumor cells.
In order to get information from single cells one need to isolate, study, and
sometimes culture them, separately. This lecture will describe some of the
advancements in genome and transcriptome analysis of single cells using
massively parallel DNA sequencing and describe in more detail an approach
to spatially identify gene expression in single cells in a tissue sections.
Next generation sequencing has revolutionized the search for disease-causing genetic alterations. Unfortunately, the task of distinguishing the handful of causative mutations from the multitude of harmless polymorphisms
remains daunting. We have developed a system that permits the study of all
types of mutations in any gene of choice. The system is based on the generation of stable human cell lines that simultaneously and inducibly express
a cDNA encoding the protein carrying the mutation of interest and shRNA
against the endogenous mRNA. In this way, the endogenous wild type protein can be replaced with a variant expressing any mutation and, if desired,
also a tag for affinity purification or visualization. In this presentation, I shall
focus on polymerase-delta and MLH1, which have been found to be mutated
in familiar and sporadic colon cancers.
ES4.2
Aging and cancer: The impact of DNA damage
J. H. J. Hoeijmakers;
Dept. of Genetics, Erasmus MC, Rotterdam, Netherlands.
Inherited defects in nucleotide excision repair (NER) removing helix-distorting DNA lesions are associated with cancer predisposition in xeroderma
pigmentosum and neurodevelopmental deficits and segmental progeria in
Cockayne syndrome and trichothiodystrophy (TTD). Mutations in single
NER genes, such as XPD, are linked with all three disorders. Various single and double NER mouse mutants reveal that the severity of specific repair defects strictly correlates with the acceleration of selective premature
aging features, whereas the type of DNA repair defect determines the kind
of progeroid symptoms and/or cancer susceptibility. Microarray, functional
and physiological studies revealed that persistent DNA damage, like caloric
restriction, down-regulates the IGF1/GH-, lacto- and thyrotropic hormonal
axes and upregulates anti-oxidant defenses, favoring maintenance at the
expense of growth. This survival response links accumulation of DNA damage and IGF1 control of life span. Micro- and mRNA expression profiling of
normal, accelerated and delayed aging also revealed a clear parallel with the
expression changes triggered by persistent transcription-blocking DNA lesions. These findings strongly support the DNA damage theory of aging. We
will present phenotypes of conditional DNA repair models targeting aging to
selected organs, parallels with Alzheimers disease and the effect of nutritional interventions on the life span of progeroid repair mutants.
ES5.1
Genomic View of Mosaicism and Disease
N. B. Spinner, L. K. Conlin;
The Childrens Hospital of Philadelphia and The Perelman School of Medicine, at The
University of Pennsylvania, Philadelphia, PA, United States.
ES5.2
Revertant mosaicism in skin disease
Back to index
M. F. Jonkman, A. M. G. Pasmooij;
University Medical Center Groningen, Groningen, Netherlands.
Revertant mosaicism (RM) refers to the co-existence of cells carrying disease-causing mutations with cells in which the inherited mutation is genetically corrected by a spontaneous post-zygotic event. RM in skin was first
reported by us in 1997 in the genetic disorder epidermolysis bullosa (EB),
which is characterized by lifelong fragile skin that easily forms blisters and
erosions. In a patient with generalized atrophic benign EB, caused by compound heterozygosity at the COL17A1 locus, we found several patchy areas
of healthy skin and provided molecular proof that the keratinocytes in the
clinically unaffected skin were corrected by a gene conversion event, and
consequently produced normal type XVII collagen. Mutations in as many as
18 genes can result in EB. Five of these genes have shown to revert: KRT14
encoding keratin 14 in EB simplex, LAMB3 encoding the 3 chain of laminin332, and COL17A1 encoding type XVII collagen in junctional EB, COL7A1 encoding type VII collagen in dystrophic EB, and FERMT1 encoding kindlin-1
in Kindler syndrome. RM was also found in other heritable skin diseases:
dyskeratosis congenita, and in ichthyosis in confetti (ichthyosis variegata)
induced by increased homologous recombination of KRT10. Similar examples of natural gene therapy by RM have been described in Bloom syndrome, leukocyte adherence deficiency type 1, Wiskott-Aldrich syndrome, and
RAG1-deficient severe combined immunodeficiency. This natural gene therapy phenomenon manifests as normal appearing skin areas surrounded
by affected skin. Although initially thought to be rare, RM is now considered
relatively common in genetic skin diseases. To address the issues relevant
to RM, we will discuss the following questions: 1) What is the incidence of
RM in heritable skin diseases? 2) What are the repair mechanisms in RM? 3)
When do the revertant mutations occur? 4) How do you recognize revertant
skin? 5) Do the areas of RM change in size? The answers to these questions allow us to acquire knowledge on these reverted cells, the mechanisms
of RM, and utility of the reverted cells to the advantage of the patient. The
revertant skin could potentially be used to treat the patients own affected
skin. Revertant skin cells can be used for transplantation by means of: 1)
own skin biopsies, 2) cell suspension, 3) cultured epithelial cell sheet, 4) induced pluripotent stem cells reprogrammed as epithelial sheet for skin grafting or as haemopoietic stem cells for infusion. Transplantation of revertant
skin biopsies has already succesfully been performed in a patient with EB.
ES6.1
Strategies for rare disease gene discovery in the era of nextgeneration sequencing
K. M. Boycott1, F. S. Alkuraya2;
1
Childrens Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa,
ON, Canada, 2King Faisal Specialist Hospital and Alfaisal University, Riyadh, Saudi
Arabia.
17
The 1998 IVD Directive regulates the market for diagnostic tests within the
EU, setting out standards for the design and manufacture of in-vitro diagnostic devices (IVDs) and providing mechanisms for the oversight of these
standards. The current system is inflexible, unresponsive and does not do
a good job of protecting patients from harm. In September 2012, the European Commission launched a proposal for a complete overhaul of the IVD
regulations. While welcoming these proposals as a move towards a flexible,
transparent and proportionate system which would bring the EU closer to
international best practice, EuroGentest and ESHG have also expressed concerns about a number of provisions which do not provide effective regulation of the burgeoning commercial market for genomic diagnostics, including
direct-to-consumer genetic testing. We have made detailed submissions to
the Commission, to MEPs and to regulatory bodies to improve the proposed
regulation in these areas. Tests manufactured and used within health institution labs are exempt from the current IVD Directive - the so-called health
institution exemption. EuroGentest has proposed that the health instituti-
18
Back to index
N. Brison, B. Bayindir, P. Brady, L. Dehaspe, S. Ardui, J. Van Houdt, H. Van Esch, E. Legius,
T. De Ravel, K. Devriendt, J. R. Vermeesch;
Center for Human Genetics, UZ Leuven, Leuven, Belgium.
The presence of cell-free fetal DNA in the maternal circulation has allowed
for the development of methods for non-invasive detection of fetal chromosomal aneuploidies. Non-invasive prenatal testing (NIPT) thus avoids
miscarriages due to invasive sampling of fetal material. We developed an
innovative, fast, cost efficient workflow and high throughput analysis pipeline. For validation, cell-free DNA of 297 maternal blood samples were collected from women between 9-15 weeks of gestational age who were also
undergoing invasive sampling for increased risk of fetal aneuploidy. Whole
genome sequencing was performed on cell-free DNA sequencing libraries
on a HiSeq2500 using the fast mode 50bp single-ends. In addition to the
Z-scores, traditionally the main measure for non-invasive aneuploidy detection, we defined different new parameters which allow for a higher accuracy aneuploidy detection. Moreover, the use of Z-scores across sliding bins
enables the distinction to be made between maternal and fetal CNVs. This
approach resulted in 100% specificity and sensitivity for trisomy 21 and 18
detection (trisomy 21, n=17; trisomy 18, n=7). This analysis pipeline has
been clinically implemented and accredited (ISO15189). An overview of our
clinical experience, currently standing at 500 samples, will be provided. In
addition to the traditional trisomies, we will present data on other aneuploidies and clinical management thereof.
C01.2
Clinical Validation of Noninvasive Prenatal risk assessment for fetal
sex chromosome aneuploidies in maternal plasma using Direct
ANalysis of Selected Regions (DANSR) assays
Fetal cell-free DNA (cffDNA) in maternal plasma enables screening for fetal aneuploidy using next generation sequencing technologies. We have
previously described using DANSR assays for biochemical analysis of chromosomes 13, 18, and 21, combined with the Fetal fraction Optimized Risk
for Trisomy Evaluation (FORTE) algorithm to compute the risk of trisomy
with high sensitivity and specificity. We have developed additional DANSR
assays for the X and Y chromosomes and have applied the FORTE algorithm
on a two blinded sets of samples with and without sex chromosome aneuploidies (SCA). We report clinical validation results on HarmonyTM Prenatal
Tests ability to detect fetal SCA.
609 samples comprised the two sets. All subjects provided informed consent. Matching fetal karyotype results from invasive testing were available
for all subjects. Samples were processed as previously described with lab
and analysis personnel blinded to fetal karyotype. FORTE models were built
against monosomy X, XXX, XXY, XYY, and XXYY genotypes. Harmony results
were compared against fetal karyotype.
All samples that passed standard Harmony QC metrics generated a sex chromosome result (100%; 95% CI: 99.4-100%). All were concordant with karyotyping for fetal sex (100%; 95% CI: 99.4-100%).
Directed analysis of cffDNA is accurate for risk assessment of non-mosaic
fetal SCA. This is the largest fetal SCA validation study to-date. This demonstrates ability to expand Harmony to genetic conditions besides trisomies
13,18 and 21.
Karyotype
Sensitivity (95%
CI)
Specificity (95%
CI)
47, XYY
100%(43100%)
3 of 3
100%(99.3100%)
C01.3
mtDNA mutations variously impact mtDNA maintenance throughout
the human embryofetal development
S. Monnot1, S. Rondeau1, P. Vachin1, E. Herzog2, B. Bessieres1, N. Gigarel1, D. Samuels3,
L. Hesters2, N. Frydman2, G. Chalouhi1, M. Rio1, A. Rotig1, A. Benachi2, L. Salomon1, A.
Munnich1, J. Bonnefont1, J. Steffann1;
1
Hopital Necker, Paris, France, 2Hopital Beclere, Clamart, France, 3Vanderbilt Medical
Center, Nashville, TN, United States.
Back to index
inherited with high transmission risk, resulting in requests for preimplantation or prenatal diagnoses. These procedures are hampered by our poor
knowledge on the pathophysiology of mtDNA mutations during human development. Specifically, how mtDNA mutations impact the mtDNA content?
We collected oocytes, embryos, placentas and fetal tissues at various stages
of development, from controls and carriers of m.3243A>G (MTTL1, MELAS),
m.8344A>G (MTTK, MERRF) and m.8993T>G (MTATP6, NARP). We devised
a test assessing simultaneously mtDNA copy number (CN) and mutant load
in single cells.
MtDNA CN increased from the germinal vesicle to the blastocyst stage in
m.3243A>G cells, suggestive of a mutation-dependent induction of mtDNA
replication, that may compensate for the respiratory chain dysfunction.
Analyses of placentas showed that mtDNA CN significantly increased in
m.3243A>G at 11-GW, becoming identical to controls at delivery, probably
owing to a placental energy demand maximal at the end of the 1st trimester.
Analyses of fetal tissues showed that mtDNA CN was similar in m.3243A>G
vs control tissues apart from the lung; lower in m.8993T>G muscle, heart,
and liver vs control. Mutant loads were identical in all tissues from a given
fetus, indicating an absence of mutant load/mtDNA content correlation.
These data highlight the complex relationships between mtDNA mutations
and mtDNA content, depending on mutation types, mutant loads, cell types
and development stages. Transcriptome and mtDNA replication studies
should help us in unravelling the molecular bases of these observations, of
particular relevance for therapeutic approaches in mtDNA disorders.
C01.4
Whole-genome single-cell haplotyping, a generic method for
preimplantation genetic diagnosis
19
The implementation of non-invasive prenatal testing (NIPT) of Down syndrome (DS) in national health care systems requires decision-making about
the possible restriction of NIPT to high-risk pregnancies, the timing of NIPT,
and the combination with other tests. In this study, we combined ethical exploration with a decision-analytic model. We compared three implementation strategies: (1) restriction of NIPT to high-risk pregnancies on the basis
of the combined test (CT) (risk for DS 1:200); (2) NIPT for all women at 13
weeks of gestation; (3) NIPT for all women at 10 weeks of gestation. Comparing strategy 1 to strategy 2, 95% fewer women underwent NIPT, and false
positive 1st trimester screening results and invasive procedures were reduced by 95% and 37%, respectively. This was at the expense of a decrease in
DS detection of 11% before 15 weeks of gestation and 4% throughout the
entire pregnancy (3 DS cases/100,000 pregnancies). Comparing strategy
2 to strategy 3, NIPT was avoided in an additional 4% of pregnancies that
would have resulted in miscarriage. Furthermore, delaying NIPT to 13 weeks
allows for immediate confirmation by amniocentesis, and may be beneficial
to women who may feel overloaded with information in 1st trimester. As
NIPT currently does not detect all prenatal abnormalities, it should be offered subsequent to or alongside a nuchal translucency (NT) measurement. In
all cases of fetal anomaly on ultrasound (including NT enlargement), broad
genetic testing after invasive procedure should still be performed.
C01.6
Whole genome sequencing and analysis in prenatal screening: ethical
reflection
G. de Wert, W. Dondorp;
Maastricht University, Maastricht, Netherlands.
Non-invasive prenatal testing (NIPT) for fetal abnormalities currently focuses on Downs syndrome. It is to be expected, however, that it will become
possible to broaden the scope of screening with NIPT. As proof of principle,
it has been demonstrated that the complete fetal genome can be sequenced
using fetal DNA from maternal plasma, potentially allowing prenatal whole
genome analysis.
This scenario makes a proactive ethical reflection of utmost importance.
Normative issues to be addressed include the following: First, the prerequisite of proportionality. Do the possible benefits of whole genome sequencing and analysis in prenatal screening outweigh the possible harms and
disadvantages? Secondly, would it be possible to fulfill the requirement
of informed consent, given the wide range of possible outcomes of whole
genome prenatal screening? Could so-called generic consent, based on pretest information about general categories of test outcomes, be accepted as a
sound variant of informed consent ? And thirdly: if whole genome prenatal
screening would result in the birth of children whose genotype has been
elucidated prenatally, the childs right not to know may be violated, i.e. the
right of the future, competent, person to decide about predictive testing for
later onset diseases. How to handle, then, possible conflicts between future
parents right to know and future childrens right not to know?
This presentation offers a systematic exploration of these issues, aimed at
contributing to adequate ethical guidance for whole genome prenatal screening.
C02.1
A novel variant in the SLC9A9 gene influences disease activity in
interferon-beta treated multiple sclerosis patients
1,2
1,2
1,2
20
Back to index
ted from HC. For this reason we performed a trans eQTL analysis in 211
CD14+ monocytes isolated from HC followed by pathway analyses using
three independent softwares. All of them enlighten an enrichment of IFNrelated pathways of genes trans-regulated by rs9828519. We observed in
the rs9828519GG samples an up-regulation of genes involved in IFN signalling, including STAT1, JAK1, STAT5B, but also an impairment of the final
effects of IFN, with an upregulation of osteopontin and a downregulation
of IL10. Here we report a new genetic marker that affects the response to
IFN in MS patients supported by experimental evidences of the involvement in the IFN pathways.
C02.2
High throughput sequencing in sporadic forms of steroid-resistant
nephrotic syndrome: heterogeneous genetic alterations can predict
resistance to treatments
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C02.6
Collaboration to integrate genomics into clinical care: a
demonstration evaluation
M. H. Willemsen1, V. Tucci2, T. Kleefstra1, A. Hardy3, I. Heise3, S. Maggi4, W. WissinkLindhout1, A. Vulto-van Silfhout1, B. de Vries1, Z. Iqbal1, H. Brunner1, W. Nillesen1, H.
Yntema1, H. Hilton3, M. Simon3, S. Tsaftaris5, H. van Bokhoven1, A. Constestabile6, T.
Nieus6, A. Raimondi6, B. Greco6, D. Cantatore6, L. Gasparini6, L. Berdondini6, A. Bifone7, J.
Veltman1, L. Peart-Vissers1, A. Gozzi7, S. Wells3, P. Nolan3;
1
Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands, 2Istituto Italiano
di Tecnologia, partment of Neuroscience and Brain Technologies, Genova, Italy, 3MRC
Harwell, Harwell Science and Innovation Campus, Oxfordshire, United Kingdom,
4
Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia,
Genova, Italy, 5Instituto Italiano di Tecnologia, Center for Nanotechnology Innovation,
Pisa, Italy, 6Department of Neuroscience and Brain Technologies - Istituto Italiano di
Tecnologia, Genova, Italy, 7IMT Institute for Advanced Studies Lucca, Lucca, Italy.
21
Different genes of the glycosylphosphatidylinositol anchor synthesis pathway, PIGV, PIGO, and PGAP2, have recently been implicated in hyperphosphatasia-mental retardation syndrome (HPMRS), also known as Mabry syndrome, a rare autosomal recessive form of intellectual disability with characteristic additional phenotypic features. We developed a diagnostic gene
panel for targeting all known genes of the GPI-anchor synthesis pathway
to screen patients matching these features, and detected compound heterozygous and homozygous mutations (c.439dupC, c.914A>G, c.314C>G) in
PGAP3, a gene that is involved in the GPI-anchor maturation, in two unrelated patients with developmental delay, elevated serum alkaline phosphatase,
seizures and particular facial anomalies. Our functional studies show that an
impairment of the later GPI-anchor remodeling steps also causes HPMRS. In
addition we analyzed the mutation spectrum of all four genes as well as the
associated phenotypic spectrum in a large cohort of individuals diagnosed
with HPMRS. Among this cohort biallelic PIGV mutations were identified in
about 50 % of unrelated families whereas mutations of the other involved
genes are less frequent. Our findings demonstrate that the severe end of the
clinical spectrum presents as a multiple congenital malformation syndrome
with a high frequency of Hirschsprung disease as well as vesicoureteral, renal, and anorectal malformations. At the other end of this spectrum HPMRS
could present as apparently non-syndromic form of intellectual disability.
Taken together with recent data, HPMRS displays a heterogeneous etiology
caused by impairment of different steps of the GPI-anchor synthesis and is
associated with a marked clinical variability regarding additional malformations and growth patterns.
22
Back to index
C03.4
The significance of small copy number variants in neurodevelopmental disorders
Despite abundant evidence for pathogenicity of large CNVs in neuro-developmental disorders (NDDs), the individual significance of genome-wide
rare CNVs <500 kb has not been well elucidated in a clinical context. By high
resolution chromosomal microarray analysis, we investigated non-polymorphic exonic CNVs sizing 1-500 kb in a cohort of 714 patients with NDDs, of
which 8.8% had an obvious large disease causing CNV. Excluding recurrent
false positive sites, we detected 96 small CNVs, of which 58 (60.4%) could be
confirmed by secondary testing.
Six of the confirmed de novo or likely de novo CNVs were clearly pathogenic
affecting the recurrent microdeletion region in 17q21.31 or OMIM morbid
genes (CASK, CREBBP, PAFAH1B1, SATB2). Two further de novo CNVs affecting single genes (MED13L, CTNND2) were instrumental in delineating novel recurrent conditions. In addition, two unreported homozygous deletions
were found likely pathogenic: An intragenic deletion of ACOT7 which likely
defines a novel autosomal recessive disorder, and a deletion of the genes
PREPL and C2orf34 representing a new variant of the hypotonia-cystinuria
syndrome. Four of the inherited CNVs affecting previously reported sites
(16p11.2, AUTS2, NRXN3, GRM8) were also considered likely pathogenic. In
total 14 (24.1%) of the small CNVs were categorized as pathogenic or likely
pathogenic (median size 130 kb), nine (15.5%) as likely benign and the rest
remained unclear. These results verifies the diagnostic relevance of genome-wide rare CNVs <500 kb (overall ~2%; 1.1% de novo, 0.3% homozygous,
0.6% inherited) and their inherent potential for discovery of new conditions
enabling better characterization of NDDs.
C03.5
Rare large CNVs are associated with intellectual disability, education
level, and female fertility in general population
A. Reymond1, K. Mnnik1, R. Mgi2, A. Mace3, A. Maillard3, H. Alavere2, A. Kolk2, L.
Leitsalu2, A. Ferreira1, M. Noukas2, J. S. Beckmann3, S. Jacquemont3, Z. Kutalik3, A.
Metspalu2;
1
Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland,
2
Estonian Genome Centre, University of Tartu, Tartu, Estonia, 3Department of Medical
Genetics, University of Lausanne, Lausanne, Switzerland.
To investigate the burden of the rare large CNVs in the general population, we analyzed the Estonian Genome Centre cohort. It is a longitudinal,
prospective, population biobank linked to comprehensive personal, educational, medical and daily life data of 5% of the Estonian adult population.
Within a subset of 6819 individuals, we identified 60 carriers of known
genomic disorder lesions, equivalent to a prevalence of 1% in the general
adult population. Their phenotypes are reminiscent of those of carriers of
identical rearrangements identified in disease cohorts. Importantly some of
the associated traits appear to have been previously overlooked due to agedependent penetrance.
We then generated the genome-wide map of rare autosomal CNVs and identified a total of 697 carriers of CNVs 250 kb with MAF 0.05%. While
duplication carriers are equally distributed in both sexes, we observe a significant bias towards female carriers of deletion. This biased mutational
burden supports the notion that females are somewhat protected from
neurodevelopmental disorders. Cumulatively, these CNV carriers show a
significant increase in the prevalence of intellectual disability and a decrease in the rate of higher-achieved education level, as well as a decrease of
female carriers fertility. In addition to CNV size, these effects are associated
with the number and quality of encompassed genes and pronounced in
carriers of deletions and duplications that encompass 3 and 10 genes,
respectively. Our results suggest that large rare variants significantly impact
life quality of carrier identified from non-clinical cohorts.
C03.6
Altered neuronal network in iPSC derived cortical neurons from
patients with MECP2 duplication syndrome
We previously showed that increased dosage of methyl-CpG-binding protein-2 (MeCP2) leads to a severe neurodevelopmental disorder in males, designated as the MECP2 duplication syndrome (MIM#300260). The increased
dosage of MeCP2 is the result of a copy number gain at Xq28, including the
MECP2 gene and results in severe to profound neurodevelopmental delay
with onset at birth, limited or absent speech, hypotonia, epilepsy, autistic
behavior and motor dysfunction. We developed induced pluripotent stem
cells from 3 patients with MECP2 duplication syndrome carrying different
duplication sizes, to study the impact of increased MePC2 dosage in human
neurons. Differentiation of these iPSCs into neurons of cortical identity showed modulation in the expression of progenitor genes like BLBP and FOXG1
and cortical genes like RELN, CTIP2, TBR1 and VGLUT. Cortical neurons
derived from Mecp2dup-iPSCs had more synapses, and altered network
synchronization as well as dendritic complexity. Next, we tested a series of
epigenetic drugs for the ability to rescue neuronal defects and validated two
HDAC inhibitors as potential clinical candidates. Our model recapitulates
early stages of the human MECP2 duplication syndrome and represents a
promising cellular tool to facilitate therapeutic drug screening for severe
neurodevelopmental disorders.
C04.1
EIF2AK4 mutations cause pulmonary veno-occlusive disease, a
recessive form of pulmonary hypertension
Back to index
Congenital heart defects are the most common birth defect worldwide and
a leading cause of neonatal mortality. Left ventricular outflow tract obstruction (LVOTO) is an important subtype, with lesions on the most severe end
of this spectrum being significantly life limiting. Although mutations in several genes have been associated with LVOTO in the majority of cases the cause is still unknown. In an individual with LVOTO and a balanced chromosome translocation, the highly conserved NR2F2 was found to be interrupted.
Whole mount studies in mice showed Nr2f2 to be expressed in the atria and
in human fetal samples, expression was also confirmed in the heart including
in atrial tissue. A further study conducted exome sequencing in 13 parentoffspring trios and 112 unrelated patients with nonsyndromic atrioventricular septal defects, a further important subtype of CHD for which the
genetic architecture is poorly understood. Five rare missense variants (two
of which arose de novo) were found in NR2F2, a very significant enrichment
(P=7.710-7) compared to 5,194 controls. A further two CHD families were
identified with other variants in NR2F2; a de novo substitution disrupting a
splice donor site and a 3bp insertion that co-segregated in a multiplex family. NR2F2 encodes a pleiotropic developmental transcription factor, and
decreased dosage of NR2F2 in mice has been shown to result in abnormal
heart development. Furthermore, using luciferase assays, it was shown that
all six coding sequence variants observed in patients significantly alter the
activity of NR2F2 target promoters.
*These authors contributed equally to this work
C04.3
Loss of alpha1 beta1 soluble guanylate cyclase, the major nitric oxide
receptor, leads to moyamoya and achalasia
Moyamoya is a cerebrovascular condition of unknown mechanism characterized by a progressive stenosis of the terminal part of the internal carotid arteries (ICA) and the development of abnormal moyamoya vessels leading
to stroke. We describe a novel autosomal recessive disease leading to severe
moyamoya and early onset achalasia in 3 unrelated families. Using genetic
linkage and exome sequencing we identified in all 3 families homozygous
mutations of GUCY1A3, the gene encoding the alpha1 subunit of soluble
guanylate cyclase (sGC), the major receptor for Nitric Oxide (NO). Platelet
analysis showed a complete loss of the mutated protein and an unexpected
stimulatory role of sGC within platelets.The NO/sGC/cGMP pathway is a major pathway controlling vascular smooth muscle relaxation, vascular tone
and vascular remodeling. Our data suggest that alterations of this pathway
may lead to an abnormal vascular remodeling process in sensitive vascular
areas with low blood flow and shear stress,such as ICA bifurcations. These
data provide treatment options for these patients. They also suggest that investigation of members of this NO/sGC/cGMP pathway is warranted in both
non syndromic moyamoya and isolated early onset achalasia. Data accepted
in the Am J Hum Genet.
23
Next Generation Sequencing enables simultaneous screening of multiple genes for multiple patients in a single run. We designed a panel of 111 genes
known to be associated to CMs to study 94 unrelated patients (80 with Hypertrophic Cardiomyopathy, HCM; 18 with Dilatative Cardiomyopathy, DCM
and 6 with Arrhythmogenic Cardiomyopathy, AC). Targeted resequencing
was performed on Illumina platform (98,13% of the regions with a depth of
coverage of 20X or more, mean coverage on target of 530X). A mean of 1016
variants were found for each patient. Rare (frequency <0.05), non-synonymous, loss- of- function and splice-site variants were defined as candidates.
Pathogenic or likely-pathogenic variants were all confirmed by Sanger and
cosegregation was tested when possible. Excluding titin missense variants,
we identified 48 variants (27 novel) in sarcomeric or associated genes in
48/70 HCM patients (68%), with 14% of complex genotype. MYH7, MYBPC3 and TNNI3 resulted the high-yield genes; 19 additional candidate variants (13 novel) in desmosomal and ion-channel genes in 14 patients (20%)
were identified in this group. We identified 10 candidate variants (7 novel)
in 7/18 DCM patients (39%) and 5 candidate variants in 3/6 AC patients
(50%). A targeted protocol allowed the identification of likely pathogenic
variants in a large proportion of patients with CMs, irrespective of phenotype. The unexpected finding of rare non synonymous variants in desmosomal and ion-channel genes among HCM patients raises important issues
regarding their role as previously unappreciated modifiers of the disease,
potentially relevant to risk prediction and counseling.
C04.6
Causal relationship of body mass index with cardiometabolic traits
and events: a Mendelian randomization analysis
24
Back to index
McMaster University, Hamilton, ON, Canada, 6Mount Sinai School of Medicine, New
York, NY, United States, 7University Medical Center Utrecht, Utrecht, Netherlands,
8
Durrer Center for Cardiogenetic Research, Utrecht, Netherlands, 9University of Virginia,
Charlottesville, VA, United States, 10Fred Hutchinson Cancer Research Center, Seattle, WA,
United States.
5
Studies (Individuals)
Regression coefficient
(95%CI)
6 (20,677)
3 (12,758)
Metabolic
Fasting glucose (mmol/l)
Fasting insulin (% difference)
Inflammation
C-reactive protein (% difference)
Interleukin-6 (% difference)
Fibrinogen (% difference)
Blood pressure
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Lipids
HDL-C (mmol/l)
LDL-C (mmol/l)
Triglycerides (% difference)
Surrogate marker of CHD
Carotid intima medial thickness
(% difference)
Events
Type 2 diabetes
Coronary heart disease
Stroke
7 (24,319)
5 (9,885)
6 (19,041)
6 (24,943)
6 (23,364)
6 (24,761)
6 (30,136)
6 (30,137)
3 (6,260)
Studies (Cases/
Individuals)
7 (4,407/31,844)
7 (6,073/ 26,193)
6 (3,813/ 23,782)
C05.1
Compound inheritance of a low-frequency promoter deletion and a
null mutation in a new gene causes Burn-McKeown syndrome (BMKS)
D. Wieczorek1, W. G. Newman2, T. Wieland3, T. Berulava1, M. Kaffe3, D. Falkenstein1,
C. Beetz4, S. Douzgou2, J. Clayton-Smith2, S. B. Daly2, S. G. Williams2, S. Bhaskar2, J.
Urquhart2, B. Anderson2, J. OSullivan2, O. Boute5, E. Graf3, J. C. Czeschik1, A. J. van
Essen6, F. Hazan7, A. Hing8, A. Kuechler1, J. Lemke9, C. Marques Lourenco10, U. Hehr11, B.
Horsthemke1, T. Meitinger3, J. Burn12, H. Ldecke1, T. M. Strom3;
1
Institut fr Humangenetik, Essen, Germany, 2Manchester Centre for Genomic Medicine,
Manchester, United Kingdom, 3Institute of Human Genetics, Mnchen, Germany,
4
Institut fr Klinische Chemie und Laboratoriumsdiagnostik, Jena, Germany, 5Service
de Gntique Clinique, Lille, France, 6Department of Genetics, Groningen, Netherlands,
7
Department of Medical Genetics, Izmir, Turkey, 8Seattle Childrens Hospital, Seattle,
WA, United States, 9Abteilung Humangenetik, Bern, Switzerland, 10Neurogenetics Unit,
Sao Paulo, Brazil, 11Department of Human Genetics, Regensburg, Germany, 12Institute of
Genetic Medicine, Newcastle University,, Newcastle upon Tyne, United Kingdom.
Back to index
(ER) Ca2+ sensor. The effect of the mutation on the structure of STIM1 was
investigated by molecular modeling, and its effect on function was explored
by calcium homeostasis experiments. We show that STIM1 p.R304W variant
may affect the conformation of the inhibitory helix and unlock the inhibitory state of STIM1 molecule. Results obtained from calcium imaging experiments using transfected cells together with the fibroblasts from one affected
patient were in agreement with impairment of calcium homeostasis. The
p.R304W mutation probably causes a constant Ca2+ release activated Ca2+
channels (CRAC) opening leading to a permanent entry of calcium in many
cell types. Our results are in agreement with already published models for
STIM1 structure and activation. We conclude that p.R304W mutation in
STIM1 may be the cause of the Stormorken syndrome.
C05.4
TashT is a novel mouse model that phenocopies both the variable
penetrance and male sex-bias of Hirschsprungs disease
Neural crest cells (NCC) are progenitors of diverse cell types such as peripheral neurons and glia as well as melanocytes. Via an insertional mutation
screen for loci affecting NCC, we identified several mouse lines that combine
defects in pigmentation and formation of the enteric nervous system. One of
these lines, named TashT, displays an aganglionic megacolon phenotype in a
subset of homozygotes and, most interestingly, almost exclusively in males.
This is highly reminiscent of human Hirschsprungs disease, a neurocristopathy with an incidence of 1/5000 newborns and a currently unexplained
4:1 male-to-female bias.
We localized the TashT transgene insertion site in a gene desert containing
multiple highly conserved elements on chromosome 10. Migration assays
as well as time-lapse imaging showed that megacolon is due to defective
NCC migration within the gut mesenchyme, a defect generally more severe
in males than females. At the molecular level, RNAseq analysis of TashT enteric NCC notably revealed upregulation of many genes encoding secreted
proteins and downregulation of several X-linked genes. This analysis also
identified the novel gene Fam162b as a strong candidate for being the TashT
causative gene. Fam162b is located near the transgene insertion site and
reporter gene as well as 3C assays suggest that this TashT overexpressed
gene is normally repressed in NCC via long range interactions with some of
the highly conserved elements near the transgene insertion site.
Altogether, our results demonstrate that the TashT line represents a unique
mouse model that will help understand the male sex bias of Hirschsprungs
disease.
C05.5
WNT pathway downregulation and Cornelia de Lange Syndrome
The cohesin complex is formed from a multi-subunit core and their associated regulatory proteins.
Genetic variants within components of the cohesin complex (NIPBL, SMC1A,
SMC3, RAD21, PDS5, ESCO2, HDAC8) are believed to be responsible for a
spectrum of human syndromes known as cohesinopathies that includes
Cornelia de Lange Syndrome (CdLS), a multiple malformation syndrome
affecting almost any organ and causing severe developmental delay. The cohesin complex has a canonical role in cell division and a non-canonical role
in gene expression regulation. Cohesinopathies seem to be caused by dysregulation of specific developmental pathways downstream of mutations in
cohesin components. However, it is still unclear how mutations in different
components of the cohesin complex effect the output of gene regulation.
In this study, zebrafish embryos and patient-derived fibroblasts were used
to analyze abnormalities underlying CdLS, focusing on SMC1A, the second
most commonly mutated gene responsible for the disease. We show that the
knockdown of smc1a in zebrafish impairs neural development, increases
apoptosis and specifically downregulates the canonical Wnt pathway that
can be rescued by chemical activation suggesting a therapeutic potential for
CdLS. The same downregulation of the WNT pathway is observed in SMC1Amutated patient fibroblasts. Previously we have demonstrated that the haploinsufficiency of NIPBL, the cohesin gene mutated in Cornelia de Lange
Syndrome, produces a similar phenotype in zebrafish and in CdLS patients.
Finally, the double-knockdown of nipblb/smc1a in zebrafish shows a synergistic effect for the canonical role of cohesins in cell cycle and survival.
25
Cornelia de Lange syndrome (CdLS) is a highly variable multisystem disorder with a broad phenotypic spectrum. Most typically-affected individuals
carry de novo heterozygous loss-of-function mutations in NIPBL. Mutations in other components of the sister chromatid cohesion system, SMC1A,
HDAC8, SMC3 and RAD21, result in phenotypes that overlap with CdLS but
which can be highly atypical. We carried out trio-based exome sequencing in
ten unrelated individuals with the diagnosis of atypical CdLS, who had previously been screened for intragenic mutations in the known CdLS genes and
for genomic deletions or duplications. Using a likelihood-based approach,
we identified nine de novo variants in eight cases, including de novo lossof-function mutations in NIPBL (c.3150delA [p.Glu1050Aspfs*20]) and KMT2A (c.3649G>T [p.Glu1217*]), an essential splice-site mutation in PUF60
(c.604-2A>C) and a missense mutation in NAA10 (c.247C>T [p.Arg83Cys]).
Furthermore, de novo, probably damaging missense mutations were identified in PIK3C3, PDCD6IP, UNC45A, NUP210 and CELF3. We then re-sequenced these genes in 85 of our mutation-negative CdLS and CdLS-like cases
by AmpliSeq-Ion Torrent sequencing, which revealed three additional lossof-function mutations in KMT2A. We also detected the aforementioned de
novo mutation in NAA10 (c.247C>T [p.Arg83Cys]) in a similarly-affected,
but unrelated individual. Molecular modelling suggests that the p.Arg83Cys
conversion is likely to alter binding of NAA10 to acetyl CoA and would result in reduced acetylation activity of the protein. Our results highlight the
genetic heterogeneity in atypical CdLS. They also demonstrate the phenotypic overlap between atypical CdLS and other dysmorphic conditions such as
Wiedemann-Steiner syndrome as shown by molecular data.
C06.1
Resolving variants of unknown significance through reanalysis of
4,978 public RNA-seq samples
26
Back to index
C06.2
The long non-coding RNA landscape of autoimmune diseases
We have recently shown that the majority of predisposing autoimmune disease SNPs are intronic or intergenic and have the potential to be regulatory
(Ricao-Ponce & Wijmenga, 2013) by affecting expression of nearby genes
(so-called eQTLs). It has become clear that non-coding RNAs are an important class of regulatory elements. Annotating autoimmune SNPs shows that
close to 10% map to long non-coding RNA genes (lncRNA). Transcriptome
analysis across 11 distinct immune cell types (granulocytes, monocytes, NK
cells, B-cells, memory-T cells, naive CD4+ and CD8+ T-cells, and four CD4+
T-helper cell populations) revealed that these autoimmune lncRNAs are significantly enriched in immune cell types. We also correlated the autoimmune SNP genotypes with expression levels (eQTLs) of both coding genes and
lncRNA genes and observed > 70% of the autoimmune SNPs to be eQTLs.
Interestingly, ~16% of these eQTL SNPs also affect lncRNAs and as high as
6% of these eQTLs are specific to lncRNAs alone. To gain a first understanding on the biological processes in which these autoimmune lncRNAs are
involved we performed pathway analysis based on co-expression profiles
of lncRNAs and protein-coding genes using 5000 publicly available RNAseq
samples. More than 90% of the autoimmune lncRNAs were significantly coexpressed with genes in cis (P = 0.009 to 4.64 x 10-92) that are at an average
distance of 245 kb. Our results show that lncRNA-eQTLs represent a novel
link between non-coding SNPs and the expression of genes, which can be
exploited to understand the process of gene-regulation through lncRNAs in
more detail.
C06.3
Population Scale Comprehensive Identification and Analysis of
Complex Structural Variation Using Nanochannel Array
Back to index
Dideoxy-based chain termination sequencing developed by Frederick Sanger is the gold standard sequencing approach used by clinical genetic laboratories. Recently, new NGS technologies found their way into diagnostic
27
Next generation sequencing (NGS) is quickly being optimized for use in diagnostics. The technologies bring challenges at the technical level, in terms of
data management and in the interpretation of results. Over the past 2 years,
guidelines have been issued by the American, Australian, Dutch and British
genetic professional societies. At EuroGentest, an expert group has been
working on compiling, integrating and completing these guidelines. For instance, we believe that defining the diagnostic utility of the NGS test is the
laboratorys first duty when preparing to offer diagnostic NGS. Second, we
introduce a scoring system for the different NGS assays depending on their
quality and comprehensiveness. This is important for patients and clinicians
to allow comparison of the diagnostic offer from the different laboratories.
It could also be used by the health care system to evaluate and reimburse
28
Back to index
the tests. This scoring system is new, as it does not feature in any other guideline. As far as reportable range is concerned, we propose the use of 3
specific percentages depending on the reference (technical target, coverage
of transcript in a gene panel, coverage with reference to the genome) which
will again allow to compare individual results within runs, between tests
and between laboratories. The guidelines propose a template for reporting
NGS results as well. Finally, the guidelines also deal with informed consent,
unclassified variants and unsolicited findings, again from the laboratory
standpoint. Similarly, they define the difference between research and diagnostics, with a practical solution to the duty to recontact.
C07.6
Clinical exome sequence performance for reporting secondary
genetic findings
The American College of Medical Genetics and Genomics (ACMG) has recommended the reporting of clinically actionable incidental genetic findings
in the course of clinical exome testing. Specifically, the pathogenic findings
of 56 specific genes with known clinical importance should be reported. However, this assumes that exome sequencing returns data of sufficient quality
using methods that were not validated for clinical use. To address this issue,
we surveyed the potential false negative rate of mutations in the 56 ACMG
genes. We retrospectively, analyzed forty-four exome datasets from four different exome capture kits and two-sequence platforms. The exome methods
were examined for their ability to detect clinically relevant mutations in the
56 ACMG genes. A total of 17,774 pathogenic nucleotide variants are annotated in the Human Gene Mutation Database (HGMD) for the 56 genes,
and data was examined for depth of coverage in the exome datasets. Overall, the four-exome methods had inadequate depth of coverage for accurate
base calling ranging from 5.2% to 34.8% of the pathogenic variant positions. At least one gene in each exome method was missing >40% of the pathogenic variant positions. The worst performing method was predicted to
miss >90% of clinically significant variant positions in the genes TMEM43,
PCSK9, KCNQ1 and LMNA. The heterogeneous and occasional poor depth of
coverage across this set of 56 genes illustrates the opportunity for further
innovation in standardizing clinical NGS methods. Implementation of the
ACMG incidental findings guideline requires recognition of the substantial
possibility of reporting false negative results.
C08.1
Smc1a cohesin gene mutations in colorectal precancerous lesions
Mutations in the E-cadherin gene, CDH1, account for 40% of hereditary diffuse gastric cancer (HDGC) cases. The genes responsible for the remaining
cases of HDGC, as well as other familial gastric cancers (FGC) are currently unknown. We examined a large family with FGC and no CDH1 mutations
using a combination of genetic mapping with high-density SNP arrays and
exome sequencing and identified that the cancer is associated with a germline coding variant (p.P946L) in mitogen-activated protein kinase kinase
kinase 6 (MAP3K6). A somatic second-hit (p.H506Y) was present in DNA ob-
Back to index
29
Genetic variants in the SHANK2 gene have been previously reported in patients with intellectual disability and autism spectrum disorder. Our study focused on the analysis of SHANK2 variants associated with schizophrenia. We
analyzed all exons and exon-intron boundaries of the ProSAP1A_AB208026
isoform of SHANK2 by Sanger sequencing in a cohort of 481 schizophrenia
patients (177 trios and 304 singleton patients) and 374 unaffected individuals. We detected ten missense variants that affect protein structure which
are only present in the patient group.
We used mutation prediction tools that are based on evolutionary protein
conservation and chemical properties of amino acids to select the four most
promising variants. In silico investigation is helpful to estimate a functional
relevance of mutations, but cannot substitute the functional analysis itself.
To analyze the functional impact of the four selected mutations, we conducted overexpression and knockdown-rescue experiments in primary hippocampal neurons from rat with a major focus on morphological changes of
the neurons. Another major point of our study was to investigate the effect
of different SHANK2 isoforms and schizophrenia mutations on the actin
structures. We used COS-7 cells as a model system and live cell TIRF (Total
Internal Reflection Fluorescence) microscopy for imaging of actin structures. Additionally, we also performed actin polymerization assays to measure in vivo F-actin/G-actin ratio in our mutants compared to SHANK2 wild
type. With these functional tests we were able to show for the first time a
functional effect of SHANK2 variants which were identified in schizophrenia
patients.
C09.2
Exome sequencing of familial parkinsonism in Scandinavia
30
Back to index
Rett syndrome is due to de novo mutations in MECP2, CDKL5 or FOXG1 genes. MeCP2 and FoxG1 are transcriptional regulators; CDKL5 encodes for a
Back to index
their expression in Central Nervous System and/or neuronal function, previous reported involvement in a known genetic disease and the interaction
to genes/protein responsible for a resembling phenotype. By this approach
we have validated by Sanger sequencing 57 variants corresponding to 38
genes. In 8 cases parents analysis has proved the de novo origin of the SNVs,
which occur in GABA receptors, Sodium and Potassium neuronal channels,
synaptic vesicular releasing factor and a transcriptional regulator. Our data
confirm exome targeted NGS as a promising tool to disclose novel pathogenetic mechanisms leading to RTT.
C10.1
PLS3 mutations in X-linked osteoporosis and fractures: unraveling a
new bone regulatory pathway
Humans display structural and functional asymmetries in brain organization, strikingly manifested through language and handedness. While we
understand the biology of body asymmetries, the molecular basis of brain
laterality is still unknown. We report a genome-wide association study
(GWAS) for a quantitative measure of handedness and dexterity (pegboard)
in individuals with dyslexia (n = 728). The most strongly associated variant,
rs7182874 (P = 8.6810-9), is located in PCSK6, a gene known to activate
NODAL, which is required to regulate left/right body axis determination. A
novel approach for GWAS pathway analysis, based on gene-set enrichment
strategies, showed that left/right asymmetry pathways are associated with
handedness in both the dyslexia and a general population (n = 2666) cohorts. In particular, genes involved in corpus callosum development were
enriched among the GWAS top hits. Furthermore, different markers at the
PCSK6 locus were found to be associated with a measure of handiness in a
completely independent study. The dyslexia-specific marker-trait association could be the result of an epistatic effect. Recent findings show that dyslexia candidate genes play a role in ciliogenesis, an important developmental
process at the basis of left/right structural asymmetries determination. We
propose that handedness is a polygenic trait controlled in part by the molecular mechanisms which establish left/right body asymmetry early in development, which in turn influence brain midline development and might be
implicated in neurodevelopmental disorders. We are now investigating the
molecular mechanisms underlying this association using neuronal stem cell
and zebrafish models.
C09.6
Exome sequencing to disclose potential new pathogenetic variants in
Rett patients without mutations in the known Rett genes
Mutations in the X-linked gene MECP2 are the main cause of classical Rett
syndrome (RTT), while 28% of patients with early onset seizures and a
small percentage of those with congenital variant forms carry mutations in
CDKL5 and FoxG1 genes, respectively. About 5% of classical and 40-50% of
atypical RTT patient remain without a molecular diagnosis. Aiming at disclosing novel candidate genes , a cohort of 31 Rett patients with both classic
and atypical phenotype and unascertained genetic defect was processed .on
a HiScan Illumina platform by True Seq Exome Enrichment. As most mutations in the known RTT causative genes discovered up to day have a de
novo origin, we adopted this model and filtered heterozygous variants, prioritizing those predicted to introduce a frameshift or stop codon or splice or
missense changes. Prioritization of the interesting genes took into account
C10.2
Mutations in plastin 3 cause osteoporosis with fractures.
Overexpression of PLS3 and other F-actin bundling proteins influence
skeletal development in zebrafish and mice
Osteoporosis affects a large proportion of the human population, particularly women after menopause. Recently we reported that pathogenic lossof-function variants in the plastin 3 (PLS3) gene, localized on Xq23 are causative of familial osteoporosis with fractures. Furthermore, a rare variant in
PLS3 (rs140121121) associated with a 2-fold increased risk for fractures
among elderly heterozygous women in two large cohorts from Rotterdam
(Van Dijk et al, NEJM, Oct 2013). PLS3 is an ubiquitously expressed actin-
31
C. T. Gordon1, F. Petit2, P. Kroisel3, L. Jakobsen4, R. Zechi-Ceide5, M. Oufadem1, C. BoleFeysot1, P. Nitschke1, A. Munnich1, S. Lyonnet1, M. Holder-Espinasse1, J. Amiel1;
1
Universit Paris Descartes, INSERM U-1163, Paris, France, 2Hopital Jeanne de Flandre,
Centre Hospitalier Regional Universitaire de Lille, Lille, France, 3Institute of Human
Genetics, Medical University of Graz, Graz, Austria, 4Copenhagen University Hospital,
Herlev, Denmark, 5Hospital for Rehabilitation of Craniofacial Anomalies, University of
Sao Paulo, Bauru, Brazil.
32
Back to index
ted to interfere with enzymatic cleavage of the EDN1 pro-protein. The other
two cases involved patients with dominantly-inherited isolated QME; they
harboured heterozygous EDN1 mutations - a premature stop in one case
and a missense mutation affecting a highly conserved residue of the mature
EDN1 peptide in the other. The nature of the mutations and the different
modes of inheritance suggest that heterozygous loss of function mutations
in EDN1 cause isolated QME and that homozygous hypomorphic mutations
cause ACS. These are the first reported mutations of EDN1 in humans and
suggest that ACS and QME are a phenotypic continuum resulting from impaired EDN1-EDNRA signaling.
C10.5
Defects in TAPT1, involved in Axial Skeletal Patterning, Cause a
Complex Lethal Recessive Disorder of Skeletal Development
TAPT1 encodes Transmembrane Anterior Posterior Transformation-1 protein. TAPT1 is evolutionarily conserved, with similar orthologs from yeast
to vertebrae. ENU mutagenesis of TAPT1 resulted in embryonic lethality of
murine homozygotes, with posterior to anterior transformations of thoracic
and lumbar vertebrae. The mechanism by which this ubiquitously expressed
protein causes a specific patterning defect is unknown. We combined homozygosity mapping with exome sequencing for a Moroccan family with three
lethal fetuses affected with fractures of ribs and long bones, undermineralized skull and axial skeleton, hydramnios with ascites and dilated ventricles. We identified a homozygous c.1108-1G>C mutation in TAPT1, causing
in-frame skipping of exon 10. A second TAPT1 mutation was identified by
direct sequencing in a Syrian pedigree with three lethal fetuses with fractures and multiple congenital anomalies of brain, face, heart and lungs. These probands were homozygous for a missense mutation in TAPT1 exon 9
(c.1058A>T, p.Asp353Val). In both families, a diagnosis of recessive osteogenesis imperfecta (OI) was initially proposed. Both variants affect the second
luminal loop of TAPT1. Proband fibroblast type I collagen has broadened
electrophoretic mobility of alpha-chains in the cell layer, suggesting abnormal post-translational modification of collagen and a relationship with OI
forms. Immunocytochemical staining of dermal fibroblasts revealed colocalization of TAPT1 with the centrosomal protein gamma-tubulin at the
basal body of the cilium, suggesting TAPT1 might have a ciliary function. The
novel autosomal recessive skeletal defects reported here underscore the importance of the cilium in embryonic bone formation. Functional studies with
siRNA, zebrafish and expression studies are ongoing.
C10.6
ZIC1 mutations cause coronal craniosynostosis and learning disability
S. R. F. Twigg1, J. A. C. Goos2, I. Westbury1, S. J. McGowan1, M. van Dooren2, A. M. W.
van den Ouweland3, P. J. van der Spek4, .. 500 Whole-Genome Sequences (WGS500)
Consortium5, S. A. Wall6, I. M. J. Mathijssen2, E. Pauws7, A. O. M. Wilkie1,6;
1
Weatherall Institute of Molecular Medicine, Oxford, United Kingdom, 2Department
of Plastic and Reconstructive Surgery, Erasmus MC, Rotterdam, Netherlands,
3
Department of Clinical Genetics, Erasmus MC, Rotterdam, Netherlands, 4Department
of Bioinformatics, Erasmus MC, Rotterdam, Netherlands, 5Wellcome Trust Centre for
Human Genetics, Roosevelt Drive, Oxford, United Kingdom, 6Craniofacial Unit, Oxford
University Hospitals NHS Trust, Oxford, United Kingdom, 7UCL Institute of Child Health,
London, United Kingdom.
Introduction
High levels of ADHD symptoms during childhood carry risk of worse academic performance and can impact on employment and earnings in adulthood. Polygenic score analysis was used to show that common risk alleles
for clinical ADHD contribute to the risk of having higher ADHD symptoms in
the general population (Martin et al. in press). We have used polygenic score
analysis to investigate the contribution of common risk variants for clinical
ADHD on educational performance and IQ in the general population.
Methods
Academic performance was assessed using results from Key Stage 3 national tests and externally marked GCSE examinations in 6,385 children from
the Avon Longitudinal Study of Parents and Children (ALSPAC). Polygenic
risk scores were calculated for ALSPAC children and their mothers based on
the results of an ADHD GWAS (Stergiakouli et al. 2012).
Results
ADHD polygenic scores on the children were associated with worst educational outcomes as represented by both time points and also with lower IQ
scores at age 15.5 (see Table). Moreover, ADHD polygenic scores on the mothers were associated with lower IQ in the mothers and worst educational
outcomes in the children (see Table).
Discussion
Our results suggest that the same genetic variants that are relevant for an
ADHD diagnosis are also implicated in impaired academic performance in
the general population and lower IQ score in both children and adults.
Outcome
Score achieved at key stage 3
Capped GCSE points
IQ at age 15.5
Mothers IQ
N
6385
6298
3858
2313
p value
4.2x10-6
4x10-4
2.4x10-4
0.025
C11.2
Polygenic risk score analysis shows shared genetic aetiology between
AN and five other psychiatric disorders
L. M. Huckins1, K. S. Mitchell2, L. Thornton3, WTCCC3 Consortium, GCAN Consortium, D.
A. Collier4, P. F. Sullivan5, C. M. Bulik5, E. Zeggini1;
1
Wellcome Trust Sanger Institute, Hinxton, United Kingdom, 2Department of Psychiatry,
Boston University, Boston, MA, United States, 3Dept. of Psychiatry, University of North
Carolina at Chapel Hill, Chapel Hill, NC, United States, 4Institute of Psychiatry, Kings
College London, London, United Kingdom, 5Department of Psychiatry, University of
North Carolina at Chapel Hill, Chapel Hill, NC, United States.
Anorexia nervosa (AN) is marked by extremely low body weight and intense fear of gaining weight. We evaluated shared genetic determinants of
AN and commonly comorbid psychiatric disorders (PsyD) by testing whether polygenic risk scores derived from genome-wide data of other PsyD
can predict AN status. We obtained allele risk scores for major depressive
disorder (MDD), bipolar disorder (BPD), autism (AUT), attention deficit hyperactivity disorder (ADHD) and schizophrenia (SCZ) from the Psychiatric
Genomics Consortium (PGC). We divided each of these sets into 10 Pt significance level thresholds. The test set comprised AN cases and controls from
a published Wellcome Trust Case Control Consortium 3 AN GWAS study. For
every test set sample, we produced a polygenic risk score as a weighted sum
of risk allele scores. Logistic regression was used to assess whether each
PsyD polygenic score predicted AN case-control status. We computed pseudo R^2 values, and compared these to the values obtained when randomly
permuting case-control status to measure the proportion of variance in AN
explained by each PsyD risk score. Our pseudo R^2 values were ~0.5-1%,
comparable to pseudo R^2 values in a recent PGC polygenic score analysis
of five PsyD. We computed an empirical p-value for every significance threshold and found significant pseudo R^2 values at the lowest Pt threshold (Pt
Back to index
<0.001) for AN vs AUT (p=0.0009), MDD (p=0.009), and SCZ (0.0008), and
at Pt <0.01 for BPD (p=0.0004). We demonstrated for the first time a shared
genetic aetiology between AN and other PsyD using genome-wide data.
C11.3
Efficient estimation of pairwise genetic correlations between
hundreds of quantitative traits from population samples of thousands
of individuals
M. Pirinen1, C. Benner1, T. Lehtimki2, J. G. Eriksson3,4,5, O. T. Raitakari6,7, M. Jrvelin8,9,10,
V. Salomaa3, S. Ripatti1,11,12;
1
Institute for Molecular Medicine Finland, Helsinki, Finland, 2Department of Clinical
Chemistry, Fimlab Laboratories, University of Tampere School of Medicine, Tampere,
Finland, 3Department of Chronic Disease Prevention, National Institute for Health and
Welfare, Helsinki, Finland, 4Department of General Practice and Primary Health Care,
University of Helsinki, Helsinki, Finland, 5Unit of General Practice, Helsinki University
Central Hospital, Helsinki, Finland, 6Department of Clinical Physiology and Nuclear
Medicine, University of Turku and Turku University Hospital, Turku, Finland, 7Research
Centre of Applied and Preventive Cardiovascular Medicine, University of Turku and
Department of Clinical Physiology and Nuclear Medicine, Turku University Hospital,
Turku, Finland, 8Department of Epidemiology and Biostatistics, MRC Health Protection
Agency (HPA) Centre for Environment and Health, School of Public Health, Imperial
College London, London, United Kingdom, 9Institute of Health Sciences, University of
Oulu, Oulu, Finland, 10Biocenter Oulu, University of Oulu, Oulu, Finland, 11Wellcome Trust
Sanger Institute, Hinxton, Cambridge, United Kingdom, 12Hjelt Institute, University of
Helsinki, Helsinki, Finland.
33
Selection constantly filters out genomes with an excess of slightly deleterious variants (SDVs) maintaining the fitness of human population. In
case of consanguineous marriages high level of homozygosity, observed in
offspring, uncovers the damaging effect of many SDVs variants making the
purifying selection more effective. We hypothesize that healthy offspring
of consanguineous marriages carry a deficit of these variants as a result of
the embryonic selection acting against the high genetic load. Using 50 genotyped pedigrees with consanguineous marriages we revealed a signature of
purifying selection. First, by means of modified transmission disequilibrium
test we observed a genome-wide deficit of rare derived alleles, transmitted
from consanguineous parents to their healthy offspring. Because rare derived alleles are enriched in deleterious variants we assume, that the deficit
of rare derived alleles could be a signature of embryonic purifying selection,
which filters out low-fitted genomes with high load of mutations. Second,
we expect that haplotypes, which are rarely seen in long Runs Of Homozygosity (ROH) harbor loci with high load of recessive mutations. Comparing
the density of slightly deleterious variants inside and outside ROH we present an approach and the first results describing transmission of haplotypes
identical by descent in consanguineous marriages. Further investigation of
the genetic load of long ROH and the pattern of their inheritance can help to
allocate mutations causing recessive autosomal diseases.
C12.1
Next generation sequencing as a reliable and efficient technique to
identify mutations in patients with retinal dystrophies
34
Back to index
We developed a diagnostic NGS pipeline to identify mutations in 170 genetically and clinically unselected RD patients. Underrepresented regions
were examined by Sanger sequencing. We found mutations in 50 to 60%
of retinitis pigmentosa and approximately 80% of syndromic (Bardet-Biedl
or Usher syndrome) cases. Seventy-one novel mutations in 40 genes were
identified, among which the genes USH2A, EYS, ABCA4, and RHO were more
frequently affected by pathogenic sequence alterations. Occasionally, cases
carried mutations in more than one RD-associated gene suggesting modifier
effects of the additional variants. We further found possible dominant denovo mutations in sporadic RD cases imposing difficulties for counseling of
patients and families. Exome sequencing showed similar detection rates in
11 additional research-based RD cases confirming that both approaches are
efficient to identify mutations in known disease genes. In contrast to panel
sequencing, exome analysis provided the bases to search for gene candidates for RD.
In conclusion, NGS-based mutation analyses provide reliable and cost-efficient approaches to investigate genetically heterogeneous diseases like RD.
C12.2
New Hereditary hearing loss (HHL) genes/mutations identified by
High throughput sequencing and genotyping in the Italian and Qatari
populations.
To overcome the remarkable genetic heterogeneity of HHL an extremely powerful 3 steps gene-identification strategy was designed. STEP1 consists
in a screening of 96 HHL genes by targeted re-sequencing (TS) on Ion Torrent PGM (3487 amplicons). Positive cases contribute to define an accurate
molecular epidemiology picture while negative ones undergo a combination
of STEP2 (linkage studies) and STEP3 (whole exome sequencing) or directly
STEP3 (depending on pedigree size).
Illumina protocols were used for STEP2 and STEP3. Sequencing variants
were annotated/filtered according to standard pipelines. This strategy was
applied to a first series of 28 Italian and Qatari families. STEP1 characterized 43% of all families and led to the identification of several novel alleles in known HHL genes (Table1a). STEP2 identified four new HHL loci
(Table1b), while STEP3 had already led to the discovery of 2 new genes
(BDP1 and TBLY1) and it is in progress in the remaining 14 families. Briefly,
p*2625Gluext*11 mutation resulting in an elongation of 11 residues of the
BDP1 protein was identified in a recessive family. Bdp1 inner ear expression
was confirmed by immunohistochemistry. In a pedigree showing a Y-linked
pattern, the predicted pathogenic D69V mutation was identified in TBLY1
gene. Functional studies are in progress to confirm its pathogenicity. This
strategy proved to be very successful by explaining several unsolved cases.
Updated data will be presented and discussed
Table 1
a
2nd
mutation
described
worldwide
Novel
mutation
Novel
mutation
Novel
mutation
Novel
mutation
Novel
mutation
Novel
mutation
Novel
mutation
Novel
mutation
Novel
mutation
Novel
mutation
Novel
mutation
b
Origin
inheritance
cDNA change
Amino acid
change
Italy
dominant
c.1057G>C
pG353R
Italy
Italy
Qatar
Qatar
Italy
Qatar
Qatar
Qatar
Qatar
Qatar
Qatar
dominant
c.775G>C
p.G259R
p.T340R/p.
P431S
Gene
P2RX2
(Faletra et
al. 2013)
TECTA
TMPRSS3
recessive
c.1019C>G/c.1291C>T
recessive
c.453_455delCGAinsT
GGACGCCTGGTCGGG p.E152GfsX81 MYO15A
CAGTGG
recessive
recessive
c.1588G>T
c.G989A
p.E530X
LOXHD1
p.R330K
POU3F4
p.L603H
TMC1
recessive
c.G1334A
p.R445H
TMC1
recessive
c.G634A
p.G212S
TRIOBP
recessive
recessive
recessive
recessive
Markers Chromosome
RS7388463 to
New locus
chr8
RS12678154
c.T1808A
c.C2170T
c.A1837G/c.T2A
c.C1294G
LOD SCORE
3.327
p.R724C
p.M613V/p.
M1K
p.L432V
OTOF
LOXHD1
WFS1
chr9
4.1413
chr7
2.1
chr14
4.2
C12.3
Disclosure of false disease genes - an underestimated potential of
targeted and genomewide NGS: The example of MYO1A and deafness
type DFNA48
MYO1A is considered the gene underlying autosomal dominant non-syndromic hearing loss type DFNA48, based on six missense variants, one small
in-frame insertion and one nonsense alteration. By next-generation sequencing (NGS) targeting 66 deafness genes in 109 hearing-impaired patients,
we identified three families (including a consanguineous one) that provide
strong evidence against a causative role of MYO1A in inherited deafness:
Two novel nonsense mutations (p.Tyr740* and p.Arg262*) and a previously
described missense mutation were identified not only in the index patients
in heterozygous state, but also in unaffected relatives. The hearing deficit in
these families was clearly due to mutations in other deafness genes, MYO7A,
EYA1 and CIB2, respectively. All but two of the altogether ten MYO1A mutations have been annotated in dbSNP, and population frequencies (dbSNP,
1000 Genomes, Exome Sequencing Project) above 0,1% contradict pathogenicity assuming a dominant model. Moreover, one healthy individual
from the consanguineous family was homozygous for the nonsense mutation p.Arg262*, compatible with a previously reported homozygous Myo1a
knockout mice lacking any overt pathology. We conclude that MYO1A is dispensable for normal hearing and may even represent a non-essential gene,
adding to the list of erroneous disease genes that is growing rapidly with
increasing availability of data from large-scale sequencing projects. Data
from individuals born to consanguineous parents are particularly valuable
because they are enriched for homozygous loss-of-function variants, with
the potential to unmask false disease genes. Their identification is urgently
needed to improve mutation databases and avoid pitfalls in diagnostics and
genetic counseling.
C12.4
AON intravitreal injections to manipulate splicing in retinal cells
Purpose: Leber congenital amaurosis (LCA) is the leading cause of hereditary blindness in children. CEP290 encodes a ciliary protein important to
photoreceptor connecting cilium assembly and function. The CEP290 intronic c.2991+1655A>G change is the most common LCA-causing mutation
(10%). It introduces a cryptic exon in the mRNA encoding a premature termination codon. Recently, we made the proof-of-concept of antisense oligonucleotides (AON)-mediated exon skipping to correct the splicing in patient
fibroblasts which recovered ability to ciliate. The purpose of this study was
to make the proof-of-concept of exon-skipping in vivo using intravitreal injections of AONs. Methods: AONs were designed to skip mouse Cep290 exon
36. Variable concentrations of 6-FAM-AONs were injected into the vitreous
of C57BL/6J mice. Retinal sections and mRNA were prepared during 30 days
post-injections (dpi) to follow i) the distribution of oligonucleotides across
cellular layers and ii) exon skipping efficiency. Results: The most efficient
AONs identified by in vitro analyses were injected in the vitreous of animals.
Excellent correlation between the efficiency of exon skipping and AON injected dose has been demonstrated. Histological analyses revealed a wide
distribution of AON in all retinal cell layers at least until 30 dpi. A linear
amount decrease of mRNA lacking exon 36 was measured but still detectable at 30 dpi. Conclusion: Here we report that single intravitreal injection of
AON allows efficient and persistent exon skipping in retinal cells. Hence, this
strategy may be regarded as an attractive alternative to gene replacement
therapy for 10 % of patients affected with LCA.
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C12.5
Mutations in the tricarboxylic acid cycle enzyme, Aconitase 2,cause
either isolated or syndromic optic neuropathy with encephalopathy
and cerebellar atrophy
Inherited optic neuropathy has been ascribed to mutations in mitochondrial fusion/fission dynamic genes, nuclear and mitochondrial DNA-encoded
respiratory enzyme genes or nuclear genes of poorly known mitochondrial
function. On the other hand, enzymopathies of the tricarboxylic acid cycle
(TCA) have been reported to cause severe encephalopathies or isolated retinitis pigmentosa, but no TCA-cycle enzyme deficiency has been hitherto
reported in isolated optic neuropathy. Studying a series of five patients with
optic atrophy, we found homozygous or compound heterozygous missense and frameshift mutations in the gene encoding mitochondrial aconitase
(ACO2), a TCA-cycle enzyme, catalyzing interconversion of citrate into isocitrate. Retrospective studies using patient-derived cultured skin fibroblasts
revealed various degrees of deficiency in ACO2 activity but also in ACO1 cytosolic activity. Our study shows that autosomal recessive ACO2 mutations
can cause either isolated or syndromic optic neuropathy. This observation
supports the view that optic atrophy is a hallmark of defective mitochondrial energy supply and that extra-ocular involvement is not related to severity
of the enzyme deficiency.
C12.6
Isolated foveal hypoplasia with secondary nystagmus and low vision
is associated with a homozygous SLC38A8 mutation
35
36
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tools are being used by HCPs to overcome poor literacy in some families.
We liaised with HCPs to develop a guidelines handbook to help with the
management of common genetic disorders at a local level. We produced a
series of common clinical scenarios that HCPs might encounter to help with
advising on relative risk and genetic testing procedures. The two-day Clinical Bioinformatics course aimed to update our laboratory staff to enable
translation of our research findings into our local diagnostic laboratory.
Our educational seminars covered topics such as carrier testing, intellectual disability, consanguinity, rare disease research and the role of advocacy. Discussions with seminar attendees suggest that there is a significant
knowledge deficit when it comes to genetics in healthcare. However, encouragingly, HCPs voiced an overwhelming interest in gaining more knowledge
about genetics and its application to their practice. We plan to continue our
efforts and develop eLearning tools to support the integration of genetics
into mainstream medicine.
C13.4
Unanticipated results in whole exome study: weve still a lot to learn
C. Skrzynia, J. M. ODaniel, D. Marchuk, K. Lee, J. S. Berg, J. P. Evans;
University of North Carolina, Chapel Hill, NC, United States.
Mutations in SCN5A are associated with Brugada syndrome, LQTS, atrial fibrillation and other conditions with risk of sudden cardiac death. Evidencebased guidelines exist to significantly improve or avert the mortality and
morbidity associated with these conditions. As such the ACMG included SCN5A on its list of incidental findings which it recommends should be returned as part of all whole-exome/genome sequencing tests. We outline three
challenges faced in the return of unanticipated SCN5A variants: (1) Defining
pathogenicity in the presence of reduced penetrance and a highly heterogeneous grouping of conditions; (2) Balancing potential psychosocial harm to
patient/family with result disclosure vs. possible medicolegal ramifications
of not reporting this type of results; and (3) Psychosocial impact of a risk for
sudden cardiac death in the absence of a personal or family history context.
Through the NCGENES whole exome sequencing study, these challenges are
being explored in a cohort of ~500 patients. Incidental, medically actionable
findings are reported and this is emphasized during the pre-test counseling.
We present here our experience with incidental finding of SCN5A mutations
detected in 8 of 145 (5.5%) participants. Each variant, previously reported
as pathogenic, was independently reviewed and discussed with members
of the molecular analysis team to reach consensus regarding interpretation
of its pathogenicity. Upon review we determined there was convincing evidence for only one to be considered pathogenic and reported. The remaining
were not reported, though future information may change the assessment of
these interpretations.
C13.5
The stepping stone approach towards the Genetics Clinic of the Future
D. Schuurbiers1, G. Bertier2, M. Radstake3, P. Bauer4, C. Bock5, P. Borry6, A. Bredenoord7, A.
Brookes8, X. Estivill2, R. Hennekam9, L. Johnston10, H. Kriinen11, N. Knoers12, F. Lescai13,
R. Maalman14, F. Nielsen15, G. van Ommen16, C. Oosterwijk17, J. Paschall18, B. Prainsack19, J.
Saarela20, R. Sudbrak21, M. Swertz22, H. Teare23, T. Vrijenhoek12,24;
1
Responsible Innovation Collective, Delft, Netherlands, 2Centre for Genomic Regulation,
Barcelona, Spain, 3Centre for Society and the Life Sciences, Faculty of Science, Radboud
University, Nijmegen, Netherlands, 4Institute of Medical Genetics and Applied Genomics,
Rare Disease Center Tuebingen, University of Tuebingen, Tuebingen, Germany, 5CeMM
Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna,
Austria, 6Department of Public Health and Primary Care, KU Leuven, Leuven, Belgium,
7
Julius Center, Department of Medical Humanities, University Medical Centre Utrecht,
Utrecht, Netherlands, 8Department of Genetics, University of Leicester, Leicester,
United Kingdom, 9Department of Pediatrics, Academic Medical Center, University of
Amsterdamt, Amsterdam, Netherlands, 10Institute of Genetic Medicine, International
Centre for Life, Newcastle University, Newcastle upon Tyne, United Kingdom, 11National
Institute for Health and Welfare, Helsinki, Finland, 12Department of Medical Genetics,
University Medical Centre Utrecht, Utrecht, Netherlands, 13Department of Biomedicine,
Human Genetics, Aarhus University, Aarhus, Denmark, 14Ruben Maalman Illustrations,
Rotterdam, Netherlands, 15DNA Digest, London, United Kingdom, 16Department of
Human Genetics, Leiden University Medical Center, Leiden, Netherlands, 17European
Genetic Alliances Network, Soest, Netherlands, 18European Bioinformatics Institute,
Hinxton, United Kingdom, 19Department of Social Science, Health and Medicine, Kings
College London, London, United Kingdom, 20University of Helsinki, Helsinki, Finland,
21
Dahlem Centre for Genome Research and Medical Systems Biology, Berlin, Germany,
22
Genomics Coordination Center, University of Groningen, University Medical Center
Groningen, Groningen, Netherlands, 23HeLEX Centre for Health, Law and Emerging
Technologies, Nuffield Department of Population Health, University of Oxford, Oxford,
United Kingdom, 24Center for Genome Diagnostics, Utrecht, Netherlands.
Routine application of genomics in the clinic faces many cross-cutting, controversial challenges. To map them, we have introduced the concept of the
Genetics Clinic Of the Future (GCOF). Moreover we have developed a stepping stone approach to meet these challenges, comprising of field laboratories (the stepping stones) that address controversial, multi-dimensional
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I. Maya1, L. Basel-Vanagaite2,3,4, E. Taub1, A. Koifman5,6, D. M. Behar7, R. TomashovMatar1, R. Sukenik-Halevi8, D. Marom3,9, A. Reches10, M. Shaohat1,3;
1
Raphael Recanati Genetics Institute, Rabin Medical Center, Beilinson Campus, Petah
Tikva, Israel, 2The Raphael Recanati Genetic Institute and Felsenstein Medical Research
Center, Rabin Medical Center, Petah Tikva, Israel, 3The Sackler Faculty of Medicine,
Tel Aviv University, Tel Aviv, Israel, 4Pediatric Genetics, Schneider Children s Medical
Center of Israel, Petah Tikva, Israel, 5Institute of Human Genetics, Soroka University
Medical Center, Beersheba, Israel, 6Ben-Gurion University of the Negev, Beersheba,
Israel, 7Rambam Medical Center, Haifa, Israel, 8Meir Medical Center, Kfar Saba, Israel,
9
Pediatrics A, Schneider Children s Medical Center of Israel, Petah Tikva, Israel,
10
Souraski Medical Center, Tel Aviv, Israel.
Genome-wide association studies (GWAS) of quantitative electrocardiographic (ECG) traits in large consortia identified over 50 loci associated with
QRS, RR, QT and PR intervals, with even more loci discovered in ongoing
HapMap-based meta-analyses in larger sample sizes. We hypothesized that
imputation from sequencing-based reference panels may help in identifying
new loci as the contribution of lower-frequency variation has not yet been
extensively characterized. In the current study, we meta-analyzed GWAS results from 30,000 samples on the QRS, RR, QT and PR intervals after imputing 19 million SNPs from the Genome of the Netherlands (GoNL) reference
panel (998 unique haplotypes). This approach proved successful; in addition to many known loci, we identified seven novel locus-trait associations. Of
the seven novel loci, three were for PR, three were for QT and one was for
QRS. Two were novel trait/locus combinations for loci previously observed
for other ECG traits. Several others are involved in ion handling crucial to
cardiac electrophysiology. The dense coverage of the genome (~seven times
more SNPs than HapMap-based studies) also enabled us to study known loci
in much finer detail. In conclusion, we show that larger reference panels
with more accurate haplotype estimates and denser SNP data enable us to
find new loci and fine-map previously known ones.
C13.6
Teaching Genomic Medicine to Physicians - this is our responsibility
as medical geneticists
Background: Due to new discoveries in genomic medicine, and the transition of genetic knowledge from research laboratories into clinical practice,
an increasing number of medical societies incorporate recommendations
regarding genomic tests and therapeutics into routine clinical guidelines.
Primary care practitioners have inadequate knowledge and skills in medical genetics and many are unaware of the technical, ethical, legal and psychosocial implications of genetic testing. Methods: We initiated a genomic
education program for the purpose of providing physicians from different
medical fields advanced knowledge in genomic medicine . We emphasized
the main take-home messages for physicians, defined as: risk calculation
for various genetic diseases, recognition of the mode of inheritance from
the pedigree, guidelines for decision-making on which molecular tests to
use, interpretation of test results and their clinical implications. Results:
To date, 82 physicians have participated in 6 courses of our genomic education program, which included lectures, workshops and guided tours in
genetic laboratories. In the pre-course examination the average score was
56% (range 20%-80%), whereas in the post-course examination it was
79% (range 40%-100%). The average improvement in score as a result of
the course was 21% (range 0%-80%). The physicians who participated in
our program reported a very high level of satisfaction from the theoretical
and practical knowledge they aqired as well as the concept of a one-week
update course. Conclusion: A one-week genomic education program is an
effective strategy to update primary care physicians regarding the advances
in Genomic Medicine in order to improve their care of patients.
C14.1
Insights into the genetic architecture of anthropometric traits using
whole genome sequence data
E. Zeggini, UK10K consortium;
Wellcome Trust Sanger Institute, Hinxton, United Kingdom.
Body weight and fat distribution measures are associated with increased risk
of cardiometabolic disease. As part of the UK10K study, we have investigated
the genetic architecture of anthropometric traits in 3,538 individuals with
6.5x whole genome sequence (WGS) data from the ALSPAC and TwinsUK
cohorts. Variants discovered through WGS, along with those from the 1000
Genomes Project (1KGP), were imputed into additional individuals from the
ALSPAC and TwinsUK cohorts with GWAS data (total sample size 9,979). We
investigated association between anthropometric traits and 8.6 million low
frequency and common variants (MAF>0.01). We are in the process of obtaining in silico replication of prioritised signals. In interim replication analysis
across ~15,000 samples, 43 out of 66 novel signals for BMI have the same
direction of effect in the replication cohorts (p-value=0.0093). We examined
the concordance of the direction of effect at established loci for each trait.
Out of the 31 established independent loci for BMI that were present in our
data, 28 have the same direction of effect (p-value=2.3e-06). For weight, 10
out of 11 known loci (p-value=0.006), and for height 151 out of 172 loci (pvalue<2.2e-16) have the same direction of effect, respectively. We estimated
C14.2
Genome of the Netherlands imputation identifies seven new loci for
quantitative ECG traits in meta-analysis of 30,000 samples
C14.3
Genome-wide association analysis identifies a new gene involved in
salt perception and liking
A. Robino1,2, N. Pirastu1,2, C. Mansfield3, D. Hwang3, D. R. Reed3, P. Gasparini1,2;
1
Institute for Maternal and Child Health - IRCCS Burlo Garofolo, Trieste, Italy,
2
University of Trieste, Trieste, Italy, 3Monell Chemical Senses Center, Philadelphia, PA,
United States.
37
Comprehensive DNase I hypersensitive site (DHS) mapping by the ENCODE project identified all classes of cis-regulatory elements. Recent studies
showed enrichment of complex trait GWAS noncoding hits in DHS. However,
GWAS hits only partially explain complex trait heritability. The discovery of
rare variants located in regulatory regions specific to immune cells should
explain part of this heritability. Our approach combines selective DNA capture of relevant regulatory regions coupled to next-generation sequencing.
Genome-wide DHS mapping data from the ENCODE and NIH Roadmap Epigenomics Projects were used to select regulatory regions of immune cells.
We designed a custom DNA capture panel (Roche SeqCap EZ developer)
to target these selected regions, as well as exonic and HLA regions. Captured DNA (163Mb) is indexed (5-fold per lane) for sequencing on Illumina
HiSeq2000 system, yielding average coverage of ~28x. We first applied the
Immunoseq. assay to 30 healthy individuals from Sweden (Uppsala BioResource), where we have existing transcriptome, methylome and ChIP-seq
data from three primary immune cells (monocytes, B-cells and T-cells). The
capture panel was also applied to 150 trios from the SaguenayLac-SaintJean asthma familial collection (complex trait with immune and inflammatory components). We observe an enrichment of variants in the vicinity
of allelicly differentially expressed genes (p<1e-10) as well as potential
impact of rare conserved variants on allelic differential expression of genes (p<0.05). This highlights the 1) potential impact of rare variations on
functional mechanism in immune cells and 2) power of our targeted capture
approach to identify novel rare variants in regulatory regions.
C14.5
Exome array analysis in >30,000 Europeans establishes a functional
role for G6PC2 and identifies novel coding variants influencing
glycaemic traits
To identify coding variants associated with fasting glucose (FG) and fasting
insulin (FI) levels, we analysed exome array data in up to 33,407 non-diabetic individuals of European ancestry. We identified multiple glucose-lowering coding variants in G6PC2, which resides in an established FG genomewide association study (GWAS) locus. Conditional single-variant association
analysis established the presence of two coding variants at exome-wide
significance (P<5x10-7) in this gene, one common (PCOND=7.1x10-10, 48.1%
MAF, p.V219L) and one rare (PCOND=1.3x10-11, 0.8% MAF, p.H177Y), both
were independent of the GWAS SNP at this locus and of each other (PV219L
=4.0x10-7 and PH177Y COND on V219L=3.7x10-10). Gene-based analysis
COND on H177Y
(PSKAT=8x10-10) highlighted another coding variant p.Y207S (MAF=0.5%)
with evidence of association with FG. In vitro studies of these G6PC2 variants showed a reduction in protein expression by 98% (p.H177Y, p<0.001),
100% (p.Y207S, p<0.001) and 43% (p.V219L, p<0.01) compared to wildtype in HEK293 cells, with similar results in INS1E cells. Protein expression
was rescued with a proteasome inhibitor implying degradation of unstable
G6PC2 proteins via the ubiquitin-proteasome pathway. Expressed variant
proteins localized as expected to the endoplasmic reticulum. We also identified two genes not mapping to previously reported GWAS loci in which
coding variants were associated with FG (GLP1R: P=4.4x10-7, 1.6% MAF,
p.A316T) or FI (URB2: P=3.8x10-7, 0.1% MAF, p.E594V). These data establish
a functional role for G6PC2 in FG homeostasis and identify novel glycaemic
trait loci. They do not support a role for low-frequency or rare coding variants in explaining previously reported association signals at GWAS loci.
C14.6
Transethnic association study of IBD identifies novel risk loci and
shows pervasive sharing of genetic risk factors across populations
38
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There are now over 163 loci associated with inflammatory bowel disease
(IBD) susceptibility in European populations, though little is known about
their contribution to disease risk in non-European populations. Using the
custom Immunochip array, we genotyped 9,846 individuals of East Asian
(Japan, South Korea, Hong Kong, China and UK individuals of East Asian
descent), Indian and Indo-European (Iranian) descent. Combining these
with the existing cohort of 81,248 individuals of European descent (Jostins
et al. Nature 2012), we identified 14 novel IBD susceptibility loci at genomewide significance (P<510-8). The majority of IBD risk loci were shared between both Europeans and non-Europeans, and differences in the amount of
variance explained in disease liability at each locus were driven by a combination of differences in allele frequencies and effect sizes. For instance, the
variance in Crohns disease (CD) liability explained by four independent risk
variants at TNFSF15 was 19 times greater in East Asians than Europeans, a
reflection of the risk-increasing alleles being more common and having larger odds ratios in East Asians. Aggregation of all SNPs assayed on the Immunochip revealed significant genetic correlation (0.4-0.85) for IBD between
European and non-European populations. Together, this study increases the
total number of IBD susceptibility loci to 177 and demonstrates the pervasive sharing of genetic risk factors across populations, though there exists
heterogeneity in the variance explained per locus.
C15.1
BCAP31 mutations cause a new X-linked syndrome with deafness,
dystonia, central hypomyelination and disorganization of the Golgi
apparatus
BAP31 is one of the most abundant proteins of the membrane of the endoplasmic reticulum (ER). It is a chaperone protein involved in several
pathways, including ER-associated protein degradation, export of proteins
from the ER to the Golgi, and programmed cell death. BAP31 is encoded by
the BCAP31 gene located in Xq28. It is highly expressed in neurons. We identified loss of function mutations in BCAP31 in seven individuals from three
different families and one missense mutation in another fourth family. These people suffer from intellectual and motor disability, dystonia and sensorineural hearing loss with abnormal white matter. The association of these
clinical signs define a new X-linked syndrome. In primary fibroblasts from
patients, we found that the absence of BCAP31 changed the morphology of
the ER and disorganized the Golgi apparatus in a significant proportion of
cells. We demonstrated that the constitutive deficiency of BCAP31 did not
activate the response to unfolded proteins (UPR) nor triggered cell death.
Rather, our data suggest that the absence of BAP31 affects ER and Golgi apparatus metabolism and that BAP31 plays an important role in ER-Golgi exchanges. These results provide a molecular basis for a new Mendelian syndrome and connect intracellular protein trafficking with a severe congenital
neurological disorder.
C15.2
Mutations in KPTN Cause Macrocephaly, Neurodevelopmental Delay,
and Seizures
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Cytogenetically visible chromosomal translocations are a unique resource as they can pinpoint strong effect genes. Here we report a mother and
daughter with borderline intelligence and learning problems within the
dyslexia spectrum and two apparently balanced reciprocal translocations;
t(1;8)(p22;q24),t(5;18)(p15;q11). By low coverage mate-pair whole genome sequencing we were able to pinpoint the genomic breaks to within a 2kb
window and the DNA breakpoints were then verified by PCR and Sanger sequencing. The 5p breakpoint was located between exon 7 and 8 of CTNND2.
By genome wide array comparative genomic hybridization we identified an
additional individual with similar phenotypic presentation and a maternally
inherited 163kb microdeletion exclusively involving CTNND2. The microdeletion at 5p11.2 is present as mosaicism in the mother and absent in the
patients healthy siblings.
Inappropriate migration of cortical neurons has been postulated as a potential mechanism for various neurological disorders including dyslexia, given
the potential role of CTNND2 in neuron motility, we were particularly interested in assessing defects in neuronal migration in vivo. Knockdown of
one of two zebrafish CTNND2 orthologues using antisense morpholino oligonucleotides showed mostly normal development of the neuronal clusters.
However, analysis of a subpopulation of forebrain neurons expressing GFP
under the islet promoter (isl:GFP) in knockdown embryos showed the presence of misplaced isl:GFP cells between the telencephalon and diencephalon
indicative of a defects in migration. This suggests that defective migration of
subpopulations of neuronal cells due to loss of CTNND2 could be part of the
underlying mechanism behind the cognitive dysfunction in the patients.
C15.6
Novel (ovario)leukodystrophy related to AARS2 mutations
39
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Child Health, London, United Kingdom, 3Molecular Medicine Unit, UCL Institute of Child
Health, London, United Kingdom, 4GOSgene, UCL Institute of Child Health, London,
United Kingdom, 5Group on the Molecular and Cell Biology of Lipids, Department of
Medicine, University of Alberta, Edmonton, AB, Canada, 6University Clinic of Paediatrics,
Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 7Clinical Genetics
Department, Great Ormond Street Hospital, London, United Kingdom, 8Radiology
Department, Great Ormond Street Hospital, London, United Kingdom, 9Department of
Paediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok,
Thailand, 10Department of Medical Genetics, The Childrens Memorial Health Institute,
Warsaw, Poland, 11Department of Biology and Medical Genetics, University Hospital
Motol and 2nd Faculty of Medicine, Prague, Czech Republic, 12Centre de Gntique
Humaine, Universit de Franche-Comt, Besanon, France, 13Cutis Laxa Study Group,
University of Franche-Comt, Besanon, France, 14Department of Pediatrics, University
of Washington, Seattle, WA, United States, 15Seattle Childrens Craniofacial Center,
Seattle, WA, United States, 16Department of Pediatrics, University of Torino, Torino,
Italy, 17Histopathology Department, Great Ormond Street Hospital for Children,
London, United Kingdom, 18Neural Development Unit, UCL Institute of Child Health,
London, United Kingdom, 19Reta Lila Weston Institute, UCL Institute of Neurology,
Queen Square, London, United Kingdom, 20Department of Molecular Neuroscience,
UCL Institute of Neurology, Queen Square, London, United Kingdom, 21Laboratrio de
Anemias Congnitas e Hematologia Molecular, Hospital Peditrico, Centro Hospitalar e
Universitrio de Coimbra, Coimbra, Portugal.
Lenz-Majewski syndrome (LMS) constitutes a unique rare disease characterised by the association of generalised sclerosing bone dysplasia, intellectual disability, cutis laxa and distinct craniofacial, dental, and distal limb
anomalies. By using whole-exome sequencing in four patients and selecting
variants according to the predicted dominant de novo etiology model, we
identified causative heterozygous missense mutations in PTDSS1 gene,
which encodes phosphatidylserine synthase 1 (PSS1). Subsequently, three
additional patients were screened by Sanger sequencing and similar mutations were found. In total, five different missense mutations affecting four
specific PSS1 amino acids were identified in the seven patients studied to
date. PSS1 is one of two enzymes involved in production of phosphatidylserine. Phosphatidylserine synthesis was increased in patients intact fibroblasts and end-product inhibition of PSS1 by phosphatidylserine was significantly reduced. Therefore, these mutations cause a gain-of-function effect
associated with regulatory dysfunction of PSS1. In support of this model,
craniofacial defects similar to those found in LMS, were observed following
expression of mutant but not wild-type PTDSS1 in zebrafish. Interestingly,
total phospholipid cellular content was similar between patient and control
fibroblasts. Ongoing lipidomic studies suggest that the identified mutations
cause abnormal fatty acid composition of individual subclasses of membrane phospholipids. In conclusion, we have identified LMS as the first human
disease caused by disrupted phosphatidylserine metabolism and one of
the few examples of gain-of-function mutations affecting an enzyme. Our
results bring important insight to the understanding of phosphatidylserine
metabolism and point to an unexplored link with bone development and
homeostasis.
C16.1
A congenital disorder of glycosylation, with lymphopenia,
neutropenia, and skeletal dysplasia, caused by mutations in the gene
encoding phosphoglucomutase 3 (PGM3).
Human phosphoglucomutase 3 (PGM3) catalyzes the conversion of GlNAc6-P into GlcNAc-1-P, during the synthesis of UDP-GlcNAc, a sugar nucleotide
critical to multiple glycosylation pathways. PGM3-defects have earlier been
associated with hematopoietic deficiency in a mouse model. We identified
three unrelated patients with recurrent infections, congenital leukopenia
including neutropenia, B and T cell lymphopenia, and progression to bone
marrow failure. Whole exome sequencing revealed novel, deleterious mutations in the PGM3 gene in all three subjects, delineating a new type of
congenital disorder of glycosylation (CDG). Functional studies of the human
immunodeficiency-associated PGM3 gene variants in E.coli cells demonstrated reduced PGM3 enzyme activity with decreased phosphate group transfer from position GlcNAc-6-P to GlcNAc-1-P. Two of the 3 patients had skeletal anomalies with short stature, brachydactyly, and intellectual disability,
however these additional features were absent in the third patient, showing
the clinical variability of the disease. Two patients were transplanted using a
cord blood hematopoietic stem cell and a matched-related donor bone marrow; both had successful engraftment and correction of neutropenia and
lymphopenia. We define PGM3-CDG as a novel cause of treatable genetic immunodeficiency, document the power of whole exome sequencing in gene
discoveries for rare disorders, and illustrate the utility of genomic analyses
in studying combined and variable phenotypes.
C16.2
Lenz-Majewski syndrome: disturbed phosphatidylserine metabolism
causes intellectual disability and a sclerosing bone dysplasia
40
C16.3
Homozygous FIBP truncating mutation in a new multiple congenital
anomalies syndrome with overgrowth, macrocephaly, Iris coloboma,
and learning disabilities
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Sciences and Technologies, University of Salento, Lecce, Italy, 4PhD Program, Scienze
della Riproduzione e dello Sviluppo, University of Trieste, Trieste, Italy.
Kabuki syndrome (KS) is a multiple congenital malformation syndrome characterized by facial features, skeletal anomalies, dermatoglyphic abnormalities, mental retardation, and postnatal growth deficiency. Heterozygous mutations in the KMT2D gene are detected in 60% of Kabuki patients. KMT2D
gene encodes a H3K4 histone methyltransferase that plays important role in
the epigenetic control of active chromatin states modulating the expression
of genes essential for embryogenesis and development. A subset of KS individuals was recently identified with mutations in the chromatin modifier
KDM6A. We performed a mutational screening on 303 Kabuki patients by
direct sequencing, MLPA, and qPCR, detecting 133 patients with KMT2D and
four with KDM6A mutations. Among the KMT2D mutations we identified
46 missense variants across the entire length of the KMT2D gene, 16 were
inherited from an apparently asymptomatic parent.
Aim of this study is to ascertain the pathogenicity of KMT2D missense mutations through an integrative analysis of bioinformatics tools and biochemical
and cellular assays. We used an innovative in silico approach that combines
comparative analysis and motif/domain search tools with threading/homology modeling protocols to predict functional/structural effect of KMT2D
missense variants and identify and confirm potential pathogenic mutations.
Due to the huge size of KMT2D gene, we devised a strategy where minigenes carrying KMT2D missense variants were generated. We evaluated their
potential pathogenicity effects by measuring KMT2D enzymatic activity and
expression level of KMT2D known target genes. Our strategy should offer a
valuable support to estimate the real deleterious effect of KMT2D missense
variants, an issue in diagnostic counseling.
C17.1
A dominant mutation in CHCHD10 causes neurodegenerative disorder
with mitochondrial DNA instability
V. Paquis-Flucklinger1,2, S. Bannwarth1, S. Saadi1, A. Chaussenot2, E. Genin1, K. Fragaki1,
L. Berg-Alonso1, S. Lacas-Gervais3, V. Serre4, A. Verschueren5, C. Rouzier1, G. Aug1, C.
Cochaud2, F. Lespinasse1, J. Pouget5;
1
IRCAN, UMR CNRS 7284/INSERM U1081/UNS, Nice, France, 2Department of Medical
Genetics, CHU Nice, Nice, France, 3Joint Center for Applied Electron Microscopy, Nice,
France, 4UMR7592 CNRS, Paris, France, 5Department of Neurology, Timone hospital,
Marseille, France.
Mutations in OPA1 and MFN2, two genes encoding membrane proteins involved in mitochondrial dynamics, are responsible for mitochondrial DNA
(mtDNA) instability disorder with Autosomal Dominant Optic Atrophy
(ADOA) plus phenotype. We report a large family with a late-onset complex phenotype including motor neuron disease, cerebellar ataxia, cognitive
decline and myopathy. Muscle biopsy showed ragged-red and COX negative
fibres with combined respiratory chain deficiency and abnormal assembly
of complex V. The multiple mitochondrial DNA (mtDNA) deletions found in
skeletal muscle revealed a mtDNA instability disorder. By whole-exome sequencing (WES), we identified a missense mutation (c.176C>T; p.Ser59Leu)
in the CHCHD10 gene that encodes a coiled-coil helix coiled-coil helix protein, whose function was unknown. We show that CHCHD10 is a mitochondrial protein located in the intermembrane space and enriched at cristae
junctions. Patient fibroblasts carrying the CHCHD10 mutation present with
a respiratory chain deficiency and a fragmentation of the mitochondrial network. Furthermore, we show that overexpression of CHCHD10S59L triggers
mitochondrial fragmentation in HeLa cells, thus confirming the deleterious
effect of this mutant on mitochondrial morphology and network. DRP1K38A, which is resistant to fission, did not modify the mitochondrial fragmentation observed in cells expressing CHCHD10S59L, suggesting that the
CHCHD10 mutant leads to impaired fusion activity. This work, suggesting
that CHCHD10 plays a role in mitochondrial fusion and/or in maintenance
of cristae morphology, highlights the critical role of mitochondrial dynamics
in terms of human disease and mitochondrial genome stability.
C17.2
Decoding Mitochondrial Disorders using Exome Sequencing
Mitochondrial disorders are a heterogeneous group of diseases characterized by faulty oxidative phosphorylation. Despite good progress in the field,
most disease causing mutations still have to be identified. We applied whole
exome sequencing in 300 unrelated individuals with juvenile-onset mitochondrial disorder. In a quarter of patients, we detected mutations in known
disease genes. In another quarter of patients, we identified mutations in ge-
41
MNGIE is an autosomal recessive disease caused by deficiency of the enzyme thymidine phosphorylase (TP), resulting in systemic accumulation of
nucleosides thymidine (Thd) and deoxyuridine (dUrd), and mtDNA deletions, depletion and dysfunction. We aimed to use ex vivo lentiviral vector (LV)
hematopoietic stem cell (HSC) gene therapy to deliver human TP enzyme in
Tp-/-Upp-/- double knockout mice, a model for MNGIE disease. To that end, LV
transduced Tp-/-Upp-/- HSCs containing the native cDNA sequence (TP) or
codon optimized (TPco) driven by the phosphoglycerate kinase (PGK) or the
spleen focus forming virus (SFFV) promoter were transplanted in sublethally irradiated Tp-/-Upp-/- mice. Wild type mice had detectable but very low
levels of enzyme activity in blood cell fractions (0.07 0.03nmoles/h/mg,
N=4), 1 month post transplantation, enzyme activities increased at least 90fold in LV recipient mice (LV-TP and LV-TPco = 1504 and 964 nmoles/h/
mg respectively, N=4), with a 400-fold increase observed in recipients of LVSFFV-TPco (4505, N=4), resulting in reduction of Thd and dUrd in plasma
and urine. TP activity was detectable in brain of gene therapy-treated mice
14 months after treatment (average of 1.2-fold in LV-TP and LV-TPco compared to wild type and 36-fold for LV-SFFV-TPco), which significantly reduced
the nucleoside levels.High molecular chimerism (76.58.2 % donor chimerism, N=12) with low LV vector copy numbers (1.011.1VCN/donor derived
cell, N=12)were achieved.Overall, HSC gene therapy resulted in stable TP expression and long-term biochemical correction without geno-phenotoxicity
in MNGIE mice to be further optimized to develop a clinical protocol to treat
MNGIE patients.
C17.4
Deletion of a distant-acting enhancer near C16ORF91 underlies
recessive congenital diarrhea
We studied eight patients from seven families of Jewish Iraqi origin, suffering from congenital diarrhea, historically defined as intractable diarrhea
of infancy syndrome (IDIS). Next-generation sequencing and SNP arrays
identified two structural genomic deletion alleles near C16ORF91, leading
to a homozygous deletion of a 1500bp intergenic critical region (V) shared
by all patients. The V region contained a segment with vertebrate evolutionary conservation and transcription factor binding clustering. V was
shown by mouse embryo transgenic assay to harbor a robust enhancer,
active in specific sub-regions of stomach, duodenum and pancreas at early
developmental stages (E11.5-14.5). In a mouse V deletion strain, homozygous offspring had on average 36% smaller post-natal weight as compared
42
Back to index
Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare developmental disorder affecting children with segmental features of aging, including thin skin, loss of subcutaneous fat, alopecia, osteopenia and accelerated cardiovascular arteriosclerotic disease, leading to death at the mean
age of 13 years. The syndrome is due to a mutation in the gene encoding
lamins A/C, leading to the production of an aberrant lamin A precursor
called progerin. Intranuclear progerin accumulation exerts multiple toxic
effects which ultimately lead to organismal premature aging. Preclinical
data provided proof-of-principle that the combined use of zoledronate and
pravastatin (ZoPra) could ameliorate several disease parameters, including
Back to index
analyzed 106 patients (mostly males and sporadic cases) with molecularly
undiagnosed ID. One third of them harbored autistic features.
We identified causative mutations in 26 patients: sixteen in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA,
MECP2, SLC9A6, SLC16A2, PHF8), 10 de novo in autosomal genes (DYRK1A,
GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We detected likelycausative mutations requiring additional validation in 5 patients (in NLGN3
for instance). Our findings confirm MED13L as an ID gene, but raise doubts
on SHROOM4 or SRPX2. We identified causative mutations in syndromic genes in patients deviating from the classic phenotype. Some genes were hit
more than once suggesting they correspond to more frequent conditions.
The identification of mutations in 25-29% of patients proves the diagnostic
efficiency of this strategy, higher than CGH and comparable to trio-exome
sequencing at lesser cost. We have now updated our captured gene list to
270 genes, and will report on its use.
C18.3
Comprehensive NGS based diagnostics in over 1000 patients with
epileptic disorders
43
Clinical exome sequencing is an unbiased test that is used for straight-forward causative mutation detection, or for the discovery of novel (allelic)
gene - disease associations. Often, it leads to broader disease spectra, or reverse phenotyping. Here, we describe the use of clinical exome sequencing
for a group of 76 probands with ataxia or spastic paraplegia. Since these
patients have had extensive testing (4.5 genes on average) before inclusion,
mutations were not anticipated in the common genes. In a two-tier analysis, variants in known disease genes were analysed first, followed by analysis of the full exome data set if no causative mutations were identified.
Subsequently, segregation analyses, enzyme tests or reverse phenotyping
tests have confirmed or excluded the pathogenicity of most variants. In those 76 patients, we have thus detected causative mutations in 16, and likelycausative in another 9, all in the known disease genes. Some of the identified diseases are very rare or even the second case described. Furthermore,
another 8 putatively-causative mutations were detected in genes outside
the known disease genesl, and await confirmation (functional tests or other
families with mutations in those gene). Some of the latter diseases may be
allelic disorders, while others may be novel. In conclusion, exome sequencing for these neurological disorders has resulted in a molecular diagnosis
in one-third of the patients, and has provided a candidate gene identification
in approximately 1/10. More importantly, it has also provided some patients
with a molecular diagnosis that was both unanticipated and otherwise not
revealed.
C18.6
WES detects disease causing SNVs and CNVs in Primary immunodeficiencies
44
Back to index
C19.1
Constitutive Activation of PRKACA in Adrenal Cushings Syndrome
Endogenous hypercortisolism, referred to as Cushings syndrome, is associated with excess morbidity and mortality. Corticotropin-independent
Cushings syndrome is caused by tumors or hyperplasia of the adrenal cortex.
We performed exome sequencing of ten cortisol-producing adenomas and
matched control tissue to identify somatic mutations and evaluated recurrent mutations in candidate genes in adenomas of additional 171 patients.
We further performed genome-wide copy number analysis in 35 patients
with cortisol-secreting bilateral hyperplasias. We studied the effects of these genetic defects both clinically and in vitro.
Exome sequencing revealed somatic mutations in the PRKACA gene, which
encodes the catalytic subunit of cyclic AMP-dependent protein kinase (PKA),
in 8 of 10 adenomas. Overall, PRKACA somatic mutations were identified
in a total of 22 of 59 adenomas (37%) from patients with overt Cushings
syndrome; these mutations were not detectable in patients with subclinical hypercortisolism (n=40) or in other adrenal tumors (n=82). Among 35
patients with bilateral cortisol-producing hyperplasias, 5 carried a germline copy number gain of the chromosome 19 region, including the PRKACA
gene. In vitro studies demonstrated impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, while cells from patients
with germline chromosomal gains showed increased protein levels of the
PKA catalytic subunit; in both instances, basal PKA activity was increased.
This study links genetic alterations of the catalytic subunit of PKA to human disease. Germline duplications of this gene result in bilateral adrenal
hyperplasias, whereas somatic PRKACA gain-of-function mutations lead to
unilateral cortisol-producing adrenal adenomas.
C19.2
A mutation in SEC61A1 causes autosomal dominant interstitial
kidney disorder associated with anemia and growth retardation
Progressive familial intrahepatic cholestasis (PFIC) is an autosomal recessive disorder manifesting as early-onset cholestatic liver disease. In 1998
mutations in three genes encoding membrane transporters were identified.
However, 30% of patients remain without genetic diagnosis. Combined
targeted resequencing (TRS) and whole-exome sequencing were used to
analyse a cohort of 33 children, from 29 families, with chronic cholestatic
liver disease and no mutations in known PFIC genes. Most were from consanguineous families. Homozygous mutations in TJP2 were identified in
12 individuals of 8 families; all were predicted to abolish translation. TJP2
encodes a cytosolic component of cell-cell junctional structures, linking
integral membrane proteins such as claudins with actin filaments. Immunohistochemistry and western blotting of liver tissue from these patients
showed a complete absence of TJP2 protein. Claudin-1 was not localised,
but showed no alteration in protein level. Tight junctions were abnormal
on transmission electron microscopy. A further 53 cholestatic patients were
then examined by TRS. Mutations in TJP2 were identified in 8 families. All
mutations were protein-truncating mutations, except for three families with
missense mutations. One missense mutation, p.His788Leu, was found in 2
unrelated families with severe early-onset cholestasis, which subsequently
remitted. Previously a single, incompletely penetrant, missense mutation
was identified in TJP2 in Amish patients with isolated hypercholanaemia. In
conclusion, a complete absence of TJP2 causes severe cholestatic liver disease, with destabilization of tight junction structures, whilst missense mutations in TJP2 can lead to less severe phenotypes. These findings highlight the
importance of TJP2 in the maintenance of liver integrity.
C19.4
Identification and functional characterization of ESR2, a new disease
gene for 46,XY disorders of sex development (DSD)
Back to index
frame deletion in ESR2 in this patient, c.541_543del (p.Asn181del). The deleted amino acid is located in the DNA-binding domain and is highly conserved. This deletion was absent in an ethnically matched control population.
ESR2 mutation screening in a 46,XY DSD cohort revealed an additional heterozygous mutation c.251G>T (p.Gly84Val). Prediction programs suggest an
effect on protein function (SIFT, Polyhen, Mutation Taster). In addition the
affected amino acid is conserved until fruitfly.
Immunohistochemistry in an 8-weeks old human male embryo showed ERbeta expression in the hindgut and the eyes, which might recapitulate the
systemic manifestations of the index case.
Dose-response assays with DPN, ERE-luc and TK-3xEREluc constructs, and
ER-beta wild type and mutant constructs in HEK293T cells showed a differential transcriptional activation of the ER-beta mutants.
In conclusion, our study sustains a role of ESR2 as novel disease gene for
syndromic 46,XY DSD. It is expected that further functional studies of these
ER-beta mutants will provide insights into their molecular consequences
and into the role of ESR2 in the pathogenesis of 46,XY DSD.
C19.5
LRP5 variants associated with development of polycystic kidney and
liver disease
Alport syndrome (ATS) is a clinically and genetically hereditary nephropathy, that is often associated with deafness and ocular lesions. Monogenic inheritance models are well known, with semidominant X-linked inheritance,
linked to COL4A5 gene, or autosomal dominant or recessive inheritance, linked to COL4A3 or COL4A4 gene. The increased availability of high throughput
sequencing permits identification of kindreds with pathogenic mutations in
more than one disease-gene, also exploring digenic inheritance models. We
have found 6 kindreds, in which the proband had 2 pathogenic mutations in
different Alport associated genes. In 4 out of 6 cases it was shown that each
45
De novo germline mutations create the genetic variability that is the driving
force of species evolution. However, they also can cause sporadic genetic
diseases if they affect critical genomic regions. Whole Genome Sequencing
(WGS) of parent-offspring trios allows us for the first time to study the result of mutational processes in a single generation in full detail. Here we
report on the rate and pattern of de novo mutations based on WGS of 50
trios at high (80x median) coverage.
We identified an average of 58 de novo mutations (DNM) per trio (range
32-84), 2,883 DNMs in total. By using the segregation of informative SNPs
we determined the parental origin for 678 DNMs, and found a paternal/maternal ratio of 4 to 1 (79%/21%), consistent with previous data. Using the
parental origin of these DNMs we found a significant correlation between
the paternal age and the number of paternal DNMs (Spearmans p=0.006)
as well as a suggestive results for the correlation between maternal age and
the number of maternal DNMs (p=0.08).
We find that DNMs do not occur completely random in the genome, but are
spatially clustered within individuals (observed=28 (0.9%), expected=1.3
(0.004%), p-value<10-16) and have a sequence context that is enriched for
CpGs (observed=482 (20.6%), expected=58 (2.6%), p-value<10-16). In conclusion, our study provides insight into the parental origin and distribution
of de novo mutations throughout the human genome.
46
Back to index
C20.3
Study of the regulatory landscape of SHOX in 503 LWD and ISS cases
uncover a key role of the upstream cis-regulatory element CNE-3
The human sex chromosomes differ in sequence, except for the pseudoautosomal regions (PAR) at both the short and the long arm, denoted as PAR1
and PAR2. The boundary between PAR1 and the unique X and Y sequences
was established during the divergence of the great apes. During a copy number variation screen, we noted a paternally inherited chromosome X duplication in 15 independent families. Subsequent genomic analysis demonstrated that an insertional translocation of X chromosomal sequence into
the Y chromosome generates an extended PAR. The insertion is generated
by non-allelic homologous recombination between a 548 bp LTR6B repeat
with two paralogues located within PAR1 as well as at a distance of 105
kb from the PAR boundary on the X chromosome. The identification of the
reciprocal deletion on the X chromosome in one family and the occurrence
of the variant in different chromosome Y haplogroups, demonstrate this is
a recurrent genomic rearrangement. In contrast to its perceived evolutionary stability, we demonstrate that a PAR1 length polymorphism exists in the
human population. This finding represents a novel mechanism shaping sex
chromosomal evolution.
C20.5
Comparative proteomic analysis of different fragile X syndrome cell
lines
Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, is caused by absence of the FMRP protein due to expansion over 200
repeats of the CGG tract at the 5 UTR of the FMR1 gene and subsequent
The endoplasmic reticulum (ER) is an organelle where proteins are synthesized and modified. ER stress occurs when there is an excess of misfolded
proteins, and if the stress persists, then apoptosis is induced. Here we study RNA editing as a part of the ER stress response and how RNA editing
modulates the cellular response to promote cell survival. We sequenced the
RNA and DNA of B-cells from 10 individuals before and following 2 and 8
hours of tunicamycin treatment to induce ER stress. By comparing the RNA
and DNA sequences, we identified 15,823 A-to-G editing sites in 1,523 genes
as targets of RNA editing by ADAR (Adenosine Deaminase Acting on RNA).
Among these sites, 314 showed significant changes in editing level following
ER stress (p-value<0.01; ANOVA). These sites are found in genes known to
be involved in ER stress response: RNA processing, protein transport and
apoptosis. For example, sites found in XIAP (X-linked Inhibitor of Apoptosis)
show a 3-fold increase in editing level. Furthermore, ADAR knock-down increases apoptosis following ER stress, suggesting that RNA editing is induced to promote cell survival. The effect of ER stress on editing level is not
limited to canonical ADAR editing, as we also detected other types of differences between the RNA and DNA sequences whose levels change following
tunicamycin treatment. In this presentation, we will describe RNA sequence
modification as a critical step in ER stress response and cell survival.
C21.1
Mutations in POGLUT1, encoding protein O-glucosyltransferase 1,
cause autosomal dominant Dowling-Degos disease
Back to index
(c.11G>A (p.Trp4*), c.652C>T (p.Arg218*), c.798-2A>C) in these five individuals, all of which are in POGLUT1, encoding protein O-glucosyltransferase
1. By further screening of POGLUT1 in our unexplained cases of DDD, we
identified six additional mutations. Immunohistochemistry of skin biopsies of affected individuals with POGLUT1 mutations showed significantly
weaker POGLUT1 staining in the upper parts of the epidermis compared to
healthy controls. Immunoblot analysis revealed that translation of the wild
type POGLUT1 and a mutated form with an amino acid substitution led to
the expected size of about 50 kDa, while a nonsense mutation led to a truncated protein of about 30 kDa. Immunofluorescence analysis identified a colocalization of the wild type protein with the endoplasmic reticulum and a
notable aggregating pattern for the truncated protein. Protein modeling and
transcript analysis supported the pathogenicity of the identified mutations.
Recently, mutations in POFUT1, encoding protein O-fucosyltransferase 1,
were also reported to be responsible for DDD. Both POGLUT1 and POFUT1
are essential regulators of Notch activity. Our results emphasize the important role of the Notch pathway in pigmentation and keratinocyte morphology.
C21.2
The phenotypic spectrum of SHOC2 c.4A>G (p.Ser2Gly)
47
Loss of function mutations in PAK3 contribute to non-syndromic X-linked intellectual disability (NS-XLID) by affecting dendritic spine density and morphology. Linkage analysis in a three-generation family with affected males
showing intellectual disability, agenesis of corpus callosum, cerebellar hypoplasia, microcephaly and ichthyosis, revealed a candidate disease locus in
Xq21.33q24 encompassing over 280 genes. By sequencing all coding exons
of the X chromosome, we identified a single novel variant within the linkage
region, affecting a conserved codon of PAK3. Biochemical studies showed
that, similar to NS-XLID-associated lesions, the predicted amino acid substitution (Lys389Asn) abolished the kinase activity of PAK3. In addition, it conferred a dominant negative function to the protein that drives the syndromic
phenotype. The in silico tridimensional model of the mutated proteins and
in vitro inhibition of protein neosynthesis by cycloheximide treatment revealed that NS-XLID mutations make the inactive kinase unstable and prone
to degradation, while Lys389Asn confers stability to PAK3 protein, which
escapes its physiologic degradation. Finally, in vivo studies in zebrafish embryos showed that PAK3N389 leads to a perturbation of the MAPK signaling
and to alterations of cerebral and craniofacial structures, through an uncontrolled kinase-independent function. Our data expand the spectrum of phenotypes associated with PAK3 mutations, characterize a novel mechanism
resulting in a dual molecular effect of the same mutation with a complex
PAK3 functional deregulation, and provide evidence for a direct functional
impact of aberrant PAK3 function on MAPK signaling, with the generation
of a phenotype that could be included within the clinical spectrum of Rasopathies.
C21.5
Activating mutations in RRAS underlie a phenotype within the
RASopathy spectrum and contribute to leukaemogenesis
48
Back to index
RASopathies, a family of disorders characterised by cardiac defects, defective growth, facial dysmorphism, variable cognitive deficits and predisposition to certain malignancies, are caused by constitutional dysregulation
of RAS signalling predominantly through the RAF-MEK-ERK cascade. We
report on two germline mutations in RRAS, a small monomeric GTPase
controlling cell adhesion, spreading and migration, underlying a variable
phenotype with features partially overlapping Noonan syndrome, the most
common RASopathy. We also document that somatic RRAS mutations rarely
occur in juvenile myelomonocytic leukaemia, a childhood myeloproliferative/myelodysplastic disease caused by upregulated RAS signalling, defining
an atypical form of this haematological disorder rapidly progressing to acute myeloid leukaemia. Two of the three identified mutations affected known
oncogenic hotspots of RAS genes, and conferred variably enhanced RRAS
function and stimulus-dependent MAPK activation. Expression of a RRAS
mutant homolog in Caenorhabditis elegans enhanced RAS signalling, and engendered protruding vulva, a phenotype previously linked to a RASopathycausing SHOC2 mutant. These findings establish a functional link between
RRAS and RAS signalling, and reveal an unpredicted role of enhanced RRAS
function in human disease.
C21.6
A New Mouse Model for Costello Syndrome
Costello Syndrome (CS) is a distinctive rare multisystem disorder comprising characteristic prenatally increased growth retardation, coarse facial
features, redundant skin with deep palmar, plantar creases and papillomata
of later onset. CS patients present also laxity of small joints, tight Achilles
tendons, cardiac malformations, and developmental delay. The primary cause of CS was associated to the germ line activation of H-Ras oncogene, with a
common missense mutation G12S in 80% of the patients. Here we describe
the generation and the consequent phenotypic characterization of a genetically engineered mouse model of CS, by introducing the oncogenic G12S
mutation by homologous recombination into the mouse Ras gene. The effect
of the H-Ras G12S mutation was evaluated on behavioral, visual, metabolic,
cardiac and histological traits in young adult animals. The behavioral analysis revealed that H-Ras G12S mutant males displayed reduced locomotor
activity, accompanied by decreased muscle strength and altered motor coordination performance. In addition, the cardiac exploration revealed that HRas G12S mutants exhibit a hypertensive phenotype combined with tachycardia. In conclusion, the H-Ras G12S mutant mice showed a polysyndromic
phenotype reproducing some of the CS features observed in patients. The
future study of the here-described CS mouse model should have a significant
impact of our understanding of CS disease. The use of H-Ras G12S mutant
mice as a CS mouse model opens up new fields of investigation to better
understand the pathophysiology of the disease and to evaluate drugs dedicated to the reduction of the disease associated symptoms.
C22.1
The impact of reporting exome and whole genome sequencing:
Predicted frequencies of primary, secondary and incidental findings
based on modelling
The American College of Medical Genetics and Genomics (ACMG) has released practice guidelines recommending reporting of incidental findings
(IFs) from exome and whole genome sequencing by Massively Parallel (Next
Generation) Sequencing for multiple conditions. Policy statements from additional agencies are still being developed, with many attempting to take
into consideration the predicted increase in workload of reporting IFs and
secondary findings. We describe the effects on rates of various diagnostic
findings of changing the sensitivity, the specificity, the implications of varying diagnostic criteria and a priori prevalence, and of increasing the number of included conditions.
Methods: We developed a simple mathematical model based on binomial
There has been a discussion regarding the ethical acceptability of genetic testing of children for years, resulting in the majority view that minors should
only be tested for early onset disorders where treatment or preventive options exist. Two principles underlie this consensus: first, the beneficence-based
best interest standard that urges physicians to test for clinically relevant and
actionable results. Second, the childs right to an open future principle that
urges physicians not to test for adult onset disorders and carrier status, in order to preserve the childs future autonomy right to make its own decisions,
also concerning the possible obtainment of genetic information.
The emergence of next generation sequencing (NGS) technology seems to
challenge the previous consensus. There now is a growing list of commentators and examples from practice showing disagreement on whether conditions that do not have immediate consequences for the health of the child
should be disclosed to parents. The American College of Medical Genetics
and Genomics for example recently proposed to relinquish the current distinction between pediatric and adult genetic testing policy, thereby abandoning the childs right to an open future, while the American Academy of
Pediatrics maintains the previous consensus.
In this paper we explain the normative rationale that underlies the current
debate on a child- versus family centred genetic testing policy and argue that
the right to an open future should remain a leading ethical principle in pediatric genomics.
C22.3
Implementation of a duty-to-recontact system in molecular and
clinical genetics: perspectives from professionals and patients
Advances in DNA-diagnostic techniques and NGS are leading to more diagnoses, but also to interpretation problems. Many findings cannot be interpreted yet, but may prove medically relevant in the future. Currently, there
is no moral or legal obligation to recontact former patients. Many geneticists
nevertheless consider it desirable to recontact patients when important
new information arises. The UMCG (Groningen, NL) is investigating whether
to develop a recontacting system in clinical genetics and how one could be
implemented. We explored how professionals and patients from our university hospital feel on these two issues. We organised a focus group discussion
with 12 professionals (clinicians, clinical geneticists and laboratory staff)
and two group discussions with 3 and 5 patients, respectively, in which we
discussed the desirability and requirements for successfully implementing
recontacting. Both professionals and patients agreed that recontacting is
desirable and implementation could be successful if the following requirements are met: (1) provide a guideline describing the responsibilities of
molecular geneticists, genetic counsellors, and patients. This would also
specify what information requires recontacting, which information should
be given priority, and how recontacting should take place; (2) availability
of a lab-based databank containing up-to-date genetic information and the
possibility to automatically match new genetic information, e.g. on former
variants of unspecified significance, with patients/groups for which the
information is important; (3) patients preferences regarding recontacting
(e.g. what type of information and which recontacting medium to use); (4)
development of e-health devices facilitating automatic recontacting.
Back to index
C22.4
International views on sharing incidental findings from whole
genome research
Whilst genome-wide sequencing in a research setting may be used to explore the genetic basis of a phenotype it also offers the chance to opportunistically screen for additional results unrelated to the research project but
relevant to the participants future medical health (termed incidental findings, IFs).There is a wealth of medical and ethics literature supporting the
feedback of IFs, yet there are limited empirical work offering a voice from
both professional and public stakeholders directly affected by this.
A cross-sectional, web-based survey investigated the attitudes of 6944 individuals from across 91 countries towards searching for and sharing IFs.
Participants included 4961 members of the public, 533 genetic health professionals, 843 non-genetic health professionals and 607 genomic researchers.
Eighty percent of participants believed that IFs from sequencing studies
should be made available to research participants if they want them. Treatability and perceived usefulness of the data were important with 98% personally interested in learning about life-threatening conditions that were preventable. However, only 31% of participants thought genomic researchers
should actively search for IFs that were not relevant to their research study.
Genetic health professionals were the most likely to take this view (OR =
3.09, CI 2.23-4.28, P < 0.0001). This may be due to their appreciation of the
complexities involved in translating genomic data in the clinic. Participants
felt that genomic researchers should be able to focus on their research question without being forced to actively search for IFs, potentially at the expense of their study.
C22.5
Newborn screenings and whole genome sequencing: the real need of
a genuine public involvement
M. Tomasi1, A. Santosuosso2;
1
University of Trento, Trento, Italy, 2University of Pavia, Pavia, Italy.
In the last decade newborn-screening programs and whole genome sequencing are making their way in clinical practice.
The question we focus on is whether the intrinsic persistence in time of genetic characters (monitored in newborn-screening programs), once combined both with the rules governing bio-banks which store those information
and the natural lifelong evolution of personal individuality (from infancy to
old age), creates an apparently inextricable set of problems.
In this paper we have the ambition simply to draw a list of them in order to
open up a space for a public interdisciplinary discussion about new arising
challenges that need to be framed and faced by public policies.
Some examples are:
- The complex and not yet unraveled relationship between the gradual evolution of the interested person and epigenetic developments.
- Regulations of parents opting in/out screening programs and the chances
of modifying their choices during time.
- The involvement of relatives and future siblings.
- The (im)possibility of a real opt out during the phase of reconsent.
- Difficulties arising from the organizational rules of biobanking in relation
to the described long lasting process of consent.
- Privacy issues deriving both from the stable relationship between person
and collected data (impossible anonymousness) and the possibility of control over informational flows.
We maintain it is necessary to foster a real engagement of community in genuine discussion on a) benefits of a universal system of genetic monitoring
and b) ethical and legal implications for personal rights.
C22.6
Current Developments in the Regulation of Direct-to-Consumer
Genetic Testing in Europe
49
50
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ABSTRACTS POSTERS
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ESHG POSTERS
P01.002-M
Chromosome methylation and hydroxymethylation patterns
are dynamically reprogrammed during human preimplantation
development
We analyzed the distribution of 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in metaphase chromosomes from IVF-produced human
triploid zygotes and morphologically abnormal cleavage stage embryos. To
obtain metaphase chromosomes, zygotes and embryos were treated with
0.1% colchicines, 0.9% sodium citrate and fixed with freshly prepared 3:1
methanol:acetic acid. Then, QFH/AcD-banding technique was applied and
cytosine modifications were detected by indirect immunofluorescence.
In zygotes, parental sets of chromosomes had reversed patterns of methylation and hydroxymethylation. Paternal sets, detected by the presence
of chromosome Y, contained little 5mC, but were enriched in 5hmC, while
maternal chromosomes were heavily methylated and contained little 5hmC.
Thus, active demethylation in zygotes involves both parental genomes, but is
more intensive in paternal genome. Metaphase chromosomes from zygotes
had band-specific distribution of 5hmC: R-bands, but not G-bands and pericentromeric heterochromatin, were enriched with 5hmC. In contrast, 5mC
did not demonstrate band-specific distribution. In 3-cell embryos, chromosomes with sister chromatids differed both in the levels of 5hmC and 5mC
were identified. Number of these asymmetric chromosomes in blastomeres decreased with each cleavage division up to the blastocyst stage. At the
blastocyst stage asymmetric chromosomes were rarely encountered. This
advocates for the replication-dependant loss of 5hmC and 5mC.
Thus, maternal and paternal chromosomes demonstrate reverse patterns
of 5hmC and 5mC at the zygote stage. The number of both methylated and
hydroxymethylated chromosomes decrease during cleavage divisions, suggesting replication-dependant loss of modified cytosine.
Supported by RFBR, Administration of St. Petersburg, OPTEC grant and stipend from RF President.
P01.003-S
Single-tube multiplex-PCR panels of highly polymorphic STR markers
for application to prenatal and pre-implantation genetic diagnosis of
alpha- and beta-thalassemia and fragile X syndrome
M. Chen1, A. S. C. Tan1, H. Law2,3, S. S. Chong1,4;
1
National University of Singapore, Singapore, Singapore, 2KK Womens and Childrens
Hospital, Singapore, Singapore, 3Duke-NUS Graduate Medical School, Singapore,
Singapore, 4National University Hospital, Singapore, Singapore.
Alpha- and beta-thalassemia and fragile X syndrome (FXS) rank among the
most common monogenic disorders globally. Prenatal diagnosis (PND) and
pre-implantation genetic diagnosis (PGD) of these disorders are usually
performed by direct mutation detection. Flanking polymorphic markers
provide alternative indirect mutation detection through linkage analysis,
or serve to corroborate direct mutation testing results. Currently, limited
numbers of linked markers have been optimized and validated for PND/
PGD of these diseases. We performed in silico mining to identify 24, 99 and
122 short tandem repeats (STRs) within 1 Mb on either end of the HBA, HBB
and FMR1 loci, respectively. Markers with low heterozygosity (HET) and/or
polymorphism information content (PIC) after preliminary analysis on 16
anonymous DNAs were dropped, prior to large scale testing on 288 or 480
anonymous DNAs. The 9-plex HBA STR set (0.68PIC0.92; 0.71HET0.93;
10alleles34) can be further multiplexed with the Y1 box or other HBA2
exonic fragments for simultaneous deletion/point mutation detection with
linkage analysis, for either HbBarts or HbH disease. The 15-plex HBB STR
set (0.70PIC0.89; 0.74HET0.90; 10alleles23) is further multiplexed
with additional HBB exonic fragments to allow simultaneous thalassemia
major mutation detection with linkage analysis. The 13-plex FMR1 STR set
(0.40PIC0.86; 0.49HET0.87; 6alleles17) can be analyzed in parallel
with FMR1 CGG repeat expansion mutation detection for FXS, using aliquots
of whole genome amplified (WGA) product. All three single-tube multiplexPCR STR assays have been optimized for use on genomic DNA, single cells,
as well as WGA products of single cells.
P01.004-M
Two unusual unbalanced X chromosome rearrangements: a case
report
M. Exlerova1, V. Wernerova1, V. Horinova1, I. Slamova1,2, H. Filkov2, V. Vallova2, P.
Kuglik2, J. Sobotka3, P. Texl1;
1
Sanatorium Helios ltd., Laboratory of Medical Genetics, Brno, Czech Republic,
P01.005-S
Anti Mllerian Hormone (AMH) association analysis on about 1,000
caucasian women highlights 2 suggestive loci
51
ABSTRACTS POSTERS
wed a 1.34 Mb sized loss on chromosome 4p (4pter-->16.3) and a 32.48 Mb
sized gain on chromosome 7p (7pter-->14.3). Therefore, the final karyotype
of the patient was defined as 46,XX,der(4)t(4;7)(p16.3;p14.3). The parental
karyotypes were normal. In this case, we describe for the first time, as far as
we know, the clinical and cytogenetic analysis of a patient with concomitant
occurrence of partial of Hirschhorn (4p-) syndrome and partial trisomy 7p.
This report suggests that the array CGH would be a valuable diagnostic tool
for identifying the origin of small additional genetic materials.
P01.007-S
Preimplantation genetic screening of copy number variations
(CNVs) using oligonucleotide-based array comparative genomic
hybridization
Chromosome aneuploidy is the most prevalent genetic abnormality in human embryos and represents the leading genetic cause of miscarriages. In
view of this fact, diagnosis of embryos for chromosome abnormalities using
array-CGH, i.e. preimplantation genetic screening (PGS), is suitable way to
improve clinical outcomes in patients undergoing in vitro fertilization (IVF).
In our work we analyzed of the whole genome profiles from trophoectoderm cells in 23 patients and 9 healthy donors (DEM). Overall, we evaluated
118 embryos using oligonucleotide-based CytoSure Single Cell Aneuploidy
Array 8x15K. The copy number abnormalities (CNAs) were found in 23.7%
of all embryos. While incidence of CNAs in DEM embryos was 11.8% of
samples, CNAs in patients embryos occurred in 28.6% of samples. Aneuploidies of chromosomes were observed in 26.7%, segmental imbalances
were proved in 6.8% of embryos. In patients cohort, we found overall 35
different CNAs. The most common aneuploidies were trisomy 21 and 13
(both 8.3%), whereas the most frequent losses of genetic material were
monosomy 8 (12.5%) and 18 (8.3%). Two patients had embryos with complex aneuploidy. Segmental CNAs were found in 5q, 8p, 8q, 14q and 16p. In
DEM embryos, we observed two 2 structural CNAs (gain 13q; loss 7p) and
2 cases with aneuploidies (trisomy 19, monosomy 18). This study shows
that oligonucleotide array is novel progressive tool for sensitive genomewide analysis of chromosome instability and opens the route towards highresolution preimplantation screening which improves selection of embryos
and implantation rates. Supported by OP VK CZ.1.07/2.4.00/31.0155 and
CZ.1.07/2.3.00/20.0183
P01.008-M
Copy Number Variation (CNV) in azoospermic males
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The discovery of fetal DNA in maternal plasma has provided a new source of
fetal genetic material that can be safely obtained from maternal blood and
successfully processed for non invasive genetic diagnosis (NIPD). We describe a protocol for non-invasive prenatal diagnosis of thalassemia with a
next generation sequencing approach and Ion Torrent technology. Eighteen
experiments of NIPD of -thalassemia have been carried in cfDNAs of women who underwent PD for -thal. In total, we have amplified and sequenced 47 amplicons mapping in the beta-globinic gene cluster and further 3
amplicons (ZFX/ZFY, SRY and TSPY1) useful to detect fetal sex and fractional fetal DNA. In the validation phase we have included the parental DNAs
and trophoblast DNA to infer parental haplotypes inherited from the fetus.
In the meanwhile we have developed a protocol to infer parental haplotypes
without fetal information. The method is based on haplotype selection by
allele-specific long-range PCR (LR-PCR) with allele-specific primers with
the 3 base complementary to the mutated or to the normal allele for the
most common -thalassemia mutations. These reactions were performed
with an high fidelity polymerase able to amplify genomic targets extending
both downstream and upstream the causative mutations. By this approach,
the 69.4% of fetal haplotypes, mainly of paternal origin, has been correctly
identified in the cfDNAs processed. Following these encouraging results, we
are working in order to improve the performance of our platform by extending the analysis to a higher number of SNPs and by developing specific
algorithms of analysis.
P01.011-S
Prenatal diagnosis of severe X-linked chondrodysplasia punctuata in
8 female fetuses
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ABSTRACTS POSTERS
P01.012-M
Fetal Fraction estimate in twin pregnancies using directed cell-free
DNA analysis
This study examined the methylation difference in AIRE and RASSF1A between maternal and fetal DNA, and the implication of this difference in the
identification of free fetal DNA in maternal plasma and in prenatal diagnosis
of trisomy 21. Maternal plasma and amniotic fluid samples were collected
from 30 singleton pregnancies. Methylation-sensitive restriction enzymes in
digestion of differential maternal-fetal methylation followed by fluorescent
quantitative PCR (MSRE + PCR) were employed to detect trisomy 21. Diagnosis of trisomy 21 was established according to the ratio of fetal-specific
AIRE to RASSF1A that are hypermethylated in maternal plasma and are not
digested with methylation sensitive restriction enzymes. All of the results
were approved with karyotype results. Based on the data from 22 euploidy
pregnancies, the 95% reference interval of the fetal AIRE/RASSF1A ratio in
maternal plasma was 0.33-1.77, which was taken as the reference value for
determining the numbers of fetal chromosome 21 in 30 pregnancies. Firstly, 18 from 22 euploidy pregnancies were detected euploid correctly and 4
cases incorrectly so the early sensitivity rate was 81.81% (18/22). But by
repeating the test with better digestion, the four cases made correct results
so the final sensitivity rate was 100% (22/22). All of the eight trisomy 21
pregnancies were diagnosed with this method correctly so the specificity
of this method was 100%. Also with performing STR-Typing and checking
paternal alleles in maternal plasma and comparison with maternal alleles
in 16 loci, the protocol of cell free fetal DNA extraction from plasma were
confirmed.
P01.014-M
Non-invasive EXamination of Trisomy (NEXT) Study: Directedcell-free
DNA analysis versus 1st trimester combined screening for Trisomy 21
risk assessment in a large Routine pregnancy population
Non-invasive prenatal testing (NIPT) with cell-free DNA (cfDNA) is highly accurate for fetal trisomy evaluation in high-risk pregnancies. Routine
pregnancy population NIPT performance has not been evaluated in a large
prospective study. Our objective was to compare NIPT with directed cfDNA
Back to index
analysis to first trimester combined screening (FTS) for trisomy 21 risk assessment in a general pregnancy population.
This prospective multi-center blinded cohort study compared HarmonyTM
Prenatal Test, a directed cfDNA test, with FTS using first trimester PAPP-A,
hCG and nuchal translucency measurement. Women with a singleton fetus
presenting in the first trimester for routine prenatal screening for fetal aneuploidy were eligible. Participants had both FTS and Harmony. FTS results
were provided as part of routine care. Participants and care providers were
blinded to Harmony results, calculated as probability scores. Pregnancies
were followed for newborn outcomes. Invasive test results or neonatal
phenotype,with karyotype confirmation in cases of suspected aneuploidy,
were used for trisomy 21 identification. Harmony, FTS results and outcomes
were reported to an independent data coordinating center. Primary outcome was comparison of the area under the ROC curve for trisomy 21 test
performance of the Harmony and FTS.
18,955 women were enrolled across 38 centers in USA, Canada and Europe
from March 2012 to April 2013. The mean maternal age was 30.6 (18-52)
years. The mean gestational age was 12.4 (10-14.3) weeks. Follow-up is
complete.
Study results will be presented. Implications for use of NIPT for trisomy 21
risk assessment in the general pregnancy population will be discussed.
P01.015-S
A case report of a high level 46,XX/46XY true chimerism without
clinical effect in a healthy female who gave birth to healthy twins after
IVF
K. Adamov1, M. Godava1, R. Vrtl1, J. Dostl2, J. Ehrmann3, Z. Slobodov3, M. Kvapilov1,
H. Filipov1, P. apkov1, R. Vodika1;
1
Department of Medical Genetics and Foetal Medicine, University Hospital and Palacky
University, Olomouc, Czech Republic, 2Center of Assisted Reproduction Department of
Obstetrics and Gynecology, University Hospital and Palacky University, Olomouc, Czech
Republic, 3Department of Clinical and Molecular Pathgology, University Hospital and
Palacky University, Olomouc, Czech Republic.
Methylation of the CpG islands is a common epigenetic marker of gene repression. Monoallelic and parental-specific DNA methylation pattern at the
Imprinting Control Region 1 (ICR1) and 2 (ICR2) regulates the expression
of IGF2/H19 and KCNQ1/CDKN1C domains at the imprinted locus 11p15.5.
Alterations of ICR1 and ICR2 methylation state are common in BeckwithWiedemann (BWS) and Silver-Russell (SRS) syndromes and a robust molecular investigation is crucial to support phenotypic evidences, particularly
in prenatal diagnosis for BWS. Since it is known that cell culture conditions
could per se modify the epigenetic signature of the cells, we aimed to compare ICR1 and ICR2 methylation profile in fresh chorionic villus samples
(CVS) with the corresponding cell cultures (CVC) to verify whether methylation at ICRs is stable after cell culture. By pyrosequencing we analyzed 9 CVS
and their relative CVC from healthy pregnancies that underwent prenatal
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ABSTRACTS POSTERS
diagnosis for maternal age. The range of methylation levels of ICR1 and ICR2
in control CVS was previously reported by our group (ICR1: 38-48%; ICR2:
37-47%). Herein, we found that the mean methylation percentage of ICR1
and ICR2 remains stable after cell cultures: CVS= ICR1: 44% 1,7%; ICR2:
42% 1,8%, CVC= ICR1: 44% 2,3%; ICR2: 42% 3,1%. The high stability
of such genomic imprinted regions makes them useful to be investigated in
prenatal diagnosis.
P01.019-S
Prenatal diagnosis: chromosomal microarray in fetuses with
increased nuchal translucency
Chromosomal microarray has significant advantages over standard metaphase karyotyping for detecting large chromosomal imbalances and alterations smaller than 10 MB in size. The method has besome an important
diagnostic instrument in the prenatal setting for pregnancies with abnormal
ultrasound findings. In our clinical setting (The Central Region, Denmark),
more than 90 % of pregnant women receive combined 1st trimester screening and 2nd trimester anomaly scan via the public health care system. Since January 2013 we have used a two-tiered approach for invasive diagnostics of all foetuses with nuchal translucency > 3.5 mm (99 percentile) in 1st
trimester pregnancies. This consists of a fast prenatal analysis for common
aneuplodies by QF-PCR followed by chromosomal microarray on samples
with normal QF-PCR test results. The analysis methods used are the Elucigene QST*R kit (GenProbe) and comparative genomic hybridization-based
microarrays (SurePrint G3 Human CGH microarray 180K, Agilent). DNA
was extracted directly from chorionic villus samples using the Maxwell
system.
We demonstrate the usefulness of microarray testing in this clinical setting
based on our laboratory experiences.
P01.020-M
Clubfoot as an indication to perform array-cgh on prenatal diagnosis
even when isolated?
54
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flow. 100% of assayed CNV regions (n=34) were detected using a reference
set of 31 samples with known chromosomal aberrations. Low-pass wholegenome sequencing data, with approximately 0.01x read coverage, allowed
the rapid 10 hour analysis of aneuploidies from research samples with
extremely low initial input DNA amountseven from a single cell. Using a
control set of 10 samples with known chromosomal aberrations, 100% of
the copy number changes were found, ranging from gains or losses of whole chromosomes to subchromosomal alterations tens of megabases (Mb) in
size. The Ion PGM System minimizes the high cost and complexity of nextgeneration sequencing and, with Ion Reporter Software, facilitates userdefined CNV and aneuploidy detection, with three sensitivity options so that
copy number analysis workflows can be tuned to achieve desired levels of
sensitivity and specificity.
P01.022-M
Fetal intracerebral hemorrhage and cataract: think COL4A1
The COL4A1 gene encodes the alpha1 chain of type IV collagen, a crucial
component of nearly all basement membranes. Mutations in COL4A1 were
first associated with cerebral microangiopathy and familial porencephaly
and have later been implicated in a clinicopathologic broad-spectrum affecting the brain, eyes, kidneys and muscles. Recently, COL4A1 mutations
have also been identified prenatally in fetuses with intracranial hemorrhage
(ICH). We report two additional prenatal cases of COL4A1 mutations in fetuses with ICH and cataract.
Case 1: Fetal ultrasound examination (US) at 23 weeks gestation (WG) showed left cataract, left ventriculomegaly and hyperechogenic lesion of basal
ganglia. Fetal magnetic resonance imaging (MRI) at 32 WG confirmed the
subependymal hemorrhage affecting the left hemisphere.
Case 2: Fetal US examination at 31 WG showed hyperechogenic lesion in left
hemisphere with thalamic echogenicity and bilateral cataract. Fetal MRI at
32 WG showed a left-sided periventricular parenchymal hemorrhage and
mild ventriculomegaly.
In both these cases, the involvement of COL4A1 was evoked because congenital cataracts had been previously reported in association with ICH in
pediatric cases.
The sequencing of COL4A1, performed on fetal DNA after termination
of pregnancy, evidenced two heterozygous novel missense mutations
c.2317G>A (p.Gly773Arg) and c.3005G>A (p.Gly1002Asp) in fetuses 1 and
2 respectively.
The two cases reported here show that the COL4A1 mutation should be envisaged in fetuses with prenatal ICH especially in the presence of lens abnormalities at US examination. Molecular confirmation of a COL4A1 mutation
may have important implications for the outcome of the pregnancy and for
genetic counseling.
P01.023-S
Validation And Clinical Application Of A Next-Generation Sequencing
(Ngs)-Based Protocol For 24-Chromosome Aneuploidy Screening Of
Embryos
ABSTRACTS POSTERS
protocol revealed 76(39.6%) euploid blastocysts. Following transfer of 50
embryos, 30 women had a sustained pregnancy (63.8% clinical pregnancy
rate/ET; 64.0% implantation rate).
This is the first study reporting extensive validation and clinical application
of NGS for PGS purpose, allowing identification and transfer of euploid embryos resulting in healthy pregnancies. Evidence of accuracy demonstrates
that NGS represents a reliable high-throughput methodology for comprehensive aneuploidy screening, capable of detecting whole chromosome aneuploidies and segmental changes in embryos, with the potential to revolutionize preimplantation diagnosis.
P01.025-S
A new case of Aperts syndrome detected prenatally in Romania
In the 2010 - 2014 period 689 chorionic villus sampling (CVS) were successfully performed. QF-PCR and a complete chromosomal examination
(karyotyping) was a standard examination schema for all samples. Abnormal results were found in 184 samples (26.9 %). Totally 165 (89.7 %) cases out of these 184 pathological results has been announced by QF-PCR
(most frequently an autosomal aneuploidy) prior to karyotyping. This is in
contrast to 19 abnormal findings detected only due to classic chromosomal
examination (9 balanced aberrations, 9 mosaics of both autosomes and
gonosomes, 1 unbalanced aberration). In the same period of time 102 CVS
(with normal karyotype and QF-PCR results) were evaluated by SNP-array
(Illumina). Well-defined pathogenic microdeletions or microduplications
were detected in 7 CVS (6.8 %) indicated from following reasons: increased
nuchal translucency (5 cases), heart defect (1 case) and anal and esophageal
atresia (1 case).
In our study, an examination of CVS based on QF-PCR method only would
let 19 abnormal findings (2.5%) undetected out of which only 10 (1.45 %)
are defined as pathological (9 mosaics of both autosomes and gonosomes,
1 unbalanced aberration). In all 10 cases we suppose that these aberrations
would be detected by SNP-array evaluation. In addition to it, re-examination
of CVSs with normal karyotypes and QF-PCR results by SNP-array allowed
us to detect another 7 submicroscopic pathological abnormities. Based on
these data we consider the classic karyotype examination to be still an invaluable part of the CVS testing procedure.
P01.027-S
Invasive prenatal diagnosis in bizkaia: a 20-year experience
Erandio, Spain.
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55
ABSTRACTS POSTERS
numerary derivative chromosome, partly consisting of chromosome 13 material: an example of unexpected findings with NIPT. In 2013, thousands of
Dutch women had NIPT performed abroad, at their own expense. In one of
these women, who had received normal NIPT results, fetal anomalies were
seen at the 20 week ultrasound. Invasive testing showed a trisomy 18. NIPT
was repeated in our centre prior to amniocentesis, and it clearly indicated
a trisomy 18. Placental and fetal biopsies, investigated with FISH after termination of pregnancy, confirmed this finding. Data of the initial NIPT could
not be shared with us, which prohibited tracing of the source of this false
negative finding. False negative, false positive and unexpected results will
be obtained with NIPT. For safeguarding and examining follow up samples,
leading to a further understanding of possible underlying biological causes,
close interaction between specialists involved is essential. This is also vital
to ensure adequate care and counseling for couples confronted with such
results.
P01.030-M
Dyscordant Chromosomal Finding in Monozygotic Twins
P. Losan, M. Hasch;
Genetika Plzen, Pilsen, Czech Republic.
Genetic disorders are of the most common reasons of early pregnancy loss.
Chromosome abnormalities can be found in up to 70% karyotypes of chorionic villi after miscarriages of both spontaneous and IVF pregnancies.
Here we find out if embryo quality is a sufficient criterion of successful
pregnancy outcome after IVF, and identify the relationship between the embryo morphology and chromosomal pathology in spontaneous abortions or
missed miscarriages.
We analyzed 45 samples from patients underwent dilation and curettage after IVF. Chromosome preparations were made using direct material processing, GTG-stained or FISH was performed on chromosomes
13,16,18,21,22,X,Y.
Embryo transfer (ET) was performed at the day 5 of embryo development.
In the most (76%) cases 2 embryos per woman were transferred. All embryos were divided into groups based on their quality. Day 5 blastocysts AA
were defined as excellent; AB, BA, BB as good; BC, CB, C as poor quality.
Chromosome abnormalities were found in 53% cases. In these cases 25%
were ET of excellent embryos, 63% were ET of good, and 12% were ET of
poor. In abortions with normal karyotype 33% of ETs were with excellent
embryos, 66% with good. Control group was 53 cases of live birth after IVF
at the same period of time. In these successful pregnancies 41% were ETs
with excellent embryo quality, which is significantly higher than in pathology group. Otherwise, embryos of excellent and good quality also need preimplantation genetic diagnosis, because chromosomal anomalies in more
than 70% cases could be identified using this technique.
P01.032-M
ESX1 gene expression is a predictive marker of residual
spermatogenesis in azoospermic males
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ESX1 mRNA expression was previously investigated by our group in testicular biopsies of infertile men and correlated to the presence of residual
spermatogenesis in Non Obstructive Azoospermic (NOA) patients. Here, we
further deepen this issue investigating by real-time PCR ESX1 expression in
both testicular fragments (TF) and seminal fluids (SF) of 78 NOA men. The
aim was to verify whether a positive ESX1 expression in TF or even in SF
was predictive of a successfully sperm recovery at TESE/microTESE. Concerning TF, ESX1 mRNA levels: 1) significantly decrease with the increasing
of spermatogenesis defect, as classified by histology; 2) are higher in dilated
tubules, likely containing spermatozoa, compared to thin ones. In addition,
the presence of a positive or negative testicular expression of ESX1 strongly
correlates (p<0.0001) with positive or negative sperm recovery at surgery.
Regarding SF, ESX1 mRNA expression was detected in the ejaculate of 44/56
azoospermic men, at lower levels than normospermic men (p<0.05). No significant differences were found in ESX1 expression levels among samples
with different degree of spermatogenic failure, based on histological classification. Regarding spermatozoa recovery and ESX1 expression in SF, the
two variables were concordant in 33/56 (59%) of cases. The overall data
reinforce a correlation between a positive ESX1 mRNA detection and residual spermatogenesis in testes of NOA patients, indicating a role of ESX1 as
predictive spermatogenesis molecular marker. We can speculate that in SF
discrepancies between ESX1 expression (+) and sperm retrieval (-) could be
attributed to limitations in surgery techniques for sperm recovery.
P01.033-S
The importance of ftal pathology. Regional assessment report for
Montpellier for the years 2010 to 2012
P. Blanchet, N. Bigi, Y. Musizzano, S. Doutre, D. Genevieve, P. Sarda, M. J. Perez;
Genetic Center - Arnaud de Villeneuve Hospital, Montpellier, France.
M. Barasoain1, G. Barrenetxea2, I. Huerta1,3, M. Tlez1,3, A. Carrillo2, C. Prez2, E. OrtizLastra4, J. Gonzlez4, B. Criado5, M. Arrieta1;
1
Department of Genetics, Physical Anthropology and Animal Physiology. Faculty of
Science and Technology. University of the Basque Country, Bilbao, Spain, 2Center for
Reproductive Medicine and Infertility Quirn Bilbao, Bilbao, Spain, 3Virgen de Begoa
Clinical Analysis Laboratory (Medikosta), Erandio, Spain, 4Department of MedicalSurgical Specialities. Faculty of Medicine. University of the Basque Country, Bilbao,
Spain, 5Cooperativa de Ensino Superior Politecnico e Universitario, Porto, Portugal.
Primary ovarian insufficiency (POI) is an ovarian dysfunction defined as irregular menses and elevated gonadotrophin levels before or at the age of 40
years. Several genes have been reported as having significance in POI but
the FMR1 (intermediate and premutation alleles) is one of the most important genes associated with it. The FMR2 gene has also been related with the
development of this condition. A group of 68 women with POI and 47 control women from the Basque Country has been analyzed. Considering the
FMR1 gene, the number of women carrying at least one allele with >35 CGG
ABSTRACTS POSTERS
repeats (intermediate and premutation alleles) was statistically higher in
patients (26.47% vs. 0%). The patient group was divided in three categories
concerning their ovarian condition. Among patients with amenorrhea and
elevated FSH levels, the frequency of alleles between 35 and 54 CGG was statistically higher than in controls (15% vs. 0%). This frequency is also statistically higher among women with irregular menses and elevated FSH levels
(11.11% vs. 0%). Regarding the FMR2 gene, small alleles with fewer than 11
repeats have been associated with premature ovarian failure. The frequency
of these alleles in this study is not statistically different (3.68% vs. 3.19%).
The data suggest that carrying more than 35 CGG repeats in the FMR1 gene
might be related with the development of POI. However, the FMR2 gene has
not a clear association with this ovarian dysfunction.
P01.035-S
Follicle stimulating hormone receptor gene alterations and ovarian
response to gonadotropins in Iranian infertile women
One of the major effort in medical genetics is the development of non-invasive prenatal diagnosis, aimed to replace current invasive methodologies. It
is well known that the circulating free fetal cells may be employed as a new
alternative source of DNA for prenatal diagnosis, in order to avoid the risks
associated to invasive diagnosis. The cell free fetal DNA (cffDNA) in maternal plasma or serum allows the development of non-invasive method for
the detection of chromosomal and molecular diseases. The same approach
can be even applied to determine fetal sex and the risk for X-linked genetic
disorders.
To date, several techniques, such as PCR, Real Time PCR, Next Generation
Sequencing, have been employed for cffDNA analysis. Here we propose a
new molecular technology (Plexor HY System), based on Real Time PCR
for fetal sex determination. The Plexor technology takes advantage of the
specific interaction between two modified nucleotides and the reduction
in fluorescence. The qPCR approach allows the simultaneous quantification and detection of fetal sex in pregnant women. Peripheral blood samples were obtained from 50 pregnant women at the 12th to 14th gestational
week and the cffDNA was isolated from maternal plasma.
Determination of fetal sex of all samples demonstrated a concordance of
100% with the results obtained by traditional method (karyotyping). We
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FSHB gene transcription is the rate-limiting step for FSH production. The
FSHB -211 G/T single-nucleotide polymorphism (SNP) is the only genetic
variant which has a major effect on serum FSH concentrations in men. The
aim of this study was to evaluate the effects of this FSHB SNP on male infertility. The SNP was analyzed in 83 men with oligoasthenozoospermia/
azoospermia (group 1) and in 82 normozoospermic controls (group 2). Genotyping of SNP was performed by Real-time PCR with TaqMan Genotyping
Assay. The FSHB genotypes frequencies were: 74.4% (GG), 22.7% (GT) and
2.9% (TT). The distribution frequency of heterozygous and homozygous T
carriers was significantly different between the two groups: 65.8% of GT
heterozygotes and 100% of TT homozygotes were found in group 1 (Ztest, p=0.0057). The T allele frequency was statistically different in the two
groups: 18,6% and 7,9% in group 1 and 2, respectively (Z-test, p=0.007).
Moreover, the FSH serum levels were differently distributed with a trend
from the highest values in the GG genotype to the lowest levels in the TT
genotype (Kruskall-Wallis test: p=0.037). The T allele was associated with
significant declining levels of LH, testosterone (GG+GT vs. TT and GG vs.
TT (p<0.05, ANOVA) and sperm concentration (p<0.05, Median test). These results corroborate the observation that the highly conserved promoter
regions of the FSHB gene have a regulatory function on the transcription
of this gene and demonstrate convincingly that the promoter variant may
be involved to the modulation of testicular functions both in healthy and
infertile men.
P01.038-M
Noninvasive prenatal diagnosis of Huntington disease in the
Netherlands; a validation study
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ABSTRACTS POSTERS
Hypophosphatasia (HPP) is a clinically diverse condition characterized by
defective bone and/or teeth mineralization in the presence of low activity of
serum and bone alkaline phosphatase. Perinatal lethal and infantile forms
of HPP have autosomal recessive mode of inheritance with prenatal ultrasound and postnatal x-rays showing extreme skeletal hypomineralization
that often results in neonatal death. Intrafamilial variability for autosomal
recessive conditions is rare and HPP is not an exception; however, occasionally unexplained variations have been noted among affected siblings. We
report a family with extreme variability in the perinatal presentation of HPP.
The couple had two pregnancies affected with HPP confirmed by DNA analysis showing compound heterozygous mutations in the ALPL gene. Fetal ultrasound in the first affected pregnancy showed short long bones, fractures
and demineralization of the skull at 16 weeks gestation. The second affected
pregnancy had normal skeleton and normal growth of long bones at both
16.5 and 19.5 weeks gestation ultrasound. Both pregnancies were terminated in the second trimester. Fetal autopsy on the first fetus confirmed the
prenatal ultrasound findings while external examination and x-rays on the
second fetus showed few significant findings of perinatal HPP. This case emphasizes that in HPP prenatal diagnosis should rely on molecular analysis
rather than ultrasound and suggests that other genetic or prenatal environmental factors can significantly modify the phenotype even in known lethal
skeletal dysplasias.
P01.040-M
Additional Genetic Testing in case of increased Nuchal Translucency
and normal Karyotype
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Microsatellite analysis of genomic DNA extracted from tissues of both fetuses as well as from the blood of both parents revealed loss of heterozygosity
for two markers located inside the deleted region. Missing alleles originated
from the mother.
The results of molecular and cytogenetic studies strongly suggest that the
maternal gonadal mosaicism could be the cause for the increased risk of
recurrence in described case.
The possibility of germline mosaicism is of particular concern when counselling the parents of a child with a de-novo chromosomal abnormality.
P01.042-M
Gene Expression Patterns in a disturbed Karyotype: Keys to the
Clinical Conundrum of Klinefelter Patients
Klinefelter Syndrome (KS) is the most common sexual chromosome abnormality (47,XXY) and represents the first genetic cause of male infertility. The
mechanisms leading to KS testis degeneration are still unclear and no therapy is so far available for affected patients.
The present study is aimed to unravel information about molecules playing a
key role in the disruption of the spermatogenesis.
Gene expression profiles analysis of KS azoospermic testis versus normal
testis, could provide useful information about the molecular basis of the alteration of the spermatogenesis.
Transcriptome analysis was performed carrying out gene expression profile
by a whole genome microarray approach on testis biopsies obtained from 6
azoospermic non-mosaic KS men and from 3 controls, for a total of 12 experiments. T-test and False Discovery Rate were used to evaluate differentially
expressed genes. Identified transcripts were analysed by Ingenuity Pathways
Analysis software to disclose genes biological functions.
Data analysis revealed the differentially up- and down-expression, in KS
testis versus the control ones, of 656 and 247 genes related to Endocrine
system development and function, Lipid metabolism, Reproductive disease,
Free radical scavenging, and Cell death.
ABSTRACTS POSTERS
Take together these data show the presence of several genes involved in testis
microenvironment deregulation leading to the spermatogenesis failure.
This information, associated with an early diagnosis could help to unravel
possible therapeutic targets for testis failure prevention and limitation.
P01.044-M
Immunoelectron microscopy reveals massive expression of laeverin
in microvesicles in preeclamptic placentas
Preeclampsia is a pregnancy-specific syndrome characterized by hypertension and proteinuria. It complicates 5-10% of pregnancies and is a major
cause of maternal mortality world-wide. Laeverin (aminopeptidase-Q) is a
plasmamembrane-bound aminopeptidase that is specifically expressed in
the human placenta. We reported previously that the mRNA levels of laeverin gene are significantly up-regulated in preeclamptic compared to healthy
placentas, indicating that laeverin might play a role in the pathophysiology
of preeclampsia. Immunofluorescence studies have indicated that laeverin
protein is highly expressed in the cytoplasm, instead of the plasmamembrane, of villous trophoblasts in preeclamptic placentas. In this study, we used
immunoelectron microscopy to investigate the subcellular localisation of
laeverin protein. Ultrathin sections of high pressure freezed (Tokyasu method) tissue samples of four placentas (two from healthy women and two
from women with severe preeclampsia) were fixed and labeled with primary antibody raised against laeverin, followed by antibody conjugated with
gold particles. Double labeling with markers for endoplasmatic reticulum
(ER) and Golgi apparatus (GA) were also performed. We found laeverin in
the ER, GA and massive expression in microvesicles within the cytoplasm of
villous trophoblasts, extracellular space and in the fetal capillaries in preeclamptic placentas. Since the ER and GA in normal placentas did not show
any evidence of accumulation of laeverin, we hypothesize that dysregulation
of transport or folding of laeverin might have a role in the development of
preeclampsia.
P01.045-S
Leri Weill syndrome findings in an infertile man with 45,X/46,X,derY,
t(Y;Y)(p11.2;q11.21) and SHOXY deletion
K. Yararbas1, B. Saglam Ada2, F. Alp2, B. Cavdarli3, A. Ocal2, S. Gurkan1, G. Karatas1, I.
Akin1, K. Bilecen1, E. Laleli Sahin1, A. Tukun1;
1
Duzen Laboratories Group, Istanbul, Turkey, 2Etlik Zubeyde Hanim Women Health
Teaching and Research Hospital, Ankara, Turkey, 3Ankara Numune Teaching and
Research Hospital, Ankara, Turkey.
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There are two common polymorphisms in luteinizing hormone beta-subunit (LH) gene, which form the variant LH (v-LH). These polymorphisms
change the amino acid sequence (Trp8Arg and Ile15Thr) of the protein. The
hormone with v-LH (Arg8/Thr15) was shown to have higher bioactivity
(tests in vitro) but shorter lifetime in vivo. The presence of v-LH is associated with reduced womans fertility, menstrual disorders and abortions.
Female IVF patients with at least one v-LH allele tend to be hypo-responders to controlled ovarian hyperstimulation. Thus, we propose that women
with v-LH would be in a lower risk to develop an ovarian hyperstimulation
syndrome (OHSS).
In our study, 102 fertile male-controls, 149 fertile female-controls and 58
patients affected by OHSS type III-V were analyzed. The genotype was determined by restriction fragment length polymorphism (RFLP). The prevalence of v-LH allele was 10,1% in female controls, 14,7% in male controls
and 6,0% in OHSS patients.
There was no statistical difference between Czech control men and women
in genotype or allelic frequencies (P=0,22; P=0,12, respectively). The Czech
population frequency of v-LH is closest to the prevalence from Iceland, Italy and The Netherland.
There was no difference between female controls and OHSS patients in genotype or allelic frequencies (P=0,41; P=0,25, respectively). The protective
effect of the v-LH from OHSS was not confirmed, but this needs to be verified in a larger cohort.
Supported by grants IGA NT13770-4/2012, project for conceptual development of research organization 00064203 and OPPK CZ.2.16/3.1.00/24022.
P01.047-S
Genetic basis of male infertility: Belorusian Centrum of Reproductive
Medicine data.
S. Zoukovskaya, K. Mosse, N. Rumiantsava, L. Savenko;
Center of Reproductive Medicine, Minsk, Belarus.
The X-chromosome genetic content is predicted to be important in spermatogenesis but its role in male infertility remains unknown. We investigated
whether X-linked copy number variations (CNVs) might have clinical significance in idiopathic infertile men. We previously detected by a-CGH a number of CNVs including 16 patient-specific gains and 3 recurrent deletions on
Xq, exclusively (CNV67) and prevalently (CNV64; CNV69) found in infertile
patients. Among the 14 gains, we selected 5 for further qPCR analyses on a
larger study population including 276 idiopathic infertile patients and 327
normozoospermic controls. The difference in duplication load was statistically different (p=1.65x10-4). PAR1-linked DUP1A displayed the highest
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ABSTRACTS POSTERS
frequency (1.44% of patients) and we hypothesize it may cause spermatogenic failure by disturbing meiotic X-Y pairing or by affecting the correct
regulation of a gene potentially influencing spermatogenesis (PPP2R3B).
Concerning the 3 deletions, 627 patients and 628 controls were tested for
each deletion with PCR+/. CNV64 and CNV69 were significantly more frequent in patients than controls (p>0.05). CNV67 was detected exclusively
in patients (1.1%) and was maternally transmitted. The oligozoospermic
phenotype of one carrier versus his normozoospermic non-carrier brother
strongly indicates a pathogenic effect of the deletion on spermatogenesis.
MAGEA9, an ampliconic gene reported as independently acquired on the
human X chromosome with exclusive physiological expression in the testis,
is likely to be involved in CNV67. Our investigation further indicates an association between X-linked CNV burden and spermatogenic impairment and
identifies the first X chromosome-linked recurrent deletion and duplication
with clinical significance.
P01.049-S
A novel m.9588G>A missense mutation in the mitochondrial COIII
gene as a cause of low sperm motility in asthenozoospermic Tunisian
infertile men
S. Baklouti-Gargouri1,2, M. Ghorbel3, A. Sellami4, F. Fakhfakh5, L. Ammar-Keskes5, L.
Ammar-Keskes5;
1
Laboratory of Molecular, Human Genetics, Faculty of Medicine, University of Sfax, Sfax,
Tunisia, Sfa, Tunisia, 2Laboratory of Histology, Sfax Faculty of Medicine, University
of Sfax, Tunisia., Sfax, Tunisia, 3Laboratory of Molecular, Human Genetics, Faculty of
Medicine, University of Sfax, Sfax, Tunisia, SfaX, Tunisia, 4Laboratory of Histology, Sfax
Faculty of Medicine, University of Sfax, Tunisia., sfax, Tunisia, 5Laboratory of Molecular,
Human Genetics, Faculty of Medicine, University of Sfax, Sfax, Tunisia, sfax, Tunisia.
Oligoasthenoteratospermia (OAT) is a condition characterized by the presence of sperm with abnormal morphology. The causes of OAT are unknown
in most cases. Routine genetic examination of patients with nonsyndromic
male infertility (Y-chromosome microdeletions and CFTR mutations) does
not cover this phenotype. Given the hundreds of genes identified as essential
for male fertility in animal models it is probable that substantial fraction of
cases with genetically caused OAT remains undetected. Our study aims for
mutation screening of selected candidate genes which were linked to OAT in
animal models.
We selected candidate genes whose mutation in model organism interfering
with spermatogenesis and was demonstrated like OAT. We selected CAPZA3,
CDC42, CDC14B, CNTROB, CSNK2A2, GOPC, HOOK1, HRB, OAZ3, ODF1, RIMBP3
and SPATA16. We performed genealogy, karyotyping and mutation analysis of
CFTR gene and Y-chromosome microdeletions. Control group are men with
normospermia. PCR amplified exons of the selected genes were sequenced
using GS Junior next generation sequencer.
To date we accumulated 131 cases and 88 controls. Sequencing is completed
for 41 patients and 8 controls. We found 6 persons with chromosomal aberration and one man with Y-chromosome microdeletion. We detected 115 known
sequence variants and 18 novel variants (in CDC42, CNTROB, HOOK1 and RIMBP3). The clinical significance of the novel variants is uncertain, however, we
identified two likely damaging frameshift variants (in CNTROB and RIMBP3).
The study is supported by Ministry of Health of the Czech Republic grant No.
NT/12269-5
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P01.051-S
Transcriptomic profiling of spermatozoa of infertile men with
abnormal sperm morphology
The pathogenesis of oligo/astheno/teratospermia is unknown in most patients. Especially, the proportion of genetic and environmental factors has
been hard to tease apart. However, transcriptomic or proteomic approaches
can identify common dysregulated genes or pathways in sperm, that are
substantial for their function, regardless the ultimate etiological factor.
We analyzed twelve semen samples according to WHO criteria (2010). Four
of the participants were fertile donors, clinically and andrologically normal,
who had achieved pregnancy in a period less that twelve months and got a
live born baby in the last year. Seven samples were from infertile patients examined in our clinic that presented with oligospermia and abnormal sperm
head morphology, including decapitation. We performed genome-wide expression analysis using Affymetrix HuGene2.1ST microaarays.
The principal component analysis revealed substantial overlap between
normal and abnormal samples. These results indicate that despite the common result of morphologically abnormal spermatozoa, the underlying causes of the infertility might be diverse.
Surprisingly four genes were markedly different between fertile and infertile sperm samples: an uncharacterized gene, a small nucleolar RNA gene, a
microRNA gene and RIPK4. Protein kinase RIPK4 is known to phosphorylate
the adaptor protein DVL2 (dishevelled 2) to activate the Wnt signaling pathway. In fact, the ortholog DVL1 has a role in regulating spermiogenesis via
Wnt signaling.
The project was supported by University of Buenos Aires, Science and Technology, no. UBACYT-CB06, to G.M.; and Ministry of Health of the Czech Republic no. NT12269-5.
P01.052-M
Decrease of meiotic cohesins with maternal age in human oocytes
ABSTRACTS POSTERS
miRNAs in heart development, measure miRNA concentration and expression in maternal blood.
Peripheral blood samples were collected from 53 women, 27 of them had
healthy and 26 had fetuses with congenital heart defects. Blood samples
were centrifuged and miRNA was extracted from plasma. MiRNA concentration was calculated by Nanodrop spectrophotometer. By using Gene Ontology and miRBase databases we searched for those miRNAs which can be
detected in plasma and are associated with chromosomal and congenital
heart defects. These criteria were fulfilled by let-7c miRNA of chromosome
21. qRT-PCR was carried out to validate the miRNAs expression.
There was no significant difference between the miRNA concentrations
in the two groups (6.05 ng/l vs 5.36 ng/ l). We found significant differences in the let-7c concentrations between the control and patient group
(1.123.19 ng/l vs. 0.000470.00083 ng/l; p<0.0001).
Fetal-derived miRNAs are part of the free nucleic acids found in maternal
plasma, expression studies reveals new opportunities for congenital heart
defect research and diagnosis. According to our study let-7c seems to be a
potential biomarker for fetal CHD.
P01.055-S
Overexpression of mir-21 and mir-221 in preeclamptic placentas
Preeclampsia is a multisystem disorder with partial genetic and immunological etiology; the apoptosis is thought to play a role in the development of
preeclampsia. The microRNAs are small noncoding molecules involved in
regulation of cellular processes including apoptosis. Here we designed a96well reaction plate for the analysis of 21 microRNAs in duplicates which
are known to be involved in the apoptosis regulation and are expressed in
placenta - pro- apoptotic miR-1, let-7c, let-7g, mir-200c, mir-143, mir-205,
mir-122, mir-409-3p, mir-449, mir-708, mir-149, mir-204,mir-133, antiapoptotic - mir-214, mir221, and mir 222, and miRNAs with both the antiapoptotic and apoptotic targets mir29a and mir29c. The normalization was
performed against RNU44. The microRNAs were extracted from placental
tissues obtained after delivery from preeclamptic women and healthy controls. After stem-loop primer reverse transcription in one tube, the samples
were analyzed on the plates and evaluated by deltadelta Ct algorithm. The
aberrantly expressed miRNA were validated by TaqMan MicroRNA Assay by
relative quantitation with the standard curves The analysis of 21microRNAs
revealed overexpression of mir-122, mir-21, mir-221, mir- 29a, mir- 29c
and mir-449 The validation was performed on 13 additional preeclamptic
placental samples collected after delivery and 6 placental samples from
normal delivery; a significant overexpression of mir-21 and mir-221 with
p=0.0055 (CI95% 25.2 to 123.47) and p=0.0047 (CI95% 12.16 to 57.20)
was observed, respectively. Although the validation on larger number of
samples is necessary, the analysis of miRNA deregulation can help in the
identification deregulated signaling in preeclamptic placentas for detection
of new biomarkers.
P01.056-M
Analysis of micrornas expression profile in human placentas from
pregnancies complicated by preeclampsia
A. S. Glotov1, V. S. Pakin1, E. S. Vashukova1, M. M. Danilova1, E. S. Mihailin2, O. N.
Arzhanova1, V. S. Baranov1;
1
Otts Institute of Obstetrics&Gynecology, St.-Petersburg, Russian Federation, 2State
Budget Institution of Health Maternity hospital 10, St.-Petersburg, Russian
Federation.
Preeclampsia is a main cause of maternal and neonatal mortality and morbidity However, the origin of this disease is rather obscure so far. MicroRNAs
(miRNAs) as a key a regulators of gene expression in cell proliferation, apoptosis, and embryogenesis, whereas their abnormal expression has been
suggested in various common disorders. The objective of this study was
to compare expression profiles of miRNAs in placentas from preeclampsia
patients with these ones from normal term pregnancies. The expression
profiling of five patients was performed with Ion Torrent semiconductor
sequencing. Fourteen miRNAs (miR-181a-5p, mir-143, miR-143-3p; miR126-5p; mir-369, mir-136, miR-136-5p, mir-142, miR-142-3p; mir-516a-1,
mir-516a-2, mir-519a-1, mir-518c and miR-518c-3p) were significantly
overexpressed in preeclampsia placenta compared to these ones in the controls. The results are important prerequisite for further molecular studies of
genetic and epigenetic contributors in preeclampsia.
The work was supported by grants of Russian Federation President
16.120.11.5773-MC.
P01.057-S
miRNA as potential universal fetal marker in NIPT
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Introduction: Circulating miRNA molecules are currently studied as potential diagnostic markers in many physiological and pathophysiological conditions such as oncological diseases, heart diseases and pregnancy. The aim of
our study was to determine if circulating miRNAs can be used as potential
universal fetal marker in NIPT. Methods: Circulating miRNAs were isolated
from plasma of pregnant women and miRNA samples were divided into two
groups depending on fetal gender. Firstly, expression levels of 91 miRNAs
were established in three female and three male samples by two-step RT-qPCR. Five miRNAs (miR-378, miR-122, miR-20b, miR-320a, miR-320b) whose
expression levels significantly differ between two groups were chosen for
further analyses. Subsequently, expresion levels of five selected miRNAs
were evaluated in 14 female and 15 male samples by two-step RT-qPCR. Differences between groups were tested using Student t-test with p<0.05 was
consider as statistically significant. Results: After the first part of this study
where panel of 91 miRNAs were investigated, five miRNAs significantly differ in their expression levels between two sample groups. However, there
were no significant differences in expression levels of five selected miRNAs
between sample groups, when expression levels of these miRNAs were determined in a greater number of samples. Conclusion: Our study showed,
that none of evaluated miRNA molecules in this work is not suitable as the
universal fetal marker. However, since our work addressed only limited
number of miRNA molecules, for determination if circulating miRNAs can
be used as universal fetal marker, further studies are needed.
P01.058-M
Arthrogryposis multiplex congenita caused by compound
heterozygosity to Ashkenazi founder mutation and a novel splice
mutation in NEB gene
Background: Nemaline myopathy (NM) is a heterogenous disorder. A homozygote 2502bp deletion in NEB - had been detected in 1:40,000 Ashkenazi Jews (AJ). Most cases have congenital hypotonicity that might progress,
but arthrogryposis is relatively rare.
Patients: Three affected siblings with arthrogryposis and an increased
nuchal fold / cystic hygroma in two, were detected at 11-15 weeks of gestation. Parents were AJ with non-contributory family history.
Methods and Results: Histopathology with electron microscopy of muscle
samples suggested NM on two siblings. DNA sequencing confirmed compound heterozygosity to the known Ashkenazi mutation (maternal) and
splice mutation c.9619-2A>G (paternal) predicted to disrupt the intron 66
splice acceptor site.
Discussion: The splice site mutation revealed in our patients, has been
previously detected in two unrelated affected AJ with clinical stigmata of
arthrogryposis ( Ludtke et al. ICHG 2011; Yonath et al. Prenatal Diagnosis,
2012). The detection of this mutation in several AJ families suggests it is
either hot spot site or a new AJ founder mutation. Further studies should be
performed to validate its carrier frequency among this population. Also, the
severe stigmata in affected patients harboring this splice mutation, suggests
its critical role in disease pathogenesis. In line with this, when arthrogryposis detected prenatally, screening of the NEB should be considered.
P01.059-S
Familial chromosomal translocation t(3;5)(q26.2;p14): clinical
characterization of affected members with neural tube defect as a
common clinical feature
A 26-years-old woman was referred for prenatal diagnostics because of aggravated obstetric anamnesis. Her first child (male) was born at 34 weeks
of gestation via S/C due to polyhydramnios and hypotrophy. Hypotonia,
dysmorphic facial features and multiple congenital malformations were
observed, including hypertelorism, epicanthal folds, micrognathia, hypoplastic cerebellum, congenital heart defect, optic nerve dysplasia, micropenis,
hypospadias, syndactyly, and suspected spina bifida occulta. The newborn
died at age of 1 month. According genealogy, probands mother had a miscarriage at the 14th week of gestation and stillborn at 26 weeks of gestation
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ABSTRACTS POSTERS
with uncertain gender and myelomeningocele.
During this pregnancy at the 27th week of gestation ultrasound scan showed polyhydramnios and multiple congenital anomalies in the fetus. On the
31st week of gestation the fetus died in the uterus. Autopsy findings were
pulmonary atresia, transposition of the great vessels, horseshoe kidney,
cerebellum hypoplasia, hydrocephalus, clubfoot, and spina bifida. Subtelomeric FISH was performed to the proband and balanced translocation ish
t(3;5)(qter-,pter+;pter-,qter+) was detected. Karyotype was 46,XX,t(3;5)
(q26.2;p14).
The clinical phenotype of affected family members is similar and could
result from the combination of dup(3q) and del(5p) syndromes. Only few
cases of (3;5) translocations have been reported to date. Detailed clinical
description of affected family members brings new data for the characterization of this rearrangement.
P01.060-M
The Effect of Radiofrequency Waves on Pregnant Mice in association
with Genes involved in Neuronal Migration
Non-invasive prenatal testing (NIPT) of cell-free fetal DNA (cffDNA) for fetal aneuploidy risk assessment has been shown to be both highly sensitive
and highly specific. False positive rates can be as low as 0.1%. However, discrepancies between positive NIPT result and fetal karyotype on chorionic
villus sampling or amniocentesis may occur. The source of aneuploidy may
be due to maternal mosaicism, maternal malignancy, true fetal mosaicism, a
demised co-twin, an anembryonic sac, confined placental mosaicism.
We present a case of a 38-year-old woman, pregnant in 16 gestational weeks,
referred to our Unit for amniocentesis due to positive NIPT for trisomy18.
The ultrasound scans were unremarkable.
DNA was extracted from uncultivated amniocytes, amplified with commercial QF-PCR kit Aneufast (Molgentix SL, Barcelona, Spain) and analyzed on
ABI 3130xl. Karyotyping was performed on cultured amniocytes using standard protocol.
QF-PCR and cytogenetic analysis showed normal results, trisomy 18 was
excluded.
The pregnancy was still ongoing, with no pathological findings on routine
ultrasound scans. Cytogenetic and molecular-genetic analyses are to be
done after delivery.
Since fetal cell-free DNA mostly originates from invading trophoblast cells,
this false-positive result may be due to mosaicism confined to the placenta
(CPM). CPM occurs in 2% of the viable pregnancies and before the introduction of NIPT was most often detected by direct analysis of chorionic villus
sampling.
Our case illustrates that follow-up with diagnostic testing of chorionic villus
sampling and/or amniotic fluid for abnormal NIPT results should be performed and that pre- and posttest counseling is important.
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P01.062-M
Preliminary experience with the management of positive results of
non invasive prenatal testing (NIPT) in a reference centre
During the past year techniques enabling non-invasive prenatal testing for
the most common trisomies have become available clinically. An increasing
number of women have been offered the test in connection with their first
trimester screening, regardless of their level of risk. Pregnancies with a positive test are often referred to our Centre for genetic counseling and invasive procedures as recommended. To this end, we have created a flow-chart
to assist these patients who are not receiving enough information at time of
sampling. Clinical aspects of the test, its limits and reasons for false positives are discussed and a guaranteed follow-up is scheduled to collect data at
time of birth. Between July 2013 and January 2014 6 patients with positive
NIPT (2 trisomy 21, 1 trisomy 18, 1 monosomy X, 1 47,XXY, 1 triple X) were
referred. All patients underwent an amniotic fluid procedure and only one
case of trisomy 21, which had fetal anomalies at ultrasound, was confirmed.
Normal results were obtained in the other 5. Two term placentas have been
studied, the others being ongoing pregnancies. Since false positive cases of
chromosome aneuploides after NIPT may have different underlying biological causes ,(the majority, however, are due to placental mosaicism) we believe that the cytogenetic analysis of term placentas should be acquired in all
such cases so as to increase our knowledge about the relationship between
cffDNA and the fetal genome. Genetic counselling should always be carried
out before and after testing to reduce misinterpretation of test validity.
P01.063-S
Non-invasive prenatal detection of a mosaic trisomy 16
The presence of cell free fetal DNA in the maternal circulation has allowed
for the development of methods, which allow for non-invasive detection of
fetal chromosomal aneuploidies. Our laboratory offers non-invasive prenatal
testing (NIPT) with an innovative pipeline that utilizes all of the information
provided by shallow depth whole genome sequencing to identify possible
aneuploidies of all chromosomes. We have encountered one case, referred
because of high trisomy 21 risk (1/14), with unusually high Z-score (5.7)
for the chromosome 16 indicating the presence of a trisomy 16. Follow-up
with direct FISH and array analysis of amniotic fluid cells showed a lowlevel mosaicism (between 7 and 20%). The finding of low level mosaicism is
contrasting with the high Z-score which suggests the presence of trisomy 16
in the majority of the cells. This discrepancy could be explained by confined
placental mosaicism and, if so, should be taken into account in subsequent
counseling. Whereas the gynecologist suggested interruption, the family decided to continue the pregnancy with expert ultrasound monitoring of the
fetal development following clinical genetic counseling.
P01.064-M
Non-invasive detection of fetal deletions or duplications by low
coverage massively parallel sequencing
C. ZHANG, W. Xie, X. Pan, F. Chen, Y. Gao, W. Wang;
BGI-Shenzhen, Shenzhen, China.
To report the performance of fetal chromosomal deletions/duplications detection by the massively parallel sequencing (MPS) of maternal plasma DNA,
we recruited 1,324 participants and performed the MPS test in a doubleblind study. The peripheral blood of each participant was obtained before
invasive sampling at 10-28 weeks gestation with a median of 21 weeks.
Plasma DNA was extracted and sequencing. About 0.08 fold sequencing data
per sample was generated and the bioinformatics analysis was performed
using FCAPS algorithm. Deletions / duplications, ranged from 3.07 Mb to
26.98 Mb, were suspected in 17 of the 1,324 samples, of which 16 were consistent with the results of fetal karyotyping / aCGH. In one case the suspected abnormality was not confirmed by karyotyping, representing a false
positive case. No false negative case was observed in the remaining 1,307
low-risk samples. The sensitivity and specificity for detection of fetal chromosomal deletions / duplications were100% and 99.92%, respectively. Our
study demonstrated the MPS-based test is feasible and of high sensitivity
and specificity in detecting fetal chromosomal deletions / duplications.
ABSTRACTS POSTERS
P01.065-S
Nonivasive prenatal KEL genotyping using TaqMan Real time PCR and
by capillary electrophoresis minisequencing
Introduction: There are two reasons for establishing a methodology for noninvasive determination of KEL genotypes in early pregnancy. To identify
fetuses which are at risk of hemolytic disease of fetus and newborn by alloimmunized pregnant women and to prevent alloimmunization during pregnancy. Aim: KEL noninvasive determination of fetal genotype from cffDNA in
plasma KEL negative pregnant women using TaqMan assay and minisequencing (SNaPshot). Project is supported by IGA MZ CR: NT12225. Material and
methods: 1) TaqMan assay involving region of KEL (K/k) polymorphism and
region of AMELY gene as an internal control was tested in DNA samples from
leukocytes of k/k, K/K and k/K men. 2) SNaPshot: Determination of sensitivity threshold KEL calibration was performed using a dilution series. It
was tested 141 samples of cffDNA from maternal plasma in the first trimester. Results: 1) TaqMan assay: KEL genotypes determined from leukocyte
DNA were distinguishable, but there was fluorescence background. So there
wasnt able to exactly distinguish between false positive fluorescence background and fetal DNA admixture. 2) SNaPshot: On the basis dilution series it
was possible to detect less than 0,78% admixture of K allele corresponding
DNA concentration of 0,04 ng /l. Seven fetuses with allele K were found
in 113 k/k mothers, which corresponds to about 4-5 % of the population
frequency. It wasnt possible to determine KEL genotype at 8 fetal samples
due to KEL maternal heterozygous genotype. Conclusion: SNaPshot assay is
more suitable to distinguish fetal KEL genotyp, than TaqMan assay.
P01.066-M
Feasibility of utilization of semiconductor based low coverage
genomic sequencing in noninvasive prenatal trisomy 21 detection
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Over the past year, noninvasive prenatal screening (NIPS) based on massively parallel sequencing to align and count DNA fragments floating in the plasma of pregnant women, has become clinically available into prenatal care as
an advanced screening test. We report here a case of discordant results in
fetal sex determination between NIPS and ultrasound examination. The patient was referred to our clinical genetic service at 23 week of gestation due
to discrepancies between NIPS results performed in a different center and
fetal ultrasound examination. In particular, a first NIPS report was of a normal female fetal karyotype but subsequent ultrasound examination showed
a fetal male gender. The result of a second NIPS analysis on a new plasma
sample offered and performed by the same center was of increased risk for
Turner syndrome. After genetic counseling, amniocentesis was performed
at 23 wog. FISH analysis with a probe targeting SRY gene showed a single
signal for SRY onto an almost intact X chromosome, revealing an X-Y cryptic
chromosomal translocation: 46,XX.ish der(X)t(X;Y)(p22.3;p?)(SRY+). ArrayCGH showed a subtelomeric deletion of about 2.28 Mb in Xp22.33 and the
presence of the short arm of Y chromosome. To exclude the possibility of
phenotypic consequences due to haploinsufficiency of SHOX gene, MLPA
subtelomeric analysis was carried out. These case support the recommendation that NIPT should be regarded as a screening test and offered in the
context of genetic pre-test counseling as well as post-test counseling for
screen positive individuals. Diagnostic testing is recommended to confirm
positive NIPS results.
P01.069-S
Older mothers perspectives on non-invasive prenatal testing for fetal
abnormalities and adult-onset conditions in the United Kingdom
L. M. Jackson, A. OConnor, L. Goldsmith, H. Skirton, Applied Health Genetics Research
Group;
Plymouth University, Plymouth, United Kingdom.
Since the 1970s there has been a steady increase in England and Wales in
the number of women having a baby at the age of 35 or over. Birth rates for
this group have risen from 6% in 1974 to almost 20% in 2012.
The use of new technologies such as non-invasive prenatal testing and genome or exome sequencing may result in the opportunity for women to request
fetal testing for a range of conditions. While there is considerable support
from prospective parents and health professionals for non-invasive prenatal
testing for single gene disorders, ethical concerns have been identified. It
has been suggested that prenatal testing discriminates against people with
disability and there are fears it may become routine. Detecting multiple genetic conditions in the fetus may facilitate detection of adult-onset conditions. This has serious implications for parental decision making.
63
ABSTRACTS POSTERS
In this study we aimed to explore the attitudes of mothers aged 35 or over
towards new diagnostic techniques for prenatal diagnosis of foetal abnormality. Women who had given birth in the UK within the last 12 months
were recruited using a novel recruitment strategy via Twitter. Here we present the initial findings relating to NIPT and testing for adult-onset conditions. Mothers considered the reduced risk to the pregnancy and extra time
to make a decision as being major advantages of NIPT whilst voicing concerns regarding health service readiness and test accuracy. Concerns about
testing for adult-onset conditions included increased psychological burden
on parents and respecting the childs autonomy.
P01.070-M
Prenatal diagnosis and outcome in Noonan Syndrome
Osteogenesis Imperfecta (OI) is a heritable connective tissue disorder, characterized by a wide clinical spectrum, ranging from few fractures in mild
cases to perinatal lethality. During pregnancy, if severe forms are usually
easily early recognizable, the mild and moderate forms may lead to notoriously difficulties in the evaluation of the postnatal prognosis. Objective: to
evaluate the contribution of prenatal imaging, family history and molecular
analysis in the postnatal prognosis in osteogenesis imperfecta. Methods: to
analyze retrospectively the prenatal features (fetal ultrasound examination
and 3D CT-scan), family history and postnatal outcomes (phenotype and genotype) of 30 cases diagnosed antenatally as OI in our hospital. Results: In
15 cases, the severe fetal presentation led to pregnancy termination. Among
the 15 remaining cases that are still alive, 4 are considered as severe, 6 as
moderate and 5 as mild. The family history appears to be the best prenatal
predictor of postnatal severity. We emphasize the sensitivity of the 3D-CT
scan. Molecular screening was not helpful in the prognosis given a priori to
the parents. No correlation between the severity of skeletal phenotype and
the genotype was demonstrated a posteriori. Importantly, in 3 cases with
marked bowing of the long bones, a significant improvement of the bone deformities was observed after birth. Conclusion: OI is today easily diagnosed
during pregnancy based on the improvement of the recent imaging tools;
however, the postnatal prognosis is still often difficult to evaluate, highlighting the value of a family history.
P01.072-M
Prenatal diagnosis of de novo partial trisomy 4q3lqter and partial
monosomy 22q13.3qter
K. Crkvenac Gornik, I. Tonkovic Djurisevic, M. Miklos, S. Huljev Frkovic, L. Ljiljana, R.
Lasan Trcic, D. Begovic;
Division of Genetics, Department of Pediatrics, University Hospital Centre Zagreb,
Zagreb, Croatia.
64
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ABSTRACTS POSTERS
(e.g. thalassemia and sex selection or thalassemia and HLA typing).
Implantation was succeeded to pregnancies and child births in 3 families
and one is still pregnant. The birth outcome was a twin healthy boys for
hemophilia A (born on May 2011), a triplet boys for gender and aneuploidies selections (born on December 2013), a carrier boy for -thalassemia
was born on February 2014 and another twin hemophilia A pregnant at 8
weeks.
P01.076-M
Pre-case haplotyping of the two monogenic diseases in one family in
Gennet: Marfan syndrome and SMA
Genetic pre-case haplotyping (PGH) is an important constituent of the preimplantation genetic diagnosis (PGD). In our center we have performed
haplotyping studies for 86 different monogenetic diseases of all types - Xlinked, autosomal recessive, autosomal dominant, translocation and small
deletions. The list of diagnosis for which we can offer PGD is being continually extended. PGD is available for a large number of monogenic disorders.
For most couples requiring PGD, common pre-case haplotyping analysis
of disease-associated haplotype is derived from family anamnesis. We are
presenting a case in which affected haplotype will be determined by standard PGH analysis . In this family 2 affected haplotypes were detected which
are associated with AD disease - Marfan syndrome - OMIM # 154700 and
AR disease- Spinal Muscular Athrophy(SMA) - OMIM # 253300. During the
first PGD cycle in the year 2013 (11.2013): 11 cumulus-oocyte-complexes
(COCs) were retrieved. 9 blastomeres were biopsied. All tested embryos
were analysed .2 embryos were affected of SMA , 3 embryos were affected
of Marfan syndrome .2 embryos were diagnosed as monosomy of chr.5 carrying high risk paternal haplotype SMN1 gene. 2 embryos did not show PCRamplification. The embryos were not re- analysed because they didnt show
optimal development. In this first PGD cycle no embryos were transferred
. While there was no transfer in this case during the first PGD cycle, PGD
still increases the chances of families at risk of transmitting serious genetic
disorders to plan a healthy offspring.
P01.077-S
Targeted capture and massively parallel sequencing for PGD of
monogenic diseases
Carriers of balanced translocations are at high risk for unbalanced gametes which can result in recurrent miscarriages or birth defects. Preimplantation genetic diagnosis (PGD) is often offered to select balanced embryos.
This selection is currently mainly performed by array comparative genomic
Back to index
The aim of the study was to verify the possibility to predict preeclampsia
(PE) after the 20th week of gestation by reliable cut-off levels based on PlGF
analysis at the time of pregnancy termination within weeks 24-41. Control PlGF levels for weeks 9-19 were derived from 800 cases; 100 PE sera
comprised: 17 - PE weeks 24-29, 24 (30-33), 34 (34-37) and 25 (38-41).
PlGF was examined by DelphiaXPress Tm platform (PerkinElmer). Reliable
PlGF control levels (3, 5, 25, 50, 75 and 95 percentiles) were established for
weeks 9-19. For 1st trimester PE risk determination commercial software
was used, for weeks 14-19 levels < 3 or 5 pecentiles were applied. Inverse relationship of decreased PlGF levels to the onset of PE and its clinical
impact was documented. PlGF median level increase during pregnancy is
significantly retarded. Levels of PlGF for PE (weeks 24-29) correspond to
medians of the 11th week, PE (weeks 30-33) to week 15 and intermediate/late PE to week 17. PlGF cut off levels for 100%/95% detection rate
success for PE weeks 24-29,30-33, 34-37, 38-41 are 61.6/46.2, 267.8/19.5,
262.3/180.3, 168.3/146.0 (all pg/ml). Combination of 1 st trimester PE
screening with software prediction of early intermediate and late PE with
2nd/3rd trimester PlGF examination could improve the complex care not
only for PE, but also for other adverse risks of HELLP syndrome, SGA, preterm delivery and IUGR including increased efficacy of 1st trimester aneuploidy screening. Supported by FN Motol (00064203, Modern Therapy), IGA
NT13770 and OPPK CZ.2.16/3.1.00/24022.
P01.080-M
Integrative transcriptome-based approach for association studies:
identification of new genetic markers for preeclampsia
Preeclampsia is a common pregnancy-specific disorder with unknown etiology diagnosed in 5-17% of pregnancies. It is the leading cause of maternal
and perinatal morbidity and mortality. Our prior genome-wide transcriptional profiling of placental tissue led to a novel set of 63 preeclampsia candidate genes (differentially expressed genes, (Fold Change >1.5, FDR<0.1)). In
this report, we present preliminary study on the role of variability in some
of these genes in the genetic susceptibility to preeclampsia. We analyzed 51
tagging single nucleotide polymorphisms (tagSNPs) in 12 genes (ANKRD37,
BCL6, BHLHE40, CCSAP, CORO2A, DGKG, GPT2, PLIN2, RDH13, SIGLEC6, SYDE1 and ZNF175) in 514 patients with preeclampsia and 627 women with
uncomplicated pregnancies from Russian, Buryat and Yakut populations
using MassArray iPLEX (Sequenom). We have detected significant associations for preeclampsia with tagSNPs in PLIN2, BHLHE40, DGKG, RDH13,
SYDE1 genes in Russian and Buryat population. In Yakut population, only
three genes (BHLHE40, CORO2A, GPT2) are associated with increased risk
of preeclampsia. tagSNPs in ANKRD37 and ZNF175 genes were associated
with preeclampsia in Buryat population only. Interestingly, we found an association with preeclampsia for twenty out of the fifty-one studied polymor-
65
ABSTRACTS POSTERS
phism. This results demonstrate the high informative value of the integrative approach in studies of the genetic components of preeclampsia and show
that allelic variations of the differentially expressed genes in placental tissue
are associated with preeclampsia in different ethnic groups. Nevertheless,
the clinical significance of these findings remains to be determined. This
work was supported by the Russian Foundation for Basic Research (grant
14-04-01467).
P01.081-S
Molecular understanding of the FMR1 premutation and Fragile
X-associated Premature Ovarian Failure
C. Caslini, A. Crespi, G. Pietrini, A. Marozzi;
MEDICAL BIOTECHNOLOGY AND TRANSLATIONAL MEDICINE, Milan, Italy.
66
Back to index
A 31-year old woman was referred at 20+6 weeks gestational age because of abnormalities at the standard anomaly scan. Advanced ultrasography
showed a double outlet right ventricle with transposition of the great arteries and a hypoplastic left ventricle. Furthermore a mandibular retrognathia
was noticed. Amniocentesis was performed for RAD and array analysis. The
pregnancy was terminated at the parents request and external post-mortem
examination revealed mild dysmorphic facial features including mild micrognatia and low set ears. SNP-array analysis showed a duplication deletion
rearrangement in the long arm of chromosome 20. The interstitial duplication was 20,5 Mb and the adjacent deletion was 240 kb in size. Karyotyping
revealed a ring chromosome 20 in all analysed cells. Fluorescence in situ
hybridisation analysis demonstrated that the duplication was inverted. Parental chromosomes were normal indicating that this unbalanced rearrangement occurred de novo. Only a few cases with a ring chromosome with a
telomere deletion and an additional inverted duplication have been described so far. These inv dup del chromosomes are the result of an asymmetric breakage of a dicentric chromosome that has been formed to stabilize
a broken chromosome. The ring formation represents a new mechanism, in
addition to telomere capture, through which inv dup del chromosomes can
stabilize. In conclusion this study presents a prenatal case with a ring chromosome 20, which presented with ultrasonographic anomalies initially.
P01.085-S
A 3 years worldwide experience with Prenatal BACs-on-BeadsTM for
prenatal diagnosis in over 9.500 pregnancies
Chromosomal microarrays are the gold standard in prenatal cases with ultrasound abnormalities+normal karyotype. In low-risk pregnancies their
use is still controversial because of their diagnostic yield in relation to
that of variants of unclear significance. Consequently some laboratories,
for low-risk pregnancies, adopted PNBoBsTM (in combination with karyotype) a transitional test investigating 9 critical-regions associated with
dominant microdeletion syndromes with known genotype-phenotype correlations. We present the experience of 12 worldwide laboratories using
PNBoBsTM+karyotype. The purpose was to evaluate the usefulness of PNBoBsTM in pregnancies with low a priori risk of microdeletion/microduplication syndromes (advanced maternal age,AMA; maternal anxiety,MA; increased risk after maternal serum screening for Down syndrome,MSS-DS;
soft marker/s). 9648 samples were analyzed: 17.4% AMA, 22.2% MSS-DS,
8.1% MA, 36.0% ultrasound abnormalities and 7.1% for unknown origin.
Successful rate was 96.7%; false negative incidence 0.56% related to chro-
ABSTRACTS POSTERS
mosome abnormalities not covered by PNBoBsTM but detectable by karyotype. In 0.31% of cases PNBoBsTM was reflexed by FISH for further characterization. Overall abnormal results (PNBoBsTM+karyotype) were observed
in 9.1% of cases; a microdeletion/microduplication was retrieved in 0.7%
of cases (n=69); the majority of them (68.1%) involved Di George syndrome critical-region (deletion=32; duplication=15). 23 cryptic imbalances
were found in low risk pregnancy leading to an additional diagnostic yield
of about 1/246 in the low-risk pregnancies; 41 were detected in high risk
pregnancies providing a 1.8% of additional detection rate (1/54). We will
present incidence of cryptic unbalances stratified by indication for prenatal
diagnosis that was surprisingly higher than expected basing on theoretical
incidence (1/1700).
P01.086-M
Detection of 7p14 deletion in a fetus with aortic coarctation: genetic
and fetal morphology data
L. Cardarelli1, I. Mammi2, F. Di Giovanni3, E. Nalesso1, S. Gomirato1, L. Michelotto1, K.
Marchioro1, M. Duca1, G. Abatangelo1;
1
Lab Citotest - Consorzio GENiMED, Sarmeola di Rubano (PD), Italy, 2Dolo Hospital ULSS 13, Dolo (VE), Italy, 3Mirano Hospital - ULSS13, Mirano (VE), Italy.
We present the case of a fetus with aortic coarctation suspected by ultrasounds at 26th gw, in which an interstitial deletion 7p12.3p14.2 was identified on amniotic fluid. The deleted region, confirmed by FISH and aCGH analyses, spanned in the range chr7:36,671,630-46, 039.765bp, and involved
several genes, as GLI3, ccm2, GCK, txndc3.
Decipher db shows at least 5 subjects with a microdeletion involving this
region, all with delayed psychomotor development, dysmorphisms and abnormalities of the extremities. One of the subjects also presented coarctation of the aorta.
The literature also reports some cases of del(7)(p14) and Greig Cephalopolysyndactyly, an autosomal dominant disorder associated with GLI3 aploinsufficiency, characterized by a distinct combination of craniofacial, foot and
hand malformations. Digilio et al reported a deletion 7p14 with cardiac anomalies (non-compacted myocardium, VSD, ASD and aortic valve dysplasia).
Five other subjects, with cytogenetically visible deletions 7p14, had more
severe findings as microcephaly, dysmorphism and mental retardation.
After several genetic counseling sessions with the equipe of the prenatal
center, the parents decided for ToP.
Fetal autopsy identified cranio-facial dysmorphisms, a complex CHD with
aortic coarctation, ASD, VSD and left ventricle hypertrophy, Meckels diverticulum, accessory spleen and horseshoe kidney.
This case underlies that ultrasound, genetic testing (including aCGH) and
genetic counselling should be included in the protocol of pregnancies with
fetal malformations, once again reinforcing the necessity for the different
specialists involved in prenatal diagnosis to cooperate in the identification
of a complex fetal phenotype, to offer a prognosis that may help parents in
their pregnancy options.
P01.087-S
Prenatal diagnosis and Preimplantation Genetic Diagnosis for
inherited cardiac diseases in the Netherlands: an overview
Back to index
means. The families opting for PND or PGD for inherited cardiac diseases
have experienced a severe phenotype or a severe family history of SCD. Nevertheless, the number of patients continuing with PND and PGD is low.
P01.088-M
High resolution chromosomal microarrays in prenatal diagnosis
significantly increase diagnostic power
Objective: The objective of this study was to determine for the first time the
reliability and the diagnostic power of high-resolution microarray testing in
routine prenatal diagnostics. Methods: We applied high-resolution chromosomal microarray testing in 464 cytogenetically normal prenatal samples
with any indication for invasive testing.
Results: High-resolution testing revealed a diagnostic yield of 6.9% and
1.6% in cases of fetal ultrasound anomalies and cases of advance maternal
age (AMA), respectively, which is similar to previous studies using low-resolution microarrays. In 3 (0.6%) additional cases with indication AMA an aberration in susceptibility risk loci was detected. Moreover, one case (0.2%)
showed an X-linked aberration in a female fetus, a finding relevant for future
family planning. We found the rate of cases, in which the parents had to be
tested for interpretation of unreported copy number variants (3.7%), and
the rate of remaining variants of unknown significance (0.4%) acceptably
low. Of note, these findings did not cause termination of pregnancy after expert genetic counseling. The 0.4% rate of confined placental mosaicism was
similar to that observed by conventional karyoytyping and notably involved
a case of placental microdeletion.
Conclusion: We conclude that high-resolution prenatal microarray testing is
a reliable technique that increases diagnostic yield by at least 17.3% when
compared with conventional karyotyping, without an increase in the frequency of variants of uncertain significance.
P01.089-S
Noninvasive detection of a balanced fetal translocation from maternal
plasma
S. Kim1, T. Jensen1, D. van den Boom2, C. Deciu1, M. Ehrich2;
1
Sequenom Laboratories, San Diego, CA, United States, 2Sequenom Inc., San Diego, CA,
United States.
67
ABSTRACTS POSTERS
where the brain is too small since birth but otherwise normally formed. It
results from an insufficient production of mature neurons during neurogenesis. Many genetic defects have been identified in primary microcephaly,
the common pathobiological endpoint being an alteration in the cell-cycle
timing and fate determination of neural progenitors. ASPM is the most frequently involved gene in the Microcephaly, Primary Hereditary (MCPH), autosomal recessive phenotype. Detailed prenatal and postnatal phenotypic
reports are currently lacking. We report on a Turkish family where the second child, a 36 months old boy, presented with typical MCPH. The parents
were first cousins, and the mother was in the 9th week of her third pregnancy. Next generation sequencing of the probands DNA showed a homozygous ASPM mutation, a 1bp insertion in exon 18 causing a frameshift and
premature stop codon, c.6513dupA (p.Val2172SerfsX7). Both parents were
heterozygous for the mutation. They opted for prenatal diagnosis, and amniocytes DNA showed homozygosity for the mutation. The pregnancy was
continued, and the parents declined further examinations. This case illustrates the power of high throughput sequencing for rapid prenatal diagnosis of
MPCH, where phenotypic correlations should eventually help understand
how MCPH genes shape various parts of the cortex, central gray matter, and
other parts of the encephalon.
P01.091-S
Identification of rare CNVs involving genes acting in oocyte
maturation and differentiation in a cohort of patients affected by
Primary Ovarian Insufficiency
68
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Conventional karyotyping of the LTC demonstrated a non-mosaic 45,X. Since no abnormal findings were detected on ultrasound, a confined placental
mosaicism was suggested as a possible explanation for the discordant findings. A subsequent amniocentesis revealed a normal male genotype with
QF-PCR. Karyotyping, on the contrary, demonstrated a mosaic pattern with
45,X and a structural rearranged Y chromosome, probably an i(Yp). Additional FISH confirmed the presence of an isochromosome of the short arm
of the Y chromosome. The case presented here demonstrates that caution
should be taken when conflicting results are observed in CVS - even if a normal profile is obtained with QF-PCR in a follow-up amniocentesis. This case
further illustrate that mosaicism for an abnormal cell line can result in a
normal QF-PCR profile.
P01.093-S
Persistent Elevated Population-based Rates of Congenital
Malformations (CM) and Elevated Whole Body Counts (WBC) of 137Cs
among Pregnant Women in the Polissia Region of the Rivne Province
in Ukraine.
W. Wertelecki1,2, B. Ievtushok1,3, L. Yevtushok1,3, N. Zymak-Zakytnia4,5, Z. Sosyniuk1,3, S.
Lapchenko6, I. Kuznetsov6;
1
OMNI-Net Ukraine Birth Defects Programs, Rivne, Ukraine, 2Division of Dysmorphology
and Teratology, University of California-San Diego., San Diego, CA, United States, 3Rivne
Regional Medical Diagnostic Center, Rivne, Ukraine, 4OMNI-Net Ukraine Birth Defects
Programs, Khmelnytsky, Ukraine, 5Khmelnytsky Perinatal Center, Khmelnytsky, Ukraine,
6
OMNI-Net Ukraine Birth Defects Programs, Lutsk, Ukraine.
Repeated implantation failure (RIF) is the main problem after using assisted
reproductive techniques (ART). The main causes of RIF as a multifactorial problem include decrease in endometrial receptivity, defects of embryo
or combinational. Successful embryo implantation depends on trophoblast
proliferation, migration and invasion to the endometrium, all associated
with vascular endothelial growth factor (VEGF) as the major protein in
stimulating of angiogenesis. This study aimed to determine the association betweenVEGF+405G/C polymorphism and RIF in infertile women. The
patients group included 74 women with >3 RIF and the control group consisted of 149 healthy fertile women. Genotypes and allele frequencies of
VEGF+405G/C polymorphism were determined by PCR-RFLP method and
verified by Sanger sequencing. The frequencies of GG, GC and CC genotypes
in patients group were 31.1%, 48.6% and 20.3%, respectively while those frequencies in controls were 2.0%, 47.0% and 51.0% respectively. The
frequency of GG genotype was significantly higher in patients than controls (p<0.001). CC genotype frequency was higher in controls than patients (p<0.001). The frequency of GC genotype did not show any difference
between groups. C as the wild allele was more frequent in controls while
frequency of G as the mutant allele was higher in patients (p<0.001). The
VEGF+405 G/G genotype may influence embryo implantation and lead to
ABSTRACTS POSTERS
RIF in ART candidates. Since this is the second report on association of this
polymorphism with RIF, further studies in different ethnic populations require for determining this association.
P01.095-S
Gene-gene interactions and the risk of recurrent miscarriages in EGVEGF and its receptor genes
M. Su;
Department of Obstetrics and Gynecology, National Cheng-Kung University Hospital,
Tainan, Taiwan.
Introduction: Recurrent miscarriage(RM) is defined as two or more consecutive pregnancy losses before 20 weeks of gestation which is an important clinical problem, with an incidence of 1%-3% among couples wishing
to have children. There are several factors in the etiology of RM. One of the
main genetic causes involve in the pathogenesis of RM is balanced chromosomal rearrangements in one or both of partners. In 4%-8% of couples with
RM, at least one of the partners has chromosomal abnormality. After chromosomal abnormalities, thrombophilia was identified as a major cause of
RM, with a rate of up to 40%, especially in the first half of pregnancy.
Methods: The patients group included 1100 Iranian couples(2200 individuals) referred to Royan Institute between 2004 and 2013. Karyotyping was
performed using standard cytogenetic techniques. Besides, thrombotic gene
polymorphisms were studied in 128 women who had a normal karyotype
.The results were compared with 70 healthy women as control group.
Results: Abnormal karyotypes were found in 124 people, 83 women
(3.77%) and 41 men (1.86%). The frequencies of FV Leiden, ProthrombinG20210A, MTHFRC677T and MTHFRA1298C mutations in patients were
10.93%,4.68%,43.75% and60.15%, respectively. These frequencies in control group were 2.85%,2.85%,34.28% and5.71% respectively.
Conclusion: Sex chromosome mosaicism is the most commonly detected
chromosomal abnormality in couples with RM who are candidates for offering PGD.Patients with combined thrombophilic mutations are at higher
risk for RM than women without these mutations.The most important issue
with hereditary thrombophilias is the prevention of maternal thrombosis.
Key words: Recurrent Miscarriage,Thrombophilia,Karyotype
P01.097-S
Polymorphisms in oxidative stress related genes and recurrent
miscarriage
I. V. Volodin1, M. B. Khadzhieva2;
1
Moscow State Universitet, MOSCOW, Russian Federation, 2N.I. Vavilov Institute of
General Genetics, MOSCOW, Russian Federation.
Recurrent miscarriage (RM) is one of the important problems of modern reproductive medicine. RM affects approximately 1 5 % of all couples trying
to conceive. Various factors have been identified that influence miscarriage,
including parental chromosomal abnormalities, endocrine dysfunction, and
Back to index
others. However 50% of cases fail to reveal an identifiable cause and are
therefore classified as idiopathic.
We investigated the association of polymorphisms in oxidative stress related genes with idiopathic RM. 331 idiopathic RM patients and 197 controls
were genotyped for ABCB1 rs1045642, CYP1A1 rs1048943 and rs4646903,
COMT rs4680, CAT rs17880664, GCLC rs17883901, GPX4 rs713041, NRF2
rs6721961, SOD2 rs4880, and OGG1 rs1052133. A protective effect of COMT
rs4680-G allele on RM was shown in individual SNP analysis: P = 0.0016, OR
= 0.47, 95% CI 0.29 - 0.75. The multi-factor dimensionality reduction (MDR)
approach revealed gene-gene interactions for ABCB1, COMT, GPX4, and
OGG1 genes. Cumulative gene risk score analysis demonstrated that more
than three risk alleles in the genes ABCB1, COMT, GPX4, and OGG1 were associated with idiopathic recurrent miscarriage P = 1.2 x 10-3, OR =1.97, 95%
CI 1.31- 2.97. In silico data interpreting by GeneMANIA analysis revealed
genetic, physical, pathway and coexpression networks for these four genes.
The current study shows that cumulative effects of genetic variability in oxidative stress-related genes may play a role in the recurrent miscarriage with
no known etiology.
P01.098-M
Results of conventional karyotyping and 5 thrombophilic gene
mutations in a Turkish population with recurrent pregnancy loss
Y. zen, H. Grkan, H. Tozkr, S. Ulusal, E. Gnc, D. Eker;
Trakya University, Edirne, Turkey.
Recurrent pregnancy loss (RPL) is the spontaneous loss of clinically established intra-uterine pregnancy before the fetus has reached viability
Here we present the cytogenetic data of couples and prevalence of 5 thrombophilic gene mutations among 175 women referred with RPL to our center.
Chromosome analyses were performed in 179 couples with two or more
consecutive miscarriages before 24 weeks gestation between 19.10.201131.12.2013 using GTL banding. MTHFR c.677C>T and c.1298A>C, Factor II
c.20210G>A, FactorV (Leiden) c.1691G>A and plasminogen activator inhibitor-1 (PAI-1) 4G/5G genotypes were determined using SNP primers designed by the manufacturer (NLM Diagnostics, Italy). Melting curve
analysis has been performed with labeled probes using Real-Time PCR method (Qiagen, Rotor Gene).
Table 1 shows the detail of the cytogenic findings of couples with RPL. The
frequencies of studied 5 thrombophilic gene mutations in women referred
with RPL were summarized in Table 2.
The prevalence of parental chromosomal aberrations was greater in our
study (8.4%) than in most studies in the literature, which quote a 3% - 5%
prevalence.
Comparing our results of 5 thrombophilic gene mutations with other studies we suggest considering the mutations in FV Leiden G1691A, MTHFR
C677T and PAI-1 4G/5G genes in women with RPL.
Table 1: Details of cytogenic findings of couples with RPL
Cytogenic
findings among men
n (%)
by conventional
karyotyping
156
46,XY
(87,1%)
Cytogenic
findings among women by
conventional karyotyping
n (%)
46,XX
152
(84.9%)
45,X
[2]/46,XX [48]
2 (1,11%)
45,X[6]/46,XX[94]
1 (0,55%)
45,X[4]/46,XX[96]
45,X[5]/46,XX[95]
45,X[3]/47,XXX[2]/49,XXXXX[1]
/46,XX[94]
46,XX,
inv(9)(p11q13)
46,XX,
t(1;6)(p11;q11)
46,XX,
t(11;18)(p13;q11.2)
45,XX,
rob(13;14)(q10;q10)
46,XX,
16qh+
46,XX,
21pss
46,XX,
21ps+
46,XX,
9qh+
46,XX,
1qh+
1 (0,55%)
1 (0,55%)
1 (0,55%)
2 (1,11%)
1 (0,55%)
1 (0,55%)
1 (0,55%)
3 (1,67%)
2 (1,11%)
1 (0,55%)
2 (1,11%)
3 (1,67%)
47,XXY[3]/46,XY[47]
46,XY,
t(4;10)(p14;p13)
46,XY,
t(2;3)(q35;q25.2)
46,XY,
t(1;8)(q25;q22)
46,XY,
t(6;14)(p23;q24)
46,XY,
inv(9)(p11;q13)
46,XY,
inv(9)(p12;q13)
46,XY,
inv(5)(p15;q31)
46,XYqh+
46,XY,
9qh+
46,XY,
1qh+
46,XY,
22pstk+ps+
46,XY,
21pss
46,XY,
9qh+, 16qh+
1 (0,55%)
1 (0,55%)
1 (0,55%)
1 (0,55%)
1 (0,55%)
4 (2,23%)
1 (0,55%)
1 (0,55%)
7 (3,91%)
3 (1,67%)
1 (0,55%)
1 (0,55%)
2 (1,11%)
1 (0,55%)
69
ABSTRACTS POSTERS
Table 2: Frequencies of 5 thrombophilic gene mutations of women with RPL
FII
MTHFR
FV Leiden
MTHFR1298 PAI-1
Prothrombin
677
Homozygous
151
81
Homozygous 5G
168 (96%)
70 (40,2%)
Wild Type
(86,3%) (46,2%)
28 (27%)
Heterozygous
23
82
Heterozygous
7 (4%)
79 (45,4%)
Mutation
(13,1%) (46,8%)
4G/5G 40 (39%)
Homozygous
Homozygous 4G
1 (0,6%) 12 (7%) 25 (14,4%)
Mutation
35 (34%)
175
175
175
174
103
P01.099-S
The role of HLA-genes in complex immunogenetic preconditions for
idiopathic recurrent pregnancy loss
K. Sosnina, O. Terpylyak, D. Zastavna, N. Helner;
Institute of Hereditary Pathology, NAMS of Ukraine, Lviv, Ukraine.
70
Back to index
C. J. Dommering1, L. Henneman1, A. Van der Hout2, M. A. Jonker3, A. C. Moll4, H. MeijersHeijboer1, DNA-diagnostic laboratories the Netherlands;
1
Department of Clinical Genetics, VU University Medical Center, Amsterdam,
Netherlands, 2Department of Genetics, University Medical Center Groningen, University
of Groningen, Groningen, Netherlands, 3Department of Mathematics, Faculty of Sciences,
VU University, Amsterdam, Netherlands, 4Department of Ophthalmology, VU University
Medical Center, Amsterdam, Netherlands.
Background Since the 1980s the genetic cause of many hereditary tumor
syndromes has been elucidated. As a consequence, carriers of a deleterious
mutation in these genes can opt for prenatal diagnoses (PND). We investigated uptake of PND in the Netherlands for retinoblastoma (Rb) and compared this with use of PND for five other hereditary tumor syndromes, i.e.
familial adenomatous polyposis (FAP), Von Hippel-Lindaus disease (VHL),
hereditary breast ovarian cancer (HBOC), neurofibromatosis type 2 (NF2),
and Li-Fraumeni syndrome (LFS). Methods A questionnaire was mailed to
all DNA-diagnostic laboratories assessing: 1) Number of independent mutation positive families identified until January 2013 2) Number of PNDs performed for these syndromes until that date One-sided Fishers-exact test was
used to compare uptake of PND for Rb with the other five tumor syndromes.
Results 11.8% of mutation positive Rb families used PND, whereas uptake
for the other syndromes was between never and 6.5%. For Rb PND was used
both by couples with a 50% risk and by healthy couples with a child with
a de novo mutation (2-3% risk). Overall uptake for PND was significantly
higher for Rb than for FAP, HBOC and NF2. If just Rb couples with a 50% risk
were taken into account, only a significant difference was found between Rb
and FAP and HBOC. Conclusion Large differences in the use of PND between
tumor predisposing syndromes were observed. Highest uptake was observed for Rb and other childhood hereditary tumor syndromes, which is of
relevance for physicians caring for these families.
P01.103-S
Double Robertsonian translocation of chromosomes 13, 14 and 15: a
case report
A. Vizule1,2, I. Grinfelde2,3, J. Bars3, A. Stamere4, I. Teilane4;
1
Faculty of Continuing Education, Rga Stradi University, Riga, Latvia, 2Medical
genetics clinic, University Childrens Hospital, Riga, Latvia, 3Prenatal diagnostic
department, Medical genetics clinic, University Childrens Hospital, Riga, Latvia,
4
Laboratory Department, University Childrens Hospital, Riga, Latvia.
Introduction. Robertsonian translocations (RT) are among the most common balanced structural rearrangements in humans that occurs in the
acrocentric chromosome pairs (chromosomes 13-15, 21-22). Robertsonian
translocations comprise complete centromere fusion of the long arms of
two acrocentric chromosomes, and the two short arms are lost.
Materials and methods. Trisomy 13 with double RT was characterised by
standart karyotype analysis and fluorescence in situ hybridization method
(FISH) with probes for chromosomes 13, 18, 21, X and Y with critical regions (RB1, D18Z1, D21S259/D21S341/D21S342, DXZ1, DYZ3) from Abbott
Molecular, Inc. Vysis AneuVysion DNA Probe Kit.
Case report. A pregnant 22-year old woman was referred to the prenatal genetic counseling after ultrasonography performed at 13 weeks of gestation
revealed congenital fetal abnormalities, including polydactyly of both upper
limbs and bilateral cleft lip and palate. First trimester biochemical screening showed decreased pregnancy-associated plasma protein A (PAPP-A)
and human chorionic gonadotropin (HCG), calculated risk for trisomy 18
ABSTRACTS POSTERS
was 1:105. Triple marker screening showed decreased alpha-fetoprotein
(AFP) and HCG, and normal free estriol levels. Risk recalculation with the
FMF-2012 software (Fetal Medicine Foundation) adjusted risk for trisomy 13 showed 1:55. Diagnostic amniocentesis performed at 15/16 weeks
of gestation revealed trisomy 13 by FISH and later 45,XX,+13,der(13;14)
(q10;q10),der(14;15)(q10;q10) using standart karyotype analysis.
Conclusions. Currently the estimation of recurrence risk for future pregnancies is impossible because parents refused to investigate their karyotypes.
There could be several possible explanations for double Robertsonian translocation.
P01.104-M
Sex chromosome classification by cell free DNA analysis of maternal
plasma in a general obstetrical population
C. M. Coffeen, P. L. Devers, D. Chudova, K. Jones, R. P. Rava, A. J. Sehnert;
Illumina, Redwood City, CA, United States.
E. Pompilii;
Medical Genetics Unit, St Orsola Malpighi Hospital, University of Bologna, BOLOGNA,
Italy.
Back to index
P01.106-M
Prenatal detection of a case with Simpson- Golabi-Behmel syndrome
as a consequence of GPS3 and GPS4 gene duplications
N. Kokalj Vokac1,2, N. Tul Mandi3, D. Krgovi1, A. Blatnik1, F. Mujezinovi1;
1
University Medical Centre Maribor, Maribor, Slovenia, 2Medical Faculty, University of
Maribor, Maribor, Slovenia, 3University Medical Centre Ljubljana, Ljubljana, Slovenia.
We describe the case of Simpson-Golabi-Behmel syndrome (SGBS) discovered prenataly. Fetal scan in 23rd week of pregnancy identified male fetus with
macrosomia, macrocephalia, dilatation of 3rd and 4th ventricle, hyperechogenic gut, agenesis of corpus callosum, cistical dilatated interhemispheric
fissura, macrosomic kideny and liver, and kidney polyhydramnion.
Amniocentesis was performed. Molecular kariotyping using ISCA 4x180K
arrays reveled two intersticial microduplications on Xq26.2 in size of 574
kb and 115kb. A larger microduplications encompassed four OMIM genes,
including whole GPC4 gene. A smaller microduplications was located within
the GPC3 gene and embraced exons 6 and 7 of the longest transcript of this
gene. CNVs were inherited from the mother.
Persons with this SGBS are frequently affected by embrogenic tumors of kidneys. Also the mother had Wilms tumor in her childhood. The pregnancy was
terminated because of ruptured amniotic membrane and amniotic leakage.
An autopsy of the infant confirmed organomegaly seen on ultrasound. In
addition, a ventricular septal defect, a complete agenesis of the corpus callosum, cerebellar hypoplasia were observed. Distinct facial features were
also present, including hypertelorism, short nose with broad nasal bridge,
macrostomia and macroglossia, nail hypoplasia, and an extra rib.
GPC3 and GPC4 are the two genes in which mutations are known to cause SGBS. There are only few reports on duplications of GPC3 gene and one
report of duplication of CPG4 gene causing SGBS in the literature. Mostly
deletions and mutations of GPC3 gene are described. In our presentation
possible role of two genes involved in SGBS is discussed.
P01.107-S
The correlation between transcript expression levels of nuclear
encoded and mitochondrial encoded genes in single human oocytes
during oocyte maturation
Introduction: Impairment of human oocyte maturation during oocyte maturation is a cause of infertility in infertile women. Therefore, Oocyte maturation is important in successful reproductive outcome of assisted reproduction technologies (ART). Mitochondria,which are the most organelle in the
oocytes,have in important role during oocyte maturation. Little is known
about mitochondrial genomes during oocyte maturation .This aim was to
identify the correlation between transcript expression levels between mitochondrial and nuclear encoded genes included the cytochrome C oxidase1
(MT-CO1) gene and the nuclear respiratory factor 1(NRF1) as well as the
mitochondrial transcription factor A (TFAM) genes , which using by singlecell real-time PCR during human oocyte maturation. 27 consenting women
aged 21-35 years, with male factors were selected for ovarian stimulation
and ICSI procedures.
Results:at the germinal vesicle (GV) stage oocytes ,no significance correlation between the relative expression levels (P>0.05),whereas there was
significant correlation between the relative expression levels of nuclear
(TFAM,NRF1) and mitochondrial (MT-CO1) encoded genes at the stages of
metaphase I (MI)and metaphase II (MII)stages of oocytes (P<0.05).
Conclusion:human oocyte maturation is associated with the increased correlation between transcript expression levels of nuclear ( TFAM , NRF1) and
mitochondrial (MT-CO1)encoded genes. Thus, any defect correlation between transcript expression levels of nuclear mitochonderial genes leads to
impaired developmental oocyte competence.
P01.108-M
Prenatal molecular diagnosis of skeletal dysplasias - a single center
experience
T. Schramm, S. Minderer, K. P. Gloning, C. Daumer-Haas, M. Shoukier, M. Lindner, C.
Bagowski;
Prnatal-Medizin Mnchen, Munich, Germany.
71
ABSTRACTS POSTERS
The diagnosis of a substantial number of the most frequent skeletal dysplasias can be confirmed in a short period of time by molecular genetic analysis
of the involved genes (e.g. thanatophoric dysplasia, diastrophic dysplasia,
campomelic dysplasia, Ellis-van Creveld syndrome or hypophosphatasia).
We present a retrospective analysis of 300 cases diagnosed prenatally by an
expert team of experienced sonographers and human geneticists. The study
was carried out at a single tertiary center during the last 20 years.
We demonstrate clinical findings and molecular genetic data and construct
work-ups for the diagnosis of ,,difficult cases (e.g. short rib-polydactyly syndromes, Filamin B associated skeletal dysplasias). Molecular genetic testing
was conducted in 186 cases. We were able to establish a final diagnosis in
118 cases - which is equivalent to a detection rate of 65%. Some cases are
exampflified with clinical, radiological, pathological and molecular genetic
data.
We want to point out the importance of molecular genetic diagnosis for
confirming the clinical diagnosis of skeletal dysplasias and providing exact
information for genetic counselling.
P01.109-S
Susceptibility loci for neurodevelopmental disorders - genetic
counseling and pregnancy management
L. Govaerts, M. Srebniak, K. Diderich, M. Joosten, S. Riedijk, S. van der Steen, S. van der
Steen, F. de Vries, D. Van Opstal, R. Galjaard;
Erasmus Medical Center, Rotterdam, Netherlands.
Objectives: SNP genomic array may detect susceptibility loci for neurodevelopmental disorders (SL), with possibly an increased but unquantified phenotypical risk. This study evaluates the effect of releasing the SL to pregnancy management. Psychological aspects will be reported separately.
Method: Every patient received pre-test counseling with all pathogenic results will be communicated.
The post-test genetic counseling concentrated on the phenotype of the particular SL, its incidence in the normal and affected postnatal population and
the difference between postnatal and prenatal ascertainment. Targeted parental array testing was offered. Extensive ultrasound (US) examination was
offered when the SL (postnatally ascertained) was associated with physical
abnormalities. Psychological help was available in all cases if needed.
Results: In 36 cases out of 2108 ongoing pregnancies a SL was found.
1. Two couples with an increased risk for aneuploidy and no US abnormalities opted for TOP. Both couples already had a bad feeling about the pregnancy before testing. They received psychological help. Pathological examination (N=1) revealed no structural abnormalities.
2. For parents with fetal US abnormalities the SL was usually considered as
less important. The US abnormalities were the reason for TOP.
3.The inherited nature of SL seemed reassuring.
Conclusions: In 5,5 % (2/36) of the SL cases the pregnancy was terminated
due to the presence of the SL. Further follow up of the families and their
children is needed to evaluate the significance of prenatal diagnosed SL, to
offer early intervention when neurodevelopmental disorders emerge and to
evaluate the psychological impact of prenatally discovered SL.
P01.110-M
Origin of multilocus methylation defects of imprinted genes in firsttrimester spontaneous abortions
72
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Materials and methods Data were used from the birth defects registry Eurocat Northern Netherlands. We included malformed foetuses and children
born between 1997-2010 (N=5709). Of those, 5249 were born to fertile
couples and 460 to subfertile couples (83 after IVF, 95 after ICSI, 282 conceived naturally after a time to pregnancy >12 months). We analysed whether
parental subfertility was associated with de novo mutations or microdeletions.
Results From the 5709 malformed cases, 156 (2.7%) had a monogenic
condition resulting from a de novo mutation and 61 (1.1%) had a de novo
chromosomal microdeletion. Parental subfertility was not associated with
de novo mutations (OR 0.88, 95%CI 0.47-1.63) or microdeletions (OR 1.04,
95%CI 0.41-2.60). Subgroup analyses showed that IVF or ICSI alone were
not associated with a de novo event either. Adjustment for paternal and maternal age did not change the results.
Conclusion Parental subfertility, IVF and ICSI are not associated with de
novo mutations or microdeletions in offspring. These results suggest that
the previously established increased risk of congenital anomalies after IVF/
ICSI is not explained by an increase in de novo events.
ABSTRACTS POSTERS
P01.113-S
Prenatal detection of TAR syndrome
Small supernumerary marker chromosomes (sSMC) are a group of structurally rearranged chromosomes that cannot be identified by conventional
cytogenetic techniques. Prenatally sSMC are present in about 0.075% of the
tests and postnatally in 0.044% of live born children. An overall risk of an
abnormal phenotype is about 30%.
We report on a 31-year-old woman (first pregnancy, after IVF) referred for
genetic counselling because of increased foetal nuchal translucency (5,5mm
at CRL 55,9mm) and increased risk of trisomy 21 (1:11) and trisomy 18
(1:207) in first-trimester combined prenatal screening. Ultrasound scanning at 17th week of gestation revealed no heart defects but an increased
nuchal fold, left-sided diaphragmatic hernia and hydronephrosis.
Cytogenetic studies of amniocytes showed a karyotype as follows:
48,XX,+mar1,+mar2. Parents karyotypes were normal. To determine the
origin of the markers, array comparative genomic hybridization (aCGH) was
used (Agilent SurePrint G3 Human CGH Microarray Kit 4 x 180K platform). Tetrasomy of the region 13q31.3 to 13q34 - arr[hg19] 13q31.3q34(92507936115092648) was showed.
The pregnancy was terminated at 18th week of gestation. Post mortem examination revealed dysmorphic facial features, head and body disproportion (head diameter: 8cm, CRL 12,5cm), wide neck, ambiguous genitalia.
No other malformations of the internal organs, except listed above, were
found.
It is known that a risk of an abnormal phenotype associated with de novo
sSMC derived from chromosome 13 (or 14, 21, 22) is about 7%. Molecular
characteristic of sSMC is important for genetic counselling and genotype/
phenotype correlation.
P01.115-S
Additive effect of thromobhilic gene polymorphisims is associated
with recurrent pregnancy loss
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73
ABSTRACTS POSTERS
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P01.120-M
Partial Trisomy 6q detected in Prenatal Diagnosis
P01.118-M
Performance of in-house non-invasive aneuploidy test using benchtop
next generation sequencing system
Since the discovery of cell-free fetal DNA in 1997 by Dennis YM Lo, the main
goal was formulated - a reliable method for non-invasive fetal aneuploidy
detection. The arrival of next generation sequencing technologies finally
gave scientists a proper tool to reach this goal. Today, so called large next
generation sequencing instruments are used for the so called non-invasive
prenatal aneuploidy testing. Few years ago, so called benchtop next generation sequencers were introduced, which allowed researchers worldwide to
start adopting wide range of novel methods benefiting from next generation
sequencing. However, they were generally considered not to be sufficiently
parallel for non-invasive aneuploidy test.
In our study, we used one of benchtop next generation sequencers, the MiSeq by Illumina, to test its ability to detect aneuploidy. Plasma DNA from
pregnant was isolated, fragment libraries were prepared and sequenced
in multiplex setup. Data was analysed using in-house pipeline. A set of 20
samples (average read count 2 870 322 with SD 603 708) high risk group
pregnancies with confirmed euploid fetus was used for training of our analysis pipeline. Analysis of testing set including 5 samples with T21 fetus resulted in 100% specificity and 100% sensitivity. Based on our results we believe that performance of benchtop next generation sequencers is sufficient
for non-invasive prenatal aneuploidy testing. In addition, the inherently higher flexibility of benchtop systems could be of benefit for short turnaround
and low sample throughput demands. This research was supported by ERDF
grant with ITMS 26240220067.
P01.119-S
12 Mbp chromosomal gain on 7p detected prenatally without major
dysmorphic features
We describe a prenatally detected 12 Mbp chromosomal gain on 7p. Amniocentesis was performed due to risk for a chromosomal abnormality after
first trimester screening.
Cytogenetic analysis revealed a duplication on chromosome 7p in the
foetus. A SNP array was carried out showing a 12 Mbp gain - ISCN:
arr[hg19]7p21.3p15.3(13,146,026-25,348,884)x3.
A literature review revealed one report from Miller et al. (Am J Med Genet
1979;4(4):323-32), describing a man with a duplication of the segment
7p21 to 7p25 with severe mental deficiency but normal growth.
In order to exclude a balanced rearrangement of the parents, conventional
chromosome analysis was performed. The mother showed the same duplication as the foetus. A SNP array confirmed the cytogenetic result.
The mother presented without major dysmorphic signs and showed no intellectual disability. She had finished basic education.
Pregnancy was continued due to missing ultrasound abnormalities and the
inheritance of the maternal duplication.
After caesarean section at 34+6th gestation week the premature infant presented with a weight of 2340g (P38), length 46.0cm, OFC 31.7cm and APGAR 9/10/10.
The slightly cyanosed girl showed mild facial dysmorphisms as deep set
ears, mild retrognathia and mild hyperplasia of the clitoris. No further clinical abnormalities were reported. 19 days after birth the girl left the hospital
in a good general state of health (weight 2486g). Two subsequent controls
revealed no major clinical abnormalities.
The case report shows the importance of testing parents after a pathological
prenatal result and that a 12 Mbp 7p duplication not necessarily causes a
major malformation.
74
P01.121-S
Assessment of chromosomal changes in trophectoderm cells in
relation to maternal age
Mean
Mean No
No of
No of
No of whole
Mean No of
No of
of whole
abnormal segmental chromosomal
chromosomal segmental chrom.
embryos changes
changes
changes
changes changes
/ No of
/ No of
/ No of
per abnormal
per
per
analyzed chrom.
chrom.
embryo
abnormal abnormal
embryos changes
changes
embryo
embryo
30/60
45/83
38/83 (46%)
2.8
1.5
1.3
(50%)
(54%)
63/94*
(67%)
55/70**
(79%)
80/221
(36%)
49/235
(21%)
141/221+
(64%)
186/235++
(79%)
3.5
4.3
1.3
0.9
2.2
3.4
P01.122-M
Our contribution to rescue line hypothesis for a case of Turner
syndrome.
ABSTRACTS POSTERS
tive CVS was 46,XY. The long-term CVS cultivation revealed 45,X as well as
QF-PCR and FISH. The same results were found in post mortal examination
of muscule cells cultures. These findings suggest that in trophoectoderm
initiated mosaicism with the 46, XY clone and loss of Y chromosome must
occurred after fertilization. XY clone persisted in the embryo as a minor cell
population and its prevalence increased in further chorion formation. It explains that in QF-PCR analysis of biopsied samples 46, XY represented at
least 80% cells, while the second line 45,X was under detectable limit for
this method (less than 20%). Our findings confirm recently published data,
describing similar situation for 45,X genotype derived from 46,XX. This rescue line implies an origin of this disorder in mitotic loss. We also proved
that syncytiotrophoblast is a strong candidate for the location of the rescue cell line. Most probably this may not be detected in extensive search for
cryptic mosaicism. Our casuistic point out the importance of complex molecular genetics testing in native and long-term cultured fetal cells to provide
the best reliable results allowing to make final conclusions properly. Supported by grants IGA NT13770-4/2012, project for conceptual development
of reasearch organization 00064203 and OPPK CZ.2.16/3.1.00/24022.
P01.123-S
SNP-based non-invasive prenatal testing identifies vanishing-twin
pregnancies
M. Stosic, K. Curnow, A. Ryan, E. Kirkizlar, S. Sigurjonsson, M. Hall, Z. Demko, M.
Rabinowitz;
Natera, Inc., San Carlos, CA, United States.
Back to index
9.91;p=0.032).
Comprising the allele frequencies of VKORC1 gene rs9926231 across different populations revealed that its are relatively close in Moldovan populations to those reported for Caucasians and Turkish. The allele frequencies
of VKORC1 gene rs9934438 are statistically significant different from data
available on NCBI-site, NCP, MASSource.
Conclusion: VKORC1gene rs9934438 is statistical associated with recurrent
pregnancy loss and increasing the risk of complications during pregnancy
P01.126-M
The application of post-light semiconductor-based next-generation
sequencing in clinical cases of preimplantation aneuploidy screening
(PGS) with fresh embryo transfer
K. Lukaszuk, S. Pukszta, J. Liss, C. Cybulska;
Invicta sp. z o.o., Gdansk, Poland.
75
ABSTRACTS POSTERS
individual receives half of its genetic heritage from his biological mother and
the other half from his biological father. Rare cases of twin pregnancy were
induced by fertilization from two different parents. In this case, we speak
about a superfecondation. This situation is exceptionally confirmed.
Here, we report a case of genetically confirmed superfecondation in the context of paternity test by DNA fingerprinting.
Observation: In the context of paternity, we performed a genetic study of 4
members: a mother, her 2 twin infants, and a supposed father. This study
involved the analysis of 15 STRs markers by PowerPlex 16 System kit. Results were analyzed by Genotyper v 3.7.
Results: Genetic analyzes were performed under the same technical conditions and showed that one of the twins share the same alleles with the alleged
father, while the other infant has different alleles in 11 of the 15 STRs studied. Therefore, despite that these two children are twins, their biological
fathers are different.
Discussion and conclusion: The superfecondation is a rare and special obstetric situation. It is secondary to the fertilization of two eggs from the mother by two sperm each from a different father.
In this study, we have confirmed this situation by molecular genetics tools.
However, their clinical and biological implications remain unknown.
P02.01-S
IL-8 is associated with age-related macular degeneration in Italian
samples
76
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In our diagnostic laboratory we have used NGS to analyze the 6 OCA, OA1
and HPS1 genes. This was efficient in terms of both finding mutations and
rapidity. We are designing a larger panel including all syndromic albinism
genes.
The extensive analysis of all albinism genes will allow us to better characterize gene interactions in this disease that may not be purely monogenic.
P02.03-S
Two novel (p.E930X and p.C2278X ) mutations in ALMS1 leading to
Alstrm Syndrome
T. Pieiro-Gallego, M. lvarez-Satta, S. Castro-Snchez, D. Valverde;
Departamento de Bioqumica, Xentica e Inmunoloxa, Vigo, Spain.
Alstrm syndrome (AS, OMIM#203800) is an extremely rare, autosomal recessive disorder with a significantly shorter life expectancy. It is mainly characterized by sensorineural hearing loss, early onset blindness due to retinal
dystrophy, and obesity. Other reported features include recurrent pulmonary infections, progressive renal and hepatic dysfunction, cardiomyopathy,
short stature, and endocrinological features. AS is caused by mutations in
the ALMS1 gene, which encodes a protein of unknown function that localizes to the basal bodies of cilia, playing a role in intracellular trafficking. Here
we report two AS cases born to unrelated healthy parents. The diagnosis of
AS was performed accordingly to the criteria previously defined by Marshall
et al. (2007). We ascertained two families with seemingly AS. Molecular
analysis was performed by direct sequencing of the known hot spots in the
ALMS1 gene (exons 8, 10 and 16) according to the mutational load described
in AS. We identified a homozygous mutation (c.2788G>T, p.E930X) in patient 1 and a homozygous mutation (c.6834C>A, p.C2278X) in patient 2 that
co-segregate with the disease phenotype in family 2. Both mutations cause
a truncated protein and had not been previously described in literature. We
show that direct sequencing of exons 8, 10 and 16 of the ALMS1 gene could
represent a useful tool for molecular diagnosis of AS, whilst the mutations
described here may contribute to extend the mutational spectrum in AS.
P02.04-M
Exome sequencing in 32 patients with anophthalmia/microphthalmia
and other developmental eye defects
A. Slavotinek1, S. T. Garcia2, G. Chandratillake2, T. Bardakjian3, R. Lao4, P. Tang4, E.
Wan4, D. Wu1, K. Umeda1, J. Tirch2, R. Tischler2, J. Harris2, M. J. Clark2, S. Chervitz2, A.
Patwardhan2, J. M. West2, A. Schneider3, P. Kwok4, S. Baranzini5, R. M. Chen2;
1
Department of Pediatrics, University of California, San Francisco, San Francisco, CA,
United States, 2Personalis, Inc., Menlo Park, CA, United States, 3Division of Medical
Genetics, Einstein Medical Center, Philadelphia, PA, United States, 4Cardiovascular
Research Institute, University of California, San Francisco, San Francisco, CA, United
States, 5Department of Neurology, University of California, San Francisco, San Francisco,
CA, United States.
Auditory neuropathy (AN) is characterized by an absent or abnormal auditory brainstem responses and preserved otoacoustic emissions and/or
cochlear microphonics. To date, four loci associated with nonsyndromic AN
were mapped: DFNB9 (OTOF gene) and DFNB59 (PJVK gene), responsible
ABSTRACTS POSTERS
for autosomal recessive pattern; AUNA1 (DIAPH3 gene) for autosomal dominant; and AUNX1 for X-linked. Connexin 26 mutations were also reported
in subjects with AN. The main goal of the study was to investigate genetic
mutations in patients with clinical diagnosis of AN, to verify its importance
and involvement in the etiology of AN in Brazilian patients. Clinical information and genetic evaluation of 39 patients were analyzed. We investigated
the most common causes of genetic hearing loss, including pathogenic variants in the GJB2 gene, deletions in the GJB6 gene and m.1555A>G mutation
in the MTRNR1 gene. Additionally, direct sequencing is performing for mutation screening of OTOF gene. The common Spanish p.Q829X mutation in
the otoferlin gene was not detected in our cohort. The c.35delG mutation
in the GJB2 gene was found in two patients in homozygous genotypes. However, it is not established if pathogenic variants in connexin 26 could be
involved with AN or if the otoacoustic emissions that were recorded from
these subjects only represent the residual activity of few outer hair cells that
still alive. Further investigation is needed to clarify the link between GJB2
mutations and AN. The study of AN genetic basis is extremely important to
improve the diagnosis, management, therapy and genetic counseling of the
affected subjects.
P02.06-M
Targeted high-throughput sequencing for mutation detection in
Bardet-Biedl Italian patients
I. Passerini1, S. Palchetti1, A. Mariottini1, C. Pescucci1, G. Marseglia1, S. Bardi1, M.
Benelli1, S. Falconi1, E. Contini1, A. Sodi2, F. Marin1, U. Menchini2, F. Torricelli1;
1
AOU Careggi, Florence, Italy, 2Eye Clinic, University of Florence, Florence, Italy.
Bardet-Biedl syndrome (BBS) is characterized by truncal obesity, polydactyly, hypogonadism, developmental delay, learning disabilities, progressive
retinopathy, renal disease and susceptibility to diabetes mellitus. Although
BBS is mainly transmitted in an autosomal recessive manner, few families
exhibit a tri-allelic mode of inheritance. To date, 16 different BBS genes
(BBS1-BBS16) are known and BBS1 and BBS10 show the highest mutation
frequency in BBS patients.
Six unrelated patients evaluated by standard ophthalmologic examination
and with a clinical diagnosis of BBS were analyzed by targeted re-sequencing
of 130 retinopathies-related genes on HiScanSQ Illumina platform (mean
coverage 500X). Bioinformatic analysis identified a mean of 1100 sequence
variants per sample. Filtering pipeline (exonic function, frequency, prediction and inheritance model) leads to distil a mean of 10 candidate variants
per sample. The candidate variants were independently validated by Sanger
sequencing and segregation analysis was performed.
We identified two known causative deletions in BBS9 (c.1877_1880del)
and BBS7 (c.712_715del) genes and a novel frameshift deletion in BBS10
(c.804_805del). Morever, a patient is compound heterozygote for two novel
variants in BBS10 (c.1677_1678insA and c.1220T>C); one patient present a
homozygous variant in BBS2 gene (c.209A>G) and one novel heterozygous
variant (c.538C>T) in BBS3 gene. The last patient has two variants in BBS12
gene both in homozygous status [c.1044del (novel) and c.61T>C (described)].
NGS approach has enabled us to detect variants in BBS genes. Although for
described variants and ins/del frameshif variants the genotype-phenotype
correlation is clear, in order to clarify tri-allelic variants role in BBS-onset,
further association studies are required.
P02.07-S
Neuro-developmental genes may underlie human congenital general
anosmia
The neuronal and cell-biological mechanisms underlying vertebrate olfaction have been studied in considerable detail, but there is a gap in our
understanding of the molecular genetic basis of olfactory monogenic disorders. Congenital general anosmia (CGA) affects <0.1% of the general population, and appears in isolated or syndromic forms. We have recruited a large
cohort of isolated CGA, with 66 affected families of Jewish origin. We applied
whole-exome sequencing to 43 selected individuals from 8 multiply affected
families. We identified in three members of one family with a rare X-linked
mutation in the TENM1 (teneurin 1) gene, a neuronal signal transducer that
functions in synaptic-partner-matching between olfactory sensory neuronal axons and target projection neurons in drosophila. The mutation affects
a highly conserved Proline residue in one of the YD domains of TENM1. In
another family we identified candidate variants in the genes FGFR1 (heterozygous mutation) and SEMA3A (heterozygous mutation). Affected individuals in this family obligatorily carry both mutations, suggesting a di-genic
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We report a boy affected by bilateral sensorineural deafness and palmoplantar hyperkeratosis. The pedigree suggests an autosomal dominant pattern of inheritance with variable expressivity. Molecular analysis of GJB2
was negative and GJB6 deletions were also excluded. GJB6 encodes connexin
30, which is highly expressed in the inner ear and in keratinocytes. Therefore, we sequenced the entire coding region of this gene in the proband
and identified the c.175 G>A mutation, causing a substitution of glycine 59
with an arginine residue. The same missense mutation had been previously
reported in a single patient affected by post-lingual deafness and palmoplantar hyperkeratosis (Nemoto-Hasebe et al, 2009). The G59R mutation
affects a highly conserved amino acid, it was absent in 100 healthy ethnically matched controls and segregates with the phenotype in our family. The
analysis by molecular modelling revealed that the residue is located at the
top of the channel formed by the hemiconnexons. All these findings strongly
indicate a pathogenetic role for the novel variant. To explore the functional
consequence of the missense mutation, we expressed the mutated version
of GJB6 in HeLa cells lacking connexins. We showed that GJB6 mutated protein is localized to the plasma membrane only when co-expressed with the
wild type form. This model was employed to test the electrophysiological
properties of the connexons formed by both the mutated and the wild-type
connexin 30.
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P02.10-M
Hearing and ageing: a complex genomic strategy leading to new
genes/variants identification in European and Central Asian
populations
Human MYO15A is located on chromosome 17p11.2 and encodes unconventional myosin XV, which is critical for the formation of stereocilia. Mutations
of MYO15A are associated with profound autosomal recessive nonsyndromic hearing loss (ARNSHL) in humans, and with deafness and circling behavior in Shaker-2 mice. To date, more than 50 mutations have been reported
in MYO15Amost of which have been found by linkage analysis in consanguineous families from countries like Pakistan, Turkey, Iranand India.
In present study, we used targeted genomic enrichment and massively
parallel sequencing to identify the cause of ARNSHL in probands from 94
families segregating recessive deafness. In eight probands, we identified
MYO15A mutations as the cause of ARNSHL. Of the eight mutations, 6 were
present in the homozygous state, 2 in compound heterozygous state and of
them 7 were novel. This study implicates MYO15A as an extremely important cause of ARNSHL in Iran. With a mutation frequency of 8.5% in our
cohort,MYO15A mutations are the most frequent cause of ARNSHL after
GJB2 mutations.
P02.12-M
Whole exome sequencing identifies new causative mutations in
Tunisian families with non-syndromic deafness
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Hereditary Retinal Dystrophies (RD) represent one of the most frequent genetic causes of blindness in the Western world. Because of their high clinical
and genetic heterogeneity, an exhaustive classification of RD is difficult. The
progressive forms may have an onset in early childhood, as Leber congenital
amaurosis, or later in life, as Retinitis Pigmentosa (RP). There are also described many syndromic forms, as Usher syndrome (USH), and Bardet-Biedl
syndrome (BBS).
RD can be inherited as autosomal recessive (ar) or autosomal dominant
(ad), as well as X-linked (xl), or mitochondrial traits. Nevertheless, the majority of cases are sporadic.
In this study, we analyzed 115 retinopathies-related genes by targeted nextgeneration sequencing (NGS) in a cohort of 78 patient with sporadic RP, 10
patients with USH and 10 patients with BBS.
Sequence target enrichment was performed using SureSelect XT kit (Agilent). Sequencing of each library was carried out on HiScanSQ Illumina platform (mean coverage 500X).
We obtained a detection rate of 100% for BBS patients, 80% for USH patients, and 69% for RP patients. The candidate variants were independently
validated by Sanger sequencing and segregation analysis was performed.
Were also identified 3 large deletions in the genes EYS, RPGR and USH2A,
using method CONTRA and confirmed by MLPA.
This study confirms that NGS-based mutation analyses are reliable and cost-
ABSTRACTS POSTERS
efficient approaches in molecular diagnosis of genetically heterogeneous
diseases like RD. NGS allows also a considerable reduction of costs and an
early intervention for diseases for which there are available appropriate
therapeutic strategies.
P02.15-S
Identification of three novel homozygous variants in the GJB2 gene in
Mexican patients with hereditary sensorineural hearing loss
Background: Hereditary sensorineural hearing impairment (HSHI) is a genetically heterogeneous disorder worldwide. Mutations in the GJB2 gene
are a frequent cause of hereditary SNHL. Individuals that are homozygous
for the GJB2 gene mutations manifest a wide spectrum of clinical data that
ranges from moderate to profound SNHL; this suggests the participation of
epigenetic and environmental factors in the phenotypic expression Objective: To describe three novel homozygous mutations in the GJB2 gene with
HSHI. Materials and methods: Three subjects with prelingual HSHI were included in the study. Genomic DNA was extracted by conventional methods
and all exons of GJB2 gene were analyzed through PCR and the DNA was
sequenced on an ABI 3730XL automated sequencer. Results DNA sequencing analysis showed three novel homozygous mutation in the GJB2 gene
that corresponded to p.V84M/p.F31I/p.W44X; /p.V84M/p.F31I/p.R32S
and p.E47X/p.R32S/p.S19R. Parents were tested for this molecular defect
and they present an heterozygous state that allows to confirm the recessive
inheritance pattern. These mutations were searching in 100 normal controls to discard a possible polymorphism. Conclusion: We describe three
novel varieties of homozygous mutations in patients with HSHI. All patients
presented profound hypoacusia with no other anomalies. These data show
that the genesis of HSHI is complex and that the genetic defects are greater
than expected.
P02.16-M
Regional distribution of the GJB2/GJB6 gene mutations in Mexican
population with hereditary hearing impairment
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In 2012, NMNAT1 was identified as a novel disease gene for Leber Congenital Amaurosis. The mutation spectrum contains both coding and regulatory
mutations. The starting point of this study was pseudohomozygosity of a
known NMNAT1 mutation (p.Arg237Cys, exon 5) in two unrelated Japanese
families (F1 and F2), assuming hemizygosity. Here, we aimed to identify the
putative NMNAT1 deletions.
Copy number variation (CNV) screening was performed for all exons using
qPCR. Subsequently, identified deletions were refined with additional qPCR
amplicons located in the breakpoint regions. Finally, the junction product
was amplified with long-range PCR and sequenced (Nextera XT, MiSeq, Illumina).
In F1, CNV analysis showed a heterozygous deletion of exon 4 and 5, which
was subsequently refined to a region of 13.0-18.7 kb. In F2, the amplicon
for exon 4 was deleted, whereas the copy number for exon 5, located downstream of the p.Arg237Cys mutation, was normal. Subsequent refinement at
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ABSTRACTS POSTERS
the 5end delineated the deletion to a region of 1.5-6.7 kb. Long-range PCR
revealed a band of 2.4 kb and 2.1 kb in F1 and F2, pointing to two different
deletions of approximately 16.6 kb and 4.8 kb, respectively. Sequencing analysis of the junction products is currently ongoing.
In conclusion, we report the first deletions in NMNAT1, expanding the mutation spectrum of this gene. Hence, we presume that the high number of patients in which only a single heterozygous mutation could be identified, can
be explained by structural variations, requiring additional strategies apart
from resequencing of the coding region.
P02.20-M
Prevalence and mutation analysis of NMNAT1 gene in Leber Congenital
Amaurosis in Spanish population
R. Sanchez-Alcudia1, S. Tatu1, F. Blanco-Kelly1, N. Reyes1, M. Lopez-Molina2, R. PerezCarro1, A. Avila-Fernandez1, M. Corton1, C. Ayuso1;
1
Department of Genetics, Instituto de Investigacion Sanitaria-Fundacion Jimenez Diaz
(IIS-FJD), Madrid, Spain, 2Department of Ophthalmology, IIS-Fundacion Jimenez Diaz
University Hospital, Madrid, Spain., Madrid, Spain.
Leber Congenital Amaurosis (LCA) is the most severe blinding retinal dystrophy that represents near 5% of cases. At the moment 20 genes have been
associated to this disease, more of them expressed in the photoreceptors
or in the retinal pigment epithelium, explaining around 70% of LCA cases.
The ubiquitous gene encoding nicotinamide nucleotide adenylyltransferase
1 (NMNAT1) was recently associated to this pathology using exome sequencing. The aim of this work was to screen the NMNAT1 gene in our Spanish
cohort of uncharacterized LCA families. We were selected a total of 96 LCA
families previously screened for known mutations to cause LCA using a
commercial genotyping chip with negative results. Index cases of these families were analyzed by direct Sanger sequencing of the coding NMNAT1
sequence. This screening allowed us to identify five novel mutations in the
NMNAT1 gene and to characterize 6 of the 96 total families (6.25%), carrying all of them compound heterozygous mutations. As described in other
populations, the p.E257K variation which was not found in any of the 176
Spanish controls tested, is the most frequent variation. Five additional families were found to carry the p.E257K common variation in heterozygosity
with no other apparently change in the second allele. In order to fully characterized these families other studies (RNA expression in peripheral total
blood and lymphoblast cell line, intronic sequencing and others) should be
performed.
P02.21-S
Mutation hotspot sequencing reveals RPE65 as the most frequently
mutated LCA gene in Denmark
Leber congenital amaurosis (LCA) represents the most severe and earliest
onset form of inherited retinal dystrophies, and affects 1 per 50,000 individuals worldwide. Presently, mutations in twenty genes with diverse roles in
the retina are known to be associated with LCA. The large number of LCA
genes necessitates a systematic genotyping approach in a cost- and timeeffective manner. Previous studies in LCA have described some recurrent
mutations and mutational hotspots in particular exons of LCA-associated
genes, i.e. an intronic variant in CEP290 (c.2991+1655A>G), a nonsense mutation in AIPL1 (p.W278*), a missense change in GUCY2D (p.R768W), and
various mutations clustering in exons 7 and 9 of CRB1. As gene-specific therapies are emerging, identifying mutations in RPE65 and LRAT can also be of
tremendous importance for patients. Sanger sequencing of these mutation
hotspots, and all protein-coding exons of RPE65 and LRAT in 64 Danish LCA
probands revealed one or two variants in 22 (34%) cases. Upon the identification of heterozygous variants, Sanger sequencing was performed in the
relevant genes to identify the second allele, RPE65 was shown to be the most
frequently mutated gene in Denmark (20% vs. 8% in other Caucasian populations). This is important as 9-cis-retinoid supplementation and RPE65
gene augmentation therapies have been developed.
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P02.22-M
Propensity for localized provoked vulvodynia by TRPV1 and NGF
polymorphisms
arNSHL (autosomal recessive Nonsyndromic Hearing Loss), the most frequent mode of hereditary hearing impairment, is typically severe or profound. In contrast with severe or profound hearing loss of which genetic
etiologies have been intensively investigated, relatively less attention into
mild to moderate hearing loss has been paid. This study was designed to delineate genetic contributions, if any, to moderate hearing loss in a pediatric
population using the next generation sequencing technologies. We performed whole exome sequencing (WES) for 12 unrelated children with moderate degree of hearing loss. Hearing loss from 11 of 12 probands was sporadic. We performed bioinformatic analyses of the WES data and selected the
candidate variants compatible with either autosomal recessive inheritance
pattern or de novo autosomal dominant pattern. Strong candidate variants
were detected in 6 (50%) of 12 probands, suggesting a strong genetic contribution to moderate degree of hearing loss in a pediatric population. Diverse mode of inheritance pattern was observed in this disease population
unlike in severe to profound hearing loss. Compound heterozygotes from
deafness genes, OTOGL, and SERPINB6 were detected. One proband segregated moderate degree of hearing loss as a digenic inheritance involving two
deafness genes, GPR98 and PDZ7. In addition, strong candidates from another three subjects were selected as a de novo mutation from MYH14, MYO7,
and P2RX2, respectively. This study, for the first time in the literature reveals
a strong genetic contribution to sporadic forms of moderate hearing loss in
a pediatric population.
ABSTRACTS POSTERS
P02.24-M
Identification of genes and related mutations in 10 Iranian families
with non-syndromic autosomal recessive hearing loss by whole
exome sequencing
With prevalence figures close to 0.2% at birth, hearing loss (HL) is the most
frequent sensory impairment in childhood. In developed countries, genetic
causes account for more than 60% of congenital HL, most often resulting in
non-syndromic deafness, which is usually autosomal recessive.
Hereditary nonsyndromic hearing loss (NSHL) in Iran is highly heterogeneous, and more than 50% of patients with a presumed genetic etiology lack
a specific molecular diagnosis with STR analysis. Whole-exome sequencing
(WES) has recently opened a new page in Mendelian disease gene discovery
- enabling to study autosomal recessive HL in a new way.
The aim of this study is to find more causative genes and their mutations for
NSARHL in Iranian families by WES. After ruling out any association to prevalent genes for NSARHL in Iran, ten families will be subjected to WES.
Until now, WES has been performed with genomic DNA from affected individuals of two consanguineous families with profound deafness. Analysis of
these data revealed a novel homozygous mutation in MYO7A gene in one
family but co-segregation study failed to confirm this variant as the only
cause of HL in this family. Additional clinical investigation revealed that an
intra-familial phenotypic variation and existence of both syndromic and
non-syndromic HL in this family is possible. Further studies to find the other
variants which may be associated with HL in this family, the data analysis of
the second family and also WES of remaining families are underway.
P02.25-S
Nonsyndromic hearing loss in Moravia: one fifth due to GJB2, no
mutations in SERPINB6, TMIE, COCH, ACTG1, KCNQ4, GJB3
GJB2 gene mutations are the most frequent cause of nonsyndromic hearing
loss. There are many other genes less frequently causing this disorder. The
aim of our study was to look for other genes mutated in Moravian population of patients with deafness. We have performed sequencing of GJB2 coding region on ABI3130 and (GJB6-D13S1830) detection using PCR and
gel electrophoresis in 142 patients with nonsyndromic hearing loss. Biallelic pathogenic GJB2 mutations were found in 31 patients (22%) thus explaining their hearing defect. In 9 patients (6%) only one pathogenic GJB2
mutation was found. No patient carried (GJB6-D13S1830). Sequencing of
SERPINB6, TMIE, COCH, ESPN, ACTG1, KCNQ4 and GJB3 genes was performed
on ABI3130 in 13, 13, 13, 30, 20, 14 and 30 patients without GJB2 mutation,
respectively. No pathogenic mutation was found in SERPINB6, TMIE, COCH,
ACTG1, KCNQ4 and GJB3 genes. In ESPN gene, two variants with unknown
pathogenicity were found in two unrelated patients: c.337C>T, p.Arg113Cys
(Polyphen score 1.00) and c.1797_1808delCCCACCGCCGCC, p.Pro600_
Pro603del. Both variants were inherited from parents without hearing loss.
We cannot exclude big genomic deletion/duplication of ESPN gene on the
other allele in the patients. There may also be bigenic mechanism of hearing
loss pathogenesis. So far, however, we cannot conclude that these variants
are causal in our patients. We are going to analyze WHRN gene encoding the
whirlin protein, functionally associated with ESPN gene product espin.
P02.26-M
Comprehensive genetic diagnosis of non-syndromic hereditary
hearing loss by targeted resequencing of 32 genes with use of
Haloplex design and IonTorrent PGM sequencer
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ABSTRACTS POSTERS
NSHL12 pedigree, WES coupled with homozygosity mapping analysis pointed out a known missense variant (rs74315438) in the CLDN14 (DFNB29
locus) gene, coding for the claudin 14 protein. In conclusion, we provide evidence of the usefulness of WES for the diagnosis of NSHL and increase the
knowledge on the genetic defects underlying this disease. This study was
supported by: Italian Telethon Foundation (grant#GGP11177) and Fondazione Cariplo, grant N2013-0825.
P02.29-S
Phenotypic and genotypic variability of Pendred syndrome
Background: Mutations in the SLC26A4 gene cause both Pendred syndrome [sensorineural hearing loss (SNHL), enlarged vestibular aqueduct (EVA),
and goiter] and autosomal recessive nonsyndromic hearing loss (ARNSHL)
at the DFNB4 locus. Pendred syndrome is the most common type of syndromic HL.
Materials/Patients and Methods: We performed comprehensive clinical
and genetic evaluations of 15 patients from four unrelated families from
the Ardebil Province in western Iran. After testing STR markers to confirm
linkage to the SLC26A4 locus, we sought to identify genetic mutations in SLC26A4 by Sanger sequencing all 21 exons, exon-intron boundaries and the
promoter region of this gene.
Results and Conclusions: A known frameshift mutation (965insA,
p.N322Fs7X) in exon 8 was identified in all four families. Three families
were homozygous for this mutation and one family was compound heterozygote (L597S/965insA). To date, this mutation has been reported only in
the Iranian population, so we investigated the possibility of a founder effect.
Haplotypes were constructed by genotyping seven single nucleotide polymorphisms throughout the SLC26A4 gene, thus identifying a single haplotype that co-segregated with the 965insA mutation. We conclude that this
mutation originates from a common founder in the west province of Iran.
P02.31-S
Identification by whole-exome sequencing of two novel LARS2
mutations in an Italian family with Perrault syndrome
Perrault syndrome (PRLTS) is a rare autosomal recessive disorder characterized by ovarian dysgenesis and premature ovarian failure (POF) in females,
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Plateau iris configuration (PIC) is a unique subtype of angle-closure glaucomas presenting in younger patients. PIC has been recognised as one of the
anatomical mechanisms of angle closure development along with papillary
block. The diagnosis is always based on gonioscopic and ultrasound biomicroscopy (UBM) findings. PIC-associated anterior chamber characteristics
include a normal central anterior chamber depth (ACD), a flat iris plane, an
iris root that angles sharply forward, and anteriorly positioned or large ciliary processes. Peripheral iridotomy is proposed as a treatment. Post-iridotomy complications, such as persistent angle narrowing or occlusion with
intraocular pressure elevation, give rise to the plateau iris syndrome (PIS).
An autosomal dominant pattern of inheritance with incomplete penetrance
was proposed by Etter et al. in 2006 because of a higher prevalence of PIC
in first-degree family members. To our knowledge, no familial case has been
reported to date.
Here we described a multiplex family of seven affected members in two generations. Two members have PIC and five have PIS with early-onset and severe prognosis for some of them. Whole-exome sequencing was performed
in five of the cases. Among the about 50 candidate variants left after filtering
out by segregation, frequency and pathogenicity, the four more relevant
ones -according to their function- were validated by Sanger sequencing in
the two remaining affected members. Further experimental investigations
and/or sequencing of additional families with a similar phenotype are needed to highlight the causative gene.
P02.33-S
Two Tumor Necrosis Factor promoter polymorphisms in Romanian
patients with Primary Open Angle Glaucoma Results from a pilotstudy
R. Simionescu1,2, R. Caisan3, L. Voinea2, A. Popa Cherecheanu2, C. Bara2, R. SfrentCornateanu2;
1
OCULUS, Bucharest, Romania, 2Carol Davila University of Medicine and Pharmacy,
Bucharest, Romania, 3National Hematology Institute prof C.T. Nicolau, Bucharest,
Romania.
ABSTRACTS POSTERS
in Romanian POAG patients. We assessed 258 subjects (112 POAG patients
and 146 healthy unrelated matched controls) for -857C/T (rs1799724) and
-308 G/A (rs1800629) TNF-alpha polymorphisms. These were genotyped
by Real Time PCR (Taqman SNP Genotyping Assays C_2215707_10 and
C_7514879_10 respectively, Applied Biosystems, USA). Statistical analysis
was performed using the SNPStats program for genetic association studies
(http://bioinfo.iconcologia.net/SNPstats); p-values 0.05 were considered
significant.
All studied groups were in HWE for both polymorphisms. The frequencies
of minor alleles -857T and -308A were similar in POAG patients and controls (0.22/0.10 and 0.21/0.13 respectively). There was no significant difference in SNPs regarding the allele carriage or genotype frequency between
POAG and control subjects. Three main haplotypes were constructed with
similar frequencies in patients and controls -857C/-308G/, 857T/-308G
and -857C/-308A: 0.683/0.661; 0.218/ 0.205 and 0.98 / 0.133 respectively. There was no significant association between the global haplotype and
POAG (p-value = 0.5).
Conclusion: TNF-alpha polymorphisms (-857C/T and -308G/A) seem not
to influence susceptibility of POAG. These results should be confirmed on
larger patients cohort.
P02.34-M
Genetic screening for disease-associated mutations in human retinal
diseases using whole exome sequening (WES)
A. Tiwari1, A. Bahr1, S. Feil1, J. Neidhardt1, W. Berger1,2,3;
1
Institute of Medical Molecular Genetics, University of Zrich, Schlieren, Switzerland,
2
Zurich Center for Integrative Human Physiology (ZIHP), Zrich, Switzerland,
3
Neuroscience Center Zrich (ZNZ), University of Zrich and ETH Zrich, Zrich,
Switzerland.
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solved cases was 56.7%. Interestingly, in 54% of the solved cases we were
able to detect mutations which were not previously described in literature
at the date of the medical report. 70% of these cases show exclusively a new
mutation in case of dominant or x-linked inheritance and two new mutations in case of recessive inheritance. 30% show one new and one previously
described mutation.
Therefore, we conclude that NGS is the most promising tool to identify
known as well as unknown mutations for this genetically highly heterogeneous disease entity. New mutations were found in 47 different genes. Genes most frequently affected by mutations were USH2A and EYS followed by
RPGR, PRPF31 and CDHR1.
The high number of novel mutations detected in our cohort shows the advantage of NGS over often used conventional chip-array analysis that assay
only known mutations.
P02.36-M
Splice factor-based gene therapy to correct mislocalization of the
ciliary protein RPGR in retinal degeneration
Retinal degenerations (RD) and other genetic diseases are often caused by
splice site mutations. This type of mutation accounts for approximately 20%
of all pathogenic sequence variants associated with retinal degeneration.
Previously, we reported the identification of an X-linked RD patient that showed skipping of exon 10 in the RPGR mRNA. To treat this mutation-induced
splice defects, we applied mutation-adapted splice factors. In case of the splice factor U1snRNA, the adaptation yielded an increase in its affinity towards
the mutated splice site. This novel gene therapeutic approach was highly
efficient in increasing the amount of correctly spliced RPGR transcripts in
minigene splicing assays and patient-derived fibroblasts.
In this study, we aimed at evaluating the efficacy of the U1-based therapy
on the protein level. We established an assay to locate the RPGR protein in
patient-derived and control skin fibroblasts. In control fibroblasts, we found
that the RPGR protein locates along the primary cilia including basal body,
transition zone and axoneme. In contrast, patient-derived cells showed mislocalization of the RPGR protein restricted to the basal body and transition
zone. Upon treatment with mutation-adapted U1, localization of RPGR along
the axoneme was significantly increased compared to control treatments.
We conclude that the efficacy of the U1-based gene therapy is sufficient to
correct not only misspliced transcripts but also their translated proteins.
Thus, the adaptation of U1snRNA is a promising technique to treat patients
who carry pathogenic splice defects.
P02.37-S
Homozygous deletion of glutamate receptor gene GRID2 causes new
human hotfoot mutant phenotype, characterized by early-onset
cerebellar ataxia and retinal dystrophy
It was our aim to identify the genetic cause of early-onset autosomal recessive cerebellar ataxia (ARCA) associated with retinal dystrophy in a consanguineous family.
Homozygosity mapping and copy number analysis revealed a homozygous
deletion of exon 2 of GRID2, p.(Gly30_Glu81del), in the proband, compatible
with mouse hotfoot mutant ho15J. GRID2 encodes an ionotropic glutamate receptor known to be selectively expressed in cerebellar Purkinje cells.
Here, we demonstrated GRID2 mRNA expression in human adult retina and
retinal pigment epithelium. In addition, Grid2 expression was demonstrated
in different stages of murine retinal development. GRID2 protein expression
was observed in both murine and human retina, more specifically in photoreceptor inner segments, the outer plexiform layer and ganglion cell layer.
In order to rule out involvement of mutations in another gene, whole exome
sequencing was conducted but did not reveal any other disease-causing mutations, supporting the phenotype observed here represents a single clinical
entity.
We identified GRID2 as underlying disease gene of early-onset ARCA with
retinal dystrophy, expanding the clinical spectrum of GRID2 deletion mutants, which thus far only involved cerebellar but no retinal phenotypes.
We demonstrated, for the first time, GRID2 mRNA and protein expression
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ABSTRACTS POSTERS
in human and murine retina, providing evidence for a novel functional role
of GRID2 in the retina. To the best of our knowledge GRID2 is the second
glutamate receptor gene, apart from GRM6, leading to retinal disease when
mutated. Finally, we provided further evidence for evolutionary conservation of a hotfoot fragile site between mouse and human.
P02.38-M
Homozygosity mapping and whole exome sequencing identified four
novel candidate retinal dystrophy genes
M. M. Alrashed1,2;
1
Department of clinical laboratory sciences, College of Applied Medical Sciences, King
Saud University, Riyadh, Saudi Arabia, 2Department of genetics, King Faisal specialist
hospital and research center, Riyadh, Saudi Arabia.
Inherited retinal dystrophies (IRD) are a remarkably genetically and phenotypically heterogeneous group of inherited eye diseases, with over 190 causative genes identified to date. In the highly consanguineous Saudi population,
autosomal recessive forms of IRD are thought to account for the overwhelming majority of cases. Consanguinity is known to increase the frequency
of recessive disorders since it increases the coefficient of inbreeding, which
is a measure of the percentage of the genome that is identical by descent.
Homozygosity mapping, targeted candidate gene analysis and whole exome
sequencing were used to identify the causes of IRD in the Saudi population.
Mutations in RP1 were found to be a common cause of recessive RP in the
Saudi population. Novel and previously identified homozygous mutations
in the KCNV2 gene were identified in a cohort of patients with a distinct
recessive retinal disorder, cone dystrophy with supranormal rod response,
demonstrating phenotype/genotype correlation. In addition, a founder homozygous CABP4 mutation was identified . Causative homozygous mutations were also found in the IRD genes RBP3, RDH12, CRB1, BBS4, CNGA3,
CNGB1, EYS, RLBP1, ABCA4 and PCDH12. Four novel candidate genes for
retinal degeneration were identified in this study. Potentially pathogenic homozygous variants were identified in EMC1 (c.G430A, p.A144T), KIAA1549
(c.2399_2400insAA, p.T800fs809X), GPR125 (c.C2504G, p.S835C) and
DHX29 (c.C2738T, p.A913V). In the majority of cases (31 families) the genetic cause of IRD was identified, demonstrating the power of homozygosity
mapping and whole exome sequencing.
P02.39-S
Next-generation sequencing for retinal dystrophy: two years clinical
experience
T. Gale, S. Barton, S. Ramsden, G. Black, G. Hall;
Manchester Centre for Genomic Medicine, Manchester, United Kingdom.
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Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies ultimately characterized by the loss of photoreceptors leading to
blindness. The molecular diagnosis of RP has long been hampered by the large genetic heterogeneity of this group of disorders. The clinical application
of next generation sequencing (NGS) techniques may overcome, in part, these limitations. In this study, we conducted whole exome sequencing (WES)
to uncover the genetic cause of arRP in a consanguineous Spanish family, in
which genotyping and resequencing microarrays failed to identify the genetic defect. This strategy allowed the detection of one rare homozygous mutation located in exon 1 of C2ORF71 gene (c.1795T>C, p.Cys599Arg). Although
this variant has been previously annotated (rs377190272), it has only been
detected in heterozygosity in one control individual out of 6304 exomes
from the Exome Variant Server database, being probably an asymptomatic
carrier. Moreover, the variant co-segregated with the disease in the extended family and was absent in 400 matched chromosomes. Clinically, patient
with mutation p.Cys599Arg shows signs of typical RP and resembles other
patients with missense mutations in this gene. Taking into account the high
prevalence of carriers of deleterious mutations in RP genes among the control population, and the growing number of experiments in which new variants identified by exome sequencing are reported, identifying real novel
mutations is an increasingly difficult task. All together this data allow us to
conclude that rs377190272 is the most likely causative mutation underlying
the phenotype arRP in this family.
P02.42-M
Panel-based Next Generation Sequencing as an efficient technique to
detect mutations in Italian patients with Retinitis Pigmentosa
M. Zuntini1, C. O. Pierrottet2, F. Sirocco1, A. Nicoletti1, S. El Shamieh3, I. Audo3, N.
Orzalesi2, C. Zeitz3, M. Bertelli1;
1
MAGI Human Genetics Institute, Rovereto (TN), Italy, 2Department of Ophthalmology,
San Paolo Hospital, University of Milan (MI), Milan (MI), Italy, 3INSERM, U968, Paris
F-75012, Paris, France.
ABSTRACTS POSTERS
cing. Pathogenicity of the candidate disease-causing variants in the homozygous regions was assessed by in-silico analysis.
Results: Ophthalmologic examination revealed typical features of RP in both
families. In the first family a homozygous missense variant, c.3269G>A;p.
(Arg1090Gln), was identified in SNRNP200, which previously was implicated in autosomal dominant RP (adRP) and shown to impair splicing. In
the second family a missense mutation, c.995G>A;p.(Gly332Asp), was identified in DHX38, which encodes the pre-mRNA splicing factor PRP16 and
previously has not been associated with arRP. The DHX38 variant shows a
lod score of 3.25, which is highly suggestive of linkage. Segregation analysis
indicated that both variants segregated with RP in respective families, in
an autosomal recessive manner. In-silico analysis supported the causality of
the p.(Arg1090Gln) and p.(Gly332Asp) variants.
Conclusions: Since the SNRNP200 variant p.(Arg1090Gln) appears only to
be causative when present homozygously, we hypothesize that it is a hypomorphic mutation. So far mutations in pre-mRNA splicing factor genes have
only been associated with adRP, thus this is the first report that implicates
defects in the splicing machinery proteins DHX38 and SNRNP200 to be associated with arRP.
P02.44-M
RS1 gene exon 2 deletion in a large Pedigree with X-linked juvenile
retinoschisis
Back to index
P02.45-S
Spectrum of SLC26A4 gene mutations in Slovak NSHL patients
P02.47-S
SOX2 whole gene deletion without ocular malformation but with
isolated bilateral third degree microtia : a new manifestation of SOX2related disorders?
SOX2-related eye disorders are characterized by anophthalmia and/or microphthalmia, which is usually bilateral and severe. Molecular genetic testing of this gene, including sequence analysis and MLPA (for large gene
deletions), identifies mutations in about 10-20% of individuals with such
ocular involvement. Other common findings include brain malformations,
oesophageal atresia and male genital abnormalities. Postnatal growth failure with pituitary insufficiency, seizures, sensorineural hearing loss, delayed
motor development and learning difficulties are described. Recent studies
have demonstrated a broader ocular phenotype in cases with missense
mutations, consistent with a role for SOX2 in both posterior and anterior
segment development. Severe SOX2 (OMIM 184429) mutations (whole gene
deletion/nonsense mutation) with complete loss-of-function alleles, almost
uniformly result in anophthalmia/microphthalmia.
We report the case of a newborn female, evaluated at birth for isolated bilateral third degree microtia. All investigations (cardiac, abdominal, cerebral,
ophthalmologic) were normal.
Array-CGH analysis has shown a de novo 40 kb deletion of 3q26.33
(chr3:181407035-181442290) [hg19] deleting the entire SOX2 gene.
More detailed ophthalmologic evaluations confirmed the absence of any
ocular abnormality. The girl benefits from osseous conduction hearing aids.
Is there a link between SOX2 deletion and microtia, consistent with results
85
ABSTRACTS POSTERS
of a recent study showing under-expression of SOX2 in human auricular
chondrocytes from microtia samples ? Our case also confirms the reduced
penetrance of the ocular phenotype, even in severe SOX2 mutations, and raises the question of a further broadening of SOX2-related disorders.
P02.48-M
Exome sequencing identifies POU3F4 p.Ala116fs mutation among
family with hearing loss
One of the most common genetic diseases in human is hearing loss (HL). The
majority of cases are nonsyndromic (70%) and 1-5% are nonsyndromic Xlinked. Most cases are due to mutations in a single gene. Nevertheless, DNA
diagnostics for hearing loss are challenging, since it is an extremely heterogeneous trait. Although more than 70 causative genes have been described
for the nonsyndromic hearing loss alone, diagnostic application of the scientific progress has lagged behind. Some previous reports have shown that
next-generation DNA sequencing techniques have the potential to offer a
novel testing platform that could test all known genes in a sensitive, specific
and cost-efficient manner. In this study, whole exome sequencing (WES) for
direct genetic diagnosis in NSHL was used. Sequential filtering of variants
obtained from WES, bioinformatic analyses, and Sanger sequencing validation identified premature termination p.Ala116fs mutation in POU3f4 gene
as the candidate disease-causing mutation in the family. POU3F4 belongs to
a subfamily of transcription factors, which are characterized by 2 conserved
deoxyribonucleic acid-binding domains, a 75- amino acid POU- specific domain and a 60-amino acid homeodomain, both helix-turn-helix structural
deoxyribonucleic acid-binding motifs. In this study clinical features and genetic analysis of a male child from a Polish family with congenital deafness
and POU3F4 p.Ala116fs mutation is described. Usually clinical features of
DFNX2 (DFN3) often include a mixed, progressive hearing loss, temporal
bone anomalies, and stapes fixation.
P02.49-S
Common copy number variations on the origin of disease
Introduction: It is well established the relation between CNV and intellectual disability and/or autism, as it is the existence of a large number of CNV
not associated with disease. These polymorphisms although benign, could
represent a risk of disease when occurring in homozigosity, or when combined, depending of its genetic content. Objective: We present a case of a girl
with congenital deafness, and homozygous deletion of OTOA gene, resulting
from the presence of a benign CNV in each chromosome. Method: PAMS, 8
yo was referred for genetic analysis because of congenital deafness. Conexin
26 and conexin 30 sequence analysis was done using Sanger sequencing,
conexin 30 deletions was done using MLPA. Exclusion of additional genetic
mutations was done using a proprietary mutation panel (312 mutations on
32 genes). Array CGH analysis was performed using Affymetrix Cytoscan
750K. Results: Conexin results were negative and CGC Mutation panel and
direct sequencing results demonstrated absence of amplification on OTOA.
Parents testing with array CGH revealed the presence of a CNV (heterozygous deletion) on 16p12.2 in each. Both CNV are registered on DGV as normal variants. However, each CNV encompasses the OTOA gene, thus leading
to the conclusion that the co-existence of two overlapping (individually benign) CNVs was the mechanism leading to congenital deafness on the index
case. Conclusions: This case reveals the utility of array CGH as a complement
technique in the investigation of molecular mechanisms of genetic disease
that allowed establishing the molecular diagnosis and proper genetic counseling to this family.
P03.01-S
In-house-designed synthetic probe set for MLPA identifies a novel
chimeric CYP11B2/CYP11B1 gene in a patient with 11-hydroxylase
deficiency
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mon cause of Congenital Adrenal Hyperplasia (CAH). It is caused by mutations in the CYP11B1 gene localized in 8q21, 40 kb distant from its highly
homologous CYP11B2 gene codifing for aldosterone synthase. Pathological alleles derived from the asymmetric recombination of these two genes
have been described. The formation of a chimeric CYP11B2/CYP11B1 gene
leads to 11-OHD; instead the chimeric CYP11B1/ CYP11B2 gene causes the
glucocorticoid-remediable aldosteronism (GRA). As the detection of these
rearrangement is still laborious or indirect, our objective was to project a
synthetic probe set for multiplex ligation-dependent probe amplification
(MLPA) analysis in order to simplify the detection of these chimeric genes
and other variation in the copy number of these genes. We disegned a set of
8 specific probes for both CYP11B1 and CYP11B2 genes to be used with the
commercial control kit SALSA MLPA P300 Human DNA reference (MRC-Holland). The methods was tested on 15 control samples and then applied to 6
patients with the suspition of 11-OHD. The analysis with the CYP11 probe
set has led to the identification of one copy number variation in an adult patient with adrenal rests misdiagnosed as 21-hydroxylase deficiency and not
properly treated. He resulted compound heterozygous for a novel chimeric
CYP11B2/CYP11B1 gene, with the breakpoint region locolized within intron
2, and the known A306V mutation. In conclusion, the described MLPA kit represents an optimal complement to DNA sequence analysis in patients with
11-OHD, enabling detection of deletions, duplications or chimeric genes.
P03.02-M
A Novel mutation of FGD1 in four members of a Turkish Family with
Aarskog-Scott Sydrome and growth hormone Therapy
ADPKD is the most common genetic nephron-pathology in humans, affecting about 1/1000 individuals. Aim of the present work was to define
genetic variation of PKD1 and PKD2 in Italian patients affected by ADPKD.
Analysis of PKD1 and PKD2 variation would allow to: confirm the diagnosis in clinically uncertain/atypical cases; offer genetic counseling in at risk
families; exclude the presence of a mutation in related donors for kidney
transplantation; define gene variability in Italian patients. 576 subjects has
been analyzed with a semi-automated Sanger protocol: 298 unrelated patients belonging to families and 171 relatives; 72 patients with no familiarity
and 35 cases with no data about familiarity. In 90% of the 405 probands,
variants were present: PKD1 80%; PKD2 4%; PKD1+PKD2 6%. 84.5% of the
identified variants have never been described. An average of 12 SNPs/patient in PKD1 and 2 SNPs/patient in PKD2,was observed. By combining results for truncating and known variants we classified variants as pathogenic
in 65.4% (265/405) of patients. For unclassified variants, a prediction was
attempted according to PKDB criteria. Concordance of the results obtained
with SIFT, AGVGD and PolyPhen2 allowed calling of 18 likely-neutral and 12
highly-likely-pathogenic variants. In many cases interpretation of additional
information becomes relevant (i.e. family segregation increases the score for
pathogenicity). In patients with no mutations detected, MLPA analysis has
been performed: we identified 2 patients with a huge deletion in PKD1 (ex2-
ABSTRACTS POSTERS
46); 1 patient with a deletion spanning exons 2-4 in PKD1; 1 patient with the
deletion of the whole PKD2 gene.
P03.04-M
Scattered deletion of Pkd1 in mouse kidneys causes a cystic snowball
effect and recapitulates human polycystic kidney disease
Background: Autosomal Dominant Polycystic Kidney Disease (ADPKD) patients carry a germline mutation in PKD1 or PKD2, leading to thousands of
kidney cysts. It is poorly understood why a rapid progression of the disease
is preceded by a lag-phase of several decades. Studies in which Pkd1 is inactivated in large percentages of cells in animal models, led to the presumption that inactivation of the remaining allele initiates cystogenesis, and that
the progression can be accelerated by renal injury. To mimic human ADPKD,
we lowered this percentage and found important characteristics of cystogenesis that have not been described before. Methods: The percentage of
Pkd1-deficient cells in Tamoxifen-inducible kidney-specific Pkd1-deletion
mice was controlled by varying the tamoxifen dose, visualized with reporter mice and quantified by eMLPA. Several renal injuries were applied. Cyst
progression was followed by MRI-analysis and PKD-related signaling was
analyzed by Immuno-histochemistry. Results: Interestingly, no pathological changes occurred for six months after scattered-Pkd1-deletion and renal injury did not trigger rapid PKD. However, in the following 3-4 months,
clustered cyst formation led to a severe human-like PKD phenotype. This
shift was preceded by increased pSTAT3, pCREB, pERK1/2, LCN2 and Ki67 expression near the initial cysts. Conclusions: Our data argue against
the presumption that renal injury is the major trigger for cystogenesis but
suggests that initial cysts themselves, by imposing persistent stress on surrounding tissue, triggers a snow-ball effect driving the formation of new
cysts. In addition, our model mimics human ADPKD more precisely and can
be used for pre-clinical testing.
P03.05-S
Identification and characterisation of six novel SERPINA1 null
mutations
Back to index
F. Khadangi, M. A. Kerachian;
Medical Genetics Research Center, Department of Medical Genetics, Mashhad University
of Medical Scie, Mashhad, Islamic Republic of Iran.
Background Polycystic kidney disease (PKD) can be inherited as an autosomal dominant (ADPKD) or an autosomal recessive trait (ARPKD). ADPKD
is one of the most common genetic diseases in humans affecting all ethnic
groups worldwide with an incidence of 1 in 500 to 1 in 1,000. ADPKD is genetically heterogeneous and can arise from mutations in two genes, named
PKD1 and PKD2. Mutations of PKD1 located on chromosome 16p13.3 are
responsible for 85% of cases. Aim Although there is no hotspots reported
in PKD1, most mutations are seen in the downstream exons of this gene. So,
we developed a clinical assay for PKD1 gene analysis using sequencing of 16
exons from 31 to 46 in 22 patients. Material and Method This study was
carried out with a total of 22 patients. Genomic DNA was extracted from
blood lymphocytes (5ml of whole blood) with standard methods and fragments were amplify using PCR. Screening of PKD1 mutations was performed
by direct Sequencing. Result Sequencing result of exons 44 and 45 showed
one completely pathogenic mutation causing Gln4005Arg change. Two novel non-synonymous variation including Arg4091Glu and Val4035Ilu and
five likely neutral SNP including rs10960, rs3087632, rs3087632, rs10960,
rs3087632, rs3087632 are detected. There was no any variation in exons
35, 36 and 37. Conclusion It is expected that confirmation of pathogenic
mutations can be useful for studies on disease molecular pathways. Early
and prenatal diagnosis and personalized treatments recommended for family members after genetic consoling.
P03.08-M
Molecular diagnostics of autosomal recessive polycystic kidney
disease by next-generation sequencing
87
ABSTRACTS POSTERS
P03.09-S
Dysregulation of cytoskeleton organization, carcinogenesis and
immune response pathways involved in Balkan endemic nephropathy
development
88
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P03.11-S
Whole exome sequencing reveals rare homozygous ARID1B and
heterozygous MTOR missense variants in a patient with bilateral
cystic-dysplastic kidneys and features of Coffin-Siris syndrome and
tuberous sclerosis
A. Kosfeld, M. Kreuzer, C. Scholz, V. Riehmer, D. Horstmann, L. Pape, D. Haffner, R. G.
Weber;
Hannover Medical School, Hannover, Germany.
ABSTRACTS POSTERS
incidence among the various groups. The CYP21A2 gene, is localized in a
genetic unit defined RCCX module and is considered one of the most polymorphic of human genes.
We considered new evidences about the presence of a RCCX trimodular haplotype with a CYP21A2-like gene to explain the lack of a genotype-phenotype correlation in two patients referred to our centre for a suspected Non
Classical form of CAH (NC21OH).
To identify the presence of deletions/duplications we used Multiplex Ligation Probe-Dependent Amplifications (MLPA) and to confirm the presence
of a CYP21A2-like gene downstream TNXA gene we used previously described amplification and restriction strategy followed by the sequencing of the
CYP21A2 gene downstream TNXB gene.
We validated the methods developed by recent studies and we found a good
concordance with their results. The amplification strategy, direct sequencing and restriction analysis of CYP21A1P/CYP21A2-TNXA PCR product in
association with MLPA assay and sequencing of CYP21A2 gene downstream
TNXB gene, were able to identify the presence of more than one copy of
CYP21A2 gene.
The strategy suggested is useful to screen cases where there is no genotypephenotype correlation. We validated the modified screening strategy to facilitate molecular testing of CAH patients, considering the new evidence about
possible different haplotypes.
P03.14-M
Familial mediterranean fever (FMF): genetics and role of S100A4
Back to index
the FSGS-associated PAX2 mutations perturb protein function by either affecting proper binding to DNA and transactivation activity, or altering the
interaction of the transcription factor with repressor proteins, resulting in
enhanced repressor activity. Thus mutations in PAX2 contribute to adultonset FSGS in the absence of overt extrarenal manifestations. We expand
the phenotypic spectrum associated with PAX2 mutations, which have been
shown to lead to CAKUT as part of papillorenal syndrome. PAX2 mutations
can cause disease through haploinsufficiency and dominant negative effects,
which could have implications for tailoring individualized drug therapy in
the future.
P03.16-M
Genetic and bioinformatics analysis of four novel GCK missense
variants detected in Caucasian families with GCK-MODY phenotype
Background: Isolated growth hormone deficiency (IGHD) and combined pituitary hormone deficiency (CPHD) result in significant short stature caused
by mutations in GH1, GHRHR PROP1, POUIF1 and HESX1 genes. The present
study describes the mutation profile of Growth Hormone Deficiency (GHD)
patients from India. One hundred each of patients (60 IGHD, 40 CPHD) and
controls were recruited for screening of GH1, GHRHR, PROP1 and POU1F1.
Results: Consanguinity, family history and mutations were present in 20%,
8% and 35% of the cases respectively. Five novel and four reported mutations were identified (Table-1). GHRHR mutations were the most common
change in IGHD followed by GH1 cluster deletion. A novel GH1 coding region
deletion and three promoter SNPs were identified. These SNPs are reported to induce differential transcriptional activity resulting in reduced secretion of growth hormone. Novel insertion and nonsense PROP1 mutations
identified lead to frameshift and a truncated protein leading to GHD. Novel
POU1F1 splice site mutation in three patients from the same geographical
region implies possible presence of founder effect.
Conclusion: This is the first report demonstrating genetic contribution to
GHD in our population wherein Glu72Term, the most common change and
five novel mutations were identified. Hotspot mutations reported worldwide were absent suggesting a distinct profile. Results of the study will facilitate timely diagnosis, counseling and intervention thereby reducing the
stigma associated with GHD.
89
ABSTRACTS POSTERS
Table-1 Mutations/SNPs identified in the present study
Type of
Amino acid
Nature of
S.No GHD
Gene
cDNA position
Frequency
position mutation/SNP
(N=100)
GH1 gene
1
Gross deletion
Reported
15%
cluster
Deletion of
2
GH1
Novel
2%
exons 3-5
GH1
3
rs2005171
Reported
20%
promoter
GH1
4
rs2005172
Reported
15%
IGHD promoter
GH1
(n=60)
5
rs11568828
Reported
18%
promoter
Reported
6
GHRHR
c.214G>T
Glu72Term
10%
(Nonsense)
Reported
7
GHRHR
c.527C>T
Ala176Val
3%
(Missense)
Novel
8
GHRHR
c.920insC
2%
(Insertion )
Reported
9
PROP1 c.112_124del13
2.5%
(deletion)
Novel
10
PROP1
c.45C>T
Arg16Term
2.5%
(Nonsense)
CPHD
Novel
11
PROP1
c.128InsA
2.5%
(n=40)
(Insertion)
Novel
12
POU1F1
c.605-1G->A
(Acceptor splice
7.5%
site)
P03.18-M
Exonic de novo Mutations in Sporadic Hirschsprung Disease
HSCR patients, gene burden tests and functional analysis in both cell lines
and zebra fish are currently being conducted. Interestingly, some of the genes harboring de novo mutations are members of pathways involved in the
development of the ENS and the encoded proteins interact with known key
signaling molecules. We will present all data which will enable us to make
conclusion on whether, de novo mutations in genes other that RET also contribute to the development of sporadic HSCR.
P03.19-S
RET and EDNRB mutations screening in Indonesian patients with
HSCR, functional study and its implication in diagnostic
90
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to determine causality. We screened a total of 61 HSCR children and identified 8 rare RET coding variants (R79W, R144H, P270L, R694Q, A756V,
G533S, Y1062C,D489N) and one possible splice-site variant. No rare variant
in EDNRB were identified. The mutation frequency (15%) is comparable to
previous studies. Four missense variants and one possible spice site variant
have never been reported before. Four of the nine arose de novo, while the
remaining variants are all inherited from an unaffected parent. One patient
harbor two RET coding variants, one coming from each unaffected parent.
Functional studies showed that 7 out of 8 coding RET variants resulted in
significant lower expression of the phosphorylated-RET protein. Only 4 RET
variants (R144H, R694Q, A756V, Y1062C) resulted in significantly lower
expression of phosphorylated-ERK, a downstream component of the RET
pathway, when compare to wild type RET. The possible splice-site variant
(IVS 1880-4A>G) did not disturb the splicing process and, therefore, is not
considered pathogenic. Our data suggest that 7 of the 9 identified RET variants are pathogenic and that not all rare RET variants are pathogenic, hence
functional studies are essential to prove pathogenicity.
P03.20-M
Translating genetic findings in functional defects: functionomics of
hypomagnesemia-causing genes
J. H. de Baaij, F. Arjona Madueo, E. Bongers, R. Bindels, J. Hoenderop;
Radboud University Medical Centre, Nijmegen, Netherlands.
By definition, complex diseases are caused by many different genes, but this
might hold true only at the population level. We hypothesize that within families with a high disease burden, complex diseases can arise from only a
few high-effect risk variants. Assuming that all or most affected family members share the genomic region(s) that harbor the risk variant(s), whole-genome sequencing is the most precise method for determination of identicalby-descent segments to narrow down the search space for disease genes.
Family genomics is especially powerful in multi-generation pedigrees. In
some of the pedigrees we analyze diseased individuals are separated by
more than 10 meioses each of which narrows the number of candidate alleles by approximately half, per Mendels law of segregation. Family genomics
furthermore permits identification of sequencing errors and detection of
rare variants with high confidence.
In addition to our identical-by-descent detection tools we have created an
analysis workflow to identify and score variants according to their inheritance pattern, population frequency and predicted function. We are currently analyzing over 200 families in over 30 studies that cover a wide range of
diseases, from rare congenital diseases to common neurodegenerative and
chronic inflammatory diseases. We present here our methods for identifying
high confidence candidate variants for inflammatory bowel disease in families with high disease burden.
ABSTRACTS POSTERS
P03.22-M
A hemizygous mutation in retinitis pigmentosa GTPase regulator
(RPGR) is a potential novel cause of congenital renal tract
malformations
Back to index
For decades, clinically ill-defined autosomal-dominant renal diseases originating from tubular cells and leading to tubular atrophy and interstitial
fibrosis were reported. Patients exhibit mutations in at least 4 genes: UMOD,
HNF1B, REN, MUC1, but are clinically indistinguishable all associated with
renal fibrosis and renal failure the 3rd and 6th decade of life. In contrast to
what the frequently used term Medullary Cystic Kidney Disease (MCKD)
implies, development of medullary cysts is neither an early, nor a typical
feature, as analyzed by MRI.
We now investigated 10 such families. In two large families we performed
genome-wide linkage and haplotype analyses confirming linkage to a known
3.4 Mb locus MCKD1 locus on chromosome 1q21. Targeted genomic sequencing of the complete linkage locus in affected and healthy individuals of these two families and affected individuals of further families failed to uncover
any segregating variant in any of the genes, including MUC1. The VNTR region in the coding sequence of the MUC1 was masked in these analyses due to
its high GC-content and repetitive nature. After the recent publication of one
MUC1-VNTR insertion mutation we established SNaPshot-minisequencing
confirming this MUC1 mutation in 4 families. Further 3 families carried an
UMOD mutation, leaving 3 families unsolved to date. On the basis of clinical
and pathological characteristics we propose the term Autosomal Dominant
Tubulointerstitial Kidney Disease (ADTKD) as a new name for this entity.
This new terminology should enhance recognition and correct diagnosis of
affected individuals, facilitating genetic counseling and stimulating research
into the underlying pathomechanisms.
P03.26-M
The promise and challenge of high throughput sequencing to discover
genes involved in Medullary Sponge Kidney disease
V. Palazzo1, A. Provenzano1, A. La Barbera1, E. Andreucci2, B. Mazzinghi2, M. Pantaleo2,
M. Caruso3, L. Garavelli4, G. Gambaro5, M. Materassi6, P. Romagnani6,7, S. Giglio1,2;
1
Department Of Biomedical Experimental And Clinical Sciences Medical Gentic Unit,
University Of Florence, Firenze, Italy, 2Medical Genetic Unit Meyer Childrens University
Hospital, Firenze, Italy, 3Nephrology Unit, Ospedali Riuniti di Bergamo, Bergamo, Italy,
4
Clinical Genetics Unit, Obstetrics and Pediatric Department,Arcispedale Santa Maria
Nuova Hospital, Reggio Emilia, Italy, 5Renal Unit, Department of Medicine, University-
91
ABSTRACTS POSTERS
Hospital of Verona, Verona, Italy, 6Pediatric Nephrology Unit Meyer Childrens University
Hospital, Firenze, Italy, 7Department Of Biomedical Experimental And Clinical Sciences
University Of Florence, Firenze, Italy.
Maturity-onset diabetes of the young (MODY) is a phenotypically and genetically heterogeneous group of diabetes caused by single defects in at least
thirteen genes, characterised by autosomal dominant inheritance, a young
age of onset and pancreatic -cell dysfunction. Genetic testing for MODY has
become a routine procedure allowing to set up proper treatment and discriminate from type 2 diabetes (T2D) whose symptoms are often overlapping.
We analysed, in the last 5 years, more than 300 Italian families with MODY/
T2D diagnosis by direct sequencing of genes GCK, HNF1, HNF4, IPF1 and
HNF1, and only about 40% of the cases resulted positive. Mutations in GCK
gene account for up to 80% for all Italian MODY cases according to literature
data.
We then reanalysed the negative subjects through Next Generation Sequencing technology. We excluded common, non-coding and synonymous gene
variants, and performed in-depth analysis on filtered sequence variants in
a pre-defined set of 102 genes implicated in glucose metabolism. We found,
in association with known heterozygous SNPs associated with diabetes, rare
and pathogenetic variants, demonstrating that this approach leads to a genetic diagnosis in most of patients. For example, we identified new variants
in the RFX6 gene and in two of these cases we also detected rare variants
in WFS1 and ABCC8. All patients showed a good therapeutic response to
dipeptidyl peptidase-4 (DPP4) inhibitors.
This approach may help in understanding the molecular aetiology of diabetes and in providing a more personalised treatment for each genetic subtype.
P03.28-M
A molecular genetic analysis of nephrotic syndrome in a cohort of
Tunisian families
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nes are among the main causes of early-onset and familial steroid resistant
nephrotic syndrome. This study was carried out to assess the frequencies of
mutations in these two genes in a cohort of Tunisian pediatric NS patients.
Methods:Mutation analysis was carried out by direct sequencing of the
NPHS1 and NPHS2 genes in 18 nephrotic syndrome (NS) families. This cohort included 6 families of congenital NS, and 12 infantile onset steroid resistant NS or familial cases.
Results: We detected likely causative mutations in 5 out of 6 families of congenital NS studied. A total of 5 different mutations were found in the NPHS1
gene. 3 homozygous mutations were found in the NPHS2 gene in 3 out of 12
infantile onset NS or familial cases.
Conclusions: Our results show a high prevalence of disease causing mutations in the NPHS1 (80% congenital, 30% overall) and NPHS2 (25% early
onset and 16% overall) genes in the Tunisian NS children as compared to
the European or Asian populations
P03.29-S
Gene Expressions In Glomerulopathies
ABSTRACTS POSTERS
P03.31-S
Epistasic Association of CTRC and SPINK1 Gene Variants Combination
with Recurrent Pancreatitis in Lipoprotein Lipase Deficiency
K. Tremblay, C. Dubois-Bouchard, D. Brisson, D. Gaudet;
Universit de Montral and ECOGENE-21 Research Center, Chicoutimi, QC, Canada.
Background: Lipoprotein Lipase deficiency (LPLD) is a rare autosomal recessive disease associated with severe hypertriglyceridemia and increased
risk of pancreatitis or other co-morbidities. There are important unexplained inter-individual variations in the incidence and severity of acute pancreatitis episodes in LPLD patients. Several genes involved in proteolytic
networks are associated with pancreas and lipoprotein functions and could
influence pancreatitis risk in LPLD. Objective: To evaluate the association
between two genes regulating serine proteases, chymotrypsin C (CTRC) and
serine peptidase inhibitor kazal type1 (SPINK1), and recurrence of hospitalizations for acute pancreatitis among LPLD subjects. Methods: CTRC and
SPINK1 have been sequenced in a sample of 38 LPLD patients and 100 controls. In LPLD, 18 (47%) presented a history of recurrent (5) hospitalizations for acute and severe abdominal pain, whereas 8 (21%) had not yet been
hospitalized for this condition. Comparisons between studied groups were
done with chi-square tests and multinominal regression analyses. Results:
Gene sequencing identified 15 SNPs. Genotype-stratified analyses in LPLD
subjects and controls suggested an epistasic association between rs545634
(CTRC) and rs11319 (SPINK1) SNPs combination and recurrence of hospitalizations (p<0.001) in LPLD. Regression analyses, controlling for age, gender
and smoking status, suggest that the risk of frequent and recurrent hospitalizations for acute pancreatitis is significantly increased in LPLD in presence
of this CTRC-SPINK1 SNPs combination (OR = 41.4 [CI: 2.0-848.0]; p =0.016).
Conclusion: These results suggest that modifier genes involved in protease
pathways could influence the trajectory of pancreatitis risk in LPLD.
P03.32-M
Towards introduction of exome sequencing in pediatric liver
transplant program
Study aim
Characterization of the genotype-phenotype correlation in patients with
progressive familial intrahepatic cholestasis (PFIC) and identification of
prognostic genetic parameters for long term outcome and success of liver
transplantation in order to improve individual treatment options.
Method
Genetic testing was performed for 57 PFIC index patients, including NGS panel diagnostics for 5 children with infantile cholestasis and atypical liver histopathology. Long term data (2-35 years) of 8 patients with genetically confirmed PFIC will be presented to illustrate their specific medical problems.
Back to index
Results
Most pathogenic mutations were identified in ABCB11 (56%), followed by
ATP8B1 (32%) and ABCB4 (12%); including 73% missense mutations, 17%
truncating mutations, 7% splice mutations and 3% small deletions.
Response to pharmacological therapy was insufficient in 87% of patients,
thus, biliary diversion and/or liver transplantation was performed in 75%.
One additional patient is currently listed for transplantation.
Perioperative complications observed in patients with missense mutations were mild and included bleeding and coagulation problems (n=2). Life
threatening complications (organ rejection requiring retransplantation,
death) occurred in two patients with at least one truncating ABCB11 mutation. Severe long term complications were observed in 3 ATP8B1 mutations
carriers including severe diarrhea, renal failure, deafness and polyneuropathy.
Conclusion
Truncating mutations may be associated with a higher rate of severe perioperative complications in liver transplantation, possibly due to a stronger
immune reaction towards the wild type protein. With an ongoing study we
are currently evaluating this hypothesis in a larger cohort including application of NGS panel sequencing to search for additional causative genes.
P03.34-M
Genetic analysis of PKD1 and PKD2 genes in Korean patients with
autosomal dominant polycystic kidney disease
Primary hyperoxaluria (PH) is a rare autosomal recessive disease, commonly arising in childhood with nephrolithiasis, nephrocalcinosis, chronic
renal failure. Mutations in AGXT, GRHPR and HOGA1 genes are responsible
of type 1, 2 and 3 respectively. Our laboratory, member of the European PH
consortium (OxalEurope), is the only Italian center offering these genetic
analyses.
Currently, 81 AGXT-PH1 and 1 GRHPR-PH2 patient are known in Italy.
In this study the entire coding sequence of GRHPR and HOGA1 genes and the
AGXT promoter was sequenced in 15 patients with high clinical suspicion of
PH, negative for AGXT mutations. No point mutations were detected.
One patient resulted homozygous for the c.341-81delT HOGA1 variant in
intron 2. This variant is not reported in literature and was evaluated as pathogenic by in silico prediction, generating a new acceptor splicing site (AG
in c.341-79). The minigene in-vitro assay demonstrated that this variant did
not interfere with splicing.
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ABSTRACTS POSTERS
Two patients were heterozygous for two different AGXT-promoter variants
(c.-647C>T, c.-424C>T), not reported in the scarce literature nor in 1000Genomes Database. These variants have been evaluated as not pathogenic because they do not lie in any known regulatory-transcription site.
The negative results in those patients with high clinical suspicion of PH
could be explained by undetected deletions (investigable by MLPA analysis
of the two major genes) or mutations in other genes involved in oxalate metabolism, or differential diagnosis.
P03.36-M
Screening of a large cohort of Italian patients with Albright hereditary
osteodystrophy and/or Pseudohypoparathyroidism phenotype for
subtelomeric deletions of chromosome 2
F. M. Elli1, P. Bordogna1, L. de Sanctis2, V. Boldrin1, A. Spada1, G. Mantovani1;
1
Universit degli Studi di Milano - IRCCS C Granda H Maggiore Policlinico, Milano, Italy,
2
Universit di Torino - Ospedale Infantile Regina Margherita, Torino, Italy.
Pseudo-Pendred syndrome (PPDS) is defined by the association of sensorineural deafness, hypothyroidism due to iodide organification defect, absence of inner ear malformation and absence of mutation in SLC26A4, the
gene responsible for classical PDS.
In order to determine the cause of PPDS, we performed whole exome sequencing (WES) in a family with two children affected with hypothyroidism,
developmental delay, positive perchlorate test and absence of inner ear malformation on CT-scan. Parents were healthy and non-consanguineous and
direct sequencing of SLC26A4 was normal in both patients.
WES was performed in both patients and their father. Variants were ranked
by segregation, allele frequency, protein change and degree of conservation
to assess likelihood of causation. Both patients were found to be compound
heterozygous for missense mutations (Y453D and W233C) in TPO and the
father was heterozygous for the Y453D mutation.
In silico prediction tools determined a deleterious mutational impact of
both variants on protein function. Sanger sequencing confirmed the mutation segregation and found that the mother was heterozygous W233C. The
latter mutation was novel and both were absent in the control population.
TPO encodes thyroperoxidase which catalyzes key reactions in thyroid
hormone synthesis and mutations in TPO are responsible for thyroid dyshormonogenesis 2A. Mutations in TPO have been reported in 4 patients
with hypothyroidism and deafness in a series of Israeli patients with iodide
organification defect (Tenenbaum-Rakover, 2007) but their phenotype was
not described as PPDS.
These cases together with the present report suggest that mutations in TPO
can be responsible for pseudo-PDS.
94
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P03.38-M
Renal function decay after nephrectomy: role of surgery and impact of
hypertension gene polymorphisms
L. Zagato1, S. Delli Carpini1, M. T. Sciarrone Alibrandi1, F. Trevisani2, P. Manunta2,1;
1
Ospedale San Raffaele, Milano, Italy, 2Universit Vita-Salute San Raffaele, Milano, Italy.
Renal hypouricemia is a rare heterogeneous inherited disorder characterized by impaired tubular uric acid transport with severe complications, such
as acute kidney injury. So far, more than 100 patients with a loss-of-function
mutation in the SLC22A12 gene (URAT1, OMIM #220150) and more than
ten patients with defects in the SLC2A9 gene (GLUT9, OMIM #612076) have
been described. The serum uric acid concentration in the proband was
1.1 mg/dL and expressed as an increase in the fractional excretion of uric
acid 43%. The SLC22A12 gene analysis for the patient revealed compound
heterozygous variants of p.G366R and p.R477H. Functional and immunocytochemical analysis of URAT1 mutants was performed in Xenopus laevis
oocytes. The urate uptake ability decreased to similar levels seen in mock
samples in p.G366R mutant expressed oocytes. The p.R477H variant showed almost the same activity as the URAT1 wild type. In the co-expression
samples, both variants p.WT/G366R and p.G366R/R477H lost their urate
uptake activities. Variants p.WT/R477H tended to decrease urate transport
compared to WT single expression; however, it was superior to the other two
co-expression patterns (significant to WT/G366R, P<0.05). Co-localization
studies showed an accumulation of URAT1 in the endoplasmic reticulum of
the p.G366R variant and mainly retention of wild type protein by variants
p.G366R and p.R477H. The findings suggest that not only a loss-of-function
mutation of URAT1 but also the dominant-negative effect cause renal hypouricemia via loss of uric acid absorption, partly due to protein misfolding
caused by accumulation of URAT1 protein in the endoplasmic reticulum.
Support: LH13245 and PRVOUK-P25LF1/2.
P03.40-M
Clinical reappraisal of SHORT syndrome at the light of the PIK3R1
gene discovery
ABSTRACTS POSTERS
Dficiences intellectuelles de causes rares, La Piti Salptrire, Paris, France, 9Service
de Gntique clinique, CHU dAmiens, Amiens, France, 10Service de Pdiatrie, CHU
Jean Verdier, Bondy, France, 11Service dEndocrinologie, CHU Bocage, DIJON, France,
12
Service de Gntique clinique, CHU Rennes, Rennes, France, 13UMR CNRS 6290 IGDR,
Universit Rennes 1, Rennes, France, 14Centre de Gntique et Centre de Rfrence
Anomalies du Dveloppement et Syndromes Malformatifs de lEst, FHU-TRANSLAD,
CHU Dijon, Dijon, France, 15Laboratoire de Cytogntique, CHU Dijon, Dijon, France,
16
Dpartement dEndocrinologie, Diabtologie et Nutrition, Hospices Civils de Lyon,
Centre Hospitalier Lyon-Sud, Pierre-Bnite, France, 17CENS_Centre Europen Nutrition
et Sant, Centre de Recherche en Nutrition Humaine Rhne Alpes, Unit INSERM 1060,
laboratoire CARMEN, Universit Claude Bernard Lyon, Pierre-Bnite, France, 18Service
endocrinologie, CHU Cte-de-Nacre, Caen, France, 19Dpartement dEndocrinologie,
Hpital Haut Lvque, CHU de Bordeaux, Pessac, France, 20Service de Pdiatrie 1, CHU
Dijon, Dijon, France.
SHORT syndrome is defined by its acronym: short stature (S), hyperextensibility of joints and/or inguinal hernia (H), ocular depression (O), Rieger abnormality (R) and teething delay (T). We and others recently identified the
PIK3R1 gene, encoding for the regulatory subunits of phosphatidylinositol3-kinase (PI3K) and playing a key role in insulin signaling, as responsible of
SHORT syndrome. In total, 22 cases with a PIK3R1 and a variable phenotype
have been reported. Only 15/22 patients presented at least 3 of 5 signs of the
SHORT acronym. Indeed, the Rieger abnormality was found in less than half
patients (8/21), but other ophthalmological manifestations such as hypermetropia can be present. Hyperextensibility of joints and/or inguinal hernia
(3/18) were also rarely reported. At contrary, some features not part of the
acronym are found at a high frequency: facial dysmorphism was typical with
enophtalmia (20/20), lipodystrophy (19/19), insulin resistance (11/12), as
well as diabetes mellitus in adolescence or adulthood (8/11). In conclusion,
clinical reappraisal of SHORT syndrome at the light of the PIK3R1 gene discovery permitted to revise the diagnostic criteria for SHORT syndrome. The
presence of lipodystrophy and insulin resistance permit to classify SHORT
syndrome among the rare syndromic forms of insulin resistance.
P03.41-S
Multiple SNP score associated to chronic renal disease risk predicts
the eGFR of patients with newly diagnosed type 2 diabetes
Genome-wide association studies (GWAS) have identified several loci associated with risk of cardiovascular disease (CVD), nephropathy and reduction
of glomerular filtration rate (eGFR). Moreover, albuminuria and eGFR reduction are independent predictors of CVD.
The aim of this study is to explore the relationship between SNPs and CVD
and impaired renal function phenotype in Verona Newly Diagnosed type 2
Diabetes Study (VNDS) patients.
We studied 45 CVD and 44 eGFR and/or CKD predictors SNPs in 529 GADAb negative subjects, (meanSEM age: 58.80.41 years; BMI: 30.00.22 kg/
m2; FPG: 7.170.08 mmol/L; HbA1c: 6.860.05%). The association analysis
was performed on 5 phenotypes: 3 CVD phenotypes (carotid arteries ultrasound, lower-limb arterial ultrasound, ECG) and 2 kidney disease phenotypes (eGFR, microalbuminuria). For both genetic risks was generated a genetic load score (GLS), by summing up the number of risk alleles carried by
each patient. Both GLS were considered in the analysis as natural values and
as tertiles. Cardiovascular GLS was not associated with any of the cardiovascular phenotypes (p = 0.30-0.70). Nephropathic GLS, was not associated
with albuminuria, while was significantly associated with decreased eGFR,
with the score expressed both as natural value (p <0.01) and as tertiles (respectively: 84.31.3, 81.71.5 and 78.91.5 ml/min/1.73 m2 p<0.02), even
after age, sex, BMI, antihypertensive therapy and HbA1c adjustment.
Conclusions: In patients with type 2 diabetes at diagnosis, genotype predicts
the residual glomerular function, suggesting that nephropathic genetic risk
clock starts ticking long before hyperglycemia.
P03.42-M
A genome-wide search for factors contributing to thyroid
hemiagenesis
Thyroid dysgenesis is the major cause of congenital hypothyroidism in humans. Hemiagenesis of the thyroid (TH) is a rare congenital malformation
Back to index
Trichorhinophalangeal syndrome is an autosomal dominant condition characterized by craniofacial and skeletal anomalies and three different clinical
subtypes: type I, carrying mutations in TRPS1 gene, type II, a 8q23 microdeletion syndrome with additional features such as mental retardation and
exostoses, type III, with mutations in TRPSI gene and severe short stature,
short metacarpals and normal intelligence. We report on clinical evaluation
in a 10 year old girl presenting with disharmonious short stature, facial dysmorphisms (sparse hair, bulbous tip of the nose, long upper lip), severe metacarpophalangeal shortening and normal intelligence. Hormonal and meta-
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ABSTRACTS POSTERS
bolic screening evaluation (including mucopolysaccaridosis disease) did not
reveal pathologic patterns. Since few months she suffered from pelvic pain:
an X-ray of the hip revealed left coxa vara and a round area of osteoporosis in
the femural external side ,sclerosis of femural nucleus and of the overlying
acetabular roof. Spine X-ray brought out convex scoliosis and marked left
iliac crest dysmetria, with an overcoming external subluxation of the right
hip. The CT scan confirmed these data and an orthopedic follow up started.
The peculiar face with short stature, brachidactyly, scoliosis, hip dislocation
led us to consider in differential diagnosis Trichorhinophalangeal syndrome
type 3: molecular analysis of TRPS 1 gene proved a new missense mutation (exon 7 H1233P), that was also carried by her mother, who presented a
minimum clinical phenotype (face anomalies, short metacarpals,not severe
short stature). Our study could be a further clinical contribute to the delineation of the Thricorhinophalangeal subtype III syndrome.
P03.45-S
Renal and urinary system malformations in girls with Turner
syndrome
S. Turyk, M. Sakurai, M. Ciaccio, S. Bonsergent;
Hospital Britanico de Buenos Aires, Buenos Aires, Argentina.
Turner Syndrome (TS), in which there is a loss of all or part of one X chromosome, occurs in 1 in 2500 born femals. Renal and urinary system malformations with their posterior complications such as urinary tract infections
or proteinuria have been recognized to increase in patients with TS. In this
retrospective study we report a detailed clinical history and analyzed renal
and urinary system pathology in 32 girls with TS observed between 20002010. All 32 TS patients were evaluated by renal and collecting system utrasonography and if structural renal or urinary malformations were found,
cystourethrography and centellography (DMSA or DTPA) was used. Patients
mean age at renal and urological studies was 9,8 years (2-18 years). The
cytogenetic findings in 32 patients with TS were: classic : 45,X in 18 patients (56,25%), mosaic and structural aberrations of X chromosome: in 14
patients (43,75%). The prevalence of renal and urinary system pathology
was 43,75% ( 14 patients) . The most frequent findings were urinary system
malformations 21,87% ( 7 patients) , associated with renal malformations
9,38% ( 3 patients), while 4 patients ( 12,5%) had renal malformations alone. Horseshoe kidney, malrotation or other position abnormalities, duplication of the collecting system, and different ureterovesicular obstruccion
were found. Conclusion: The early diagnosis of renal and urinary system
malformations in TS and their follow-up is crucial to reduce the morbility in
these patients. There appears to be no correlation between karyotype and
the presence or type of renal or urinary system malformations.
P03.46-M
Does summation of alleles account for genetic risk and genotypephenotype association in Type 2 Diabetes Mellitus?
N. P. Pace, A. E. Felice, J. Vassallo;
University of Malta, Msida, Malta.
Introduction: Type 2 diabetes (T2DM) and metabolic syndrome are common complex disorders with a high prevalence in the Maltese population.
The aim of this study is to further define the genetic interplay between cognate genes from metabolic and inflammatory pathways on the likelihood
of developing T2DM in adulthood and to relate the association of certain genetic profiles with defined biological and clinical endpoints. Method: Eight
hundred carefully characterised T2DM cases were recruited. Anthropometric and biochemical parameters, including serum high-sensitivity C-Reactive protein (hsCRP) levels were determined, and genotyping of 43 cognate
genes carried out. Neonatal cord blood samples were used as the control
reference population in this study. Results: Ten polymorphisms in metabolic/inflammatory pathways showed significant association with T2DM.
Three loci showed significant association with lipid profile, body weight and
hsCRP levels. hsCRP levels demonstrated a strong positive correlation with
body mass index. Genetic score analysis showed that combining multiple
genetic markers results in higher relative risks. The functional significance
of these polymorphisms is being further evaluated using targeted siRNAmediated silencing in cultured monocytes. Conclusion: A panel of ten candidate genes has consistently demonstrated significant association with type
2 diabetes and metabolic syndrome in the Maltese population. These gene
variants serve functional roles in inflammation and adipose tissue function.
A recruited cohort of untreated newly-diagnosed T2DM serves to identify
and explore genotype-phenotype association. The strong effect sizes of these alleles could be used to develop personal genetic susceptibility profiles
for T2DM leading to personalization of care and prevention of chronic complications.
96
P03.47-S
XX male sex reversal in an azoospermic proband
Back to index
ATS is characterized by hematuria, proteinuria with progression to end-stage renal disease (ESRD), eye abnormalities and sensorineural deafness. Mutations in COL4A3/COL4A4 (autosomal recessive/dominant), and COL4A5
(X-linked) have been identified as underlying cause. Compared to ATS, autosomal dominant TBMN is characterized by hematuria, minimal proteinuria
and preserved renal function. Dominant mutations have been identified in
COL4A3 and COL4A4.
A cohort of 167 patients and 49 relatives were genetically tested and clinically evaluated. In 145 patients mutations were identified, 65 of them were
novel. 54% resp. 88% of the ATS patients showed only one mutation in COL4A3 or COL4A4. Three TBMN and eight ATS patients (COL4A3 or COL4A4)
had hearing loss, but only two of the ATS patients carried two mutations. An
ATS patient with one mutation in COL4A4 showed no hematuria, but proteinuria, ESRD, and hearing loss. Two patients (COL4A5) showed isolated
proteinuria. 26% of the ATS patients (COL4A5) had hearing loss and 43% of
these presented the mutation at the 5 end of the gene.
This study comprises one of the largest genotype-phenotype correlation
in European ATS and TBMN patients. A large number of novel mutations
were detected. Many patients had only one heterozygous mutation in COL4A3/COL4A4. This might be explained either by an autosomal dominant
inheritance, the possibility of missed mutations, its function as a modifier
additionally to so far unidentified causative mutations, or a combination of
these possibilities. Interestingly, ATS/TBMN patients can also be affected by
isolated proteinuria and TBMN patients rarely develop hearing loss.
P04.01-S
Screening of 1200 FDA-approved molecules to identify
pharmacological modulators of expression of ACVR1, the gene
mutated in Fibrodysplasia Ossificans Progressiva.
ACVR1/ALK-2 encodes for a type I BMP receptor and is mutated in Fibrodysplasia Ossificans Progressiva (FOP, OMIM135100). FOP is a rare and severe
disease of heterotopic ossification, with a progressive and episodic course.
No treatment is available to the progression of the disease and great effort
is devoted to better understand the pathogenic mechanisms underlying the
dysregulated BMP pathway associated with FOP that may be targeted by in-
ABSTRACTS POSTERS
novative therapeutic approaches.
The characterization of the ACVR1 promoter region provide us with the
molecular tools to generate a cell-based system exploitable for HighThroughput Screening (HTS) of chemical compounds with potential pharmacological effect on the ACVR1 expression at the transcriptional level. The
cell system has been generated in ATDC cells by stable transfection of the
Luciferase reporter gene under the control of the ACVR1 promoter.
We describe here in detail the HTS procedure we developed and report
the results obtained with the screening of 1200 FDA-approved compounds
(Prestwick Chemical Library). We identified 18 compounds, showing an
inhibitory effect 60%, and 8 molecules with activating properties on the
ACVR1 transcription. Identified hits belong to different pharmacological
classes among which corticosteroids, PDE inhibitors, FANS. We are currently performing experimental validation of selected molecules with different
assays.
In conclusion, we present a cell-based system suitable for HTS of small
chemical compounds to target the ACVR1 transcriptional activity, thereby
modulating the downstream pathway. Screening of compounds approved
for clinical purposes, may provide candidates for a drug repositioning approach.
P04.02-M
Autosomal-recessive Adams-Oliver syndrome caused by homozygous
mutation in EOGT, encoding an EGF domain-specific O-GlcNAc
transferase
Autosomal recessive Adams-Oliver syndrome was diagnosed in three remotely related Bedouin consanguineous families. Genome wide linkage
analysis ruled out association with known Adams-Oliver syndrome genes,
identifying a single homozygosity ~1.8 Mb novel locus common to affected
individuals (LOD score 3.37). Whole exome sequencing followed by Sanger
sequencing identified only a single mutation within this locus, shared by all
affected individuals and found in patients from five additional apparently
unrelated Bedouin families: a 1bp deletion mutation in a predicted alternative splice variant of EOGT, leading to a putative truncated protein. RT-PCR
demonstrated that the EOGT predicted alternative splice variant is ubiquitously expressed. EOGT encodes EGF-domain-specific O-linked N-acetylglucosamine transferase, responsible for extracellular O-GlcNAcylation of
epidermal growth factor-like domain containing proteins, and essential for
epithelial cell-matrix interactions. F-actin staining in diseased fibroblasts
showed apparently intact cell cytoskeleton and morphology, suggesting the
EOGT mutation acts not through perturbation of cytoskeleton, but through
other mechanisms yet to be elucidated.
P04.03-S
Immunomodulation in Atopic Dermatitis: Inherited, Environmental
and Behavioral Risk Factors, and Evaluation of Biomarkers
Back to index
severe disease and a poor treatment response, such as the AD patient cohort
with extreme IgE levels (>10 000). Three candidate genes (CLDN20, SELP
and FLG2) are drawn from a systematic survey of LoF variants in human
protein-coding genes, that identified rare and likely deleterious LoF alleles
markedly enriched in the Finnish population at greater than 1% frequency.
P04.04-M
The benign joint hypermobility syndrome: guidelines for diagnosis
and management.
Introduction The hypermobility syndrome (HMS) or benign joint hypermobility syndrome (BJHS) is a frequent condition affecting many females,
interfering with their normal activities of daily life. Hypermobility is associated with an increased risk to develop a chronic pain syndrome. Patients
often seek medical help in later stages of this condition for therapy-resistant
complaints. We designed a protocol to improve the referral and management strategy for this particular patient group. Methods We report data on
patients (n=167) referred to the department of clinical genetics to exclude
rare genetic connective tissue disorders such as the Ehlers Danlos syndrome. Results Based on the data of medical history, clinical examination and
if indicated, DNA-investigations, the likelihood of an underlying hereditary
disorder in the 167 counsellees was estimated. Further DNA-investigation
was performed in 83 cases, revealing an underlying monogenetic disorder
in two cases. Conclusions Based on our experience we provide tools to distinguish between the majority of patients with BJHS and those with a hereditary connective tissue disorder. For the referring physician, the modified
scoring list according to Beighton is essential to decide whether to refer or
not. Individuals with the BJHS (Beighton scoring (BS) 4-6) should be referred to a rehabilitation specialist for treatment. In case of a BS of 7 or more,
or 6 or less with unusual features (f.e. male gender, age over 50, and/or a
positive family history) referral to a clinical geneticist is recommended for
further evaluation.
P04.05-S
A mutation in the LRP4 gene is associated with bone mineral density
in Maltese postmenopausal women
M. Formosa, J. Borg, S. Bezzina-Wettinger, R. Farrugia, A. Xuereb Anastasi;
Department of Applied Biomedical Science, Faculty of Health Sciences, University of
Malta, Msida, Malta.
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ABSTRACTS POSTERS
Medical Imaging, The Childrens Hospital at Westmead, Sydney, Australia.
Pagets disease of bone (PDB) is a common bone disease with strong genetic
component. We previously identified a PDB-susceptibility locus on chromosome 8q22 tagged by a common SNP located within the DCSTAMP gene (Albagha et al, Nat Genet 2011). To identify functional susceptibility variants, we
investigated this locus using a targeted DNA sequencing approach. A 700kb
region containing four genes (RIMS2, DCSTAMP, DPYS, and LRP12) was captured using the Haloplex target enrichment kit followed by DNA sequencing
using the Illumina Hiseq2000 platform. A total of 244 samples were sequenced (143 unrelated cases without SQSTM1 mutation, 40 controls, 48 familial
cases and 13 controls). Variants passing quality control were subjected to
multiple filters to include missense variant, those in the 5 or 3UTR or those
predicted as functional by the ENCODE database. In DCSTAMP, we detected
two novel variants (1 missense and 1 in 3UTR) that were not present in our
controls or publicly available databases including 1000 genomes. The novel missense mutation was detected in 3 cases and showed transmission in
familial cases. Two other missense mutations were only found in cases (n=
7) but they are present in 1000Genome database with MAF<0.03. No disease-specific mutations were detected in RIMS2, DPYS or LRP12). DCSTAMP,
which encodes a dendritic-cell-specific transmembrane protein, is a strong
functional candidate gene for PDB because it is required for the fusion of
osteoclast precursors to form mature osteoclasts. Our data suggest that rare
variants within this gene could influence PDB-susceptibility.
P04.08-M
Identification of mutations in DYNC2LI1, a member of the mammalian
cytoplasmic dynein 2 complex, expands the clinical spectrum of
Jeune/ATD ciliopathies
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ABSTRACTS POSTERS
to less frequently mutated genes (COL11A2, COL9A1-2): this innovation has
improved our analysis reliability and turnaround times.
Back to index
P04.13-S
Diagnostics of connective tissue disorders: NGS gets the target when
clinics wanders around
P04.11-S
About skeletal dysplasia patients carrying two Col2a1 mutations
Background and objectives: Skeletal dysplasia are usually dominant disorders due to heterozygous mutations in COL2A1 mutations. To report on four
cases carrying two COL2A1 mutations.
Methods: Bidirectional Sanger sequencing of the whole COL2A1 gene.
Results: Four patients carrying two different mutations were identified in a
series of 136 skeletal dysplasia.
Patient 11 carried a p.Thr1439>Met paternal allele and a de novo c.12672A>G. This latter may explain that she presented with a more severe form
of Kniest dysplasia than her affected father. The p.Ala145Val, predicted as
benign, might not be contributive, as the p.Gly405Asp alone was shown associated with SEDC (Meredith SP, 2007). The association of p.Arg137His and
p.Gly213Val seems to induce a different phenotype than previously observed in a patient carrying a heterozygous p.Gly213Val (Kannu M, 2011).
Conclusion: We report here the first cases of COL2A1 skeletal dysplasia with
either compound heterozygous (recessive) or complex alleles. Recessive
forms of type 2 Stickler syndrome were previously associated with COL11A1
(Richards AJ, 2013). Parents samples from families 41, 47, and 49 are under
evaluation to phase the mutations and define whether they are de novo.
P04.12-M
Identification of a novel locus for a recessive congenital myopathy by
linkage analysis in an Israeli Bedouin family
Congenital myopathy disorders (CMDs) are heterogeneous inherited diseases of muscle characterized by a range of distinctive histologic abnormalities. We have studied a consanguineous family with a non progressive
congenital myopathy. In order to pursue a molecular diagnosis in this family,
we performed genotyping on four patients, their parents and a healthy sibling using the Affymetrix GeneChip Human SNP5 array. We determined the
genotype calls by using Affymetrix GeneChip Genotyping Analysis Software
(GTYPE) and KinSNP software. Based on the consanguinity in the family, we
hypothesized homozygosity by descent of a recessive mutation as the likely
cause of the disorder. Therefore, we searched for homozygous regions consistent with linkage. Three homozygous blocks (on chromosomes 6, 10 and
14) shared by the three affected individuals, heterozygous in the parents,
and not homozygous in the unaffected sib were identified. Exome sequencing revealed a highly suggestive mutation in a gene not previously reported to be associated with myopathy in humans. The variation segregated as
expected in the family, it did not appear in dbSNP, evs or the 1000 genome
project, nor was it found in 134 Bedouin control individuals.
P04.14-M
Functional role of the Bardet Biedl Syndrome-associated gene 9 in the
pathogenesis of nonsyndromic craniosynostosis: disrupted primary
cilium in craniofacial ossification
Sagittal craniosynostosis (sNSC) is a highly prevalent craniofacial malformation, with a widely unclear etiopathogenesis. A possible involvement of the
BBS9 gene, encoding a protein located in the transition zone of the primary
cilium, has been proposed as a result of the first GWAS carried out in 2012.
Through microarray genome-wide expression profiling we have shown an
altered expression of cilium-associated genes, including BBS9, in calvarial
tissues and cells of sNSC patients. We aimed at investigating the role of BBS9
in the aberrant osteogenic phenotype of calvarial cells isolated from sNSC
patients, through gene expression analysis, immunofluorescence, gene silencing, and differentiation assays. BBS9 expression was significantly upregulated in cells isolated from fused sutures (syn-cells) compared to cells
isolated from matched patent sutures (control cells), and increased upon
5 days of osteogenic induction. Confocal microscopy showed that: syn-cells
produced less primary cilia compared to control cells; BBS9 expression was
spread throughout the cytoplasm in syn-cells, while appeared organized
in polarized structures surrounding the cilium basal body in control cells.
Upon BBS9 silencing in syn-cells, the expression of osteo-specific transcription factors (RUNX2 and OSX), and of SMO (key molecule of the hedgehog
pathway), were significantly down-regulated. The osteogenic potential of
BBS9-silenced syn-cells decreased and reverted to the physiological behaviour observed in controls. Our results suggest a functional involvement of
BBS9 and primary cilium signalling, in the aberrant osteogenic signalling
occurring at the site of premature suture closure in sNSC, proving a possible novel role of this molecular machinery in osteogenesis and craniofacial
malformations.
P04.15-S
Chromosomal aberrations in complex craniosynostosis: genetic
heterogeneity helps identifying biological pathways
Craniosynostosis (CRS) represent the most prevalent craniofacial malformation, occurring in 1 out of 2500 livebirths, either as an isolated feature
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ABSTRACTS POSTERS
(nonsyndromic CRS) or in complex phenotypes. A genetic basis can be found
in 20-30% cases of complex CRS, with extremely high heterogeneity. The
aim of this study is to describe the inventory of structural chromosomal aberrations identified through cytogenetic and molecular cytogenetic testing
(as a part of the diagnostic algorithm) in a sample of complex CRS patients.
The possible role of the genes involved in the genomic rearrangements in
CRS etiopathogenesis have been investigated in vitro, using gene expression
analysis, immunofluorescence and gene silencing assays, on calvarial stem
cells isolated from CRS patients.
In our center we have enrolled 253 patients affected by CRS, including 13
cases with complex phenotypes that did not resemble any clear known syndrome. In 9 out of 13 cases, standard cytogenetics and array CGH allowed
evidencing a chromosomal structural mutation, encompassing. multiple
alternative genomic loci (2p, 9q, 2q, 22q, 8q, 9, 10, 5, 17, and 7p, among
others). Genes involved in craniofacial development have been mapped to
those loci enabling a biological interpretation of the abnormalities. In particular, the role of developmental genes involved in the structure and function
of the primary cilium has been demonstrated in selected cases.
P04.16-M
The prostaglandinE2-pathway as a key player in the pathogenesis of
non-syndromic craniosynostosis
C. Cicione1, M. Barba1, L. Massimi2, G. Tamburrini2, G. Di Taranto1, F. Michetti1,3, M.
Caldarelli2, C. Di Rocco2, C. Bernardini1, W. Lattanzi1,3;
1
Inst. Anatomy and Cell Biology, Universit Cattolica S. Cuore, Rome, Italy, 2Dept.
Pediatric Neurosurgery, Universit Cattolica S. Cuore, Rome, Italy, 3Latium
Musculoskeletal Tissue Bank, Rome, Italy.
Introduction According to literature, the genetic cause of craniosynostosis can be identified in approximately 24% of the patients. Often, mutations
are found in FGFR2, FGFR3 and TWIST1. But mutations are also identified
in many other genes (like IL11RA, TCF12 and ERF). Those genes can be
sequenced all in parallel by Next Generation Sequencing (NGS), leading to
more complete diagnostics. Methods Syndromic craniosynostosis was defined as multisuture or unicoronal synostosis, familial craniosynostosis, or as
craniosynostosis in combination with other congenital malformations and/
or mental retardation. According to protocol, these syndromic craniosynostosis patients were tested by single-gene testing for mutations in FGFR2,
FGFR3, and TWIST1. If these genes tested negative, other genes were analyzed if applicable. In addition, a subgroup of 8 families (15 patients, 11 clinically unaffected relatives) and 8 isolated patients were sequenced by NGS
and checked for mutations in known craniosynostosis genes. Results Of our
patient cohort, 705 patients were tested by single-gene testing. Mutations
were found in 280 patients. By NGS, mutations were identified in TCF12 (1
patient, 1 unaffected carrier, 1 relative), IL11RA (1 patient, 2 relatives), ZIC1
(three patients, 1 relative) and in IDS (1 isolated patient). Conclusion By
100
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We report on a girl with widespread, rapidly progressive ectopic calcifications detected shortly after birth. Calcifications became present around most
joints, involving tendons and ligaments, but no internal organs or skin, and
eventually caused almost complete immobility of the child at 2 years. Laboratory evaluation failed to identify autoimmune disorders as well as calcium
metabolism or other biochemical abnormalities; molecular studies did not
identify any mutation in disease genes known to be involved in ectopic calcifications. Further analysis identified a de novo insertional translocation
(IT). Array-CGH analysis showed a 2p13.3 duplication which was validated
by qRT-PCR. Fluorescence in situ hybridization (FISH) confirmed the rearrangement, a der(X)ins(X;2)(q26.1;p13.3). The two breakpoints were characterized at nucleotide level by inverse PCR. The duplication on chromosome 2 encompassed nine coding genes, seven completely duplicated and two
(ANTXR1 and MXD1) partially duplicated. ANTXR1 is interrupted in IVS10;
the MXD1 coding sequence is maintained, but its 3-UTR is almost completely lost. The chromosome 2p13.3 duplication is inserted into a gene desert
on chromosome Xp26.1, between ARHGAP36 and IGSF1 genes. We suggest
the phenotype in our patient is due to a likely gain of function mechanism.
We hypothesize that the de novo insertional translocation causes a fusion
transcript or an altered regulation of a gene by position effect. Further studies are in progress to identify the pathogenic mechanism triggering this
unique phenotype.
ABSTRACTS POSTERS
P04.20-M
An 8-year-old Iranian girl with the FKBP14- related form of the
Ehlers-Danlos syndrome with severe myopathy, scoliosis but normal
hearing
Recently mutations in FKBP14 have been identified in patients with a novel variant of Ehlers-Danlos syndrome (EDS) characterized by progressive
kyphoscoliosis, myopathy and hearing loss (MIM #614557). This disorder
shares many clinical features with the kyphoscoliotic type of EDS (EDS VIA;
MIM 225400) such as congenital hypotonia, progressive kyphoscoliosis, hyperelastic skin and hypermobility of joints. However, there are also some
distinctive features like myopathy and hearing loss. While an increased ratio
of urinary lysyl pyridinoline (LP) to hydroxylysyl pyridinoline (HP) is diagnostic for EDS VIA, measurement is normal in patients with EDS caused by
mutations in FKBP14.
Here we report on an 8-year-old girl born to first cousin parents, who presented with severe congenital hypotonia, psychomotor retardation, severe
muscle weakness, progressive scoliosis, severe joint hypermobility of fingers, wrists and toes, soft skin, easy bruising, bilateral clubfoot, prominent
heel and pes planus, normal hearing, nasal speech and dysarthria. Because
of a suggestive clinical history by absence of hearing impairment our initial clinical diagnosis was EDS VIA, but normal LP/HP in urine ruled out
this condition. Therefore we sequenced FKBP14 and detected a novel homozygous c.143T>A substitution in exon 1 causing a p.Met48Lys mutation.
Hearing loss, was consistently reported in the initial cohort of patients with
this novel form of EDS, in whom nonsense mutations were found. It is compelling to hypothesize that absence of hearing impairment in our patient
might reflect a partial loss of function of FKBP14 caused by the identified
p.Met48Lys missense mutation, thus suggesting a possible genotype-phenotype correlation.
P04.21-S
Zebrafish modeling of 3GalT6-deficient Type of Ehlers-Danlos
Syndrome Stresses the Importance of Glycosaminoglycans in
Development
Proteoglycans are important components of cell plasma membranes and extracellular matrices. They are composed of glycosaminoglycan (GAG) chains
attached to a core protein through a tetrasaccharide linker region. The addition of the third residue in this linker is catalysed by galactosyltransferase II
(3GalT6), encoded by B3GALT6.
We recently identified bi-allelic mutations in B3GALT6 in several individuals from independent families with a severe autosomal recessive pleiotropic
connective tissue disorder characterized by skin fragility, delayed wound
healing, joint hypermobility and contractures, muscle hypotonia, intellectual disability and a spondyloepimetaphyseal dysplasia with bone fragility
and severe kyphoscoliosis. To characterize the function of 3GalT6 we employed zebrafish as an in vivo model. Whole mount in situ hybridization of
zebrafish embryos showed high b3galt6 expression levels in brain, retina,
pharyngeal arches and notochord epithelium, corresponding to tissues that
are affected in the human patients. A morpholino-based approach was used
to characterize the developmental effects of b3galt6 knockdown in zebrafish
embryos. Complete knockdown of b3galt6 is lethal, but partial knockdown
results in an abnormal pharyngeal cartilage phenotype and a notably reduced head and eye size. These morphological changes were accompanied by a
significant reduction in the total amount of sulfated GAG chains.
In conclusion, our results emphasize a crucial role for 3GalT6 in GAG synthesis and development. Ongoing and future experiments aim to extensively
analyze the changes in GAG composition as well as to further characterize
the way in which these changes impact embryonic development of e.g. cartilage and heart using different fluorescent transgenic reporter lines.
Back to index
P04.22-M
Familial vascular Ehlers-Danlos syndrome caused by a mutation in
COL5A1
A. F. Baas1, G. Monroe1, M. Harakalova1, v. Saskia1, D. Majoor-Krakauer2, A. BertoliAvella2, F. Moll1, A. Vink1, D. Dooijes1, A. Maugeri3, G. Pals3, I. Nijman1, E. Cuppen1, N.
Knoers1, G. van Haaften1;
1
UMC Utrecht, Utrecht, Netherlands, 2Erasmus MC, Rotterdam, Netherlands, 3VUMC,
Amsterdam, Netherlands.
Different forms of Ehlers-Danlos Syndrome (EDS) exist, with specific phenotypes and genes associated. Vascular EDS, caused by mutations in COL3A1,
is characterized by a fragile vasculature with a high risk of catastrophic vascular events at young age. Classic EDS, characterized by fragile, hyperextensible skin and joint laxity, is caused by mutations in COL5A1 and COL5A2. To
date, vessel rupture in four unrelated classic EDS patients with a confirmed
COL5A1 mutation was reported.
In the current report, we describe a familial form of clinically vascular EDS,
diagnosed in a mother and her two sons, who all three died at an early age
from arterial ruptures. Diagnostic Sanger sequencing failed to detect a COL3A1 mutation in the eldest son, our index case. Next, our probands DNA
was analyzed using a next-generation sequencing approach targeting approximately 550 genes (possibly) linked to vascular disease (VASCULOME
project). This revealed a heterozygous pathogenic mutation in COL5A1, resulting in an essential glycine substitution in the triple helix domain, nearby the C-terminus of the protein (c.4610G>T; p.Gly1537Val). This novel
mutation was present in DNA isolated from autopsy material of his brother.
No autopsy material was available from the mother, but the mutation was
excluded in her parents, siblings and in the father of her sons, suggesting
that the COL5A1 mutation occurred in the mothers genome de novo.
This is the first describtion of familial vascular EDS with a pathogenic mutation in COL5A1. We conclude that this mutation can give a similar vascular
EDS phenotype as a COL3A1 mutation.
P04.23-S
Phenotypic features of knockout mice for dermatan 4-Osulfotransferase 1 (D4ST1)-deficient Ehlers-Danlos Syndrome
(DDEDS)
Dermatan 4-O-sulfotransferase 1 (D4ST1)-deficient Ehlers-Danlos syndrome (DDEDS), caused by recessive loss-of-function mutations in CHST14, is
a recently delineated form of EDS [Dndar et al., 2009; Malfait et al., 2010;
Miyake et al., 2010; Kosho et al., 2011], characterized by a unique set of clinical features consisting of progressive multisystem fragility-related manifestations (skin hyperextensibilty and fragility, progressive spinal and foot
deformities, large subcutaneous hematoma) and various malformations
(facial features, congenital multiple contractures). Multisystem connective
tissue fragility is caused by impaired assembly of collagen fibrils through
loss of dermatan sulfate (DS) replaced by chondroitin sulfate in the decorin
glycosaminoglycan sidechains. Complete loss of DS in patients urine suggests systemic loss of DS to be the basis of the disorder. Because the patients suffer from progressive multisystem fragility-related complications,
appropriate disease modeling is indispensable in view of developing etiology-based therapy. In this study, we report phenotypic features of knockout
(Chst14-/-) mice. Frozen sperm from Chst14+/- male mice were obtained
from the Mutant Mouse Regional Resource Center, Chst14+/- mice were reproduced, and Chst14-/- mice were generated. Complete loss of DS in urine
from Chst14-/- mice was demonstrated, suggesting these mice to reflect glycobiological abnormalities in DDEDS. Chst14-/- mice lacked congenital multiple contractures, but showed perinatal lethality, reduced postnatal weight
gain, mild facial asymmetricity, thoracic kyphosis, and reduced grip power
and skin tenacity, compared with Chst14+/+ or Chst14+/- mice. These features could be therapeutic targets for etiology-based therapy such as AAV
vector-mediated gene therapy.
101
ABSTRACTS POSTERS
P04.24-M
FGF16 nonsense mutations as the underlying cause of X-linked
recessive metacarpals 4 / 5 fusion
Metacarpal 4-5 fusion (MF4; MIM%309630) is a rare congenital malformation of the hand characterized by the partial or complete fusion of the 4th
and 5th metacarpals. The anomaly occurs either as an isolated trait or part
of a genetic syndrome. Recently, using whole exome sequencing (WES), we
have uncovered the genetic cause of isolated MF4. WES was performed on
samples form a single trio of Polish ethnicity, i.e. sporadic male proband presenting with isolated MF4 and his unaffected parents. The study revealed a
nonsense mutation (c.C535T; p.R179X) in exon 3 of the FGF16 gene, which
maps to chromosome Xq21.1. The result was than confirmed by Sanger sequencing. The link between FGF16 gene mutations and MF4 was further
supported by the identification of two other truncating FGF16 mutation in
two unrelated male probands. Sanger sequencing showed that one index
case carried a nonsense mutation (c.C470A; p.S157X), whereas the other
harbored a truncating frameshift variant (c.474_477del; p.E158DfsX25).
Two out of three mothers of the probands were available for testing. Upon
assessment, they were clinically and radiologically unaffected and both carried FGF16 heterozygous mutations. All of the FGF16 disease-causing variants identified by us were located in the last exon (exon 3) of the gene,
therefore they are unlikely to result in a nonsense mediated decay. Our study
shows that truncating mutations of FGF16 are associated with X-linked recessive metacarpal 4-5 fusion and provides evidence for the involvement of
FGF16 in the fine patterning of the human skeleton of the hand.
P04.25-S
Giant cell tumor of the bone is caused by somatic mutations in H3F3A
gene while patients with giant cell tumor arising on Pagets disease of
bone shared a distinct genetic alteration
Giant cell tumor of bone (GCT) is an aggressive bone tumor caused by the
uncontrolled proliferation of the spindle-like stromal cells which promote
osteoclast-like giant cells formation responsible for the osteolitic lesions.
A recent study showed that GCT is due to recurrent somatic mutations in
H3F3A gene in the stromal cells. We analysed a cohort of giant cell tumor of
bone for the presence of H3F3A mutations identifying somatic mutations in
38 out of 44 cases (86%). In contrast, the analysis of patients with Pagets
disease of bone (PDB) associated with giant cell tumor did not show any
mutation in H3F3A gene, at both somatic and germline level, suggesting a
different genetic background. We recently reported an extended Italian family in which 4 out of 14 PDB affected members developed multiple GCTs at
pagetic skeletal sites. Clinically, all affected members had polyostotic PDB,
but subjects developing giant cell tumors showed an increased disease severity with a reduced clinical response to bisphosphonate treatment and
an increased prevalence of bone pain, deformities, and fractures. Whole
exome sequencing, in this family identifies a missense mutation in a novel
uncharacterized gene. Additional genetic analysis in 7 independent affected
patients with the same clinical phenotype, discloses the same mutation in all
patients, strongly suggesting that this clinical phenotype is due to a founder
effect. Therefore, we conclude that the GCT phenotypes associated or not
with PDB are due to mutations in different genes, suggesting that different
molecular signatures are responsible for these two phenotypes.
P04.26-M
Identification and characterization of a novel susceptibility locus for
nonsyndromic cleft lip and palate at chromosome 15q13
K. U. Ludwig1,2, A. C. Bhmer1,2, H. Peters3, D. Graf4, P. Gltepe1,2, P. A. Mossey5, R. P.
Steegers-Theunissen6,7, M. Rubini8, M. M. Nthen1,2, M. Knapp9, E. Mangold2;
1
Department of Genomics, University of Bonn, Bonn, Germany, 2Institute of Human
Genetics, University of Bonn, Bonn, Germany, 3Institute of Genetic Medicine, Newcastle
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University, Newcastle upon Tyne, United Kingdom, 4Institute for Oral Biology, University
of Zurich, Zurich, Switzerland, 5Orthodontic Unit, Dental Hospital & School, University of
Dundee, Dundee, United Kingdom, 6Erasmus Medical Center, University Medical Center,
Rotterdam, Netherlands, 7Radboud University Medical Center, Nijmegen, Netherlands,
8
Department of Biomedical and Specialty Surgical Sciences, Medical Genetics Unit,
University of Ferrara, Ferrara, Italy, 9Institute of Medical Biometry Informatics and
Epidemiology, University of Bonn, Bonn, Germany.
Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is one of the
most common congenital malformations worldwide and considered to be of
multifactorial etiology. NsCL/P shows considerable phenotypic variability
and can be subdivided into nonsyndromic cleft lip only (nsCLO) and nonsyndromic cleft lip and palate (nsCLP).
Genome-wide and replication studies have recently led to the identification
of 15 nsCL/P susceptibility loci. However, a number of additional genetic
risk factors still await elucidation. Here we used data from a recent genomewide meta-analysis (Ludwig et al. 2012, Nature Genetics) and combined
these with results from an independent European trio cohort (n=793). Integration of subgroup-information on nsCLO or nsCLP revealed rs1258763
on chr. 15q13 as a novel genome-wide significant locus associated with nsCLP (P=1.0410-08). The associated region maps in intergenically, between
the Gremlin-1 (GREM1) and Formin-1 (FMN1) genes. GREM1 is a known
antagonist in bone-morphogenetic-protein pathways which are relevant to
craniofacial genesis. Sequencing the entire GREM1 coding region in 196 patients and 196 controls did not reveal a causal variant, however, a significant
overrepresentation of rare variants within patients was observed (P=0.02).
Analyses of murine Grem1 expression during embryonic craniofacial development might suggest a functional role of Grem1 in lip and secondary palate formation. Notably, the top variant rs1258763 has previously been shown
to influence variation in facial morphology (Boehringer et al. 2011, EJHG,
Liu et al. 2012, PloS Genetics).
Our results demonstrate that increasing sample sizes and precise phenotypic information might help identifying further risk loci for genetically complex traits.
P04.27-S
Study of clinical and mutational findings in 45 Russian families with
ectodermal hypohidrotic dysplasia
T. B. Milovidova, A. V. Polyakov;
Research centre for medical genetics, Moscow, Russian Federation.
Introduction IKBKG/NEMO gene mutations are a well-known cause of Incontinentia Pigmenti (IP), an X-linked dominant neuroectodermal disorder
(OMIM #308300). IP is characterized by characteristic skin lesions and
ectodermal, ocular and central nervous system features. We collected four
female patients with a clinical IP phenotype to study their molecular and
phenotypical features.
Methods Direct genomic PCR using primers in the NEMO gene was used
ABSTRACTS POSTERS
to test for the classical exon 4-10 deletion. Sequence analysis of NEMO was
performed using standard Sanger sequencing. X-inactivation was measured
using a methylation-sensitive restriction enzyme. An Affymetrix Cytoscan
HD array was used to detect copy number variations (CNVs) according to
the manufacturers protocol.
Results The classical NEMO exon 4-10 deletion was detected in one patient
and her mother. In another patient, a 130 kb interstitial gain in chromosome
17q25.3 was identified, containing five genes (CD7, SECTM1, GPR14, TEX19
and OGFOD3). There was no skewed X-inactivation. The fourth patient was
not molecularly investigated.
Discussion We show four female patients with a clinical IP phenotype and
different genetic defects. NEMO deletions impair NF-kB activation, causing
IP cells to be highly sensitive to apoptosis. Interestingly, a novel 130 kb
gain of chromosome 17q25.3 was identified in another patient, including
SECTM1. SECTM1 is shown to induce IFN- production in vitro; IFN- stimulates apoptosis. We hypothesize that a dosis effect, caused by the SECTM1
duplication, induces cell death and thereby causes IP. This finding suggests
heterogeneity of IP and supports the role of apoptotic pathway CNVs in this
disorder.
P04.29-S
CMG2/ANTRX2 gene mutation analysis in 9 families suffering from
Infantile Systemic Hyalinosis
Infantile Systemic hyalinosis (ISH) is a severe rare autosomal recessive inheritance disease characterized by accumulation of amorphous, hyaline material in skin and other organs. It leads to skin lesions, gingival hypertrophy,
flexion contractures of the joints, digestive tract and lymph node impairment
with severe phenotypes in newborns. Beginning within the first few months
of life, it is characterized by painful joints, generalized skin thickening, papules, periorificial nodes, hyperpigmentation over the joints, osseous nodes,
failure to thrive. This disorder is progressive and usually leads to death, due
to recurrent chest infections and diarrhea, before two years of age. The diagnosis is made by histology on skin biopsy showing hyaline deposits. Deleterious mutations have been reported in Capillary Morphogenesis Gene-2
(CMG2) alias Anthrax Toxin Receptor 2 (ANTRX2). CMG2/ANTRX2 (4q21) is
composed of 16 exons and the mutations reported in several families with
ISH mostly concern exons 13 to 15. We report the causal mutations in 9 families found using Sanger sequencing. Mutations were found in 6 affected
newborns, 22 relatives, among them 6 foetus with 6 homozygote mutations,
1 composite heterozygote and 1 associated to a supposed deletion. The 8
different mutations found will be described. 5 of them are non-sens mutations and 3 are 1-2 base(s) deletions or duplications leading to frameshift,
Half of them are unknown in literature with 3 mutations in exon 1 and 10
and 1 between intron 8 and exon 9 affecting splicing. There is no treatment
for this severe disease and the prenatal diagnosis is available.
P04.30-M
Whole-exome sequencing identifies a TTN mutation in a multiplex
family with inguinal hernia
Inguinal hernia repair is one of the most frequently performed gastrointestinal surgical procedures. Over 700,000 groin hernias are repaired annually
both in the United States and Europe. Males are seven times more likely than
females to develop a hernia and have a 27% lifetime risk of developing an
inguinal hernia. Risk factors that have been associated with developing inguinal hernias include male gender, aging, pulmonary disease with chronic
cough, and other conditions that cause constant increase of intra-abdominal
pressure. Connective tissue disorders, such as Ehlers-Danlos and Marfan
syndromes, and systemic collagen subtype imbalances have also been associated with increased risk of hernia. Furthermore, several studies have
demonstrated that a positive family history is an important risk factor for
the development of primary inguinal hernia.
The aim of this study was to investigate a multiplex Estonian family with
inguinal hernia in four generations. Whole-exome sequencing was carried
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A girl born to healthy non-consanguineous parents and her three-yearyounger brother showed at birth moderate to severe respiratory insufficiency which required long-term tracheostomy in the boy. Both neonates
were large with plump facial features, narrow thoracic cage with radiographic bell-shaped configuration. Diastasis of the abdominal wall musculature
and signs of distal arthrogryposis were mild in the girl and pronounced in
the boy. Neuromotor development was delayed with independent walking
achieved at 18 months and at 5 years respectively. The mild intellectual
disability in the boy, who has persisting contractures at the wrists and ankles, may be correlated with bouts of therapy-resistant hypoxia during the
NICU admission of six months duration. At night he still needs mechanical
support with airflow.
The clinical and radiographic pattern in both sibs was compatible with paternal UPD 14. However, an imprinting defect was ruled out using microsatellite marker analysis. Recently, the ladys complaints about long lasting respiratory infections and physical tiredness prompted reexamination of this
family. SNP array analysis using an Illumina Cyto-SNP 12v2.1 chip detected a
microdeletion on chromosome band 14q32.2 in both sibs. The deletion with
a minimal size of 64 kb encompasses the lncRNA genes MEG3 and MEG8 and
several miRNAs. Each of these genes is subject to maternal imprinting. The
mother is mosaic for the same microdeletion. Molecular analysis regarding
exact size, gene content and degree of methylation is ongoing and bound to
contribute to more insights into the disease mechanisms involved in this
imprinted region.
P04.32-M
Increased frequency of F5 and F2 thrombophilia predisposing
variants in families with unilateral limb reduction defects - a pilot
study
A. Jamsheer1,2, M. Socha1, E. Olech1, A. Sowiska-Seidler1, A. Latos-Bieleska1,2;
1
Department of Medical Genetics, Poznan University of Medical Sciences, Poznan,
Poland, 2NZOZ Center for Medical Genetics GENESIS, Poznan, Poland.
Limb reduction defects (LRDs) are usually unilateral and affect 8 newborns
per 10000 livebirths. Several authors hypothesized that vascular accidents
may account for a substantial proportion of LRDs, however the basis of such
events in the fetus remains unknown. Over the past two decades, a number
of genetic thrombophilia risk factors (TRFs) have been identified and associated with the increased risk of peri/postnatal occlusive disease and with
a higher rate of pregnancy loss. Mutations c.G1691A(p.R506Q) in F5 and
c.*97G>A in F2 genes represent one of the most commonly tested genetic
TRFs. In this study, a cohort of 45 proband-mother pairs, in which the child
manifested unilateral LRDs, was recruited for the analyses. The patients
were clinically evaluated and blood of the probands and their mothers was
subjected to both F5 and F2 testing. We found either F2 or F5 heterozygous
variant (or both) in 6 mothers (13.3%) and in 4 probands (8.9%). At least
one individual carried a single (or both) mutation(s) in seven proband-mother pairs (15.6%). Within this group one proband and one mother carried
both mutations. F5 and F2 are identified in a control population with the
frequency of about 1-2%. Based on our findings, we hypothesize that there
is an excess of genetic TRFs in probands affected by unilateral LRDs and in
their healthy mothers in comparison with control population. This may contribute to the higher risk of antenatal vascular accidents and consequently
to LRDs. Further studies based on bigger samples are needed to support
our findings.
P04.33-S
Homozygous Ala529Val LMNA Mutation in Patients with
Mandibuloacral Dysplasia in a Turkish family
103
ABSTRACTS POSTERS
Medical School, Department of Histology and Embryology, stanbul, Turkey, 3stanbul
Bilim University Medical School, Department of Medical Biology and Genetics,
stanbul, Turkey, 4Genart IVF Center, Ankara, Turkey, 5Ufuk University Medical School,
Department of Medical Biology and Genetics, Ankara, Turkey, 6Hacettepe University
Medical School, Department of Pediatric Genetics, Ankara, Turkey.
Mandibuloacral dysplasia is a rare autosomal recessive disorder characterized by acroosteolysis, hyperpigmentation, mandibular and clavicular hypoplasia, bird-like facies with beaked nose and prominent eyes, delayed closures of the cranial sutures and joint contractures. Homozygous Arg527His
and Ala529Val mutationS in LMNA gene are previously reported in MAD
patients with type A lipodystrophy. Ala529Val (c.1586 C>T) missense mutation resulting in substition of alanin with valine may distrupt the formation
of salt bridge between arginine 527 and glutamate 537. Mutant lamin A and
C proteins alters the nuclear envelope, chromatin organisation and effects
the cellular processes.
Here we report a Turkish family with MAD. The proband was 36 years old
male and the first child of consanginous parents who have 3 children. He
had acroosteolysis, hyperpigmentation, mandibular and clavicular hypoplasia, beaked nose,joint contractures .Aunt of the proband has also detected as
MAD with consangineous parents. LMNA gene Exon 9 sequencing revealed
homozygous Ala529Val LMNA mutation in affected persons of this family.
Probands parents are also detected as carrier for Ala529Val LMNA mutation and the other family members were detected with normal genotype.
Previously homozygous Ala529Val mutation in patients with MAD was first
reported by Garg et al. Up to date our study was the second report about patients with homozygous Ala529Val mutation. Molecular studies of MAD patients will help to determine the genotype-phenotype correlation, variability of phenotypic expressivity of mutations and molecular basis of disease.
P04.34-M
A combined workflow for FBN1 mutation detection using next
generation and Sanger sequencing methods
K. Koczok, L. Madar, . Olh, G. P. Szab, K. Szakszon, G. Pfliegler, I. Balogh;
University of Debrecen, Debrecen, Hungary.
Melorheostosis is an uncommon, relatively benign, sclerosing bone disease, characterized by long bones hyperostosis, resembling flowing of candle wax. Skin and subcutaneous tissue can be involved. The disease may be
asyntomatic (incidental X-ray diagnosis, finding of the typical hyperostosis
pattern) or cause pain, joint stiffness or deformity. Melorheostosis is sporadic and pathogenesis is largely unknown. Occasionally in the same family
we observe individuals with melorheostosis and osteopoikilosis, disease
characterized by multiple round foci of increased bone density. LEMD3 gene
mutations have been revealed in osteopoikilosis and Buschke-Ollendorff
Syndrome (BOS), an association of skin lesions, as connective tissue naevi, and osteopoikilosis or melorheostosis. Sporadically LEMD3 mutations
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ABSTRACTS POSTERS
P04.38-M
Neurofibromatosis type 1 and Legius syndrome: differential
molecular diagnosis in children
Back to index
We report on a 60 yrs. patient with short stature (< 2nd centile), spontaneous eruption of 7 teeth only, fusion of C1 to C4 vertebrae with partial
occipito-atlantal fusion. Two of her seven siblings presented oligodontia.
Standard chromosome analysis in the proband showed a mosaic karyotype 45,X[3]/46,XX[27], which may relate with the short stature. Array-CGH
(60K, Agilent Technologies) showed a 1 Mb duplication encompassing
chromosome 7p spanning eight genes (ISPD, SOSTDC1, LRRC72, ANKMY,
TSPAN13, AGR2 and AGR3): (arr[hg19] 7p21.2p21.1(15,926,980x2,15,99
4,233-17,074,396x3,17,222,770x2). Real-time PCR on available members
showed the duplication segregated in the two siblings with oligodontia, but
it was absent in one healthy sister. We hypothesize that oligodontia is associated with the duplication of SOSTDC1 (OMIM* 609675), which is a member of the sclerostin family. The protein acts as a bone morphogenetic protein (BMP) antagonist. Mice overexpressing Sostdc1 have a reduced number
of teeth, whereas Sostdc1-/- show an increased number of teeth number and
anomalies in teeth morphology. The gene has been demonstrated to antagonize Wnt signaling in mice. Interestingly, WNT10A mutations have been
associated with oligodontia in humans. In conclusion, we speculate that the
duplication of SOSTDC1 at chromosome 7p21.2p21.1 may increase its protein level leading to oligodontia in our family. SOSTDC1 could be a new candidate gene to be screened in isolated oligodontia.
P04.42-M
Identification of a novel regulatory variant in OPTN in familial cases of
Pagets disease of bone
S. Wani, S. H. Ralston, O. M. E. Albagha;
University of Edinburgh, Edinburgh, United Kingdom.
Pagets disease of bone (PDB) is a common bone disease with strong genetic component. Previous studies have identified a PDB-susceptibility locus
on chr10p13 tagged by rs1561570 which is located within the OPTN gene
(Albagha et al, Nat Genet 2011). OPTN encodes a protein that plays a role in
multiple cellular processes including autophagy and NF-KB signalling but its
role in bone metabolism is yet unclear. Here we investigated the 10p13 locus
by targeted DNA sequencing of a 650kb region surrounding the rs1561570
which includes seven genes. A total of 260 samples were sequenced including unrelated cases and controls, 60 familial cases and controls. All cases
were negative for SQSTM1 mutations. Target capture was performed using
Haloplex kit followed by DNA sequencing using the illumina Hiseq2000
platform. Variants passing quality control measures were filtered to include
only rare coding (MAF<0.01 in 1000Genome) or regulatory variants (as predicted by ENCODE). No disease-specific missense mutations were detected
in any of the seven genes located in this region including OPTN. However,
we identified a novel rare variant located in the OPTN promoter which was
found to alter NF-KB transcription factor binding site. This novel variant had
a high call quality (Q>4200, read depth >150) and was not present in the
public databases including 1000 genomes and showed transmission from
an affected father to an affected daughter and the unaffected mother did
not carry this mutation. In conclusion our data suggest that rare regulatory
variants within OPTN influence PDB susceptibility.
P04.43-S
Evaluating susceptibility loci for nonsyndromic cleft lip with or
without cleft palate in the Arabic population: Analysis of 15 risk loci
in a new case-control sample recruited in Yemen
105
ABSTRACTS POSTERS
Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany.
Nonsyndromic cleft lip with or without cleft palate (nsCL/P) has a genetically complex etiology. In recent years, several genome-wide significant susceptibility loci for nsCL/P were identified, most of them by genome wide association studies. However, most studies have been performed in populations
from Europe and Asia, and few data are available concerning genetic susceptibility to nsCL/P in Arabic populations. The present study investigated a
newly recruited nsCL/P sample from Yemen. Twenty-four single nucleotide
polymorphisms (SNPs) representing all 15 currently known nsCL/P risk loci
were genotyped in 242 nsCL/P cases and 420 healthy controls. Single marker association analysis revealed significant associations for four loci (8q24,
9q22, 10q25, 13q31). The strongest association was for the European high
risk locus at 8q24 (Pcorrected = 5.09x10-4; heterozygous odds ratio (ORhet) =
1.74, 95% confidence interval (CI 95% CI) = 1.22-2.47, homozygous odds
ratio (ORhom) = 2.47, CI 95% = 1.55 - 3.93). Five additional loci (1q32.2, 3q12,
8q21, 17q22, 20q12) showed nominal significance that did not withstand
correction for multiple testing. Two loci (1p36, 2p21) failed to reach nominal significance but displayed a trend towards association with P < 0.1.
Although the four remaining loci (1p22, 3p11, 15q22, 17p13) failed to reach
nominal significance, the risk alleles were in the same direction as in the discovery studies. Our results suggest that four of the 15 analyzed nsCL/P risk
loci which were identified in European and Asian ethnicities significantly
confer risk for nsCL/P in Arab populations.
P04.44-M
Defective proteolytic processing of fibrillar procollagens and other
extracellular matrix proteins due to mutations in BMP1 results in a
severe form of Osteogenesis Imperfecta
106
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Osteogenesis imperfecta (OI) type V is caused by a recurrent dominant mutation (c.-14C>T) in IFITM5, which encodes BRIL, a transmembrane ifitmlike protein strongly expressed in osteoblasts, while type VI OI is caused by
recessive null mutations in SERPINF1, encoding pigment epithelium-derived factor (PEDF). We identified a 25-year-old woman with severe OI, with
normal serum PEDF but absent PEDF secretion by cultured osteoblasts. Her
SERPINF1 sequences were normal despite bone histomorphometry typical
of type VI OI. Whole exome sequencing of the proband, parents and an unaffected sibling revealed ade novo IFITM5 mutation in one allele of the proband, causing a p.S40L substitution in the BRIL intracellular domain. IFITM5
expression was normal in proband fibroblasts and osteoblasts, as was BRIL
protein level in proband osteoblasts on western blot and in permeabilized
proband osteoblasts by microscopy. Notably, SERPINF1 expression was decreased in proband osteoblasts and PEDF was barely detectable in conditioned media of proband cells. Expression and secretion of type I collagen was
similarly decreased in proband osteoblasts, confirming this OI as collagenrelated. Osteoblasts with the S40L mutation also had decreased expression
of ALP and osteocalcin, as seen in primary PEDF defects. In contrast, osteoblasts from classical type V OI have increased SERPINF1 expression and
PEDF secretion during differentiation. These data suggest (1) that the type
V OI and p.S40L BRIL are gain- and loss-of-function mutations, respectively,
and (2) that BRIL and PEDF have a relationship that connects the genes for
types V and VI OI and their roles in bone mineralization.
ABSTRACTS POSTERS
P04.48-M
A novel deletion mutation involving TMEM38B associated with
autosomal recessive osteogenesis imperfecta.
Background.Osteogenesis Imperfecta (OI) is a heritable disease of connective tissue characterized by abnormal bone mineralization and skeletal deformities. Objective. This study investigate the osteoclastogenic potential of
unfractioned peripheral blood mononuclear cells ( PBMCs) from OI patients
( mean age 10,44+/- 3,48) who received cyclical neridronate infusions for
at least 1 year, untreated OI patients and control subjects. Methods. PBMCs
from 6 patients and 6 controls were cultured in presence/absence of M-CSF
and RANKL. At the end of the culture period, mature multinucleated osteoclasts (Ocs) were identified as TRAP+ cells. By real time PCR we studied
gene expression in freshly isolated PBMC. By flow cytometry we characterized the presence of OC precursors ( CD14+/CD16+) and TNF- expression. Results. Spontaneous OC formation, without adding M-CSF and RANKL,
occurred in PBMC cultures from treated and untreated OI patients. In these
patients, the percentage of circulating OC precursors, increased respect to
the controls ( 12,5% vs 0,1%, p< 0,01). By real time PCR, we found high
levels of RANKL, TNF- and MSCF receptor, as well as decreased OPG levels,
thus leading to the increase of RANKL/OPG ratio. High TNF- levels were
also found on monocytes through flow citometry. Conclusion. We showed
for the first time the high osteoclastogenic potential of PBMCs from OI patients, treated and untreated with biphosphonate, which could be due to the
high percentage of circulating OC precursors, to the elevated TNF- levels as
well as to the increased RANKL/OPG ratio. This condition could contribute
to bone disease affecting these patients.
P04.50-M
Study of ATG2B and ATG16L1 polymorphisms in Spanish patients with
Pagets disease of bone
V. Rivero1,2, R. Usategui3,2, J. Del Pino4, R. Gonzlez2,1;
1
Centro de investigacin del Cncer, Salamanca, Spain, 2Institute of Biomedicine
Research (IBSAL), Salamanca, Spain, 3Unidad de Medicina Molecular. Facultad de
Medicina, Salamanca, Spain, 4Department of Reumatology, Virgen Vega Hospital,
Salamanca, Salamanca, Spain.
Pagets disease of bone (PDB) its a chronic skeletal disorder, that is cha-
Back to index
V. E. R. Parker1, A. Luchetti1, H. Martin1, I. Isaac1, M. J. Lindhurst2, J. Sapp2, K. KepplerNoreuil2, L. G. Biesecker2, E. R. Maher1, R. K. Semple1;
1
Cambridge University Hospitals NHS Trust, Cambridge, United Kingdom, 2National
Human Genome Research Institute (NHGRI)/NIH, Bethesda, MD, United States.
Aim: Somatic activating mutations in PIK3CA, encoded by the p110 catalytic subunit of phosphatidylinositol-3-kinase (PI3K), have been identified
in several different disorders of segmental overgrowth. PI3K is a critical
mediator of cellular growth, survival and metabolism, and is frequently
mutated in cancers. We assessed the prevalence of PIK3CA mutations in 40
patients with variable forms of mosaic overgrowth.
107
ABSTRACTS POSTERS
Methods: Screening of DNA derived from affected tissue was undertaken
using a PCR-based target enrichment with the Ion AmpliSeq Cancer Hotspot
Panel v2, and sequenced on an Ion PGM platform. Exons 2,5,7,8,10,14,19
and 21 were screened, and the detection level was 5% at 1500x depth.
Results: We identified 20 patients with somatic variants in exons 8, 10 and
21 of PIK3CA exhibiting a range of clinical phenotypes. There were no reported cases of cancer or metabolic disturbance, and 1/3 of adult patients had
minimally progressive disease from the third decade.
Discussion: Somatic activation of PIK3CA causes a diverse spectrum of disease, ranging from isolated digit enlargement to more extensive overgrowth of the limbs, abdomen, brain or blood vessels. The rate of malignancy
appears to be low, although this will need to be substantiated in larger
studies. In the future, identification of biomarkers to predict disease progression will provide critical prognostic information, whilst the possibility
of therapy with inhibitors of the PI3K-AKT pathway warrants evaluation in
clinical trials.
Number
of cases p.His1047Arg p.His1047Leu p.Glu542Lys p.Glu545Lys p.Cys420Arg p.Gly1049Arg
screened
Macrodactyly 14
4
1
1
0
0
1
Fibroadipose
9
4
2
0
0
1
0
hyperplasia
CLOVES
6
1
0
0
3
0
0
MCAP
3
1
0
0
0
0
0
KlippelTrenaunay
2
0
0
0
0
0
0
syndrome
Hemihyper3
0
0
0
0
0
0
plasia
Proteus
2
0
0
0
0
0
0
Complex
epidermal
1
0
1
0
0
0
0
naevus
syndrome
TOTAL
40
10
4
1
3
1
1
Congenital Lipomatous Overgrowth, Vascular Malformations, Epidermal Naevi, Scoliosis/
Skeletal and Spinal (CLOVES) syndrome), Megalencephaly-Capillary Malformation (MCAP)
syndrome. Tissue screened included FFPE, fresh tissue and dermal fibroblast cell lines.
P04.54-M
PRINS, the psoriasis susceptibility related non-coding RNA
contributes to various aspects of cellular stress response
We identified PRINS (Psoriasis susceptibility Related non-coding RNA Induced by Stress) and showed that it is highest expressed in the psoriatic uninvolved epidermis. According to the results of in vitro experiments PRINS
expression is induced by various stessors such as UVB, translation inhibition and microbial agents. It has been previously demonstarted by us and by
others that keratinocytes of the psoriatic uninvolved epidermis possess an
abnormal response to various stressors and respond with hyperproliferation. Therefore we aimed to understand how the altered expression of PRINS
in the uninvolved epidermis contributes to the aberrant stress response of
the keratinocytes thus to disease susceptibility. To this end we identified a
chaperon protein, nucleophosmin (NPM) as a direct interacting partner of
PRINS. We could demonstrate that UV-B irradiation induced the shuttling of
NPM from the nucleolus to the nucleoplasm and silencing of the PRINS noncoding RNA in the UV-B-irradiated keratinocytes resulted in the retention
of NPM in the nucleolus. These results suggest that PRINS is physically and
functionally linked to NPM, thus it plays a role in the NPM-mediated cellular stress response. In an other set of experiments we have demonstrated
that PRINS signals independently from the NF-kappaB signal trasduction
pathway, however its expression was induced when the keratinocytes were
treated with the inflammasome-activating poly(dA:dT).
Our results indicate that the PRINS non-coding RNA is part of a ribonucleocomplex. Its altered expression in psoriatic uninvolved epidermis could contribute to the well-established aberrant stress response of psoriatic keratinocytes and as a consequence to psoriasis susceptibility.
P04.55-S
Association of Pseudoxantoma Elasticum with renal nefrocalcinosis: a
rare manifestation of the disease
G. Parmeggiani1, M. Neri1, E. Italyankina1, A. Storari2, D. Quaglino3, S. Bigoni1, A.
Ferlini1;
1
Department of Medical Genetics, Ferrara University, Ferrara, Italy, 2Nephrology
Department, Ferrara University Hospital, Ferrara, Italy, Ferrara, Italy, 3Department of
Life Science, Modena and Reggio Emilia University, Modena, Italy, Modena, Italy.
108
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We describe a 40 years old man from Albania with clinically evident hematuria and proteinuria; renal function was normal. The peculiar renal CT findings were: small multiple calcifications at the cortical junction bilaterally.
The pedigree analysis revealed two sisters affected by an unspecified skin
disease; no consanguinity was reported in the family. The clinical genetic
examination showed yellowish papules of the skin on the neck. The association of renal calcifications in a young patient with skin lesions and the
recurrence of a skin disease in the family led us to take into account Pseudoxanthoma elasticum (PXE). Therefore an ocular examination looking for
signs of the disease was performed and angioid streaks radiating from the
peripapillary area without subretinal neovascularization signs were detected. Pseudoxanthoma elasticum (PXE), is a rare multisystem disease characterized by degeneration and calcification of elastic fibres and blood vessels. The causative gene is ABCC6, mapped on chromosome 16p13.1, which
encodes an ABC transporter protein (ABCC6) expressed primarily in liver
and the kidneys. Patients typically develop cutaneous, ocular, cardiovascular
and gastrointestinal manifestations. The molecular analysis of the ABCC6
gene detected a homozygous deletion of exons 23-29 (del23_29); this is the
most common deletion described in literature associated to PXE, resulting
in a premature stop codon with loss of 505 amino acids of MRP6 protein.
Although this renal pattern cannot be considered specific for the diagnosis
of PXE, we recommend to test PXE when both renal and skin abnormalities
are present in a patient.
P04.56-M
KIF3A is associated to arthopaty envolvement in psoriatic patients
SHOX alterations have been reported in 67% of patients affected by LriWeill dyschondrosteosis (LWD), with a larger prevalence of gene deletions
than point mutations. It has been recently demonstrated that these deletions can involve the SHOX enhancer region, rather that the coding region,
with variable phenotype of the affected patients.
Here, we report a SHOX gene analysis carried out by MLPA in LWD patients from 4 families with variable phenotype. All patients presented a SHOX
enhancer deletion. In particular, a patient with a severe bilateral Madelung
deformity without short stature showed a homozygous alteration identical
to the recently described 47.5 kb PAR1 deletion. Moreover, we identified,
ABSTRACTS POSTERS
for the first time, in three related patients with a severe bilateral Madelung
deformity, a smaller deletion than the 47.5 kb PAR1 deletion encompassing
the same enhancer region (ECR1/CNE7).
Data reported in this study provide new information about the spectrum of
phenotypic alterations showed by LWD patients with different deletions of
the SHOX enhancer region.
P04.58-M
Sjgren-Larsson syndrome: molecular characterization of the first
reported Cypriot families
Sjgren-Larsson syndrome (SLS; MIM #270200) is a rare autosomal recessive disorder caused by deficient fatty aldehyde dehydrogenase (FALDH) activity. This reduction in enzymatic activity is secondary to bi-allelic mutations
in the ALDH3A2 gene and results in impaired fatty alcohol oxidation and
accumulation of fatty alcohols and related lipid products. SLS is typically
characterized by pruritic ichthyosis, spasticity, intellectual disability and
seizures. In the present study we report, for the first time in the Cypriot
population, three apparently unrelated SLS patients. All three were homozygous for a CCC deletion at nucleotide 941 to 943 coupled with a 21 nucleotide 5-GGGCTAAAAGTACTGTTGGGG-3 insertion (c.941-942delins21bp). This genetic alteration, identified previously in two Caucasian patients,
leads to the substitution of Ala314 and Pro315 to Gly and Ala, respectively,
accompanied by the addition of six amino acids, Ala-Lys-Ser-Thr-Val-Gly
(p.Pro315AsnfsX7).
P04.59-S
Next step in collagenopathies and osteodysplasies diagnosis: NGS
Targeted re-sequencing
D. M. Valero-Hervs, A. Romera-Lpez, C. Camprub, D. Cantalapiedra, L. PrezCabornero, V. Felipe, G. Hernndez, D. Diego, C. Buades, C. Collado, R. Rodrguez de
Pablos, M. Garca-Ruz, . Rodrguez, S. Ziga-Trejos, J. Trivio, S. Santilln;
Sistemas Genmicos S.L., Paterna, Valencia, Spain.
Collagenopathies and osteodysplasies constitute a genetically heterogeneous group of diseases with low incidence, representing however an important health problem because patients require continuous specialized
care, due to their deteriorating quality of life. The molecular diagnosis is
essential to confirm clinical suspicion, but is not straightforward due to the
high number of candidate genes. NGS targeted re-sequencing is a fast and
cost-effective alternative to Sanger sequencing, as it can sequence all the
genes involved in disease development.
We designed a NGS targeted re-sequencing panel for 229 genes associated
with skeletal dysplasias, which spans 1.5Mb comprising coding exons, splice
sites and 5 and 3 untranslated regions. Once panel was validated for diagnostic use testing HapMap cell lines, these regions were sequenced in 40
patients with clinical suspicion of these pathologies. Target regions were enriched and captured using the Enrichment SureSelect system (Agilent) and
sequenced either with a SOLiD 5500 (Life Technologies) or MiSeq (Illumina)
platform. Results were confirmed by Sanger sequencing.
The analysis identified 47 variants in 24 patients, of which 8 were classified as disease-causing and 4 as probably pathogenic. Both types of variants
were either truncating mutations or glycine substitutions in collagen genes
(essential for collagen structure). The identification of these mutations allowed the molecular diagnosis and an appropriate genetic counseling in
affected families. Targeted re-sequencing is especially suited to highly heterogeneous diseases such as skeletal dysplasias, because it allows the identification of mutations in genes whose study by traditional sequencing would
be difficult and expensive, increasing diagnostic time.
P04.60-M
Mutations in DNASE1L3 and familial SLE/Hypocomplementemic
Urticarial Vasculitis Syndrome: the first italian case
Back to index
symptoms appeared (asthenia, severe anemia, C3 and C4 decrease; urticarial dermatitis; microscopic hematuria, petechiae; glomerulonephritis; splenic strokes; interstitial pneumopathy, pulmonary hypertension; mesenteric
ischemia). Interestingly, we also observed joint hyperlaxity with a peculiar
apparent contractions of interphalangeal joints (manually extendable without difficulties). Familial history revealed parents consanguinity; moreover, two other siblings were reported to show signs of an autoimmune disorder. Molecular analysis of DNASE1L3 revealed the homozygous mutation
c.289_290delAC (Thr97Ilefs*2), previously reported in three sisters, born
from consanguineous parents and affected with HUVS (Hypocomplementemic Urticarial Vasculitis Syndrome). This is the first italian reported case of
autosomic recessive SLE/HUVS caused by a mutation in DNASE1L3 gene.
P04.61-S
Spondyloenchondrodysplasia in Brazilian patients with intrafamilial
variability and a pattern of pseudo-dominance
109
ABSTRACTS POSTERS
ge of skeletal dysplasias and deciphering their molecular bases has contributed to the understanding of this complex process. Here, we report a homozygous mutation in the mitochondria-associated granulocyte macrophage colony stimulating factor-signaling gene (MAGMAS) in a novel and severe
spondylodysplastic dysplasia. MAGMAS, also referred to as PAM16 (presequence translocase-associated motor 16), is a mitochondria-associated
protein involved in preprotein translocation into the matrix. We show that
MAGMAS is specifically expressed in trabecular bone and cartilage at early
developmental stages and that the mutation leads to an instability of the
protein. We further demonstrate that the mutation described here confers
to yeast strains a temperature-sensitive phenotype, impairs the import of
mitochondrial matrix pre-proteins and induces cell death. The finding of deleterious MAGMAS mutations in an early lethal skeletal dysplasia supports a
key role for this mitochondrial protein in the ossification process.
P04.64-M
A novel missense mutation in ST14 in a patient with ichthyosis,
follicular atrophoderma and hypotrichosis
110
Back to index
EDI200 is a recombinant ectodysplasin currently in clinical trials for the correction of abnormal ectoderm development in neonates affected by X-linked
hypohidrotic ectodermal dysplasia (XLHED). For early drug trials, there is a
premium placed on identifying molecular markers of drug response likely to
predict long-term clinical benefit, especially when endpoints in XLHED such
as teeth, hair and sweat function may take months to years to ascertain. The
ectodysplasin-deficient Tabby mouse model for XLHED demonstrates a consistent and sustained phenotype response to EDI200. Through a combined
approach of qPCR and RNA-seq we have begun to map the Tabby ectodysplasin-responsive biological pathways. EDI200 at 2 mg/kg (or vehicle alone)
ABSTRACTS POSTERS
was administered by intraperitoneal injection to newborn Tabby mice, with
tail, back and footpad tissues harvested at time points ranging from two
hours to one month post-injection. Isolated RNA was subjected to analysis
by qPCR for eight genes known to contribute to the development/structure
of ectodermal placodes and appendages. Levels of expression for the ectodysplasin receptor EDAR and the downstream regulator sonic hedgehog
(Shh) were upregulated specifically at 24 hrs post-injection in all tissues.
RNA-seq analysis with DAVID bioinformatics software confirmed these results for EDAR and Shh and extended the gene set analysis to identify novel
and unexpected response pathways including those for fat metabolism and
solute transporters. This combined approach for biomarker validation and
delineation of system response biology will provide an invaluable tool set in
identifying pathways for drug targeting as well as in optimizing drug dosing
in ectodermal dysplasias.
P04.69-S
A new col5a1 genomic variant identified in an italian patient with
ehlers danlos syndrome classic type
Ehlers Danlos syndrome (EDS) is a group of heterogeneous connective tissue disorders. The clinical classification recognizes six subtypes and the
Classic type is the most frequent. Classic EDS is an autosomal dominant
disorder, characterized by skin hyperextensibility, abnormal wound healing
and joint hypermobility. It is estimated that approximately 50% of patients with classic EDS phenotype harbor mutations in COL5A1 and COL5A2
gene. We report a case of 43 Italian patient, presented to Ospedale Maggiore Policlinico (Milan) for clinical and genetic counselling. He showed: soft
and hyperextensibility skin specially at the neck, face and hands, atrophic
scars and joint hypermobility. The cDNA sequencing revealed a new single
base variation in COL5A1 gene. The mutation was a change of C for T in
exon63 and established an amminoacid substitution Leucine by Valine at
the c.1656. This mutation was located on higly conservated domain of the
protein. Sequencing of COL5A2 gene and study of the null allele of COL5A1
gene didnt showed any differences. CGH-Array platform, characterized by
a higher density of probes in those chromosomal regions that are thought
to be related to EDS, didnt revealed any variations. Bioinformatics analysis
identified that this nucleotide sequence is extremely conserved in different
species, indeed, up to date, no mutation or polymorphism have been described for this region. The identification of new genomic variant represent the
first step towards the understanding of symptom causes. Moreover, further
studies on protein structure could be necessary to understand the real properties of this mutation.
P04.70-M
Further phenotypic delineation of metatropic dysplasia
Back to index
been reported. Being a rare disorder with few reports in the medical literature, consultation with an experienced clinical geneticist may be required
before a diagnosis is made. An early diagnosis is beneficial for early referral
of the patients to the appropriate follow-up. These patients are best managed by a multidisciplinary team.
P04.71-S
A review of Molecular Genetic diagnosis of Epidermolysis bullosa
cases from Southwest Iran
Epidermolysis bullosa (EB) is a skin disorder that is divided regarding involved skin area into three main forms: Junctional epidermolysis bullosa
(JEB), dystrophic epidermolysis bullosa (DEB), and epidermolysis bullosa
simplex (EBS). EB ranges from mild to very severe forms with the age of
onset form infancy to childhood. The landscape of EB is extensive blistering
and scarring. Other signs include fused fingers and toes, joint deformities
and alopecia. EB is a rare disorder with autosomal recessive and dominant
genetic pattern. This disease is very rare in southwest Iran. From last decade
to date, 12 individuals were diagnosed for DEB. But, more clinical differential diagnosis was not possible for patients. Mutations in 4 genes COL17A1,
LAMA3, LAMB3, and LAMC2 cause the JEB disease, whereby the LAMB3
gene mutations are responsible for more than 70% of all JEB cases. In contract, mutation in the COL7A1 gene causes all three forms of DEB including
autosomal recessive HS -RDEB, non HS-RDEB, and autosomal dominant
DDEB. Mutations within exons 70-75 of the COL7A1 gene very frequent in
the DEB patients. However, three patients from southwest Iran with DEB
as preliminary diagnosis showed frame shift mutations within exons 73-74,
and another individual was positive for a novel nonsense mutation in the
LAMB3 gene. He was also affected by JEB, and not DEB. We were not able to
detect any mutation in other 8 individuals with EB. Exom sequencing or next
generation sequencing might be helpful to resolve their puzzle.
P04.72-M
Perturbation of Specific Pro-mineralizing Signalling Pathways in
Human and Murine Pseudoxanthoma Elasticum
Pseudoxanthoma elasticum (PXE) is characterized by skin, ocular and cardiovascular manifestations, due to calcification and fragmentation of elastic
fibres. Caused by mutations in the ABCC6 gene, the mechanisms underlying
this disease remain unknown. The knowledge on the molecular background
of soft tissue mineralization largely comes from insights in vascular calcification, with involvement of the osteo-inductive Transforming Growth Factor beta (TGF) family (TGF1-3 and Bone Morphogenetic Proteins [BMP]),
together with ectonucleotides (ENPP1), Wnt signalling and a variety of local
and systemic calcification inhibitors. In this study, we have investigated the
relevance of these signalling pathways as well as apoptosis and ER stress
using immunohistochemistry and mRNA expression profiling in dermal
tissues and fibroblasts of PXE patients, and the eyes and whiskers of the
PXE knock-out mouse. Apoptosis was evaluated by TUNEL staining. We demonstrate upregulation of the BMP2-SMADs-RUNX2 and TGF-2-SMAD2/3
pathway, co-localizing with the mineralization sites, and the involvement of
MSX2-canonical Wnt signalling. Further, involvement of apoptosis is shown
with activation of Caspases and BCL-2. In contrast to vascular calcification,
neither the other BMPs and TGFs nor endoplasmic reticulum stress pathways were perturbed in PXE. Our study shows that we cannot extrapolate knowledge on cell signalling in vascular calcification to a multisystemic
mineralization disease as PXE. Contrary, we demonstrate a specific set of
perturbed signalling pathways in PXE patients and the mouse model, and
propose a preliminary cell model of ECM calcification in PXE.
P04.73-S
Neonatal case of thumb duplication: case report and review
111
ABSTRACTS POSTERS
the most common anomaly of the hand. This congenital anomaly may be
isolated and sporadic or expressed with a syndromes phenotype. Within
the Oberg, Manske, Tonkin (OMT) classification thumb duplications are a
failure of formation and/or differentiation affecting the radial/ulnar axis of
the hand plate. The primary signal centre involved is the zone of polarising
activity (ZPA) in the posterior part of the developing limb bud. Sonic Hedgehog protein, which is expressed in the ZPA, plays a major role in determining radial-ulnar characteristics. Other morphogens are involved in the development of thumb duplications. Instead for an approach of management
the Wassel description is used. Case report. We describe a case of a female
newborn with a isolated hand left preaxial polydactyly. The weight at birth
was 2050g, 45 cm of lenght and head circumference 33 cm, physical examination revealed preaxial hexadactyly with thumb duplication. Extra digit
was mild hypoplasic and the patient was unable to move it independently.
Radiographs showed extra thumb containing two proximal phalanges (type
IV polydactyly, according to the Wassels Classification). Conclusion. In this
condition are recommended to value radiographs of the affected limb to
show whether the rudimentary digit contains skeletal elements, and any associated non-hand anomalies. Surgical reconstruction of the radial polydactyly is indicated, not only for the obvious cosmetic improvement, but also to
obtain a stable, mobile thumb of adequate size and appropriate shape.
P05.01-S
The gene variants in 3 end of prothrombin gene in patients with
idiopathic thrombophilia in Serbian population
Back to index
Abdominal aortic aneurysm (AAA) is multi-factorial disease with life-threatening complications due to mainly asymptomatic course of development. Vascular inflammation induced by oxidative stress contribute to pathogenesis. Inter-individual differences in response to oxidative stress are
partially under genetic control. In this study the associations between the
functional SNPs and (GT)n repeat length polymorphisms in genes involved
in the vascular response to hypoxia/ischemia: HIF1A (hypoxia inducible
factor-1) and HMOX1 (heme oxygenase-1) and the development of AAA
were examined.
The study encompassed a series of 518 AAA patients, 345 patients with
atherosclerotic aortoiliac occlusive disease (AOID) and 498 controls. The
HIF1A rs11549465C>T, rs11549467G>A and HMOX1 rs2071746A>T SNP
genotyping was performed by using predesigned TaqMan SNP-genotyping
assays. For simultaneous assessment of the HIF1A and HMOX1 (GT)n polymorphisms, the method based on multiplex-PCR with fluorescent-labeled
sense primers and fragment size analysis using DNA sequencer has been
developed.
We found, that carriers of the HMOX1 (GT)n repeat long allele (n>27) had
increased risk of developing AAA (OR=1.46 for dominant model, P=.034).
The frequency of carriers of both HMOX1 risk alleles: rs2071746T and/or
long (GT)n repeat in AAAs (58,5%) was higher as compared to AIOD (49,0%,
P=.007). On the other hand, the frequency of noncarriers in AAAs was 0.0%,
as compared to 1.3% in controls (P=.010) and 0.9% in AIOD (P=.066).
In conclusion, HMOX1 gene promoter long (GT)n repeat allele and
rs2071746T, allele related to decreased anti-inflammatory and antioxidant
capacity of heme oxygenase-1, are associated with abdominal aortic aneurysm. Supported by Polish Ministry of Sciences grant NN403_250440.
P05.04-M
Age- and sex-specific causal effects of adiposity on cardiovascular risk
factors
P05.02-M
Molecular analysis of aneurysm genes in familial and sporadic
abdominal aortic aneurysm
The role of aortic aneurysm syndromes genes in abdominal aorta aneurysms (AAA) was investigated by analysing the TGF- pathway genes TGFBR1,
TGFBR2, SMAD3, FBN1, EFEMP2, smooth muscle cells genes MYH11, MYLK
and ACTA2 and the vascular Ehlers-Danlos gene COL3A1 in a large group of
familial and sporadic AAA patients.
Sanger sequencing was performed of all coding exons and exon-intron
boundaries of the aneurysm genes in AAA patients diagnosed. Patients with
at least one affected first-degree relative with an aortic aneurysm were classified as familial AAA (fAAA). In silico analysis was used for assessment of
the clinical significance of the variants.
We found 38 different variants of unknown clinical significance (VUS) in this
study population of 72 AAA patients including 56 fAAA patients (78%) and
16 patients (22%) with sporadic AAA. In fAAA one null mutation in COL3A1
Observational studies have reported different effects of adiposity on cardiovascular risk factors between men and women and among different age
groups, but these aspects have not been investigated with regards to causality.
We estimated the causal effect of adiposity on blood pressure, lipid concentrations, glycemic indices, and markers of inflammation and liver disease in
up to 67,553 individuals using a non-weighted genetic score constituting of
32 genetic variants associated with body mass index (BMI) within a Mendelian randomization framework. All analyses were stratified by age (cut-off,
55 years) and sex.
We found similar effects of the genetic instrument on BMI in different strata.
Each additional allele of the genetic score was associated with an increase
in BMI of 0.03 SD (95% CI, 0.028-0.033; P=3x10-107). We found evidence of
a causal effect of adiposity in non-stratified analysis on blood pressure (systolic and diastolic), circulating lipids (high-density-lipoprotein cholesterol,
triglycerides), glucose homeostasis (HbA1c, fasting insulin), and markers of
inflammation (C-reactive protein, interleukin-6), and liver damage (alanine
aminotransferase and gamma-glutamyl transferase) (all P<0.05). We observed significantly larger causal estimates in younger individuals than in older
for low-density-lipoprotein cholesterol and total cholesterol (Pdiff=0.04 and
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0.02 respectively). For fasting insulin, in secondary analysis, we found a larger effect of adiposity in males than in females (Pdiff=0.01 using FTO variant
as a genetic instrument).
We provide evidence for a causal effect of adiposity on many cardiovascular
risk factors, and observe differential estimates across age and sex for insulin
and circulating lipids.
P05.05-S
Genetics in acquired Long QT Syndrome (aLQTS)
Background: Long QT Syndrome (LQTS) is an inherited arrhytmogenic disease characterized by a prolonged QT interval in association with life-threatening arrhythmias. The LQTS can be congenital (cLQTS) or acquired (aLQTS)
as an adverse response to drugs, hypokalemia or bradycardia. Mutations in
13 genes have been associated with cLQTS, while the genetic background
of aLQTS remains unclear. The aim was therefore to evaluate if a genetic
substrate may contribute to aLQTS. Methods: Through a multicentre study,
211 aLQTS probands were collected from Japan, Italy and France. They were
clinically evaluated and divided into 3 groups according to the baseline QTc
interval: true-aLQTS (females QTc<460ms, males QTc<450ms, n=112),
unmasked-LQTS (females QTc 460ms, males QTc 450ms, n=74), and
unclassified-LQTS (without ECG, n=25). Genetic screening of the 5 major
cLQTS genes was performed and mutations were compared to those of 875
genotyped cLQTS families. Results: Genetic analysis led to the identification of mutations in LQTS-susceptibility genes in 27% of aLQTS probands
(57/211). In the true-aLQTS and unmasked-LQTS, a mutation was detected in 23% and 38% of cases, respectively (p=0.04). Interestingly, KCNH2
was the most mutated gene (62% vs 15% of KCNQ1), while the frequency
of mutations in the cLQTS population were 39% and 48%, respectively. Conclusions: Genes implicated in cLQTS play an important role also in aLQTS.
The likely underlying mechanism would be a critical loss in repolarization reserve even in the presence of just a modest QT interval prolongation.
KCNH2 mutations seem to have a prominent role.
P05.06-M
+11525 C/A Polymorphism in MicroRNA Binding Site of
Angiotensinogen is Associated with Occurrence of Restenosis in
Patients After Myocardial Infarction
Introduction:
Angiotensinogen (AGT) is a key component of the renin-angiotensin-aldosterone system that plays a crucial role in blood pressure (BP) regulation.
Increased BP is a well-known risc factor for various cardiovascular diseases.
With the discovery of microRNAs, polymorphisms in 3-untranslated regions (3-UTR) of known genes became of interest. The aim of our study was
to investigate the possible association of rs7079 in 3-UTR of AGT gene with
the outcomes in patients after myocardial infarction (MI).
Methods:
Total of 652 patients (men) presenting at the emergency room with chest
pain with suspicious MI underwent selective coronarography. Diagnosis of
MI was confirmed in 571 patients. Peripheral blood for DNA isolation was
sampled during hospitalization and the genotypes were determined using
TaqMan Genotyping Assay.
Results:
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Statistically significant differences in age at onset of the first MI were observed among the genotypes of investigated polymorphism, the homozygote
genotypes preseting with MI at younger age (AA/CC vs. AC; 58.39.7 years
vs. 59.919.62 years, p = 0.04). Furthermore, statistically significant association was found between the occurrence of restenosis and AGT genotypes
(p = 0.008). The logistic regression model revealed that the CC and AA homozygotes were at the higher risk of restenosis before 45 years of age compared to AC heterozygotes.
Conclusion:
Our study shows that rs7079 homozygotes present with MI at younger age
and that the risk of restenosis is higher in these patients. This may be partially explained by altered miR-30 and miR-584 binding to 3UTR region of
AGT, as shown previously.
P05.07-S
Targeted next generation sequencing in heritable aortopathies
uncovers unexpected findings
K. Mayer, I. Vogl, T. Slupova, S. Datter, H. Klein;
Center for Human Genetics and Laboratory Diagnostics, Martinsried, Germany.
Aortopathies are characterized by aneurysms, dissections, dilation, and tortuosity of predominantly the thoracic aorta (TAA) but also the abdominal
aorta. Inherited forms comprising both syndromic and non-syndromic entities are genetically heterogeneous and phenotypically overlapping. Currently, mutations in at least 15 genes are known to cause familial thoracic aortic aneurysms (AAT3-AAT8), as well as dominant and recessive syndromic
aortopathies. Molecular testing is impeded by similar phenotypes caused
by mutations in different genes and mutations in the same gene leading to
a wide clinical variability. In this setting NGS of an aortopathy gene panel
offers an efficient alternative to conventional gene-by-gene Sanger sequencing.
We developed a NGS aortopathy multigene panel encompassing 15 genes
(ACTA2, COL3A1, EFEMP2, ELN, FBN1, FLNA, MYH11, MYLK, NOTCH1,
PRKG1, SLC2A10, SMAD3, TGFB2, TGFBR1 and TGFBR2). Coding and adjacent intronic regions were analyzed using Nextera Rapid Capture Custom
Enrichment and 2x150 bp paired-end sequencing on a MiSeq instrument
(Illumina). So far, the analysis of 20 patients with both syndromic and nonsyndromic aortic disease identified eight disease causing mutations in seven
patients in the genes COL3A1, EFEMP2 (compound heterozygous), FBN1
(2x), SMAD3, TGFB2 and TGFBR2, representing a diagnostic sensitivity of
35%. Additionally, two rare variants of unknown clinical significance were
identified in the MYH11 and TGFB2 gene. Surprisingly, two patients with
FBN1 and TGFB2 mutations were clinically diagnosed with vascular EhlersDanlos syndrome supported by ultrastructural findings of a skin biopsy. In
conclusion, patients with aortopathies will benefit from a parallel testing
approach enabling efficient and appropriate surveillance and intervention
measures.
P05.08-M
Association of Apolipoprotein E polymorphism with coronary heart
disease in Bulgarian population
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ABSTRACTS POSTERS
notypes. The current study data suggest that ApoE4 allele is a significant
risk factor for CHD in Bulgarian population. Acknowledgements: This work
was supported by Infrastructural Grant: DUNK01/2/28.12.2009 National
Complex in Biomedical and Translational Research, funded by National Science Fund, Ministry of Education and Science, Bulgaria
P05.09-S
Targeted next generation sequencing of 51 genes involved in primary
electrical disease
D. Proost1, G. Vandeweyer1, A. Rotthier2, J. Saenen3, G. Mortier1, C. Vrints3, J. Del-Favero2,
B. Loeys1, L. Van Laer1;
1
Center of Medical Genetics, Faculty of Medicine and Health Science, University
of Antwerp, Antwerp, Belgium, 2Multiplicom, Niel, Belgium, Antwerp, Belgium,
3
Department of Cardiology, Faculty of Medicine and Health Science, University of
Antwerp, Antwerp, Belgium.
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P05.11-S
Added Diagnostic Value of Multiplex Ligation-Dependent Probe
Amplification of plakophilin-2 in Arrhythmogenic Cardiomyopathy
Background: Arrhythmogenic Cardiomyopathy (ACM) is an inherited cardiomyopathy characterized pathologically by fibro-fatty infiltration and clinically by ventricular arrhythmias and an increased risk of sudden death.
Genetic testing is limited to genes encoding desmosomal components resulting in a diagnostic yield of about 50%.
Aim: The aim of this study is to search copy number variations (CNVs) in
the plakophilin-2 gene (PKP2) in order to increase the mutational detection
in ACM.
Methods: Genetic screening for all 5 desmosomal-encoding genes was carried out in 60 unrelated patients with a clinical diagnosis of ACM by Sanger
sequencing on a ABI-PRISM 3730 (Life Technologies). Genotype-negative
probands underwent Multiplex Ligation-dependent Probe Amplification
(MLPA) using SALSA MLPA kit P168 ARVC-PKP2 (MRC-Holland) and quantitative Real-Time PCR (qPCR) on a Light Cycler 480 (Roche Applied Science)
in search of large deletions/duplications in PKP2.
Results: 60% of ACM patients (n=36) resulted positive on direct sequencing, showing 5 mutations in Desmoglein-2 gene (8%), 14 in Desmoplakin
(23%), 7 in PKP2 (12%), 2 in Desmocollin-2 (3%), 2 in Plakoglobin (3%)
and 6 patients (10%) carried multiple mutations. Additional screening by
MLPA in patients without mutations in ACM-genes successfully identified
a large heterozygous PKP2 deletion, further confirmed by qPCR showing a
PKP2 copy number reduction when compared to control samples.
Conclusions: This study improved the diagnostic genetic yield in our population by nearly 2% for one gene, highlighting the usefulness of performing
additional analysis for CNVs in ACM patients.
P05.12-M
Molecular and clinical analysis of digenic inheritance in a family with
left dominant Arrhythmogenic Cardiomyopathy
K. Pilichou1, P. Sarto2, E. Carturan1, M. Cason1, E. Lazzarini1, R. Celeghin1, M. Perazzolo
Marra1, C. Furioso2, D. Corrado1, G. Thiene1, C. Basso1;
1
Dept. of Cardiac, Thoracic and Vascular Sciences, University of Padua, Padua, Italy,
2
Sports Medicine Unit, Noale Hospital, Noale-Venice, Italy.
Purpose
Mutations in Desmoglein-2 (DSG2), a heart specific cadherin, are common
ABSTRACTS POSTERS
causes of Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). A hot
spot of DSG2 missense mutations targets the consensus cleavage-site of
DSG2 pro-peptide (Arg-X-Arg/Lys-Arg) by Kex2-like proprotein-convertases. These mutations are responsible for severe phenotype associating frequent severe right ventricular dysfunction and left ventricular involvement.
In the present work, we aim to explore the pathophysiological mechanisms
of these mutations.
Methods and Results
We performed ex vivo analysis on heart samples from two mutation carriers, and in vitro analysis by expressing wild-type (WT) or mutants proDSG2-GFP fusion proteins in cellular models. First, we demonstrated that all
mutations prevented N-terminal propeptide cleavage. Using Biacore technology, we demonstrated that the presence of propeptide led to the loss of
interactions between EC1 domains of cadherins, known to interact to structure desmosomes. Uncleaved pro-DSG2 mutants were correctly addressed
to the intercellular junctions (cellular models) or at the intercalated disks
(human tissue). However, we observed at low calcium concentration a misincorporation of pro-DSG2 mutants into desmosomes associated with an
EGFR-dependant internalization of pro-DSG2 mutants and of desmosomal
partners (Plakophillin-2 and Plakoglobin) suggesting increased turn-over of
the unprocessed pro-DSG2. Mutants mis-incorporation in desmosomes was
further confirmed by western-blot in cells submitted to cyclic mechanical
stress showing an increase in mutant pro-DSG2 in the desmosome-independent soluble fraction as compared to DSG2-WT.
Conclusion
Our results strongly suggest a loss in desmosomal adhesiveness due to misincorporation of uncleaved pro-DSG2 mutant into desmosomes that might
play a central role in ARVC pathophysiology.
P05.14-M
Human Genetic Evidence that Common Variants near PIK3CG are
Associated with Atherosclerotic Plaque Hemorrhage and Vessel
Density
Aim: Atherosclerotic plaques may vary among individuals, in part due to heritable factors. However, the genetic architecture of plaque phenotypes is
largely unknown. A common variant near PIK3CG on 7q22.3 has previously
been associated with carotid plaque presence. Animal models suggest that
PIK3CG may play a role in plaque formation through neovascularization. We
hypothesized that the PIK3CG variant is associated with intraplaque hemorrhage (IPH) and vessel density in human plaques. Secondarily we focused
on characterizing the functional role of genetic variants near PIK3CG in advanced human atherosclerosis.
Methods: We collected 571 Athero-Express Biobank Study patients, and
genotyped them using Affymetrix SNP 5.0. After quality control we tested
rs17398575 for association to immunohistochemically scored IPH and
vessel density, correcting for age, gender and 10 principal components. We
used the BiKE cohort to assess the effect of PIK3CG variants on PIK3CG expression in circulating monocytes (n=95) and in carotid plaques (n=126).
Results: The reported PIK3CG variant, rs17398575 (risk allele A,
frequency=0.72), was associated with IPH (OR=1.40 [1.10-1.69 95% CI],
p=0.0271) and vessel density (=0.095 [0.0415 s.e.m.], p=0.0221).
The SNP dependent PIK3CG expression demonstrated a differential effect
in the vascular wall (p=0.783 for rs17398575) compared to monocytes
(p=0.0261 for rs17398575).
Conclusion: To our knowledge this is the first report involving the association of genetic variants to histological plaque phenotypes in humans. Further
research should focus on replicating these results and elucidating the etiology of plaque vessel formation and intraplaque hemorrhage, as epidemiological studies demonstrated these associate with cardiovascular disease.
P05.15-S
Assessment of genetic risk for cardiovascular disease in Azores and
mainland Portugal: a healthy population-based study
Back to index
Cardiovascular diseases (CVD) are a major source of morbidity and mortality worldwide, including the Azores archipelago where CVD have a higher
mortality rate when compared to mainland Portugal. In order to investigate
this question, we characterized 15 SNPs in 4 genes and 1 genomic region
associated with CVD risk: 4 in PCSK9 (rs11591147, rs11206510, rs562556,
rs505151), 4 in APOE (rs405509, rs429358, rs7412, rs439401), 4 in LDLR
(rs2228671, rs5927, rs1433099, rs2738466), 1 in USF1 (rs10908821)
and 2 in 9p21 (rs10757274, rs1333049). Genotyping was performed by
real time PCR using TaqMan Assays in a sample of 170 Azorean and 108
mainland Portuguese healthy individuals. Results demonstrate that allele
frequencies were similar in both populations; however, there were statistically significant differences for two SNPs: rs10757274 (9p21) and rs405509
(APOE; 2, p<0.05). Genotype evaluation of rs1333049 (9p21), the most replicated risk SNP for CVD, showed that 19.4% and 18.5% of Azoreans and
mainland Portuguese, respectively, present homozygosity for this risk allele.
Moreover, it is possible to observe that, although there is no statistical significance, Azoreans (9.1%) carry a higher frequency of APOE4 allele compared to mainland (8.8%). Overall, the results are suggestive of an increased
risk for CVDs in Azoreans compared to mainland; however, the joint analysis
of all variants is being carried out. Finally, the results validate the need to
study regional differences in Portugal, information that should integrate the
Portuguese National Health Plan, in order to achieve a more direct and accurate strategy in terms of prevention and community health.
P05.16-M
A comprehensive multidisciplinary approach for the validation of the
2013 diagnostic criteria in Brugada Syndrome
Background: Brugada syndrome (BrS) is a cardiac channelopathy characterized by a coved-type ST-segment elevation in the right precordial leads. The
controversy about the value of high intercostal spaces (ICSs) and the number of diagnostic leads has been settled by a recent consensus statement.
We have tested the validity of the new ECG diagnostic criteria through a
multidisciplinary approach, including the molecular one. Methods: We analyzed 114 BrS patients with a spontaneous or drug-induced type 1 pattern
recorded in one or more right precordial leads in 4th, 3rd and 2nd ICSs. The
right ventricular outflow tract (RVOT) was localized by echocardiography.
Molecular screening of SCN5A was performed through DHPLC and direct
sequencing. Results: In total, 23 patients (21%) were carriers of a diseasecausing mutation. The percentage of mutation carriers (MCs) and the cardiac event rate were similar irrespective of the diagnostic ICS (4th vs high
ICSs: MCs,23% vs 19%; cardiac event rate 22% vs 28%) and the number of
diagnostic leads (1 vs 2: MCs 20% vs 22%; cardiac event rate 22% vs 27%)
used. The concordance between RVOT anatomical location and the diagnostic ICSs was 86%. Conclusion: The percentage of SCN5A-MCs and the cardiac event rate are similar regardless of the diagnostic criteria used. The site
where the diagnostic pattern is recorded is mainly due to the anatomical
location of the RVOT. This study supports the robustness of BrS diagnosis as
achieved through the new diagnostic criteria.
P05.17-S
Mutational analysis of mitochondrial DNA in Brugada Syndrome
patients
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tients. The aim of this study was to assess a possible involvement of mitochondrial (mt) DNA variants in Brugada syndrome since their etiological
role has been already described in several cardiomyopathies.
To reach this goal, mitochondrial genome of BrS patients was sequenced
and analyzed. A specific mtDNA mutation responsible for Brugada syndrome can be excluded. However the most severe spontaneous ECG type 1
symptomatic patients show a high substitution rate in their mitochondrial
genome. These patients also share a combination of four mt single nucleotide polymorphisms (SNPs: T4216C, A11251G, C15452A and T16126C), not
found in asymptomatic subjects (either spontaneous ECG type 1 or induced)
with a low number on mtDNA SNPs.
Our evidences suggest that the detected mtDNA allelic combination and a
high number of mtDNA SNPs could represent an important cofactor in manifestation of BrS phenotype and seem to be associated to a higher risk for a
more severe clinical state of Brugada syndrome.
P05.18-M
SCN5A mutation analysis in 147 Brugada syndrome probands
SCN5A mutation analysis in 147 Brugada syndrome (BrS) probands identified 27 variants possibly associated with cardiac channelopathies, which
resulted in a genetic diagnostic yield of 17,4%. 18 (66,7%) SCN5A variants
have already been associated with BrS, 4 (14,8%) with other cardiac arrhythmias and 5 (18,5%) are novel undescribed variants. When only taking
into account the patients with a baseline BrS type I ECG (8,2%) the genetic
diagnostic yield increased to 41.6%. Interestingly, this was not observed in
patients with a BrS type II ECG (11,6%), in which SCN5A
variants seem to be either novel or associated with other arrhythmias. We
also identified a substantial number of described SCN5A mutations (> 9%)
in BrS patients clinically diagnosed by ajmaline positive testing and a family
history of BrS and/or sudden cardiac death (80,2%), demonstrating the added value of sodium channel blocker-induced ECG testing.
Segregation analysis was performed in available families of the identified
SCN5A positive BrS probands to determine genotype-phenotype correlations. In more than 66% of tested families there was an incomplete segregation of the discovered variant. Revision of all ECG data revealed that some ajmaline or baseline ECG negative BrS patients with SCN5A mutations did not
meet the stringent diagnostic criteria but demonstrated conduction disease.
These results also urge for the need to revise the clinical diagnostic criteria
for BrS, in order to stratify BrS patient groups based on more detailed phenotypic data necessary to discover novel major disease associated genes.
P05.19-S
Identification of candidate genes for Brugada Syndrome by targeted
next generation sequencing
2,5
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of each gene to that observed in repeated random sampling of healthy controls from 1000 genomes project data. Besides confirming an important role
for sodium, potassium and calcium ion channels, our results identified new
candidate BrS genes previously associated with other forms of inherited
cardiopathies, such as ANK2, RYR2, DSG2, LMNA, suggesting an overlap between different disorders; however, many patients still remained genetically
uncharacterized, prompting more extensive studies and suggesting a possible multigenic aetiology.
P05.20-S
A protein network of common susceptibility genes provides a link
between inflammation and cardiovascular disease
Genome-wide association studies (GWAS) have identified hundreds of susceptibility loci for chronic and inflammatory disease phenotypes in humans.
There is increasing evidence that chronic inflammation is a crucial driver in
the pathogenesis of cardiovascular diseases (CVD), which may be genetically determined. To understand the genetic architecture underlying chronic
inflammation and CVD we performed a systematic analysis of (1) common
risk alleles coming from published GWAS, (2) of protein-protein interaction (PPI) networks informed by (3) gene expression data with a defined
molecular target involved in the inflammatory processes promoting CVD,
myeloid-related protein (MRP) 8. (4) Through analysis of integrated haplotype scores (iHS) and FST values in HapMap phase 2 data, we investigated
whether recent selection pressure acting upon inflammatory genes affected
CVD susceptibility loci. Our findings provide significant evidence for a PPI
network (P = 0.033), which connects inflammatory and cardiovascular susceptibility genes, and establish a genetic framework of inflammatory CVD.
41.59% of PPI genes are associated with immune functions. 28.3% of integrated genes can be linked to both, an inflammatory and cardiovascular
disease phenotype. Interestingly, CDKN2B, and CELSR2/PSRC1/MYBPHL/
SORT1, unequivocally replicated CVD loci, are integrated within this network
as are several SNPs located in transcription factor recognition sequences, i.e.
NFKB1, STAT3, which are key factors in inflammation. Finally, we observed
a significant enrichment of inflammatory variants within CVD loci that are
targets of selection (P=2.001e-11 in CEU population), suggesting that recent
selective sweeps may have affected the genomic architecture underlying
CVD.
P05.21-S
Systematic screening of rare coding variants in genes involved in
cardiac arrhythmias
ABSTRACTS POSTERS
P05.22-M
Targeted oligonucleotide-selective sequencing of 101 genes from 150
patients with idiopathic dilated cardiomyopathy in Finland.
T. Alastalo1, O. Akinrinade1, L. Ollila1, T. H. Ojala1, H. Koillinen1, M. Kaartinen2, J. W.
Koskenvuo3, S. Myllykangas1, J. Tallila4, M. Gentile4, T. Heli2;
1
University of Helsinki, Helsinki, Finland, 2University Hospital Helsinki, Cardiology,
Helsinki, Finland, 3University of Turku, Turku, Finland, 4Blueprint Genetics, Helsinki,
Finland.
The strength of next generation sequencing (NGS) in both research and diagnostics is becoming increasingly evident. It can be successfully applied to
find causal mutations and confirm the clinical diagnosis in genetic cardiomyopathies. However, exome sequencing (ES) show incomplete representation and coverage of several exons, leading to clinically relevant mutations
being missed. Therefore ES will, at least for now, coexist in clinical genetic
diagnostics with other NGS-based strategies, such as targeted resequencing.
To this end, using an enrichment kit targeting 48 genes associated with hereditary cardiomyopathies and analysing 90+ patient samples, we demonstrated that the sensitivity, specificity and robustness of targeted NGS are
equal to those of Sanger Sequencing (SS). Subsequently, we constructed
an improved kit targeting 55 genes and implemented this into routine diagnostics. Using this kit, 700+ patients have been analysed, and additional
haplotype and cosegregation analyses were performed to further support
pathogenicity of potentially causal mutations. Our results show that: (1) our
approach results in significant increase in diagnostic yield, as in up to 50%
of patients (potentially) pathogenic mutations were identified; (2) critical
evaluation of the clinical diagnoses showed that higher diagnostic yields are
achieved for patients fulfilling the respective cardiomyopathy subtype criteria; (3) TTN mutations account for a significant part of the yield; (4) in >10%
of cases two or more (potentially) pathogenic mutations were identified;
(5) further haplotype and cosegregation analyses support the pathogenicity
of a significant number of potentially causal mutations. Taken together, our
gene-panel based approach largely improved genetic diagnostics in cardiomyopathies.
P05.24-M
The genetic basis of early-onset cardiomyopathies in Finland
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deletion and microduplication syndromes, are frequently associated with
congenital heart diseases (CHDs). The present work aimed to study copy
number variants (CNVs) in the 22q11.2 region of 87 CHD patients from So
Miguel Island, Azores.
The CNVs were searched in all patients using MLPA, according to MRCHolland protocol. Results showed that four (4.6%) out of 87 CHD patients
presented CNVs, which were confirmed by aCGH in patients 1 and 2, and by
FISH in patients 3 and 4. Patients 1 and 3, both affected with a ventricular
septal defect, carried a de novo 2.5 Mb deletion of the 22q11.2 region, whereas patient 2, with an atrial septal defect, carried a de novo 2.5 Mb microduplication (2:1). Finally, patient 4 showed a 2.5 Mb triplication (3:1) and
presented dysmorphic facial features, cognitive deficit, and aortic stenosis,
a clinical feature not reported in the first case described in the literature. Interestingly, the evaluation of this patients parents revealed that her non-affected father had a 2.5 Mb microduplication (2:1). Now we are investigating
by microsatellite analysis the mechanisms responsible for microduplication
and triplication.
In summary, the present study allowed the identification of very rare deletion and microduplication syndromes in Azorean CHD patients. Moreover,
we report the second patient with a 22q11.2 triplication, whose clinical features increase the symptoms that could be present. This work emphasizes
the relevance of biomedical research, since it can help paediatricians and
other professionals to better assess health care needs.
P05.27-S
Congenital heart malformations in patients with 22q11.2 deletion
syndrome
Background: Truncus arteriosus (TA) accounts for ~1% of all congenital heart defects (CHD) in live birth. The etiology of isolated TA is largely
unknown; syndromic TA is mostly associated with chromosome 22q11 deletion. Hadassah Cardiogenetic Project includes a detailed registry and biobank. Within the frame of this Project we now report the results of a study of
patients with multiple conotruncal malformations accompanied by athymia.
Methods and Results: The subjects were patients originating from two unrelated families. Following the exclusion of 22q11 deletion, exome analysis
was performed in one patient from each family. A homozygous mutation in
chr8: 23560417InsA, p.Lys152fs*0, in the NKX2-6 gene was identified in
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patients from both families. The mutation segregated with the disease in
the families and was absent from large cohorts of controls. Conclusions:
NKX2-6 encodes a homeobox-containing protein which is expressed in
mouse caudal pharyngeal arches and outflow tract at E8.0-E9.5. NKX2-6
was previously shown to be regulated by TBX1. The clear phenotype associated with a homozygous deleterious mutation in our patients, falls well
within the spectrum of the cardiac defects seen in DiGeorge syndrome, is
in agreement with NKX2-6 downstream location in the TBX1 signaling pathway and confirms NKX2-6 role in human cardiogenesis..
P05.29-S
Is Type 2 diabetes gene ABCC8 coding a subunit of KATP channel
associated with coronary artery disease in Turkish people?
Background
Cardiomyopathies are a heterogeneous group of diseases with various etiologies. The potential involvement of genes encoding desmosomal proteins,
usually associated with arrhythmogenic right ventricular cardiomyopathy
(ARCV), was preliminarily evidenced in Caucasian patients with dilated
cardiomyopathy (DCM). Accordingly, we investigated this potential genetic
overlap in a large cohort of patients with clear diagnosis of DCM and belonging to different ethnicities.
Methods
DNA from 455 DCM patients, referred to our tertiary centres in Pavia and
Cape Town, was collected following complete clinical evaluation. 290 samples (184 Caucasians; 84 black Africans; 5 Indians; 17 mixed ancestries) were
screened for the two main ARVC genes: plakophilin-2 (PKP2) and desmoplakin (DSP).
Results
Of the 290 patients tested, 9 (3.1%) were found to carry a most likely pathogenic variant, being absent in the publicly available databases (nearly
20.000 controls) and functionally relevant through 6 bioinformatic tools.
Respectively, 3 (3.6%) black Africans (1 in PKP2, 2 in DSP), 5 (2.7%) Caucasians (2 in PKP2, 3 in DSP), and 1 African patient of mixed-ancestry (1 in
DSP) were positive. Variants of unknown significance (VUS) were found in
15 patients; interestingly, one Caucasian carried two VUS in DSP, suggesting
a potential compound effect.
Conclusion
ABSTRACTS POSTERS
Our data confirm the presence of potentially damaging mutations in desmosomal protein genes in patients with DCM. The prevalence of these mutations is similar in black Africans and in Caucasians.
P05.31-S
eNOS as hypertension susceptibility gene: example from genomewide association study to functional and clinical evidences
Essential hypertension (EH) is a multifactorial disease with obscure etiology and pathogenesis. Transcriptome of patients with EH is virtually unexplored. We performed the analysis of gene expression in peripheral blood
leukocytes of patients with EH and healthy individuals using microarray
technology (RT2ProfilerTM PCR Array, SABiosciences Corporation, Qiagen)
with subsequent validation of the obtained results by quantitative real-time
RT-PCR. We discovered a group of genes with altered transcriptional activity
in hypertensive patients: CCL16, CCL17, CCL18, CCL19, CCL23, CCL8, CCR6,
CCR8, CX3CR1, CXCL1, CXCL13, ICEBERG, IL17C, IL1F10, IL1F6, IL1F9,
SPP1, CD40LG, XCR1, CCL2. Further quantitative analysis of genes with
altered transcriptional activity was performed using cDNA samples of 32
EH patients and 31 control subjects. The results have confirmed significant
differences of CCL18, CX3CR1, CXCL1, CXCL13, IL10, IL13, and CCR2 expression level between cases and controls (=0.001). Relative expression level
changes in EH patients were more pronounced for CX3CR1 gene (29.2-fold),
CXCL13 (13.8-fold), IL1F6 (12.9-fold), CD40LG (8-fold), CXCL1 (7.2-fold).
Functional analysis of differentially expressed genes was performed using
Gene Ontology Biological Process and Kyoto Encyclopedia of Genes and Genomes databases. The genes with altered transcriptional activity in EH patients were found to encode for cytokines and cytokine receptors involved
in immune response and inflammation, and their action was mediated via
cytokine signaling pathway. These findings confirm both the hypothesis of
implication of blood cells trancriptome in the pathogenesis of EH and the
hypothesis of inflammatory basis of the development of EH. The study was
supported by the RFBR grants 13-04-01561_a, 14-04-01169_a.
P05.33-S
Application of exome sequencing in differential diagnosis of pediatric
hypertrophic cardiomyopathy
L. Pezzoli1, C. Lodrini1, M. Sana1, C. Marrone2, D. Marchetti1, A. Lincesso1, K. Migliorati1,
M. Iascone1;
1
Laboratorio Genetica Medica, AO Papa Giovanni XXIII, Bergamo, Italy, 2Cardiologia
Pediatrica, AO Papa Giovanni XXIII, Bergamo, Italy.
Back to index
Recent studies suggested that resting heart rate (RHR) might be an independent predictor of cardiovascular mortality and morbidity. Nonetheless, the
interrelation between RHR and cardiovascular diseases is not clear. In order
to resolve this puzzle, the importance of genetic determinants of RHR has
been recently suggested, but it needs to be further investigated.
The aim of this study was to estimate the contribution of common genetic
variations on RHR using Genome Wide Association Study.
We performed a Genome Wide Association Study in an isolated population
cohort of 1737 individuals, the Italian Network on Genetic Isolates - Friuli
Venezia Giulia (INGI-FVG). Moreover, a haplotype analysis was performed.
A regression tree analysis was run to highlight the effect of each haplotype
combination on the phenotype. A significant level of association (p<5x10-8)
was detected for Single Nucleotide Polymorphisms (SNPs) in two genes expressed in the heart: MAML1 and CANX. Founding that the three different variants of the haplotype, which encompass both genes, yielded a phenotypic
correlation. Indeed, a haplotype in homozigosity is significantly associated
with the lower quartile of RHR (RHR58 bpm). Moreover no significant association was found between cardiovascular risk factors and the different
119
ABSTRACTS POSTERS
haplotype combinations. Mastermind-like 1 and Calnexin were found to be
associated with RHR. We demonstrated a relation between a haplotype and
the lower quartile of RHR in our populations. Our findings highlight that genetic determinants of RHR may be implicated in determining cardiovascular
diseases and could allow a better risk stratification.
P05.36-M
Different genetic background in the clinical onset of Hypertrophic
Cardiomyopathy analyzed by Next-Generation Sequencing
More than 20 genes have been correlated with Hypertrophic Cardiomyopathy (HCM), making the molecular diagnostic process time consuming,
complex and often inconclusive. To better understand the molecular bases
of HCM and to assess the utility of next-generation sequencing (NGS) into
clinical laboratory practice we designed a panel to analyze 17 HCM related
genes on PGM Ion Torrent. We selected 70 HCM patients, 35 early onset
(35 years) and 35 late onset (65 years), with a well-defined hypertrophic
phenotype.
All samples had on average 98% of target regions with coverage higher
than 20X, with a mean coverage of 600X. Common variants (MAF>5%)
were removed from the analysis, while potential splice variants and novel
single nucleotide variants were analysed with bioinformatics pathogenicity prediction programs. We identified 40 different pathogenetic mutations
(9 novel) in 9 genes: MYBPC3 (16/40=40%); MYH7 (14/40=35%); TNNT2
(3/40=7.5%); CAV3 (2/40=5%); and GLA, MYH6, TNNI3, MYL2 and MYL3
(1/40=2.5% each). The mutation detection rate was 9/35 (26%) in the late
onset and 29/35 (83%) in the early onset group (p<0.0001). Considering
only early onset patients with positive family history the detection rate was
>90%.
Our results showed a strong difference in genetic background of HCM patients for age at onset, family history and clinical features of the disease. Genetic testing with NGS was reliable and allowed the molecular diagnosis especially for cases with early onset and positive family history. These results
also demonstrated that an appropriate selection is required for molecular
testing in patients with HCM.
P05.37-S
HDAC9 gene is overexpressed in stroke patients
Aim: HDAC9 is a class IIa histone deacetylase family member and it regulates the epigenetic status of histones and therefore gene expression by catalyzing deacetylation. Risk variant rs11984041 of HDAC9 gene has been
demonstrated to be associated with large vessels stroke. HDAC9 expression
has been correlated with common carotid intima-media thickness, suggesting an association of HDAC9 gene with the atherosclerotic process, by
accelerating atherosclerosis or promoting plaque instability. In this study
we investigated the expression of HDAC9 in peripheral blood (PB) of stroke
patients (large vessel and cardioembolic) and healthy controls. Aiming to
understand the mechanism by which the risk allele is associated with large
vessel stroke, we also evaluate the effect of rs11984041 polymorphism on
gene expression.
Methods: HDAC9 gene expression analysis was performed on 26 atherothrombotic stroke patients, 26 cardioembolic stroke patients, and
20 healthy controls by Real Time PCR using Sybr Green. Polymorphism
rs11984041 was genotyped by PCR-RFLP method.
Results: A significant increase of HDAC9 gene expression was observed in
atherothrombotic and cardioembolic stroke patients compared to healthy
controls (1.390.68 vs 0.610.36; p<0.001; 1.581.19 vs 0.600.36;
p<0.001, respectively).
The genotyping of 70 individuals showed no gene expression differences
between patients carrying CC and CT genotypes.
Conclusions: The gene HDAC9 is overexpressed in stroke patients compared to controls. This result indicated that this gene, that can regulate other
genes implicated in the peripheral inflammatory reaction after stroke can
represent a new target for the development of therapeutic agents against
ischemic stroke injury.
120
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P05.38-M
Novel mutations in the ZIC3 gene cause X-linked heterotaxy
Congenital heart defects (CHD) is one of the most common congenital abnormalities in newborns, affecting almost 1% of the general population and
is associated with substantial morbidity and mortality. A small subset (3%)
of this patient population fails to correctly establish left-right patterning during embryogenesis, resulting in abnormal lateralization of the abdominal
and thoracic organs, a clinical phenotype called heterotaxy. As the heart is
the first organ to develop asymmetrically, disturbances in the left-right axis
lead to a variety of serious cardiac malformations in heterotaxy. One of the
first genes linked to heterotaxy was ZIC3, a zinc transcription factor of the
GLI superfamily, located on the X-chromosome. The ZIC3 gene is a transcriptional regulator, based on the ability to activate transcription of target genes
and to bind DNA.
Over the last 10 years we collected and diagnostically tested over 300 patients referred to our clinical genetics center with heterotaxy and/or a variety
of heart defects for mutations in the ZIC3 gene. We identified five potentially
pathogenic mutations, as well as variations in the N-terminal polyalaninerepeat. The mutations were detected in families with a clear X-linked clinical
profile. To support pathogenicity of detected mutations we performed several functional assays. We used confocal imaging to detect subcellular localization of mutated proteins and the in vivo zebra fish model to investigate the
potential laterality and/or cardiac defects of these mutated proteins.
P05.39-S
Copy Number Polymorphism study in Essential Hypertension
Essential Hypertension is a complex trait influenced by multiple susceptibility genes interacting with environmental factors. We used 2044 hypertensive cases and 1872 normotensive controls recruited over many years
across European regions within Hypergenes project and genotyped with
Illumina-1M arrays.
Cases and controls were genotyped together to avoid the batch effect for
chips and time periods. The Log R ratio of the samples for each SNP was obtained from Genome Studio and the Copy Number (CN) call was performed
in Golden Helix after quality controls. CN was estimated in each sample as
a mere signal intensity interval measured at a particular locus. Thorough
quality control to refine the signal intensity was essential before CN call.
Approximately 9% of the samples uniformly distributed between cases and
controls were removed for signal to noise ratio and waviness. The PCA analysis on wave corrected data doesnt show demarcation on sex/phenotype,
whereas the distinguishable genotyping batches were corrected. Among the
824 segments identified and discretized into 3 state CN, 13 segments were
significantly associated with the phenotype.
Odds ratio for these segments were calculated and we identified a top common variant around 1kb in 1st intron of LEPREL1 gene with an odds ratio of
1.7 for hypertension (95% CI 1.3-1.9, p-value = 3.01 x10-12). This result suggested that carriers of that CNP had a 1.7 times higher risk for hypertension
than non-carriers. Further studies are needed to confirm the association of
this CNP with hypertension in other dataset.
P05.40-M
Whole exome sequencing concentrating on the metabolome
in familiar hypertrophic- and dilated cardiomyopathy: a pilot
collaborative Czech study
ABSTRACTS POSTERS
mogenic disease or myopathies (TTN, RYR2, CALR3, MYH 7, TPM1, JPH2,
DSP, ACTC1, KCNH2, FLNC, SYNE2, CASQ2, VCL, PKP2). Three variants were
detected in MYO1C, JAG1, SYNE1 that have not been associated with CM,
thus far. Only 12/17 identified genes are included in the currently available
targeted TruSight assay. In 3/11 families the potential causative variant was
found only in one gene. The combination of two to three pathogenic variants
in CM-associated genes were found in 8/11 families. Pathogenic potential of the identified variants must be substantiated by segregation and/or
RNA/protein analyses. The failure to detect variants in two large families
underscores the limitations of NGS. Nevertheless, our preliminary data suggest, that the use of metabolome NGS in complex families may have higher
diagnostic yield than targeted approaches in CM.Supported by conceptual
research of FNM 64203, CZ.2.16/3.1.00/24022 and IGA NT13770.
P05.41-S
Identification of Mexican-specific lipid variants using a novel crosspopulation GWAS approach
Back to index
sis. Here, we describe an individual affected with HCM who carries a deletion of the entire MYBPC3 gene.
The patient was diagnosed with non-obstructive hypertrophic cardiomyopathy in his mid forties when undergoing assessment for palpitations and
hypertension. Echocardiogram revealed moderately dilated left and right
atria with mild mitral regurgitation. There was severe asymmetric left ventricular hypertrophy with interventricular septal hypertrophy. The left ventricular systolic function was normal with an ejection fraction of 65-70%.
MLPA analysis showed a deletion of all exons of the MYBPC3 gene. Subsequent microarray analysis confirmed this deletion and showed that it extended both 5 and 3 of MYBPC3 and included 3 additional known OMIM
morbid genes (DDB2, SLC39A13 and RAPSN). Unlike MYBPC3, these additional genes are implicated in autosomal recessive disorders and therefore
deletions of these genes are not expected to have clinical significance in this
patient.
Our results suggest that although large copy number variation of HCM-related genes would still be considered rare, patients highly suspected of HCM
may benefit from MLPA analysis if other deleterious variants have not been
identified.
P05.43-S
The c.912_913delTT mutation in MYBPC3 gene is a founder
mutation accounting for one-fifth of the Italian patients affected with
hypertrophic cardiomyopathy
M. De Bortoli1, C. Calore2, C. Romualdi1, A. Lorenzon1, M. Calore1, G. Poloni1, G. Rizzon2,
A. Ortile2, G. Thiene2, S. Iliceto2, P. Melacini2, A. Rampazzo1;
1
Department of Biology, Padua, Italy, 2Department of Cardiac, Thoracic and Vascular
Sciences, Padua, Italy.
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ABSTRACTS POSTERS
mutations in 7/34 patients (20,6%), 3 are novel mutations. Search for causative mutations is an integral part of the HCM diagnosis. The finding of a
causative mutation in a patient, permits the identification of family members who may have the disease in an asymptomatic way or who may develop disorders in the future and may in turn transmit the mutation to their
offspring.
P05.45-S
Semiconductor (Ion Torrent) massive parallel sequencing of the main
Hypertrophic Cardiomyopathy genes based on two tubes multiplex
amplification of DNA pools
J. Gmez, J. Rodrguez-Reguero, C. Mors, E. Coto;
Hospital Universitario Central De Asturias, Oviedo, Spain.
At least 30 different genes have been linked to Hypertrophic Cardiomyopathy (HCM), with MYBPC3 and MYH7 accounting for most of the mutations.
Due to the large size of these genes, Sanger-sequencing of single amplicons
is labor-intensive and expensive. Next generation sequencing (NGS) could
facilitate the genetic screening of the HCM-genes in large cohorts. Our purpose was to designate and validate a procedure for sequencing the 9 most
commonly mutated genes in HCM (MYH7, MYBPC3, TNNT2, TNNI3, ACTC1,
TNNC1, MYL2, MYL3, and TPM1), based on two-tubes multiplex amplification of DNA-pools (Ampliseq; 176 primer-pairs covering 15,690 bp, 98,62%
of the target coding sequence) followed by semiconductor array sequencing
with the Ion Torrent Personal Genome Machine (PGM; Life Technologies).
We created a pool with DNA from 13 patients previously Sanger-sequenced
for the coding exons plus at least 5 intronic flanking nucleotides of the nine
genes. Each patient was heterozygous for a unique rare variant (including a
small deletion); each unique allele (control variant) was thus diluted 1/26
inside the pool. We sequenced the DNA-pool in a medium capacity Ion chip
316 (semiconductor 100 Mb) array, and the data was analyzed with the
Torrent Suite software optimized to detect insertion/deletion nucleotide
changes. We successfully identified all the 13 control variants (including the
indels). In conclusion, we developed a NGS protocol for the main HCM genes.
The multiplex amplification of DNA pools would reduce the time and cost of
screening these genes at a population scale.
P05.46-M
Role of genetic testing in diagnosis and management of idiopathic
ventricular fibrillation cases
122
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Center for Medical Genetics, Faculty of Medicine and Health Sciences, University of
Antwerp, Edegem, Belgium, 2Department of Human Genetics, Radboud University
Nijmegen Medical Centre, Nijmegen, Netherlands, 3Department of Cardiology, Radboud
University Medical Center, Nijmegen, Netherlands, 4Department of Cardiology,
Maastricht University Medical Center, Maastricht, Netherlands, 5Department of Clinical
Genetics, Maastricht University Medical Center, Maastricht, Netherlands.
1
Marfan syndrome (MFS) is a multi-systemic autosomal dominant connective tissue disorder caused by mutations in the FBN1 (fibrillin-1) gene, but
approximately 10% of MFS cases remain genetically unsolved. Here, we report a new FBN1 mutation in an MFS family that had remained negative
after extensive molecular genomic DNA FBN1 testing, including denaturing
high performance liquid chromatography (DHPLC), Sanger sequencing,
multiplex ligation-dependent probe amplification (MLPA) and micro-array
analysis for copy number variation. In addition, four other aneurysm-linked
genes were screened for mutations by Sanger sequencing, TGFBR1, TGFBR2,
SMAD3 and ACTA2, without any result. Linkage analysis in the family revealed a large linked region on chromosome 15 which was confirmed by
microsatellite analysis. Cultured proband fibroblasts and subsequent cDNA
sequencing revealed a double peak pattern at the junction of exon 56 and
57. Sanger sequencing of intron 56 revealed a deep intronic point mutation
generating a new splice donor site (c.6872-961A>G; ENST00000316623).
Together with an existing cryptic splice acceptor site, this mutation results
in the integration of a 90 bp pseudo-exon between exons 56 and 57 containing a stop codon and causing nonsense-mediated mRNA decay. Although
more than 90% of FBN1 mutations can be identified with regular molecular
testing at the genomic level, deep intronic mutations will be missed and require cDNA sequencing or whole genome sequencing.
P05.48-M
Compound-heterozygous Marfan syndrome?
Introduction
Marfan syndrome is an autosomal-dominant condition often caused by mutations in the fibrilllin-1 (FBN1) gene. Here we report a family where the
proband is compound-heterozygous for two mutations in FBN1.
Case
The proband (II:1) was on suspicion of Marfan syndrome at 21 years of age.
An aortic dilation was found at age 17, during clinical investigation for rheumatoid arthritis. Family history was ambiguous: His father was operated for
an aortic aneurism at age 30, allegedly caused by endocarditis. Sequencing
using an NGS panel for Marfan and Marfan-like syndromes was undertaken.
Two mutations were found in FBN1; one previously reported as causative
for Marfan syndrome, the other novel. Results of family investigation and
clinical findings are reported in table 1.
Interpretation
Compound heterozygousity has previously been reported in Marfan syndrome. Neither of the two mutations found are present in ESP, nor found among
2000 Danish exomes. In the present case we find it more likely that the previously reported supposed pathogenic mutation found in I:2 is of no/little
phenotypical consequence. It was originally found in an isolated case of aortic dissection where no further details were given. The involved amino acid
is not evolutionary conserved. Individual I:2 is without clinical symptoms of
Marfan syndrome at age 55. The novel c.7694G>A mutation is in silico predicted to be likely pathogenic and segregates with the clinical phenotype.
Medical
Diagnosis
history
Aortic
dissection at
Marfan
Marfanoid face,
c.7694G>A;
age 36
syndrome
wrist sign, arm-span
Childhood
according to
p.Cys2565Tyr
to height ratio
(novel)
surgery
revised Ghent
1,05
for pectus
criteria
carinatium.
c.4727T>C;
p.Met1576Thr
Normal
Healthy
(previously
reported)
Marfanoid face,
wrist sign,
Marfan
c.7694G>A;
asymmetric chest,
syndrome
p.Cys2565Tyr
pes planus, myopia Aortic dilation according to
c.4727T>C;
(-7,5 bilaterally),
revised Ghent
p.Met1576Thr
striae
criteria
Height 200 cm
I:1
Mother
I:2
Proband
II:1
P05.49-S
Clinical features
ABSTRACTS POSTERS
Several loci enriched in lower frequency variants are associated
to risk factor for metabolism and cardiovascular diseases in 4,000
Italian isolated individuals
Back to index
Objective
Sudden cardiac death (SCD) is responsible for a large proportion of deaths
in young individuals. After routine post-mortem investigations, many cases
are still unexplained. Implementation of genetic investigations in forensic
medicine may increase the diagnostic rate not least in sudden unexplained
death (SUD). With next generation sequencing (NGS), genetic analysis has
become faster and more efficient, enhancing the outcome. The purpose of
the study was to explore the yield of genetic analysis using NGS in forensic
pathology and to compare the genetic findings to routinely diagnosed patients.
Methods and results
Genetic investigation was performed in 44 unrelated individuals; 15 forensic SUD cases and 29 unrelated patients diagnosed with channelopathies.
In-solution targeted sequencing capture probes were custom designed by
Roche NimbleGen, including all exons of 31 genes, and sequenced on the
Illumina MiSeq. Larger deletions and insertions in five genes were investigated with multiplex ligation-dependent probe amplification. Of the SUD
cases, 21% were found to have a probably pathogenic variant. The corresponding hit-ratio in the patient cohort was 37%. Two patients (7%) had
large deletions.
Conclusion
By NGS, it was possible to detect a probably pathogenic single nucleotide
variant in one fifth of the SUD cases, disposing to a cardiac disease. In contrast, almost half of the patients were found to carry a probably pathogenic
variant or larger deletion. Genetic investigation with NGS can be used as a
diagnostic tool in both a forensic and clinical setting.
P05.53-S
PRDM16 and MIB1 mutations are rarely identified in nonsyndromic
left ventricular noncompaction cardiomyopathy
123
ABSTRACTS POSTERS
MIB1 mutations to nonsyndromic LVNC we analysed the coding regions of
PRDM16 and MIB1 in a cohort of 41 nonsyndromic LVNC patients (39 adults
and 2 children). In PRDM16, we identified 5 synonymous variants, and five
missense variants. Two of these missense variants were found in 40 and 14
patients respectively and are considered single nucleotide polymorphisms
(SNPs). Two variants are considered likely pathogenic (c.89C>T; p.A30V and
c.1882G>A; p.D628N) and one was classified as a variant of unknown significance (VOUS: c.2290G>A; p.V764M). We did not find any mutations in the
MIB1 gene. Conclusions We identified two likely pathogenic variants and
one VOUS in the PRDM16 gene, supporting previous evidence of mutations
in the PRDM16 gene as a rare cause for LVNC. The MIB1 gene did not contribute to LVNC in our cohort.
P05.54-M
Cardiovascular malformations caused by NOTCH1 mutations do not
keep left: data on 52 mutation carriers
Rationale: The genetic basis of idiopathic pulmonary arterial hypertension (iPAH) is well recognized but still rarely utilized in diagnostic setting.
Hundreds of mutations in coding exons and introns of, at least, seven genes
have been reported to associate with idiopathic and familial forms of PAH.
Comprehensive genetic testing can improve diagnostics, prognostics and
treatment optimization of the index patient but it also allows effective screening of asymptomatic family members, which is a basis for well-informed
genetic counseling. Methods: We adopted the novel oligonucleotide-selective sequencing (OS-Seq) and developed a custom data analysis and interpretation pipeline to identify pathogenic base substitutions and insertions and
deletions in seven genes associated with PAH: BMPR2, BMPR1B, ACVRL1,
ENG, SMAD9, CAV1 and KCNK3. Our sequencing panels covered >99% of
the targeted regions with >15x coverage. We validated our diagnostic panels using reference HapMap samples with known genomes and mutations.
Results: The sensitivity of OS-Seq to detect base substitutions was >99%
and specificity was >99.9%. The accuracy to detect short INDELs was 100%.
We then analyzed 22 iPAH patient samples to identify known and novel variants potentially associating with the disease. We confirmed all candidate
124
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variants using direct sequencing. The utilized novel technology allows significant cost reduction and rapid turnaround time from sample to clinical
interpretation. Conclusion: These results demonstrate that the developed
high-throughput targeted OS-Seq platform for PAH meets the technical criteria of clinical diagnostics and is a cost-efficient tool in clinical research.
P05.56-M
Clinical and molecular characterization of Chilean patients with
Noonan Syndrome- multiple lentigines
Introduction: Noonan Syndrome-Multiple Lentigines ( NS-ML) is a rare autosomal dominant disorder, and one of the conditions known as RASopathies. PTPN11, RAF1, and BRAF are the genes known to be associated with
NS-ML. Sequence analysis of coding exons 7, 12, and 13 of PTPN11 detects
missense mutations in about 90% of individuals tested.
Objective: The aim of this study was to characterize the clinical and molecular features of Chilean patients with NS-ML. Methods: collaborative and descriptive study. Results: thirteen patients have been confirmed molecularly.
This includes two familial cases. The age at diagnosis ranged from 20 days to
33 years old. In six patients including one of the family cases, the recurrent
mutation p.Tyr279Cys in exon 7 was identified. They showed interfamilial
and intrafamiliar variable expressivity. Four patients have mutations in exon
12, three with the recurrent mutation p.Thr468Met and one with an infrecuently reported one, p.Ala461Thr. Two different mutations were identified
in exon 13, in three patients. The uncommon mutation p.Gln510Glu, was
found in two patients, both with prenatal cardiac manifestations, and a Hypertrophic cardiomyopathy (HCM) rapidly progressive. The other mutation
at codon 510 is a Gln510Pro. Discussion: The diagnosis of this condition
may be challenging because clinical features overlap with other RASopathies. Although NS-ML seems to be rare, we have diagnosed a considerable
number of patients. We developed a high index of suspicion particularly in
patients with HCM. Eventually specific mutations could be predictor of adverse cardiac events.
Grant sponsor: Pediatric Division , Pontificia Universidad Catlica de Chile
P05.57-S
The SMAD-binding domain of SKI: a hotspot for de novo mutations
causing Shprintzen-Goldberg syndrome
ABSTRACTS POSTERS
be due to mutations in the SKI gene, encoding the oncoprotein SKI, a repressor of TGF activity. Here we report eight recurrent and three novel SKI
mutations in eleven SGS patients. All were heterozygous missense mutations located in the R-SMAD binding domain, except for one novel in-frame
deletion affecting the DHD domain. Adding our new findings to the existing
data clearly reveals a mutational hotspot, with 74% (23 out of 31) of the
hitherto described unrelated patients having mutations in a stretch of five
SKI-residues (from p.(Ser31) to p.(Pro35)). This implicates that the initial
molecular testing could be focused on mutation analysis of the first half of
exon 1 of SKI. As the majority of the known mutations are located in the RSMAD binding domain of SKI, our study further emphasizes the importance
of TGF signaling in the pathogenesis of SGS.
P05.58-M
Mutations in the TSPYL1 gene are not associated with sudden infant
death syndrome in a Swiss cohort of deceased infants
Back to index
P05.60-M
Are ALOX5AP SNPs a risk or protective factor for Stroke?
The incidence of sudden cardiac death (SCD) increases with age in parallel
with coronarys diseases prevalence. In young persons and athletes, SCD
occurs in half of the cases, in the setting of genetically transmitted disorders
such as cardiomyopathies. Molecular testing performed after necropsy may
help management of families but experience in this area appears very limited. The aim is to report our experience of post mortem molecular testing
after SCD and necropsy. We studied 36 patients <40 years who died suddenly with a suspected diagnosis of cardiomyopathy, established either after
autopsy or known before death , with 6 dilated cardiomyopathy (DCM), 12
hypertrophic (HCM), 2 HCM/DCM , 1 restrictive (CMR), 14 arrhythmogenic
right ventricular cardiomyopathy (ARVC), 1 HMC and left ventricular noncompaction. Fifteen mutations have been identified in sarcomeric or desmosomal or lamin genes. The identification of these mutations had significant
impact: assessing right diagnosis in a doubtful case (HCM without LVH),
modifying the appropriate diagnosis in another case (HCM and not DCM),
providing guidance for genetic counselling and predictive genetic testing in
relatives in all situations. Technical, ethical and legal issues may however
be encountered and will be discussed. This study is one of the first series
of post-mortem molecular testing after SCD which suggests the feasibility,
efficiency and the benefit of the approach to improve the management of
families. Postmortem molecular testing must take its place in the strategy
of family care after SCD, even if a cardiomyopathy is suspected at necropsy,
since genetic findings provide additional information.
P05.62-M
Mutation detection rate and -characteristics in thoracic aortic
aneurysm (TAA) related disorders: results from next generation
sequencing (NGS) panel testing
125
ABSTRACTS POSTERS
Back to index
FBN1
TGFBR1 TGFBR2
TGFB2 SMAD3
COL3A1
ACTA2
10
1
2
3
2
3
1
9MFS
1LDS
2S-HTAD 1S-HTAD
1vEDS
Clinic
1LDS
1NS-HTAD
1NS-HTAD
1NS-HTAD 1MFS
1MFS 2NS- HTAD
Total
P05.63-S
A new COL3A1 transgenic mouse model recapitulates the clinical
features of vascular Ehlers-Danlos Syndrome
Vascular Ehlers-Danlos Syndrome (vEDS) is a severe, life-threatening heritable connective tissue disorder, characterized by translucent skin, easy
bruising, and propensity to rupture of arteries and hollow organs. The molecular basis of vEDS has been well-studied, showing a wide range of mutations in COL3A1, encoding type III procollagen. Most mutations result in a
glycine substitutions, pivotal for correct folding of the triple helical domain.
The mechanisms by which mutant type III collagen cause vascular fragility are not well understood, but factors, other than mechanical failure, are
believed to contribute to the phenotype. A Col3a1 knock-out mouse model
shows early lethality and is therefore not suitable for functional studies.
We generated a transgenic vEDS mouse model with a Col3a1 missense mutation. With a BAC transgenic approach, the Col3a1 c.547G>A (p.Gly183Ser)
mutation, a typical helical glycine substitution, was introduced into the
C57BL/6 mouse genome. Spontaneous development of open skin wounds
was observed in all transgenic males, but not in females. Expression analysis
on dermal fibroblasts revealed substantial higher expression of type III collagen in transgenic males compared to transgenic females. Biomechanical
testing of both transgenic males and females showed significantly reduced
tensile strength of the skin, aorta and colon. Transmission electron microscopy of skin and aortic tissues showed severely abnormal collagen fibrils,
intracellular spaces between smooth muscle cells and dilation of the endoplasmic reticulum (ER) , indicating excessive protein load and ER-stress.
This novel animal model provides new opportunities for in-depth analyses
of the pathogenic basis of vEDS and for possible therapeutic interventions.
P05.64-M
Genetic tests in the diagnosis of congenital vascular malformations
126
P05.65-S
Association of VAV2 and VAV3 polymorphisms with cardiovascular
risk factors in hypertensive and diabetic patients
ABSTRACTS POSTERS
suggested that patients with AA genotype in VKORC1 (-1639G>A) require
lower doses of warfarin than those with AG or GG genotype. Height and
weight did not have a significant correlation with the warfarin maintenance
dose. In addition there was no significant relationship between sex and ethnicity with the maintenance dose of warfarin (p< 0.05).
P05.67-S
Analysis of c.1166A>C polymorphism in 3UTR region of AGTR1 gene
in patients with Pulmonary Arterial Hypertension
G. Pousada1,2, A. Baloira3, D. Valverde1,2;
1
Dep. Biochemistry, Genetics and Immunology. Universidad de Vigo, Vigo, Spain,
2
Instituto de Investigacin Biomdica de Vigo (IBIV), Vigo, Spain, 3Servicio de
Neumologa del Complejo Hospitalario de Pontevedra, Pontevedra, Spain.
Pulmonary arterial hypertension (PAH; OMIM 178600) is a progressive vascular disorder characterized by pulmonary vascular resistance increase,
vascular remodelling and right heart failure. Angiotensin II is both a potent
vasopressor hormone and a primary regulator of aldosterone secretion. It is
an important factor that controls blood pressure and volume in the cardiovascular system. AGTR1 gene plays an integral role in blood pressure control, and is involved in the pathogenesis of hypertension. In this study, we
demonstrated the association between c.1166A>C polymorphism and PAH.
We included 55 PAH patients and 52 controls. Using specifically designed
primers we amplified and sequenced the 3-untraslated region of AGTR1.
We analyzed the c.1166A>C polymorphism located on 3UTR of AGTR1 gene.
By comparing genotype frequencies of controls and patients with PAH, we
obtained statistically significant differences for this SNP between the two
groups (p>0.001). The RR of developing PAH in patients with this SNP is
20.3; IC 95%; p<0.001. The statistical analysis of clinical and hemodynamics parameters between patients with or without this SNP showed significant differences in systolic pulmonary pressure (p=0.037), cardiac index
(p=0.012) and PAH subtypes (IPAH vs APAH) (p=0.028). This SNP is present
in 72.4% of IPAH patients and in 67.8% of APAH patients, producing a more
severe phenotype. This SNP only appears in 25% of control cohort.
In conclusion, this polymorphism in AGTR1 gene is more frequent in IPAH
than in APAH patients and predispose individuals to an increased risk of
developing PAH associated with a more severe phenotype.
P06.01-S
10 novel HGD mutations identified in black bone disease patients
Alkaptonuria (AKU, black bone disease) is caused by mutations in homogentisate-1,2-dioxygenase (HGD) gene leading to deficiency of HGD enzyme
activity. Clinically it leads mainly to homogentisic aciduria, ochronosis and
painful and disabling ochronotic arthritis. AKU was described by Sir Archibald Garrod as the first inborn error of metabolism in 1902, and now, more
than 110 years later, we test nitisinone as the first possible treatment of
this disorder. We analysed for HGD mutations 40 AKU patients enrolled for
the first part of this 7FP-supported project DevelopAKUre run in two study centres in Piestany (Slovakia) and Liverpool (UK). In this cohort we observed 20 different pathogenic variants, two of which were novel. The first
mutation (R53Q c.158G>A) was present in homozygous state in one patient
of Indian origin. The second one (T167I,c.500C>T) was identified in one
copy in Slovak AKU patient, further confirming the genetic heterogeneity
of AKU in this small country with increased incidence of AKU, where now
already 13 mutations are reported. Eight additional novel HGD mutations
were found in AKU patients sent to our laboratory for a routine molecular diagnostics within a frame of different collaborations. The novel R53Q
mutation identified in DevelopAKUre was found also in one Italian patient.
One mutation was identified in a patient from Brasil (M186K, c.557T>A), in
patient from India (ivs7+6T>C,c.469+6T>C), from France (F147S,c.440T>C
), and five in cases from Italy (K248E,c.742A>G// G205D,c.614G>A//
K353Q,c.1056A>G// G251D,c.752G>A// Y40S,c.119A>C)). The total number of different AKU-causing mutations as published in our HGD mutation
database is now 126.
Back to index
P06.02-M
Intrafamilial gene rearrangements in Barth syndrome leading to
different TAZ mutations in siblings
GM1-gangliosidosis and galactosialidosis are rare autosomal recessive lysosomal disorders associated with defects in the same protein complex.
GM1 gangliosidosis is caused by a deficiency of -galactosidase, whilst
galactosialidosis is associated with deficiencies of both -galactosidase
and -neuraminidase activity secondary to a defect in the protective protein cathepsin A. We have developed a dried blood spot enzyme assay for
-galactosidase and have added a reference enzyme (-galactosidase) which
is measured simultaneously as a measure of sample integrity. A panel of 300
unaffected individuals were tested to establish a normal reference range; all
5 affected GM1-gangliosidosis patients tested showed clear enzyme deficiency. -neuraminidase is an unstable enzyme which is therefore unsuitable
for dried blood spot analysis and difficult to interpret in leucocyte samples.
Differential diagnosis of GM1-gangliosidosis and galactosialidosis has traditionally been carried out by measurement of -neuraminidase in cultured
fibroblasts; however skin biopsy and cell culture can take many weeks. Nested PCR and Sanger sequencing analysis of the GLB1 and CTSA genes can be
carried out at this centre from the same dried blood spot sample as enzyme
testing, providing a rapid diagnosis from a single convenient sample.
P06.04-M
Unraveling Boucher-Neuhauser syndrome: the multifaceted
consequences of PNPLA6 gene mutations
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ABSTRACTS POSTERS
taining 6) and its role in the metabolism of lysophospholipids. Biochemical
and molecular investigations were undertook in a Brazilian kindred affected
by a complex neurological disorder, presenting mainly as a spinocerebellar
ataxia with hypogonadism and early vision loss. After molecular analysis
of genes associated with spinocerebellar ataxia and exclusion of other inborn errors of metabolism (IEMs) associated with cerebellar disease, whole
exome sequencing (WES) was performed. Mutations in the PNPLA6 gene
were identified in all affected patients. They presented with very early onset visual loss (chorioretinal dystrophy) accompanied by progressive cerebellar ataxia and primary hypogonadism. Brain MRI showed cerebellar
and pons atrophy. Peripheral neuropathy (motor axonal neuropathy) was
identified in all of them. Mutant mice with mutations in PNPLA6 gene have
shown a relentless neurological disease. PNPLA6 codifies a neuropathy target esterase (NTE) hydrolase, an integral membrane protein located on the
endoplasmic reticulum. It shows phospholipase activity, being involved in
the hydrolysis of lysophosphatidylcholine to yield glycerophosphocholine.
Boucher-Neuhauser syndrome is a clear example of the profound biological
consequences of mutations in such gene in the central and peripheral nervous system.
P06.05-S
Dissecting the genetic architecture of loci with established effects on
multiple cardiometabolic phenotypes
128
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culties and typical physical features. At 6 months she developed progressive hepatosplenomegaly, liver cirrhosis, and ascites. After a life-threatening
deterioration, she responded to aggressive management with diuretics, albumin infusions, and repeated paracentesis. She was noted to have pancytopenia with marked thrombocytopenia at 2 years of age, with platelet counts
in the range of 10-20x109/liter. She did not improve with IVIG or steroids.
She had a normocellular marrow with mild decrease of megakaryocytes and
granulocytes, which suggested a primary consumptive thrombocytopenia,
with some component of reduced megakaryopoesis. As she had become refractory to transfusion, splenectomy was performed in this medically fragile child and resulted in a moderate recovery of platelet levels. This is the
second reported case of severe thrombocytopenia in CDG1a, and the only
reported case of splenectomy with this disorder.
P06.07-S
Citrin deficiency caused by a novel mutation in the SLC25A13 gene clinical, biochemical and genetic characterization of new Caucasian
case
Mutations in MMACHC cause the most common inborn error of vitamin B12
(cobalamin) metabolism - cblC. Patients with this disease are unable to convert cobalamin into the two active forms, methylcobalamin and adenosylcobalamin; consequently, they have elevated homocysteine and methylmalonic acid levels in blood and urine. Some cblC patients also have structural
abnormalities, including congenital heart defects. In the mouse, Mmachc
has tissue and stage-specific expression during organogenesis (Pupavac,
M. et al. Mol Genet Metab. 103, 401-405 (2011)). We generated mice with
a gene-trap insertion in intron 1 of the Mmachc gene, (MmachcGt(AZ0348)Wtsi).
Mice heterozygous for this gene-trap allele were viable and fertile, but showed a 50% reduction in Mmachc protein compared to wild-type littermates.
The MmachcGt allele was inherited with a transmission ratio distortion in
a subset of matings. Homozygous MmachcGt embryos were not found after
embryonic day 3.5. These studies indicate that the Mmachc gene product is
essential for early mouse development.
ABSTRACTS POSTERS
P06.09-S
POLG mutations in Polish patients with mitochondrial disease of
unknown etiology - preliminary data
Congenital Disorders of Glycosylation (CDG) are a genetically heterogeneous group of disorders caused by the alteration in synthesis and structure
of protein and lipid glycosylation. To date, more than 60 different causes of
CDG have been defined genetically. CDG is also characterised by extremely
variable phenotypes with manifestations ranging from severe developmental delay and hypotonia beginning in infancy, to hypoglycemia and proteinlosing enteropathy with normal development. Till recently, finding the causative mutation of CDG patients has been impaired by this extreme genetic
heterogeneity: it was virtually impossible to Sanger sequence all candidate
genes. Instead, one or few genes were tentatively selected based on a combination of biochemical, cell biological and glycobiological investigations. In
order to obtain a better diagnostic yield, we designed a capture assay for
a panel of 79 genes associated with CDG type I, CDG type II and congenital muscular dystrophy-dystroglycanopathy. To evaluate the assay, targeted
sequencing was performed for 16 CDG type I and 15 CDG type II patients.
The mean coverage in the target region was about 600x and a genotype was
called for more than 97% of the targeted bases. A diagnosis was confirmed
in 8 cases. Interestingly, mutations could also be detected (and confirmed)
in the gene ALG1 that could not be assayed by genomic Sanger sequencing
due to the abundance of pseudogenes. After our targeted assay enabled an
increased diagnostic yield, the the most interesting, unsolved cases will be
subjected to exome sequencing for gene discovery.
P06.11-S
A patient with congenital disorders of glycosylation type 1q due to
mutation in SRD5A3
Back to index
ted a type 1 glycosylation defect, thus this disorder was classified as CDG1q.
We present here a 4 month old male with coarse face, hypertrichosis and
developmental delay. The baby was the fourth child of first-cousin parents.
He had narrow forehead, depressed nasal bridge, proptotic eyes, large fontanelle (6x8cm), long philtrum, thin lips, loose skin, hepatosplenomegaly
and bilateral inguinal hernia. He also had iris coloboma, glaucoma, nystagmus, corneal clouding and elevated serum transaminases. He was followed
up until 27 months of age. He had severe hypotonia and no head control at
this age. Dark pigmentation of dorsum of feet was developed. His cranial MR
imaging revealed cerebellar hypoplasia, enlargement of lateral ventricules
and frontal atrophy. Serum transferring IEF showed a type 1 pattern. Serum IGF1 and antithrombin level also were decreased. Using homozygosity
mapping followed by whole-exome genotyping, we described a homozygous
missense mutation (c.320G>A; p.Trp107X) in the SRD5A3 gene in the patient. This mutation was reported previously in a Turkish patient with hypotonia and similar eye and cranial findings.
P06.12-M
Congenital Hyperinsulinism of Infancy (CHI): hunt for new genes
Congenital Hyperinsulinism of Infancy (CHI) is a rare disorder, characterized by heterogeneity in clinical and genetic features. An inappropriate insulin secretion is responsible of hypoglycaemia, which can result in serious
neurological damage and life-long handicap. The genetic causes of CHI have
been found in genes regulating insulin secretion from pancreatic beta-cells
but in about 50 % of CHI patients the molecular defect remain unknown.
Hunting for novel CHI-causing genes we performed whole-exome sequencing (WES) on 10 CHI patients [Proverbio MC et al, 2013; doi:10.1371/journal.pone.0068740]. To pinpoint the causal mutation in a small number of
samples and to select the most promising candidate genes, we implemented
a computational strategy including: 1) a bionformatics pipeline to identify
a high quality single nucleotide variants (SNV); 2) an exome homozygosity mapping using a novel algorithm H3M2 [Pippucci T el al, 2011; doi:
10.1159/000330164] and 3) a prioritization analysis using the list of rare
and novel coding variants by mean of web tools such as ToppGene suite,
Endeavour and Gene Distiller. Our approach resulted in a selection of small
number of genes that significantly correlate with human phenotype and
molecular pathways associated to congenital hyperinsulinism and insulin
signalling. Among them we selected missense mutations affecting CDKAL1
and PIK3R1 for further investigations either at the level of protein structure
(in silico protein modelling) and in respect to their role in beta cell using
INS1-E cellular model and human pancreatic islets.
P06.13-S
Whole-exome sequencing for genetic diagnosis in congenital
hypothyroidism
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ABSTRACTS POSTERS
families harbouring known TG/TPO/DUOX2 mutations have a conclusive
genetic diagnosis and novel recessive nonsense mutations here identified
are also likely causative. Wider pedigree-genotype correlation is needed to
confirm the pathogenicity of the remaining recessive missense mutations
here identified.
P06.14-M
Efficiency of the integrated Danon disease diagnostic protocol can
be demonstrated in families affected by LAMP2 exon-copy number
variations
130
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dactyly, which is typically postaxial and rarely interdigital and can involve
all four limbs, and syndactyly of the toes. Chondrodysplasia punctata (CDP)
is specifically associated with a subgroup of DCB (Greenberg dysplasia,
CHILD syndrome, X-linked dominant chondrodysplasia punctata, male EBP
deficiency); the possible occurrence of epiphyseal stippling in SLOS, initially reported, does not appear to be confirmed. CDP is also associated with
other congenital disorders such as chromosomal abnormalities, brachytelephalangic CDP (X-linked recessive CDP, disruption of vitamin K metabolism,
maternal autoimmune diseases), peroxisomal disorders (rhizomelic CDP)
and lysosomal storage disorders. In the differential diagnosis of epiphyseal
stippling, a moth-eaten appearance of bones, asymmetry or the presence
of the common pattern of limb abnormalities are further indicators of DCB.
In conclusion, the specific differentiating radiological features of DCB are
highlighted.
P06.16-M
Regional reference range lysosomal storage diseases in the
Kazakhstan
M. Bayanova, B. Kamaliyeva, D. Samatkyzy, G. Abildinova;
National Research Center Maternal and Child Health, Astana, Kazakhstan.
ABSTRACTS POSTERS
P06.18-M
Development of a cell-based reporter assay suited for small-molecule
drug discovery in FGF23-inducible HEK293 cells stably expressing
Klotho
S. Diener1, K. Schorpp2, B. Lorenz-Depiereux1, K. Hadian2, T. M. Strom1,3;
1
Helmholtz Zentrum Mnchen, German Research Center for Environmental
Health,Institute of Human Genetics, Neuherberg, Germany, 2Helmholtz Zentrum
Mnchen, German Research Center for Environmental Health,Institute of Molecular
Toxicology and Pharmacology, Neuherberg, Germany, 3Klinikum Rechts der Isar der
Technischen Universitt Mnchen, Institute of Human Genetics, Munich, Germany.
Fibroblast growth factor 23 (FGF23) is a key regulator of phosphate homeostasis. It is of crucial importance in hereditary and acquired hypo- and hyperphosphatemic disorders. Moreover, FGF23 has emerged as a promising
biomarker for the prediction of adverse clinical outcomes in patients with
chronic kidney disease (CKD), as it might be related to mortality, cardiovascular abnormalities and disease progression. FGF23 is a bone-derived
endocrine factor, which inhibits renal tubular phosphate reabsorption by
activating receptor complexes composed of FGF receptor (FGFR) 1c and the
co-receptor Klotho. As a major signalling pathway mitogen-activated protein kinase (MAPK) pathway is employed. For the investigation of FGF23 in
an in vitro model we established FGF23-inducible HEK293 cells that stably
express Klotho (HEK293-KL). The induction of HEK293-KL cells by FGF23
was shown by detecting the activation of MAPK pathway, which could be reduced by the use of two known small-molecule inhibitors of MAPK pathway:
SU5402 and U0126. To identify novel small-molecule compounds that modulate FGF23/FGFR1c/Klotho signalling, we developed a cell-based reporter assay that is suited for high-throughput screening (HTS). The assay is
based on the AlphaScreen SureFire platform of Perkin Elmer to monitor the
phosphorylation of endogenous extracellular signal-regulated kinase 1 and
2 (ERK1/2) in cellular lysates of HEK293-KL after the induction with FGF23
in the presence of small-molecule compounds. Since increased plasma concentrations of FGF23 are the main cause of many phosphatemic disorders, a
modulation of its effect could be a potential strategy for drug discovery and
new therapeutic approaches in disorders affecting phosphate homeostasis.
P06.19-S
Gaucher disease and Langerhans cell histiocytosis
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D. Gavrilov, A. Studinski;
Departments of Medical Genetics, and Laboratory Medicine and Pathology, Biochemical
Genetics Laboratory, Mayo Clinic School of Medicine, Rochester, MN, United States.
We report a patient referred to our service due to a clinical diagnosis of Glycogen Storage Disease type 1. She is the only child of a healthy and non consanguineous couple, seen at the age of six months when hepatomegaly was
detected during a routine pediatric evaluation. Laboratorial investigation
revealed glycemia after 4-hour fasting ranging from 33 to 60 mg/dl, hypertriglyceridemia (252 mg/dl), and abnormal values of aspartate aminotransferase (132 mg/dl) and alanine transaminase (98 mg/dl). Gamma-GT, urea,
creatinine, uric acid, total cholesterol, coagulation tests, blood gases, and
CBC resulted normal. After starting treatment with frequent meals, cornstarch, and dietary restriction of lipids and sugars, she normalized neuromotor and somatic development but still presents with hepatomegaly. Genomic
DNA was extracted from peripheral blood leukocytes of the patient and both
parents by using the standard phenol/chloroform method. Exons of G6PC
and SLC37A4 genes and their flanking intron/exon junctions were amplified
by PCR using previously described primers. The fragments were directly sequenced using a MegaBACE1000 DYEnamic ET (Amersham Biosciences)
apparatus, twice on both strands, and the obtained sequences were compa-
131
ABSTRACTS POSTERS
red with the sequences available in the Ensembl genome browser. Mutation
analysis revealed that the patient is a double heterozygote for the previously
known nonsense mutation p.Arg415Ter (rs121908979; c.1243C>T) in SLC37A4, inherited from the mother, and an undescribed missense mutation
p.Gly222Glu (c.665G>A) in G6PC, inherited from the father. The molecular
analysis of the two genes suggests that the clinical picture of this patient
could be caused by digenic inheritance of GSDI.
P06.23-S
High frequency of the c. 3980 G>A (p.W1327X) mutation in AGL gene
of Tunisian patients with hepatic presentation of glycogen storage
disease type III syndrome
A. Mili1, W. Manoubi1, A. Ayadi2, A. Saad1, M. Gribaa1;
1
Laboratoire de Cytogntique, de Gntique Molculaire et de Biologie de la
Reproduction Humaines, Sousse, Tunisia, 2Service of Pediatry, hopital Tahar Sfar
Mahdia- Tunisia, Mahdia, Tunisia.
BACKGROUND: Glycogen storage disease type III (GSD-III) is an inborn error of glycogen metabolism caused by a deficiency of the glycogen debranching enzyme (AGL) .Some of the mutations appear to be population specific, whereas others are found in probands from a variety of different ethnic
backgrounds. The recurrent mutation W1327X in exon 31 was identified in
the Tunisian population, suggesting a founder effect. In this present study,
we report a phenotype-genotype correlation of this frequent mutation. METHODS: Seven unrelated Tunisian families (from MAHDIA); including 10GSD
type III patients were presented with hepatomegaly, progressive severe
myopathy and cardiomyopathy. The routine laboratory findings showed
an elevated serum aspartate aminotransferase, alanine aminotransferase,
creatine kinase and triglyceride levels. The blood lactate and uric acid levels
were within normal limits. RESULTS: The biochemical results of ten patients indicated a striking elevation of glycogen content in the erythrocytes
after several hours fasting favours type III GSD and completely decrease of
debranching enzyme activity was measured in leucocytes. Mutational analysis of the AGL gene showed a homozygous p.W1327X mutation. Study of
genotyping method, in 30 families, using four polymorphic microsatellite
markers on chromosome arm 1p21, we identified a common haplotype for
all patients originated from MAHDIA.
CONCLUSIONS: p.W1327X is the most characteristic mutation for Tunisian
patients with GSD IIIa. A common haplotype which shows the existence of a
specific effect founder of this population confirmation of GSD-IIIa in Tunisisan (from MAHDIA) patients by clinical and genetic findings.
P06.24-M
A yeast model to evaluate the pathogenicity of missense mutations
causing HHH Syndrome
132
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3-Hydroxyisobutyryl-CoA hydrolase (HIBCH) is an enzyme specific for removing CoA in the catabolic pathway for valine. So far, only 4 cases of HIBCH deficiency have been reported. In 1 case, a homozygous null mutation
in HIBCH caused congenital anomalies including a characteristic facial appearance, congenital heart disease, and multiple vertebral anomalies and
led to death in infancy. The 3 other cases, which harbored missense or splice
mutations, presented with hypotonia, neurological regression, developmental delay in infancy, episodes of ketoacidosis, and abnormal MRI findings in
the basal ganglia. Here, we describe a case of Leigh-like disease in Japanese
siblings with HIBCH deficiency who presented with developmental delay in
infancy, abnormal MRI findings in the globus pallidus, and remarkable ketoacidosis without highly increased levels of pyruvate and lactate in the CNS
during infections. The patients died before the age of 5. A new homozygous
missense mutation was identified at the substrate binging site in these patients, and their parents were found to be heterozygous for this mutation.
The levels of HIBCH activity in patient lymphoblastoid cells and HEK293
cells transiently expressing a mutant HIBCH confirmed that the patients had
HIBCH deficiency. HIBCH deficiency leads to the accumulation of the HIBCH substrate 3-hydroxyisobutyryl-CoA and subsequently an increase in the
amount of methacrylyl-CoA in the mitochondrial matrix. The methacrylylCoA then strongly binds to thiol compounds (cysteamine, cysteine, glutathione) and reduces the activity of mitochondrial enzymes by binding to
their SH residues, thus leading to a dramatically decreased reduction state
and reduced ATP production.
P06.26-M
Deleterious mutations in mitochondrial NADH dehydrogenase
subunit 4 and ATPase subunit 6 in two siblings of Leigh Syndrome
with progressive motor retardation and loss of visual function
M. Urfali, O. Ozdemir, D. Kankaya, A. Uludag, S. Yalcintepe, F. Silan;
Department of Medical Genetics, Faculty of Medicine, Canakkale Onsekiz Mart
University, Canakkale, Turkey.
We report a mitochondrial NADH dehydrogenase subunit 4 and ATPase subunit 6 mutations in two siblings from first degree relative couple with Leigh
Syndrome. Peripheral blood-EDTA samples were used for DNA isolation and
target mitochondrial genes were analysed by MLPA technique (MRC-Holland). A girl of 16 years old presented with a episode of progressive mental
and motor reardation. Until seven year she had normal apperance but at the
age of 7 year, she had developed neurological and imaging features such as;
loss of visual function, walking and speech impairment and epilepsy complaints that compatible with Leigh syndrome. We also present a 12-year-old
boy with muscle weakness, severe neurological problems, hyperactivity, developmental retardation and complete visual loss. Screening of both siblings,
based on their clinical phenotype revealed the homoplasmic deleterious
mutation in the mitochondrial DNA MT-ATP6(ligation site of probe 1206812069) and heteroplasmic deletion in NADH subunit 4(ligation site of probe
8993-8992R). The maternal mtDNA screening for target deletorious genes
is still going on. These findings are consistent with infantile Leigh syndrome
for the presented siblings.
P06.27-S
Lysosomal Storage Disorders (LSDs): Clinical and Genetic Spectrum:
Local Experience at The Eastern Province of Saudi Arabia
N. A. Al-Sannaa1, H. Y. Al-Abdulwahed1, P. M. Mathew2, P. M. Mathew2;
1
Johns Hopkins Aramco Healthcare, Dhahran, Saudi Arabia, 2Vanderbilt Childrens
Hospital, Nashville, TN, United States.
Lysosomal Storage Disorders (LSDs) are clinically and genetically heterogeneous a group of more than 50 inherited disorders, each one is caused by a
deficiency of a particular lysosomal enzyme resulting in a progressive accumulation of specific macromolecules within the lysosomes. This eventually leads to an irreversible cell damage, and multi-organs dysfunction. The
majority of LSDs are inherited in an autosomal recessive manner, with the
exception of Fabry disease and Mucopolysaccharidosis type II which follow
the X-Linked mode of inheritance. The majority of LSDs are pan-ethnic, but
some are more prevalent in specific ethnic groups.
Here, we report our local experience of LSDs at Johns Hopkins Healthcare
Main Hospital, Dhahran, The Eastern Province of Saudi Arabia from January, 1st, 1984 to December, 31st, 2013. A total of 86 patients were diagnosed
with different LSDs within this time period. ucopolysaccharidosis type VI
was found to be the most common disorder (18 %), followed by juvenile
Neuronal Lipfuscinosis (13 %). The patients are distributed according to
ABSTRACTS POSTERS
the geographic location or their tribe origin where particular disorders and
specific genotypes were identified. This information had been utilized for
asymptomatic carrier testing for those selective disorders among the high
risk population. However, this valued data could be further utilized for establishing the newborn screening of LSDs in Saudi Arabia.
P06.28-M
Changes of metabolome and lipidome in lysinuric protein intolerance
(LPI)
J. Kurko1, M. Tringham1, L. Tanner2, K. Nnt-Salonen2, M. Vh-Mkil2, T.
Hytylinen3, M. Oresic3, O. Simell2, H. Niinikoski2, J. Mykknen2;
1
Department of Medical Biochemistry and Genetics, University of Turku, Turku, Finland,
2
Department of Pediatrics, Turku University Hospital and University of Turku, Turku,
Finland, 3VTT Technical Research Centre of Finland, Espoo, Finland.
Lysinuric protein intolerance (LPI; MIM222700) is a rare autosomal recessive disorder caused by a defect of cationic amino acid transport in the
small intestine and kidney tubules. All the Finnish patients share the same
homozygous mutation c.1181-2AT in the SLC7A7 gene which encodes the
y+LAT1 amino acid transporter. LPI can be considered a multisystem disease which can be life-threatening. The main symptoms of LPI include protein
aversion, failure to thrive, osteoporosis and hepatosplenomegaly. However,
despite the homogenous mutation in the Finnish LPI patients, symptoms
may vary markedly even within one family and may include severe complications, such as alveolar proteinosis and end-stage renal disease. Some LPI
patients also suffer from combined hyperlipidemia.
Our recent microarray study revealed that also other amino acid transporter
genes than SLC7A7, including non-cationic amino acid transporters, have
changes in their expression levels in LPI patients compared to the controls.
Therefore, LPI patients seem to have wide and persistent changes in their
amino acid balance. This finding together with the fact that patients have
combined hyperlipidemia let us hypothesize that there may be hitherto uncharacterized systemic metabolic and lipid alterations in LPI. We studied
these alterations in the whole blood plasma samples of 26 Finnish LPI patients and 19 gender and age-matched controls. Global metabolomic and lipidomic analyses were performed with GCxGC-TOF and Q-TOF mass spectrometry, respectively, combined with ultra-performance liquid chromatography (UPLC). Taken together, this study will reveal the nature of system-wide
dysregulation of metabolites and lipids associated with the Finnish founder
mutation of LPI.
P06.29-S
Establishment of Zbtb16 role in metabolic syndrome by means of
single-gene congenic rat strain derivation
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P06.30-M
DNA diagnostics of MIDD and MELAS syndromes in Slovakia
133
ABSTRACTS POSTERS
ditions with a very heterogeneous clinical presentation, initiated by a maternally inherited pathogenic aberration residing in the mtDNA. Over 400
mtDNA alterations (including point mutations and deletions) implicated in
a deleterious OXPHOS are already identified, with tRNA leucine and lysine
genes being hot spot mutation regions (www.mitomap.org, updated 15th November 2013). A UK study for the occurrence of 10 frequent point mutations
in cordial blood of newborns, revealed an unexpected carrier frequency of
1 in 200. Usually, mtDNA aberrations are heteroplasmic, a term referring to
the co-existence of both wild type and variant mtDNA molecules within the
same cell of an individual and opposite to homoplasmy. We present here the
case of a young male patient with a cerebral-vascular accident, but also suffering from migraine, epilepsy and transient lactic acidosis and renal insufficiency. This clinical picture was compatible with a respiratory chain disease caused by a mtDNA mutation. First line analysis of his tRNA leucine and
lysine genes identified both the common MELAS (Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke like episodes) and MERRF (Myoclonic
Epilepsy with Ragged Red Fibers) mutations, in our patients blood. This
is a rather unique and unusual situation. To our knowledge, the presence
of both pathogenic mtDNA mutations, at clinical relevant levels, within the
same patient has never been reported before. Further molecular investigation with sequencing technologies of the leukocytes, urine epithelial cells and
other tissues of the patient and maternal relatives is in progress.
P06.33-S
A unique combination of mitochondrial ribosomal RNA variants
responsible for a form of mitochondriopathy with respiratory
complex I deficit and hypoacusia
134
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P06.36-M
Combined deletions of GALNS and PIEZO1 genes in two patients
affected by MorquioA syndrome
Mucopolysaccharidosis IVA (Morquio A syndrome) is an autosomal recessive lysosomal storage disorder, caused by the deficiency of the enzyme NAcetylgalactosamine-6 sulfate sulfatase (GALNS). The disease, albeit multisystemic, is characterized by prevalent skeletal involvement, short stature,
and cardiorespiratory complications. Here we report two new large genetic
rearrangements detected at the heterozygous level in two patients (Pt1 and
Pt2) affected by Morquio A. The deletions include part of the PIEZO1 gene
(piezo-type mechanosensitive ion channel component 1) whose mutations
were found in the dehydrated hereditary stomatocitosis, a dominant autosomal recessive syndrome. This syndrome is typically associated with silent to mild hemolysis, perinatal edema, splenomegaly, elevated bilirubin
levels and iron overload. Pt1 was found to be seemingly homozygous for the
c.1485C>G (p.Asn495Lys) missense mutation in the exon 14 of the GALNS
gene. Parents genetic analysis, haplotyping some loci in exon 14 of the
GALNS gene (in the context of a prenatal diagnosis of a patients relative),
and CGH-array revealed that Pt1 has a large deletion encompassing exons
10-14 of the GALNS gene and three upstream genes, including a part of the
PIEZO1 gene. In Pt2, heterozygous for the c.346G>A (p.Gly116Ser) missense mutation, a large deletion ablating exons 9-14 of the GALNS gene and
exon 1 of the PIEZO1 gene was also identified. The breakpoint regions were
characterised in both patients and the eventual contribution of the partial
deletion of the PIEZO1 gene on such patients phenotype was considered at
a biochemical and clinical point of view.
P06.37-S
Mucopolysaccharidosis type II: molecular analysis in a cohort of male
patients and in a manifesting female carrier
Mucopolysaccharidosis II (Hunter syndrome, MPS II) is a rare X-linked recessive disorder caused by a deficiency of the lysosomal enzyme iduronate-2- sulfatase. Major clinical manifestations of the severe form of MPSII
include coarse facial features, short stature with joint stiffness, hepatosplenomegaly, cardiomyopathy and mental retardation.
The diagnosis of MPSII based on clinical symptoms and increased excretion
of glycosaminoglycans in urine is confirmed by enzymatic assay and mutation analysis. We performed mutation analysis in 24 unrelated male patients
and found 21 different mutations: two large and four small deletions, three
small insertions, two splicing defects, two nonsense mutations, seven missense mutations and a recombination between IDS gene and pseudogene
IDS2. Eleven of the found mutations are novel.
Even though the MPSII is expected to be found in males, few symptomatic
females have been reported. We present a case of four-year-old girl with
severe phenotype of MPSII. Molecular analysis of gDNA revealed a missense
mutation c.1403G>A (p.R468Q) in heterozygous state, but only the mutant
allele was detected at the cDNA level. X-inactivation was nonrandom and
silencing preferentially the maternal X-chromosome. The mutation was not
found in periferal blood samples of both parents. These findings support
the assumption of either a de novo mutation in affected girl or a germinal
mozaicism in the girls father.
In spite of extremely rare manifestation of MPSII in females, an unrelated
female patient with skewed X-inactivation and p.R468Q inherited from her
mother was reported in the literature.
Support: IGA MZ R NT 14015, RVO-VFN64165/2012, MZ R-RVO
(E,00023761)
P06.38-M
Rare mutation in a patient with MPSII (Hunter disease)
ABSTRACTS POSTERS
Mucopolysaccharidosis type II (MPS II) is an X-linked recessive multisystem
disorder characterized by glycosaminoglycans (GAG) accumulation. Different severity of the disease has been described depending on the mutation
in the iduronat sulphatase gene (IDS). Here we present a case, with severe
MPS II detected early with an unusual duplication of the exon 7.
The child presented with a delay in reaching milestones of the first year and
recurrent febrile episodes. At 11 months he had most of the major features
of the MPSII: hoarse and hairy face, significant thoracic kyphosis, large scull,
hepatomegaly and neurodevelopmental delay. X rays of the spine were typical for MPSII. Urinary mucopolysacharides were increased as well as the
iduronatsulphatase in the urine ( 80nmol/h/ml).
Genetic analysis confirmed duplication of the exon 7 of the IDS gene in the
child and his mother who does not have any signs of the disease.
The patient has been followed for 5 years. His growth during this period
was unusual, he had height 4 SDS above the average for the age, head circumpherence was 3SD above the average. Values of growth hormone were
normal, as well as the MRI of the pituitary region.
Therapy with Elaprase (Idursulphatase) improved the joint mobility, however, the boy has severe neuro-developmental delay.
Although no significant genotype/phenotype relation has been shown in
children with MPSII, it seems that this rare gene change is associated with a
severe form of the disease.
Prenatal diagnosis in the mother is planned for the second pregnancy.
P06.39-S
Exome sequencing identifies mutations in coenzyme A synthase
causing neurodegeneration with brain iron accumulation
Back to index
is now unable to raise arms in horizontal position (61 years old). The second
brother, since 44 years of age, had exercise intolerance, cramps and pain in
lower limbs. He currently has a distal amyotrophy. Genetic analysis revealed
that also one of the two sisters presents the pL56R and pI193F mutations,
but she is still barely symptomatic. Using a functional in vitro assay, we have
observed that these mutations caused the production of ATGL proteins with
diminish lipase activity, but able to bind to LDs. This is a very interesting
family since it shows heterogeneity of clinical presentation from relatively
asymptomatic phenotype to full expression of a severe myopathy.
P06.41-S
Producing hiPS cells for disease modeling of NLSD-M
135
ABSTRACTS POSTERS
P06.43-S
An exome-wide study for obesity in Singaporean East-Asian samples
R. Dorajoo1, S. Ooi2, J. Bei1, Y. Li1, J. Foo1, H. Liany1, X. Zheng1, K. Sim1, K. Loke2, T. Aung3, C.
Cheng3,4,5, Y. Teo1,5,6, T. Wong3,4,7, E. Tai5,8,9, C. Khor1, Y. Lee2,10, J. Liu1;
1
Genome Institute of Singapore, Singapore, Singapore, 2Department of Pediatrics, Yong
Loo Lin School of Medicine, National University of Singapore, National University Hospital
System, Singapore, Singapore, Singapore, 3Singapore Eye Research Institute, Singapore
National Eye Centre, Singapore, Singapore, Singapore, 4Department of Ophthalmology,
Yong Loo Lin School of Medicine, National University of Singapore, National University
Hospital System, Singapore, Singapore, 5Saw Swee Hock School of Public Health,
National University of Singapore, National University Hospital System, Singapore,
Singapore, 6National University of Singapore Graduate School for Integrative Science and
Engineering, National University of Singapore, Singapore, Singapore, 7Office of Clinical
Sciences, Duke-NUS Graduate Medical School, Singapore, Singapore, 8Department of
Medicine, Yong Loo Lin School of Medicine, National University of Singapore, National
University Hospital System, Singapore, Singapore, 9DukeNational University of Singapore
Graduate Medical School, Singapore, Singapore, 10Singapore Institute for Clinical Sciences,
Agency for Science, Technology and Research, Singapore, Singapore.
Table1
Total samples
Sample with call-rate < 0.99
Samples with extreme heterozygozity (>
3 SD)
1st degree relatedness
PCA outliers
Samples remaining
Samples with BMI data
Controls (BMI between 18.5kg/m2 and
23.0kg/m2)
SCES
2576
67
5
38
50
2416
2403
1083
8
5
178
NA
NA
P06.44-M
Identification of new missense variations in the SH2B1 gene in obese
and lean individuals
E. Aerts1, D. Zegers1, J. K. Van Camp1, K. Van Hoorenbeeck2, G. Massa3, A. Verrijken4, I. L.
Mertens5, J. Cassauw1, K. Van Ackeren1, R. R. Rooman2, K. N. Desager2, L. F. Van Gaal5, W.
Van Hul1, S. Beckers1;
1
Centre of Medical Genetics, University of Antwerp, Antwerp, Belgium, 2Department of
Pediatrics, Antwerp University Hospital, Antwerp, Belgium, 3Department of Pediatrics,
Jessa Hospital, Hasselt, Belgium, 4Department of Endocrinology, Diabetology Diseases,
Antwerp University Hospital, Antwerp, Belgium, 5Department of Endocrinology,
Diabetology and Metabolic Diseases, Antwerp University Hospital, Antwerp, Belgium.
136
Back to index
Disorders caused by affected urea cycle enzymes are characterized by hyperammonemia, encephalopathy, and respiratory alkalosis. Ornithine transcarbamylase (OTC) deficiency is an X-linked inborn error of metabolism of
the urea cycle which causes hyperammonemia. The disorder is treatable
with supplemental dietary arginine and low protein diet. Here we report
two unrelated Bulgarian families with OTC deficiency, subjected for genetic
testing. The first patient is a 3 years old female with persisting hyperammonemia, elevated transaminase activity and elevated orotic acid excretion
in urine. The girl demonstrated behavioral and sleep disturbances. The genetic test revealed a novel frameshift mutation c.695dupT in exon 7 of the
OTC gene. The mutation is de novo, both parents are not carriers. After the
genetic verification of the disease, the patient was subjected to low protein
diet and ammonaps with a very good outcome. The second patient is a 1
year old boy with abnormal profile of organic acids (organic aciduria), undetectable citruline level and hyperammonemia. The genetic test revealed
a missense mutation c.622G>A, p.(Ala208Thr) in exon 6 of the OTC gene.
The mutation is available in the literature and inherited from the mother.
The carrier mother does not demonstrate any clinical symptoms of the disease. The presented data enlarges the spectrum of OTC gene mutations in
patients with persisting hyperammonemia. The genetic testing provides the
possibility for adequate genetic counseling and prenatal diagnostics in affected families.
P06.46-M
Biochemical diagnosis of Peroxisomal disorders by GC/MS: Egyptian
patients with X-linked adrinoleukodystrophy
M. M. Ibrahim, E. M. Fateen, A. S. A. Gouda;
National Research Centre, Cairo, Egypt.
Introduction: Peroxisomes are organelles responsible mainly for metabolism of lipids and peroxides. Lack of peroxisomes or dysfunction in any of
their normal functions is the cellular basis for human peroxisomal disorders
(PDs).
Aim of the Work: diagnosis of peroxisomal disorders among a high risk
group of Egyptian patients using gas chromatography mass spectrometry.
Subjects and Methods: Forty six patients suspected to have peroxisomal disorders were included in this study. Their ages ranged from 2 to 20 years.
They were referred to The Biochemical Genetics Department, National Research Centre from all over Egypt. Forty one (89%) were males while five
were females (11%). Parental consanguinity was positive in 28 cases (61%
out of 46). Very long chain fatty acids were quantified after extraction from
plasma of all cases using gas chromatography/mass spectrometry (GC/MS)
technique.
Results: The present study included 46 cases suspected clinically to have one
of the peroxisomal disorders; four of them (8.7%) proved to have X-linked
adrenoleukodystrophy by quantitative determination of the very long chain
fatty acids after extraction from their plasma. The other 42 cases showed
normal profile for very long chain fatty acids.
Conclusion: X-linked adrenoleukodystrophy is the only type diagnosed
among the study group of the suspected Egyptian cases. This study showed
that GC/MS analysis for VLCFA discriminates patients from controls, representing a non-invasive, reliable, specific and sensitive method for the diagnosis of peroxisomal disorders.
ABSTRACTS POSTERS
P06.47-S
X-linked adrenoleukodystrophy from Iran, reporting 8 cases and a
novel mutation
Back to index
137
ABSTRACTS POSTERS
The nuclear receptor PPAR is a key regulator of cell proliferation and differentiation, including adipogenesis. Defects in PPAR signaling are implicated in metabolic syndrome, cardiovascular diseases and cancer. The wide
number of ligands, coregulators and target genes, combined to different
protein isoforms and alternative transcripts, determines the complexity of
PPAR biological role.
Notably, our group identified a new PPARG isoform (ORF4) with a dominant negative effect toward PPAR, suggesting underestimated aspects of
its regulation. We also recently described the differential contribution of
PPARG canonical transcripts and ORF4 variants to adipocyte differentiation
of human mesenchymal stem cells. The analysis demonstrated that PPARG
transcription is regulated in a time-specific manner, through differential
usage of distinct promoters, also indicating that dominant negative isoforms
are actively transcribed and regulated throughout the adipogenesis. Moreover, during this study we identified another PPARG transcript, named 5,
with features similar to ORF4. Through transfection experiments, the usage of a PPAR agonist and luciferase assay, we demonstrated its dominant
negative activity and an altered ability to regulate cellular proliferation. In
order to understand, on a genome-wide scale, the effects of 5 isoform on
PPAR target genes expression, we also performed a transcriptome analysis by RNA-Sequencing in HEK293 cells over-expressing 5, in presence of
a PPAR agonist. Our results indicate that different dominant negative isoforms encoded by PPARG gene may have a relevant, and yet unexplored, role
in the biological function of PPAR, with a potential involvement in physiologic and pathologic conditions, of which PPAR is a key player.
P06.52-M
Association of rs780094 in GCKR with Metabolic syndrome and
related traits were confirmed in Tehran lipid and glucose study
Background: The minor T-allele of rs780094 in intronic region of glucokinase regulator gene (GCKR) is reported to be associated with a number of
metabolic traits including higher triglyceride levels and lipid metabolite
factors in GWA studies mostly European ancestry. Here we report replicating these finding in Iranian population using samples from the Tehran lipid
and glucose study (TLGS), a large population-based cohort study. Methods:
Among TLGS participant 1777 cases with metabolic syndrome according to
ATPIII criteria, were selected and compared to 3909 control. In GCKR gene,
rs780094 genotyped using the Centaurus (Nanogen) platform in DeCODE
genetics. Association of T-allele with metabolic syndrome and lipid related
variables (e.g. triglyceride, cholesterol, HDL and LDL) was tested using plink
software after age and sex adjustment. Results: Replicating previous findings TLGS participants showed that T-allele of rs780094 was associated
with higher triglyceride levels (p <108), higher cholesterol levels (p < 0.05)
and metabolic syndrome prevalence (p < 0.0025). Conclusion: Our findings
showed the association between the presence of T allele in rs780094 and
metabolic syndrome and related traits among Iranian population and confirmed previous result in other ethnicity.
P06.53-S
Sengers syndrome: case report and review of literature
138
Back to index
tious episode.
Literature does not propose systematic and consensual investigations for
bilateral congenital cataract. However, the wide range of aetiologies of
congenital cataracts implies various but specific and appropriate managements. This case highlights the interest of cardiac ultrasounds in congenital
cataract investigation.
P06.54-M
Cellular model of polyprenol reductase deficiency: useful tool for
SRD5A3-CDG study
Inborn errors of vitamin B12 (cobalamin) metabolism result in homocystinuria and methylmalonic aciduria, either alone or in combination. The most
common error, cblC caused by mutations in MMACHC, results in the combined metabolic phenotype. We recently described a biochemically similar inborn error, cblX, caused by mutations in the gene for the X-linked transcriptional coregulator HCFC1, which activates the transcription of MMACHC. The
cblX patients were originally diagnosed as cblC; however they have a severe
neurological and milder biochemical phenotype than most cblC patients (Yu
et al. AJHG, 2013, 93:5). One patient, characterized as cblC by complementation analysis, had no mutations in either MMACHC or HCFC1 by Sanger
sequencing. Clinically, he had methylmalonic aciduria, encephalopathy, profound mental retardation, seizures and tetraplegia and died at age 10. His
biochemical manifestations were mild, with higher cellular function of both
cobalamin-dependent enzymes than normally seen from cblC patients and
no homocysteine elevation. Sequencing of patient genomic DNA identified
a homozygous mutation, c.240C>G (p.F80L) in THAP11, the gene encoding
a zinc finger transcription factor that functions with HCFC1. The mutation
affects a residue located in a conserved zinc finger-containing domain, two
residues away from P78, responsible for interacting with the zinc ion, and is
predicted to be probably damaging by Polyphen-2. F80 is conserved to zebrafish and in 9/12 human THAP proteins. Transcriptome profiling showed
significant down-regulation of the same group of genes, including MMACHC,
in fibroblasts from cblX and the THAP11 patient.
P06.56-M
Rogers syndrome (thiamine responsive megaloblastic anemia
syndrome): the success of multidisciplinary approach
ABSTRACTS POSTERS
Hospital of Lithuanian university of Health Sciences, Kaunas Clinics, Kaunas, Lithuania,
4
Department of Endocrinology, Hospital of Lithuanian University of Health Sciences,
Kaunas Clinics, Kaunas, Lithuania.
In the present study we report the clinical features and the molecular genetic investigation of the tyrosine aminotransferase (TAT) gene in a young
girl from Croatia with Richner-Hanhart syndrome, mainly suffering from
photophobia, hyperkeratosis of the palmes and soles and slight neurological
abnormalities. Sequencing analysis of the TAT gene revealed a novel homozygous missense mutation c.1250G>A (p.R417Q) in exon 12, and herewith
confirmed the clinical diagnosis. Showing the first symptoms in babyhood,
at the age of 8 years it was for the first time clinically diagnosed that the
patient suffers from tyrosinemia type II and a therapy with tyrosine and
phenylalanine reduced diet has been started successfully. All symptoms disappeared within 2-4 weeks. Since that time, we have been following the girl
until today for more than ten years. She is in a good condition, and attends
the normal high school program.
P06.58-M
Neurological impairment is frequent among heterozygote women for
X-linked Adrenoleukodystrophy - A clinical, neurophysiological and
biochemical study
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Persons with unexplained early-onset stroke have been targeted for screening surveys for Fabry disease, the most common of the three X-linked lysosomal disorder, because Fabry patients with stroke are more likely to have
the life-threatening progressive cardiac and renal manifestations and would
therefore most benefit from early diagnosis and intervention with enzyme
replacement therapy (ERT). Among 173 Israeli patients with unexplained
cryptogenic stroke screened for mutations in the Fabry galactosidase A
(GLA) gene, sequencing identified four with 2-4 GLA intronic variants, one
of whose father and three sisters had the same variants. Two variants, c.64016A>G (g.10115A>G) in intron 4 and c.1000-22C>T (g.10956C>T) in intron
6, were common to all patients; only one male with a deletion in intron 2 had
low residual enzyme activity; both he and the father had decreased mRNA
expression. X-chromosome inactivation was not highly skewed in the six females. Nonetheless, these cases differ largely from those in the literature of
carriers of intronic variants with multi-system Fabry disease. These current
patients may not benefit from ERT, raising the question whether intronic
variants alone are disease-causing or whether these patients should be considered manifesting carriers of an X-linked recessive disease.
P06.60-M
A pilot study on an expanded newborn screening program in
Palestine
S. A. Khatib, A. Ayyad;
Alquds University Medical School, Jerusalem, Palestinian Territory.
Objective: Until this day newborns in Palestine are being screened for phenylketonuria and congenital hypothyroidism only. However, a number of
other metabolic diseases have been recognized amongst the population,
including amino acid and urea cycle disorders. The study was conducted
in an effort to investigate the possibility of finding such cases in newborns
throughout the West Bank of Palestine. In addition, reference ranges for a
number of amino acids and urea cycle intermediates will be established.
Study Design: A cross-sectional observational study design was used and
the study was conducted in all 12 districts of the West Bank. Convenience
sampling was used to recognize the study newly born participants. The
sample size was 4240 and an informed consent form was collected from
all parents. The study blood collection cards were collected over a one year
period by Ministry of Health staff. The study cards were shipped to the University of Liege Human Genetics Department in Belgium for analysis using
tandem mass spectrometry.
Results: Statistical analysis showed a significant relationship between weight
and some amino acid levels. A significant difference between some amino
acid levels and the districts was also observed. The reference range for each
amino acid and urea cycle intermediates tested was calculated based on the
non-parametric percentile method as indicated by CLSI C28-A3.
Conclusion: Based on our established reference ranges for each analyte the
results showed that 22.5% of the tested samples (955) had at least one amino acid level in the upper 2.5% of the population.
P06.61-S
First experience of idursulfase enzyme replacement treatment in
patients with Hunter syndrome in Bashkortostan Republic (Russia)
T. Nekrasova1, Y. Timasheva2;
1
Centre of Children Psychoneurology and Epileptology, Ufa, Russian Federation,
2
Institute of Biochemistry and Genetics USC RAS, Ufa, Russian Federation.
139
ABSTRACTS POSTERS
abnormalities, including hydrocephaly, ventriculomegaly, and white matter
lesions, as well as other symptoms, such as facial and thoracic deformities,
joint stiffness, exudative otitis media, hepatosplenomegaly and intellectual
disability. After at least 9 months of treatment, urinary glycosaminoglycans
levels had decreased, and liver and spleen size were reduced. Idursulfase
treatment was well tolerated (adverse reactions occurred in none of the patients). However, cognitive development had shown clear tendency to decline over time, resulting in severe mental retardation by the end of second
year of treatment. Our experience of enzyme replacement therapy of Hunter
syndrome provides an evidence of the beneficial somatic effects and also
suggests the importance of early diagnosis of the disease which might lead
to more favorable impact on cognitive development of patients.
P07.01-S
ACTN1 : identification of novel mutations in a cohort of Italian IMTP
patients
Anti-apoptotic genes such as Mcl-1 or survivin may be responsible for resistance to apoptosis induced by chemotherapeutic drugs. The aim of this study was to investigate whether the knockdown of Mcl-1 or survivin expression by small interfering RNA (siRNA) would sensitize HL-60 acute myeloid
leukemia cells to etoposide. Knockdown of Mcl-1 or survivin expression was
confirmed by quantitative real-time PCR and Western Blotting. The effects
of Mcl-1 or survivin down-regulation on the chemosensitivity of the cells
was assessed by the MTT assay. Cell viability and apoptosis were also determined using the trypan blue exclusion assay and the annexin V/PI doublestaining method, respectively. Transfection of siRNAs markedly decreased
the expression levels of both Mcl-1 and survivin genes in a time-dependent
manner. Down-regulation of Mcl-1 or survivin significantly inhibited the
proliferation and enhanced the chemosensitivity of the cells. Furthermore,
pretreatment with siRNAs clearly enhanced the etoposide-mediated apoptosis of the leukemic cells. Surprisingly, siRNA co-transfection had a more
antileukemic effect relative to the single siRNA transfection. Our study demonstrates that a triple approach involving siRNA-mediated silencing of the
Mcl-1 and survivin along with chemotherapeutic drugs could potentially be
used to lower the effective doses of the chemotherapeutic drugs and reduce
drug-related toxicities.
140
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P07.03-S
Role of the mutations identified in the 5UTR of ANKRD26 responsible
for an inherited form of thrombocytopenia
C. Gnan1, L. Arnoldo2, M. Faleschini3, D. De Rocco3, R. Sgarra2, G. Manfioletti2, A. Savoia1;
1
IRCCS Burlo Garofolo, Trieste, Italy, 2Department of Life Sciences, Trieste, Italy,
3
Department of Medical Sciences, Trieste, Italy.
The joint application of clinical and genetic investigations has greatly expanded the knowledge of inherited thrombocytopenias, leading to characterization of new forms, such as ANKRD26 related disease (ANKRD26-RD). ANKRD26-RD is characterized by thrombocytopenia and bleeding tendency, as
well as an increased risk of leukemia. Since the identification of ANKRD26,
as the gene responsible for the disease, 12 different mutations, all localized
in a short stretch of 22 nucleotides in the 5UTR, have been identified. To
further investigate their effect, we tested the activity of the basal promoter
together with the wt or the mutant 5UTR in a reporter assay. In a megakaryocytic cell line, mutations generated a statistically significant increase of
the luciferase activity, suggesting that the 5UTR plays a role in inhibiting
the expression of ANKRD26 during megakaryopoiesis. Consistent with this
hypothesis, ANKRD26 is expressed in human CD34+ and BFU-E but it is
hardly detectable in megakaryocytes. Bioinformatic analysis revealed putative binding sites for transcription factors in the region hit by mutations. In
order to identify these factors, we performed electrophoretic mobility shift
assay (EMSA). Among different complexes of the same size in both wt and
mutant samples, we observed an additional band generated by two mutant
probes. Experiments of super-shift EMSA are in progress to characterize the
pathogenetic factor(s) that could interfere with the physiological control of
the ANKRD26 expression.
P07.04-M
HBB gene mutation spectrum of beta-thalasemia patients from
Turkey
ABSTRACTS POSTERS
tral nervous system involvement. In this patient was identified a c.913G>A
(p.D305N) mutation in the exon 3 of the NRLP3 gene. The analysis of the
parents did not show the same mutation. Mutations at the codon 305 of
NRLP3 gene have been described but not the Asp >Asn substitution. The
second case is a 35 years old woman with FCAS. She showed recurrent episodes of skin rash, fever, arthralgia and conjunctivitis after generalized exposure to cold. In this patient the c.2113C>A (p.Q705K) mutation in exon 3
of the NRLP3 gene has been identified. Also in this case the analysis of the
parents did not show the same mutation. In conclusion, we report two de
novo mutations in NLRP3 gene in CAPS italian patients and we suggest the
molecular screening of NRLP3 gene in patients with a strong suspicious of
autoinflammatory syndrome.
P07.06-M
IPEX-like syndrome: beyond FOXP3 analysis
IPEX-like patients have clinical features resembling IPEX syndrome (Immune-dysregulation, Polyendocrinopathy, Enteropathy, X-linked) without
mutations in the FOXP3 gene (Xp11.23), that encodes a transcriptional regulator critical for the development and function of CD4+CD25+ regulatory
T cells. These cells are indispensable for the maintenance of immune selftolerance and homeostasis by suppressing aberrant or excessive immune
responses.
IL2Ralfa and STAT5b are two genes whose deficiency has been associated
with IPEX-like syndrome and both encode proteins involved in FOXP3 pathway. IL2Ralfa codes for the alfa subunit (CD25) of the receptor complex
for IL2, and the interaction between IL2-IL2Ralfa is the first step that trigger
FOXP3 trascription.
In this study we describe the identification of three different point mutations affecting IL2Ralfa one of them was recently published by our group and
the other two were never reported in literature.
All variations are missense mutations leading to a single aminoacid substitution that modify the protein structure. Two are located in exon 4 and alter
the sushi domain that seems to be fundamental for IL2-IL2Ralfa interaction, the third mutation determines the substitution of a cysteine that may
be involved in the formation of a disulfide bond, thus causing the alteration
of the tertiary structure of the receptor.
Cytofluorimetric analysis revealed total absence of CD25 expression in all
three patients, strengthening the hypothesis that these mutations cause the
disruption of the receptor structure thus leading to its degradation. Further
studies are ongoing to better characterize the consequence of these mutations from the molecular and cellular point of view.
P07.07-S
Immunophenotypic profile of erythroid extracellular vesicles
obtained from peripheral blood of patients with Diamond-Blackfan
Anemia
S. Macr1, R. Crescitelli1, E. Pavesi1, C. Botto2, P. Quarello3, C. Vizziello4, P. Notari4, U.
Ramenghi2, S. R. Ellis5, I. Dianzani1;
1
Department of Health Sciences, University of Eastern Piedmont, Novara, Italy,
2
Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy,
3
Pediatric Onco-Hematology, Regina Margherita Childrens Hospital, Turin, Italy,
4
Chemical Clinical Analysis laboratory, SCDU, Azienda Universitaria Ospedaliera
Maggiore della Carit, Novara, Italy, 5Department of Biochemistry and Molecular
Biology, University of Louisville, Louisville, KY, United States.
Back to index
Only the CD34+/CD71low population is significantly different between patients with DBA and controls (p< 0,05). This population that is always present
in healthy controls, is absent in patients with DBA. The absence of CD34+/
CD71low population in DBA patients plasma is in agreement with the low
level of erythroid progenitors in the patients BM. The area under the ROC
curve that compares patients with DBA and healthy controls is 0.92. Further
analyses are needed to ascertain whether this assay may be used in the clinics.
P07.08-M
Analysis of rRNA maturation in patients with Diamond-Blackfan
anemia
Diamond-Blackfan anemia (DBA MIM # 105650 ) is an inherited erythroid aplasia caused by mutations in 11 genes encoding ribosomal proteins of
the small (RPS) or the large ( RPL) subunit. The 28S, 5.8S and 18S rRNAs
are transcribed as a single precursor that is processed into mature rRNAs,
which form the ribosomal subunits with the RPs. Mutations in a RPS or a
RPL alter the processing of 18S or 28S and 5.8S rRNAs, respectively. The
analysis of pre-rRNA by Northern blot in cell models or lymphoblastoid cells
from patients shows different patterns of precursors depending on which
RP is mutated.
Mutation analysis of 11 genes is needed to confirm the diagnosis. We have
looked whether rRNA analysis of activated lymphocytes could improve the
DBA diagnostic approach. We extracted total RNA from activated lymphocytes of patients with DBA (4 mutated in RPS19, 1 in RPS17, 2 in RPL11, 4
in RPL5 and 5 with unknown mutations). Northern blot analysis was performed using appropriate probes. All the patients with a mutation in a RPL
showed the accumulation of a 32S precursor that is visible also by ethidium
bromide staining. Northern blot analysis allowed to discriminate patients
with mutations in RPS19 from those with mutations in RPS17. We observed
the rRNA maturation defect also in a patient with a mutation in RPL5 after
stable remission from the disease.
This is the first demonstration of rRNA abnormalities in patient lymphocytes. rRNA processing analysis is a useful approach to direct the subsequent
mutational screening.
P07.09-S
A case of factor X (FX) deficiency caused by novel mutations Q56K,
Q104X
H. Koo;
Yonsei University College of Medicine, Seoul, Korea, Republic of.
Background
Inherited deficiency of coagulation factor X(FX) is a rare bleeding disorder
with prevalence of 1 per 500,000 in general population. Mostly, the condition is associated with missense mutation, which is a valuable clue.
Case and results
A six month-old infant was hospitalized due to fever, lethargy and seizure
with no significant medical history. Symptoms were attributable to brain
abscess subsequently diagnosed. For operability evaluation, prothrombin
time (PT) and activated partial thromboplastin time tests were performed,
and the results were prolonged to 121.6 seconds and 109.1 seconds, respectively. Coagulation factor activities were also measured. Factor II, V, X activities were 80%, 99% and 1%, respectively. Factor VII, VIII, IX, XI and XII activities were all in normal ranges. FX activity of the parents was not tested due
to refusal of family study. FX of the patient was sequenced on coding regions
and exon-intron boundaries. We found 1 missense mutation and 1 nonsense
mutation of FX (c.C343A in exon 4, c.C487T in exon 5) that correspond to
amino acid substitution Q56K and stop codon at Q104, respectively. Both
amino acids are located in EGF-like-domain 1.
The patient suffered hematochezia, hematuria, subdural hemorrhage and
hemarthrosis which were all irrelevant to the operation. Bleeding was
treated with tranexamic acid and fresh frozen plasma with monitoring of FX
activity which increased from 1% to 22%.
Conclusion
The amino acid substitution of glutamine to lysine at EGF-like-domain 1 is
probably related to functional inactivation of coagulation FX. Confirmation
by expression study will be required.
141
ABSTRACTS POSTERS
P07.10-M
Study of IL-1 and IL-1RA gene polymorphisms in familial
Mediterranean fever (FMF)
Objectives. Familial Mediterranean fever (FMF) is a recessively transmitted autoinflammatory disease, caused by mutations in the MEFV gene. The
pro-inflammatory cytokine IL-1 has been implicated in the pathogenesis
of FMF and the balance between IL-1 and its receptor antagonist IL-1RA
plays an important role in the development of the disease. Since polymorphisms in the IL-1 gene cluster have been suggested to have an effect on IL1 and IL-1RA production, our aim was to determine a possible association
of specific polymorphisms in IL-1 and IL-1RA genes with susceptibility to
and/or severity of FMF.
Subjects and Methods. Forty-two genetically confirmed FMF patients and
42 controls were genotyped for IL-1(-511C/T), IL-1(-31T/C) and IL11(+3954T/C) polymorphisms by PCR- digestion. The IL-1RA VNTR was
identified by fragment-size analysis. IL-1 and IL-1RA levels were evaluated
by Luminex in supernatants of PBMC cultures of 30 FMF patients with and
without 24h stimulation of monocytes by LPS.
Results. The CC genotype and C allele at positions -31 and +3954 of IL-1
gene were observed more frequently in FMF patients than in controls. No
significant difference was observed in the genotypic and allelic frequencies
of the IL-1(-511) polymorphism and IL-1RA VNTR. FMF patients carriers
of IL-1(-31) CC genotype were associated with a 2 fold increase in LPSinduced IL-1 secretion, compared to patients carrying other genotypes.
Conclusion. These results indicate that IL-1 gene polymorphisms at positions -31 and +3954 may be associated with susceptibility to FMF. IL-1(-31)
may also contribute to the severity of the disease, probably by modulating
IL-1 secretion.
P07.11-S
Host genotype-pathogen interactions in the PBMC cells of healthy
individuals identify critical immune regulators
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A gluten free diet (GFD) is the most common diet worldwide. It is not only
an effective treatment for celiac disease, but also commonly followed by individuals with gut complaints. How a GFD affects the human microbiome
is largely unknown. We studied changes in the gut microbiome in healthy
individuals following a short-term GFD.
23 healthy volunteers followed a GFD for 4 weeks. Stool samples were collected before the start of the diet, then at weekly intervals during the GFD
and again at weekly intervals for 4 weeks on normal diet after a wash-out
period. The samples were sequenced using 454 sequencing of the 16s rDNA
(hyper variable region 3 to 4). We used closed reference picking to cluster
reads into OTUs using the 2013 GreenGenes reference. Function imputation
of the OTUs, is performed used PiCRUSt and HUMAnN.
We observed a limited difference of the GFD intervention, with the intrasubject variation being larger than the effect from a short-term diet change.
However, we did observe that family Veillonellaceae (class Clostridia) was significantly less present in the GFD samples (p=6.58e05, q=0.0132). Based
on richness of the samples we were able to define two groups which show
a difference in response to a GFD period. The lower richness group showed
more bacterial variation in composition during the diet change. Furthermore, sixteen gene pathways showed significant association to the change in
diet. In conclusion, in this we observed changes in microbiome and gene
composition associated to a GFD.
P07.13-S
A dominant-negative GFI1B mutation causes autosomal dominant
gray platelet syndrome
The genetic region between HLA-A and HLA-G covers 300 kb and is highly
polymorphic. A large-scale deletion of 50 kb between these loci associated
to HLA-A*23 and HLA-A*24 has been described. This deletion includes many
genes and pseudogene, as HLA-H.
We showed in a study conducted in a Malian population that HLA-A alleles display LD with HLA-G alleles and UTR; HLA-A*23:01:01~HLAG*01:04~UTR3 haplotype displaying the highest frequency. This strong LD
could be emphasized by a deletion occurring between those two loci, thus
reducing the recombination rate between HLA-A and HLA-G. This haplotype
ABSTRACTS POSTERS
conservation might also be the result of a specific biological effect, as HLAG*01:04 has been associated with high sHLA-G production and that HLAA*23:01~HLA-G*01:04 haplotype may constitute a risk factor for allograft
rejection in renal transplantation. Interaction with other immune effectors
as HLA-E could also be incriminated. We showed that 2 haplotypes in Tswa
Pygmies, HLA-G*01:04~E*01:03:01 and G*01:04~E*01:01 exhibited LD values.
The aim of our study is to explore the region between HLA-A and -E. We
genotyped HLA-A, -G, -H and -E alleles in 71 french samples and explore
their association.
We found that 11 haplotypes represent 75% of all the haplotypes. HLA-H
deletion was exclusevely associated with A*23~HLA*G01:04~UTR-3~HLA*E01:01 and HLA-A*24~HLA*G01:04~UTR-3~HLA*E01:03:01 haplotypes. A new HLA-H*02:04(:02) allele was descibed and associated with
A*11~HLA*G01:01~UTR-7~HLA*E01:01.
This study suggests that the HLA region between HLA-A and -E loci displays
a haplotype conservation that might be the results of biological function.
This haplotype conservation has to be further studied to understand their
clinical implication.
P07.16-M
HLA-DRB1 genotyping in romanian patients with early rheumatoid
arthritis
A. Martinescu, L. Hanzu-Pazara, M. Suta;
Ovidius University, Faculty of Medicine, Constanta, Romania.
Back to index
compared to children with a low-risk genotype. These data indicate that genotypes in the IL22 promoter enhancing IL-22 production is preferentially
enriched in psoriasis with onset before puberty and may predispose to development of psoriasis at an early age.
P07.18-M
Whole exome sequencing as a tool for detection of the genetic basis of
inherited thrombocytopenias
B. D. Johnson, G. C. Lowe, S. Drake, S. J. Fletcher, J. Ftterer, D. J. MacDonald, S. P.
Watson, N. V. Morgan, UK GAPP Study Group;
University of Birmingham, Birmingham, United Kingdom.
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ABSTRACTS POSTERS
nia caused by mutations in GP1BA, GP1BB and GP9, the genes encoding for
three subunits of the GPIb-IX-V complex, which is the platelet receptor for
von Willebrand factor (vWF). In addition to the biallelic form, a less severe
autosomal dominant form (monoallelic) of BSS is due to mutations identified so far in GP1BA or GP1BB. Except for p.Ala172Val (Noris et al, 2012),
which is relatively frequent at least in the Italian population, other mutations have been reported only in single families. In order to evaluate the frequency of the monoallelic form in our thrombocytopenic cohort, we selected 120 probands with large platelets and no mutations in candidate genes.
In 11 cases we found heterozygous mutations in GP1BA (n=4), GPIBB (n=6)
and GP9 (n=1). In addition to 4 variants previously identified in biallelic BSS
patients, the others are all novel mutations. They are classified as missense
(n=8), nonsense (n=1) or frame-shift (n=2) mutations. Segregation analysis within the families showed that only the affected individuals carry the
mutations. Functional studies are in progress to determine the effect of the
mutations on GPIb-IX-V complex formation and its capacity to interact with
vWF. Considering that half of the patients with inherited thrombocytopenia
remain without a molecular diagnosis, we could estimate that the frequency
of the monoallelic form of BSS could account for 5% of the cases. This suggest that monoallelic BSS should be always taken into consideration in the
differential diagnosis of inherited thrombocytopenia.
P07.21-S
MHC and the 1000 genomes: genotyping from exome data
The publicly available 1000 genomes (1KG) data is still a valuable source
of scientific discovery after years of its release. We have demonstrated previously that HLA typing of MHC-I genes is possible from 1KG exome data for
HapMap samples. In this study we are aiming to determine types for MHC-II
genes and for any 1KG exome samples passing certain quality control (QC)
conditions. We are presenting the generation and interpretation of these QC
values. Besides the HLA genes we are showing typing results for other polymorphic MHC genes like TAP1, TAP2, MICA and MICB. The HLA genotype
assignations were compared to classical HLA typing obtain by sequencing
techniques. Typing a diverse set of genes of MHC sheds light on linkage disequilibrium and makes it comparable to the previously known HapMap statistics. Furthermore, differences in results from different exome capturing
kits are also to be presented.
P07.22-M
SNP variants in MHC are associated with sarcoidosis susceptibility
and subgroups - a joint case-control association study in four
European populations
144
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The Nijmegen breakage syndrome (NBS) is a recessive genetic disorder, resulting in high predisposition to developing a malignancy. The 5bp deletion
(c.657-661del) in the NBN gene is founder mutation for Slavic populations.
In Ukraine 40 NBS cases were diagnosed, and in 12 cases (30%) lymphoid malignancies has been developed at age 5-12 years. It remains unclear
why the carriers of same mutation are implemented in a different morbidity.
The purpose of this study was looking for immunogenetic criteria for tumor
developing in NBS patients. Interleukin-10 (IL-10) and interferon-gamma
(IFN-) play a key role in controlling the immune response and SNPs IL-10
-1082 A/G and IFN- +874A/T respectively can significantly affect their expression. Patients with severe outcome (multiple chronic recurrent inflammation, developing of tumor, death) and moderate course of disease were
comparatively investigated. The distribution of IL-10 1082AA, 1082GG and
1082AG genotypes in cases of severe and mild outcome established as 42.9%
and 33.3%, 35.7% and 33.3%, 21.4% and 33.3% respectively. The distribution of IFN- 874AA, 874AT and 874TT genotypes in groups of patients with
severe and mild outcome was 20.0% and 29.4%, 40.0% and 58.82%, 40.0%
and 11.86% respectively. The results allow to suggest, that severe outcome
in NBS may depends on carrying of IL-10 1082AA genotype, associated with
decreased anti-inflammatory activity, and IFN- 874TT genotype, known by
increased pro-inflammatory properties. In conclusion, disruption in balance
of pro- and anti- inflammatory cytokines may strongly affect NBS course.
P07.24-M
MEFV gene mutations in pediatric patients with PFAPA syndrome in
Slovenia
Introduction: PFAPA syndrome is the most common autoinflammatory fever disorder in childhood, characterized by recurrent fever, aphthous stomatitis, pharyngitis and adenitis. Mutations in the MEFV gene are known
to cause syndrome with PFAPA overlapping symptoms (Familial Mediterranean Fever), which is common in eastern Mediterranean population, but
rarely reported in patients from Slovenia.
Objective: The aim of this study was to assess the frequency of MEFV gene
mutations in pediatric patients with PFAPA syndrome from Slovenia.
Methods: We collected clinical and laboratory data and results of genetic
testing of MEFV gene of PFAPA patients under the age of 18, who were followed from the beginning of 2006 to the end of 2013. All 10 exons and intron/
exon regions of gene were directly sequenced.
Results: In total, 91 PFAPA patients were tested for MEFV gene mutations.
All of them were under the age of 18, mean age at diagnosis was 7 years. The
ratio of women to men was 1:1.
15 patients (16%) were found to have at least one mutation. 11 patients
(12%) were heterozygote and 4 patients (4%) were compound heterozygote, 3 with R408Q/P369S and 1 with K695R/I591T mutation. The overall
number of mutation found was 19. The most frequent was K695R(26%),
followed by R408Q(16%), P369S(16%), E148Q(10%), I591T(10%),
M694V(5%), S730F(5%), A289V(5%) and A744S(5%).
Conclusion: In order to evaluate effect of these mutations on PFAPA phenotype, we are planning to determine the carrier rate in healthy Slovenian
population and evaluate genotype-phenotype correlation in MEFV gene mutation positive patients.
ABSTRACTS POSTERS
P07.25-S
A new gene involved in an autosomal dominant form of common
variable immunodeficiency (CVID)
Molecular Medicine and Science for Life Laboratory, Department of Medical Sciences,
Uppsala University, Uppsala, Sweden, 2Rheumatology, Department of Medical Sciences,
Uppsala University, Uppsala, Sweden.
1
Common variable immunodeficiency (CVID, MIM#607594) is the most common symptomatic primary antibody deficiency in adults. It includes a heterogeneous group of disorders characterized by defects in the terminal stage
of B lymphocyte differentiation, whose underlying genetic defects remain
unknown in the majority of cases.
We studied a five-generation Italian family with an autosomal dominant
form of CVID through whole exome sequencing, giving priority to the variants within a 9.2 Mb candidate genomic interval on chromosome 3q27.2q29 previously identified by genome-wide linkage analysis. Since no causative mutation was identified by whole exome sequencing, we performed a
whole genome high resolution SNP array analysis, which allowed to detect a
~ 880 kb tandem duplication in 3q27.3, located inside the candidate linkage
region and involving 8 genes: ST6GAL1, RPL39L, RTP1, MASP1, RTP4, SST,
RTP2 and BCL6.
The expression pattern analysis in control peripheral blood lymphocytes (PBL) of all genes included in the duplicated region revealed that only
ST6GAL1, RPL39L, RTP4 and BCL6 are expressed in mature circulating lymphocytes, allowing us to exclude the remaining four genes from further
investigations. Preliminary qRT-PCR analyses conducted on affected vs
unaffected subjects showed a significant upregulation of RTP4 in affected
members (t-test, p < 0.0001). This finding suggests RTP4 overexpression as
a possible pathogenetic mechanism underlying CVID in this family. RTP4 is
a Golgi chaperone and we hypothesize that it might be involved in the regulation of the Unfolding Protein Response (UPR), a key process of plasma cell
differentiation.
P07.26-M
A family based exome sequencing identifies ADA2 deficiency as a
novel cause for PIDD
Primary immunodeficiency diseases (PIDD) encompass a wide and heterogeneous group of disorders caused by mutations in genes with immune
function. The molecular mechanisms behind many forms are not yet known.
The significant clinical and immunological heterogeneity of PIDD often delay the diagnosis making treatment challenging. Few thousand individuals are expected to suffer from PIDD in Finland, where the unique genetic
background has proven to be very useful in identifying genes for monogenic
disorders. An exome sequencing analysis of a Finnish PIDD family characterized by recurrent prolonged bacterial infections complicated by basal cerebral and frontal infarcts, subarachnoidal hemorrhage and occasional small
aneurysms, has identified compound heterozygous mutations in the CECR1
gene not previously implicated in PIDD. The affected individuals carried a
splice donor site mutation leading to nonsense mediated decay (NMD) of
the allele and a rare missense variant, highly conserved and predicted deleterious. Analysis of the patient sera confirmed that the protein product
(ADA2) of the mutated gene had no measurable activity. ADA2 is a member
of the adenosine deaminase family, which also includes ADA1, a known cause of severe combined immune deficiency (SCID). ADA2 is a secreted protein which binds to cell surface via proteoglycans, may degrade extracellular
adenosine and has been shown to induce T cell-dependent differentiation of
monocytes into macrophages and stimulate macrophage proliferation. Thus
ADA2 deficiency is a plausible cause for the immunodeficiency complicated
by infarcts and subarachnoidal hemorrhage in this family. The ongoing functional analyses will shed further light on molecular defects of the disease.
P07.27-S
Genome-wide DNA methylation analysis in CD19+ B cells in Primary
Sjgrens Syndrome
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Primary Sjgrens syndrome (pSS) is a complex chronic autoimmune disease characterized by the inflammation of the exocrine glands leading to
dry eyes and impaired salivary flow. Apart from glandular symptoms, systemic manifestations such as arthritis and chronic fatigue are common, and
75% of the patients have autoantibodies in their sera. PSS patients have a
15 fold increased risk of developing B cell lymphoma. Increasing evidence
suggests an epigenetic contribution in the pathogenesis of autoimmune
diseases, including pSS. Epigenetic modifications represent a dynamic link
between genotype, environment and phenotype, by for example modulating
gene expression. As methylation of the DNA base cytosine is considered as
a prototypic epigenetic mark, our aim was to investigate the role of DNA
methylation in pSS. We performed a genome-wide DNA methylation study in
purified CD19+ B cells from 17 pSS patients and 28 healthy controls using Illumina HumanMethylation450 BeadChip arrays which cover about 486,000
CpG sites across the genome. Significant differentially methylated CpG sites
between patients and controls were identified. We found disease-associated
changes in DNA methylation in pSS and highlight pathways that may contribute to the pathogenesis of this autoimmune disease.
P07.28-M
Palmoplantar pustular psoriasis and its genetic background
Palmoplantar pustular psoriasis (PPP) is a chronic inflammatory skin disease characterized by sterile pustules, erythema and hyperkeratosis on palms
and soles. In at least 25% of PPP cases, psoriasis vulgaris (PsV) is also present and many patients suffer from psoriatic arthritis. Smoking and female
sex are more frequent in PPP compared to PsV. So far, there are no confirmed genetic risk factors for PPP. Recently, in generalized pustular psoriasis
(GPP), an extreme manifestation of pustular psoriatic disease, homozygous
and compound-heterozygous mutations in IL36RN have been identified to
be causal.The same mutations have been described to be more frequent in
a group of 139 European PPP patients.Here, we recruited a group of >140
PPP patients,most of them were female and smokers (>60%, respectively).
About half of them had a manifestational age of 40 years.We could confirm previous data showing that the frequency of the HLA-C risk allele, the
major genetic risk factor for PsV, was comparable to the frequency of controls, indicating that PPP is genetically different from PsV. We further analyzed IL36RN for mutations in coding exons and for intragenic deletions/
duplications. We identified three heterozygous carriers of mutations and no
carriers of copy number variants. Compared to a population-based control
group of 4.300 European individuals, there was no significant difference in
the frequency of IL36RN mutations.Our data indicate that PPP is genetically
distinct both from PsV and GPP. Further effort is needed to identify genetic
factors contributing to PPP.
P07.29-S
Genetic, functional and therapeutic insights into patients with rare
recurrent viral infections
Primary immunodeficiencies (PIDs) are inborn defects in the immune system affecting about 1 in 500 people. In this present study we show that
understanding the functional and/or genetic defect in patients suffering
from severe PIDs can provide us new leads for treatment. Here we studied
three patients suffering from severe recurrent viral infections with Herpes
Simplex virus (HSV). The infections were caused by HSV2 in two of the patients, with no history of other infections, while the third patient was initially diagnosed with HPV6 papillomatosis and she developed recurrent HSV
infections later in life. We isolated and stimulated PBMCs from patients and
controls, and stimulated them with various ligands among which the double
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ABSTRACTS POSTERS
stranded RNA ligand poly:IC. Cytokine production was measured in patients
and compared to controls, and a specific defect in poly I:C-induced interferon-gamma was detected. Substitution therapy with interferon-gamma was
successfully administered to all patients, leading to impressive amelioration. Additionally, we performed exome sequencing in all 3 patients, aiming
to understand the genetic basics of the underlying immunodeficiency. While
this genetic analysis did not identify one common cause of the primary
immunodeficiency, rare variants in candidate genes SYK, IFIH1 and EIF3E
were identified that may have caused the aberrant interferon signaling. In
summary. These preliminary results indicate that patients with recurrent viral HSV infections may have genetic heterogeneous forms of primary immunodeficiencies; however, they show a common functional defect in double
stranded RNA-induced IFNgamma, and common replacement therapy with
interferon-gamma was shown to be an effective treatment.
P07.30-M
Allele specific expression of HLA haplotypes associated to
autoimmune diseases
A. Zhernakova, J. Fu, D. Zhernakova, L. Franke, C. Wijmenga;
University medical centre Groningen, Groningen, Netherlands.
HLA is strongly associated with many autoimmune diseases, including rheumatoid arthritis (RA), celiac disease (CD) and type 1 diabetes (T1D). For
many diseases the association is established, but the underlying mechanisms are unknown. For example, heterozygosity for DR3-DQ2/DR4-DQ8 is a
stronger risk factors to T1D compared to homozygosity for both DR3-DQ2
and DR4-DQ8 alleles. We aimed to study the downstream effect of HLA haplotypes by looking for allele specific gene expression (ASE) and haplotype
expression quantitative trait loci (eQTLs) in HLA alleles associated to autoimmune diseases. We selected individuals heterozygous for DR3-DQ2/
DR4-DQ8 and homozygous for both alleles (T1D and CD risk haplotypes),
and individuals heterozygous for DR4-DQ8/DR4-DQ7 and homozygous for
these alleles (RA risk haplotypes). We run RNAseq to quantify gene expression and performed eQTL and ASE analysis in these individuals. In total, 90
individuals were selected for this analysis. We observed different expression
of multiple genes in HLA locus in DR3-DQ2/DR4-DQ8 heterozygous individuals, in compare with both homozygous group. In particular, HLA-DQA2,
HLA-DQB1 and HLA-DQB9 (P-value Wilcox test <0.001 for all three genes).
ASE analysis of individuals heterozygous for DR3-DQ2/DR4-DQ8 indicated
ASE effect for multiple variants located in TAP2 gene, which plays a role in
antigen presentation. In DR4-DQ8/DR4-DQ7 analysis we identified multiple
eQTLs in the HLA locus, as well as allele specific effect on SNPs located in
DOA genes.
P07.31-S
Phenotypic analysis of Peptidylarginine deiminase type 4 knock-out
mice
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P07.32-M
Identification of susceptibility loci associated with primary Sjgrens
syndrome by genome-wide association study
I. Song1,2, H. Chen3, C. Chen3, C. Chen1, J. Wu1;
1
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, 2Graduate institute
of Life Science, National Defense Medical Center, Taipei, Taiwan, 3Rheumatology,
Immunology and Allergy Division, Tri-Service General Hospital, National Defense
Medical Center, Taipei, Taiwan.
Dogs represent an excellent model to study complex genetic disorders including many autoimmune diseases that they share with humans. A systemic
lupus erythematosus (SLE)-related disease complex, which shows similarities to human SLE, has high prevalence in Nova Scotia duck-tolling retriever
(NSDTR) dogs. Genome-wide association analyses have identified five candidate loci in NSDTR containing several genes. Detailed analysis of the candidate loci identified variants that alter the expression of several of these genes.
Multiple genes involved in T-cell activation show expression differences, but
regulatory mutations also alter expression of genes such as BANK1, involved
in human SLE. As a proof of concept, the genes and pathways identified in
dogs and also genes relevant for human SLE were sequenced in 140 Swedish
human SLE patients. Patients were divided in 9 pools according to disease
manifestation and a group of healthy Swedish controls was also sequenced.
Targeted Nimblegen + Illumina sequencing of 219 genes and their regulatory elements resulted in approximately 250x coverage per individual. We
detected 4276 novel SNPs (not present in 1000genomes or dbSNP137) of
which 1258 SNPs were found only in cases. Seventeen genes showed 5
novel case-only variants. Of these, three genes were previously associated
to human SLE and 14 were novel candidate genes. From the top genes, ten
variants that show strong regulatory potential have been selected and being
evaluated for their importance for gene expression and correlation with clinical phenotypes. Several variants appear to be potential regulators of specific SLE sub-phenotypes.
P07.35-S
Deep analysis of TCR repertoires of twins by next generation
sequencing
Immune system interacts with great diversity of pathogens. Receptors involved in antigen recognition - BCRs (B-cell receptors) and TCRs (T-cell receptors) - are not encoded in genome due to its limited capacity, but generated
by V(D)J recombination. For a long time, no instrument to estimate TCR diversity in individual organism has been available. Next generation sequencing methods (NGS) have created the possibility of deep TCR profiling. Here,
we aimed to estimate the role of genetic factors in TCR repertoire formation
by sequencing TCR repertoires of monozygotic and dizygotic twins.
16 cDNA TCR libraries were generated and sequenced on Illumina platform.
Surprisingly, the overlap of amino acid sequences of CDR3 region in TCR
clonotypes was not greater in MZ twin pairs and depended on sample size
ABSTRACTS POSTERS
only. However, the number of identical clonotypes was higher for monozygotic twins in the abundant clonotypes subset, representing mainly antigenexperienced T cells. At the same time V-segment usage is more similar in
twin pairs for clonotypes before and after thymic selection.
We also showed that the ratio of clonotypes with identical CDR3 and V-segments (coding for CDR1 and CDR2) to clonotypes with identical CDR3 is
higher in twin pairs in the abundant clonotypes subset.
All these features were not observed in a dizygotic twin pair, which probably
reflects the impact of genetic factors on TCR repertoire formation.
P07.36-M
A novel StripAssay identifies genetic variants modifying betathalassemia disease severity
H. Puehringer, B. Rauscher, C. Oberkanins;
ViennaLab Diagnostics GmbH, Vienna, Austria.
Background: The clinical phenotype of patients with beta-hemoglobinopathies is extremely heterogenous, ranging from nearly asymptomatic forms
of thalassemia intermedia to severe transfusion-dependent thalassemia
major. The wide phenotypical variability is associated with the type of betaglobin mutation, the co-inheritance of alpha-thalassemia and the ability
for persistent production of fetal hemoglobin (HbF) in adult life. For the
latter, three different quantitative trait loci, accounting for 20-50% of HbF
variation, have been identified by now. Single nucleotide polymorphisms
(SNPs) in the gamma-globin gene promoter (HBG2), in the BCL11A gene
and the HBS1L-MYB intergenic region lead to increased residual HbF levels
in adults. Methods: A teststrip-based reverse-hybridisation assay was developed for the simultaneous detection of SNPs in the HBG2 (g.-158 C>T),
BCL11A (rs1447407, rs10189857), HBS1L-MYB (rs28384513, rs9399137)
genes. Results: The new StripAssay enables the concomitant identification
of genetic variants known to influence beta-thalassemia disease severity.
Based on the presence of positively modifying alleles, and combined with
alpha- and beta-globin genotyping, it allows the prediction of patients likely
to display less severe phenotypes. Favourable properties, such as the rapid
DNA extraction protocol, ready-to-use reagents and teststrips, as well as the
potential for automation of the hybridisation/detection and interpretation
steps, make the StripAssay convenient and easy to perform within less than
six hours. Conclusions: Testing for genetic modifiers influencing disease severity will lead to more specific and effective treatment, and support clinical
decisions regarding the beginning of transfusion therapy in beta-thalassemia patients. Furthermore, the knowledge about prognostic markers has
implications for genetic counselling and prenatal diagnosis.
P07.37-S
Family with inherited thrombocytopenia and homozygous pathogenic
variant in FYB gene
H. A. Hamamy1, P. Makrythanasis1, N. Al-Allawi2, A. A. Muhsin3, S. E. Antonarakis1,4;
1
Univesity of Geneva, Geneva, Switzerland, 2Univesity of Dohuk, Dohuk, Iraq, 3Jin Center,
Dohuk, Iraq, 4University Hospitals of Geneva, Geneva, Switzerland.
The genome-wide association studies on the genes related with iron metabolism has been showed that some of the genetic variants were associated
with serum iron level and erythrocyte parameters. One of these common
Back to index
variants was A736V in TMPRSS6 gene which regulates serum iron level. Inactivation of TMPRSS6 gene causes iron-deficiency anemia and impairment
of iron absorption. It has been found that A736V variant of TMPRSS6 gene
is associated with lower levels of serum iron and erythrocyte MCV with higher levels of serum hepcidin protein. It was also shown that inhibition of
TMPRSS6 has a therapeutic effect on Beta-thalassemic mice. However the
frequency of this variant among Beta-thalassemia patients is unknown. Related with these data, we aimed to investigate frequency of A736V variant
of TMPRSS6 gene among patients carrying beta-globin gene mutations. 93
patients investigated for beta-globin gene mutation with DNA sequencing
method were enrolled in this study. A736V (rs855791) variant of TMPRSS6
gene was detected by real-time quantitative PCR method. Erythrocyte parameters and hemoglobin levels were measured and their distributions according to gene variants were analyzed. Frequency of TMPRSS6 gene A736V
variant was 0.47 among all patients. There was no association between
A736V variant of TMPRSS6 gene and beta-globin gene mutations, hemoglobin levels or erythrocyte parameters. However, frequency of A736V variant
was higher among the patients having low levels of hemoglobin or erythrocyte MCV with a wild type beta-globin gene. Further studies are needed for
better understanding the relationship between of A736V variant and Betathalassemia prognosis.
P07.41-S
Novel 21 nucleotide duplication in alpha1/alpha2 globin gene
involves in a variety of hypochromic microcytic anemia from mild to
Hb H Disease
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ABSTRACTS POSTERS
and marked deficiency of blood and lung iNKT cells in patients with newly diagnosed sarcoidosis. During 4 years of disease follow-up, there was a
significant increase in expression of SLAM-SAP signalling factors, mainly
SLAMF1, SLAMF6, and FYN, and blood iNKT cells. This increase clearly correlated with improvement in patients clinical symptoms as after 4 years the
disease had gone into remission in the great majority of patients.
Our longitudinal study showed that an increase in expression of SLAM-SAP
signalling factors and iNKT cells characterizes the clinical remission of pulmonary sarcoidosis.
P07.43-S
A late diagnosis of NOMID-syndrome /CINCA-syndrome - lessons to
learn
K. Becker;
Labor Krone, Bad Salzuflen, Germany.
The case of a 10 year old boy from Germany is presented with a new diagnosis of NOMID syndrome, also known as CINCA-syndrome, the rarest and
most severe of the Cryopyrin-associated periodic syndromes (CAPS), which
are genetic syndromes of autoinflammation. The diagnosis was delayed despite many clinical clues including the typical non-itching maculopapular
rash which was present from the neonatal period, raised inflammatory markers on many occasions without detectable infectious pathogens, progressive macrocephaly with hydrocephalus, evolving short stature, progressive
optic atrophy, progressive deafness, progressive arthropathy and other nonspecific haematological and immunological abnormalities. In addition, the
patient had episodic conjunctivitis and finger clubbing. Periodic fever does
not always occur in this condition and in the case of this patient was completely absent. This patient was treated for several years with growth hormone in the absence of a diagnosis without any improvement of his short
stature. Early diagnosis of these patients is vital because treatment with interleukin -1-receptor antagonists is now well established and very effective.
Untreated, the patients may develop a destructive arthropathy, blindness,
profound deafness and renal failure secondary to amyloidosis. The macrocephaly, hydrocephalus and short stature result from the chronic aseptic meningitis. The disease is caused by mutations (mostly new dominant) in the
NLRP3 gene, which may be mosaic and thus are sometimes only detectable
by next generation sequencing.
P08.01-S
Chromosome 15q11.2 imbalances associated with neuropsychiatric
and developmental disorders - array-CGH findings in a cohort of 1000
patients
L. M. Pires1, S. I. Ferreira1, J. Ferro1, N. Lavoura1, F. Ramos2, A. Beleza-Meireles2, J. B.
Melo1,3,4, I. M. Carreira1,3,4;
1
Laboratrio de Citogentica e Genmica, Faculdade de Medicina da Universidade de
Coimbra, Coimbra, Portugal, 2Servio de Gentica Mdica, Hospital Peditrico, CHUC,
Coimbra, Portugal, 3CIMAGO Centro de Investigao em Meio Ambiente, Gentica e
Oncobiologia, Coimbra, Portugal, 4CNC- Centro de Neurocincias e Biologia Celular,
Universidade de Coimbra, Coimbra, Portugal.
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P08.02-M
Overgrowth and developmental delay associated with a 200 kb
deletion in 16p11.2 in two families
Due to a high density of segmental duplications, the short arm of chromosome 16 is prone to a number of recurrent rearrangements. A common 600
kb microdeletion or -duplication in 16p11.2 has been associated with autism, intellectual disability, schizophrenia, and mirrored weight and head
circumference phenotypes. An adjacent, but separate distal 200 kb region in
16p11.2, which contains the SH2B1 gene, has been associated with isolated
obesity as well as developmental delay. Both aberrations are associated with
high variability and incomplete penetrance.
We now report on two families harboring the 200 kb microdeletion in
16p11.2. Both index patients were initially referred with suspected Sotos
syndrome due to tall stature, obesity and developmental delay. After normal
NSD1 testing molecular karyotyping showed the 200 kb deletion in 16p11.2
in both patients as well as in patient 2s brother who had mild obesity and
unspecific moderate intellectual disability. In both families the deletion was
maternally inherited. Interestingly, patient 2, but not her cognitively more
severely affected brother, additionally harbored a microduplication 1q21.1,
which has been recurrently associated with variable and incompletely penetrant developmental delay, intellectual disability, behavioural anomalies and
large head circumference. Both aberrations were inherited from the mother,
who was obese but otherwise healthy and without cognitive problems. These two families further characterize the variable spectrum of phenotypes
associated with the 200 kb microdeletion in 16p11.2. Our findings in family
2 also show that not even the co-occurrence of two ID-associated microaberrations necessarily leads to cognitive impairment.
P08.03-S
Clinical and molecular characterization of a patient with de novo 2.8
Mb deletion of 17q24.2-q24.3
Microarray methods contributed to the identification of many rare microdeletion syndromes including that associated with deletions of 17q24.2-q24.3,
characterized mainly by growth retardation, microcephaly, developmental
delay, speech delay, intellectual disability, feeding problems, and teeth and
facial abnormalities. Seven patients have been described in detail since
2008, and additional cases with only limited clinical information are listed
in databases. It becomes evident that not all 17q24.2-q24.3 deletions share
a single common region of overlap.
We report a 12-year-old girl with a normal karyotype who shows clinical
features consistent with the syndrome. SNP array analysis revealed a 2.8 Mb
long deletion of 17q24.2-q24.3 (chr17:65,281,651-68,017,013; hg19). FISH
analysis confirmed the deletion and showed its de novo origin. The deletion affects 18 protein-coding RefSeq genes including PRKAR1A, MAPK2K6,
and the ABCA gene cluster. Some of the genes could be candidates for the
clinical symptoms and some are predicted to exhibit haploinsufficiency, for
instance PRKAR1A, encoding a postsynaptic density protein associated with
Carney complex, or BPTF producing a transcript in fetal brain which binds
FMRP and encodes a transcriptional regulator. Only much smaller population copy number polymorphisms are present in the region. The deletion of
our patient overlaps two groups of previously reported deletions. Despite
the proximity of the deleted regions, the candidate genes could be distinct.
Identification of additional cases will help to define the genes involved and
their role in predisposition to neuropsychiatric phenotypes, as well as the
emerging genetic heterogeneity within the 17q24.2-q24.3 microdeletion
syndrome.
Supported by CHERISH, NT/14200, 00064203 and CZ.2.16/3.1.00/24022.
P08.04-M
Deletions of 1p34.3, encompassing the AGO1, AGO3, and AGO4 genes, in
five children with microcephaly and intellectual disability
P. Prontera1, M. Tokita2,3, P. Chow2,3, G. Mirzaa2,3, N. Dikow4, B. Maas4, B. Isidor5, C. Le
Caignec5, L. Penney6, G. Mazzotta7, L. Bernardini8, A. Battaglia9, D. Earl2,3, E. Donti1;
1
Medical Genetics Unit, University and Hospital of Perugia, Perugia, Italy, 2Division
of Genetic Medicine, University of Washington, Seattle, WA, United States, 3Seattle
Childrens Hospital, Seattle, WA, United States, 4Institute of Human Genetics, Heidelberg
University, Heidelberg, Germany, 5Gntique Mdicale, Centre Hospitalier Universitaire
de Nantes, Nantes, France, 6Department of Pediatrics, Dalhousie University, Halifax, NS,
Canada, 7School of Specialization in Childhood Neurology and Psychiatry, University
of Perugia, Perugia, Italy, 8Mendel Laboratory, IRCSS Casa Sollievo della Sofferenza
Hospital, San Giovanni Rotondo, Foggia, Italy, 9The Stella Maris Clinical Research
Institute for Child and Adolescent Neurology and Psychiatry, Calambrone, Pisa, Italy.
ABSTRACTS POSTERS
Small RNAs (miRNA, siRNA, and piRNA) regulate gene expression through
RNA interference (RNAi), a process that has emerged as a fundamental principle of normal cellular function. The Argonaute (AGO) proteins are critical
mediators of the RNAi pathway and constitute a highly conserved family of
genes found in almost all eukaryotes. Four AGO genes are present in humans, three of which (AGO 1, 3, and 4) reside in a cluster on chromosome
1p35p34.
The possible effects of germline AGO mutations or dosage alterations in humans is not known, however, animal models deficient for AGO proteins display developmental brain defects including a reduction in total number of
neurons and glia. Moreover, different studies have established that miRNA
and siRNA are prevalently or exclusively expressed in the brain, where they
play an essential role in the development and function of the central nervous
system (CNS).
We describe five patients with hypotonia, microcephaly, intellectual disability, and facial dysmorphisms, in whom array-Comparative Genomic Hybridization revealed overlapping de novo microdeletions of the chromosomal
region 1p34.3. The minimal critical region is a segment of approximately
694 Kb that encompasses the AGO1, AGO3, and AGO4 genes.
We propose that the neurocognitive deficits present in these patients are
due to deletion of the 1p34.3 region and resulting haploinsufficiency of
several AGO genes. It seems plausible that the 1p34.3 deletion syndrome
may eventually be recognized as a neurodevelopmental disorder associated
with RNAi deregulation, however, further investigation is necessary to prove
this.
P08.05-S
Two brothers with 2q23 microdeletion syndrome inherited from
their mosaic father
Patient 1, the older brother, is 13 years old and has moderate intellectual
disability. He communicates by single words, pointing and some signing. He
walked at 4 years. He uses diapers, suffers from constipation and manifests
aggressive outbursts and stereotypic movements. His sleeping pattern is disturbed by periods of awakeness. He has teeth enamel erosions. His twin
sister, with Apert syndrome, died at 2 years. Patient 2, the younger brother,
is 6.5 years and has moderate intellectual disability. He speaks a few words
and infrequently combines two words. He has a heart murmur, enamel teeth
erosions and periodic sleep problems. He is restless and active.
ArrayCGH of the patients and father showed a deletion of
chr2q23.1(148705701-148926786 bp), including the MBD5, considered
critical in the 2q23.1 microdeletion syndrome. By FISH the deletion in the
father was found to be a mosaicism found in 73% of lymphocytes. Assessment of the mosaicism level in additional tissues is ongoing. The father received speech therapy in childhood and has dyslexia. He completed secondary
school with low-average marks, and did not finish high school. He has been
working as a manual worker in the same factory for 18 years. In patient
2 and his father aCGH detected also a chr15q26.1(9022468-90255068 bp)
deletion, including PLIN1. PLIN1 mutations cause dominant familial partial
lipodystrophy type 4, but the effect of PLIN1 haploinsufficiency is unknown.
Patient 2 and his father, but not Patient 1, are obese.
To our knowledge this is the first familial case of the 2q23 microdeletion
syndrome.
P08.06-M
Microdeletion of 8q21 region: clinical and molecular analysis based
on a new case
M. Kucharczyk1, K. Iwanicka-Pronicka2, M. Kugaudo1,3, A. Cielikowska1, A. JezelaStanek1, K. Chrzanowska1, M. Krajewska-Walasek1;
1
Department of Medical Genetics, The Childrens Memorial Health Institute, Warsaw,
Poland, 2Department of Audiology, Phoniatrics and Otolaryngology, The Childrens
Memorial Health Institute, Warsaw, Poland, 3Department of Child and Adolescent
Psychiatry, Medical University of Warsaw, Warsaw, Poland.
Submicroscopic deletion of 8q21.11 is a rare cause of intellectual disability, developmental delay and craniofacial dysmorphy. Hypotonia, impaired
balance, sensorineural hearing loss, abnormal behaviour as well as mild
fingers and toes anomalies are frequently observed. To date, 13 cases, including 5 from the same family, have been clinically and molecularly characterized. Here we report on the case of a 14-year-old girl with moderate mental
retardation, numerous dysmorphic features (a round face with full cheeks,
high forehead, ptosis, long and downslanting palpebral fissures, short philtrum, Cupids bow of the upper lip, down-turned corners of the mouth, micrognathia, high palate, low-set and prominent ears, and short neck), short
stature and overweight. At birth, microtia of the right ear with external auditory duct stenosis and atrial septal defect were diagnosed. Muscle tone was
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We report the case of a girl affected by mild mental retardation, macrocephaly and microcytic anemia. Clinical examination did not reveal significant
dysmorphic features, except for frontal bossing and mild hypertelorism.
Also her mother presents with mild intellectual disability.
Array-CGH analysis detected a 493.1 Kb telomeric deletion on chromosome
16p13.3, which was found to be maternally inherited. Further examination
of mothers medical history revealed that she was also affected by microcytic anemia, which had been treated with oral iron therapy without benefit.
The deletion involves the two alpha-globin genes (HBA1 and HBA2), explaining the haematological defect.
Deletions of 1.5 and 2 Mb involving the telomeric short arm of chromosome
16 cause the contiguous gene syndrome ATR-16 (MIM #141750) characterized by alpha-thalassemia, mental retardation and variable dysmorphic
features (downslanting palpebral ssures, mild hypertelorism, broad nasal
bridge, small ears and a short neck with webbing). Haploinsufficiency of
SOX8 (MIM #605923) is thought to be responsible for intellectual disability.
The deleted region in our patient does not include SOX8, whereas it comprises 31 genes whose function is still partially unknown, except for HBA1
and HBA2.
To our knowledge this is the smallest 16p13.3 telomeric deletion characterized by mental retardation and microcytic anemia, narrowing the critical
region and pointing at other candidate genes for intellectual disabilities.
Moreover this is the first case of inherited ATR-16 syndrome.
149
ABSTRACTS POSTERS
P08.09-S
Intellectual disability and autistic behavior due to de novo
microduplication of Xq28 involving part of the AFF2 (FMR2) gene. Is
this a plausible explanation?
150
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Massachusetts Eye and Ear Infirmary, Boston, MA, United States, 3Department of
Ophthalmology, Harvard Medical School, Boston, MA, United States.
ABSTRACTS POSTERS
P08.14-M
Mutation in CAPN10 Causing Intellectual Disability in Two
Independent Iranian Families with Overlapping Phenotypes
Brjeson-Forssman-Lehmann syndrome (BFLS) is a rare X-linked mental retardation syndrome. BFLS was first described in 1962, and in 2002 the PHF6
gene at Xq26 was identified as the responsible gene.
Approximately 20 cases have been reported with mutations in the PHF6
gene. At least 12 different PHF6 mutations have been found in both familial
and sporadic cases, predominantly missense and truncation mutations. Five
recurrent mutations have been reported, which arose independently.
The main clinical features are normal birth weight, feeding problems and
hypotonia in infancy, developmental delay, mild/moderate mental retardation, large ears, short toes, small genitalia, gynaecomastia, truncal obesity,
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Engineering of the mouse germline has long been used to create targeted
mutants. The TALEN-mediated approach based on embryo microinjection
of TALENs was shown to result in up to 50% efficiency of germline mutated
offspring. As TALENs can modify one or two copies of the target gene either before or after genome replication, mutant genotypes identified in the
founder generation may represent heterozygous, compound heterozygous
or mosaic state. The high efficiency could allow the generation of mouse
models for diseases caused by dominant de novo mutations. Here we tested
this approach to generate a mouse model for X-linked dominant neurodegeneration caused by de novo mutations in WDR45. After microinjection of
TALEN mRNA surviving Embryos have been transferred in groups of 10 into
the oviducts of 3 pseudopregnant females resulting in 19% mutated founders (5/26). Mutations are deletions between 10 and 57bp in length with a
predicted frameshift in 80% of mutations. From the in situ design and delivery of the TALENs (~1month), over in vitro testing in cell culture to the in
vivo injection into embryos (~2months) until the genotyping of the founder
mutants it only takes 4-5 months. Hence, this presents an innovative tool
to investigate de novo arising mutations in mosaic status which would be
embryonic lethal.
P08.18-M
NR2F1 mutations cause optic atrophy with intellectual disability
Optic nerve atrophy and hypoplasia can be primary disorders or can result
from trans-synaptic degeneration arising from cerebral visual impairment
(CVI). CVI comprise a class of disorders of the projection and/or interpretation of visual input in the brain, and symptoms can consist of low vision,
visual field defects and abnormal visual behavior. Here we report six individuals with CVI and/or optic nerve abnormalities, born after an uneventful
pregnancy and delivery. The affected individuals show mild to moderate
intellectual impairment, without specific facial dysmorphisms. In three
patients large optic disc excavations were seen, which are, until now, only
reported in CVI caused by perinatal damage. In four of the affected persons
de novo heterozygous missense mutations in NR2F1 were identified. In the
two other affected individuals SNP array investigations revealed heterozygous deletions on 5q14.3q15 of 0.84 Mb and 2.83 Mb encompassing 4 and 5
genes, respectively, including NR2F1.
NR2F1 encodes a nuclear receptor protein that regulates transcription. A
luciferase reporter assay showed that missense mutations in the zinc-finger DNA-binding domain and the putative ligand-binding domain decrease
NR2F1 transcriptional activity. Additionally, previous studies in KO mice has
indicated that the protein is important for neurodevelopment, including
oligodendrocyte differentiation (partly in vitro experiment), cortical patterning, and guidance of thalamocortical axons, as well as eye and optic nerve
development.
151
ABSTRACTS POSTERS
In conclusion, these findings indicate that NR2F1 plays an important role in
the neurodevelopment of the visual system and that its disruption can lead
to optic atrophy with intellectual disability.
P08.19-S
Chromosomal microarray analysis of patients with intellectual
disability, autism or multiple congenital anomalies presenting for
genetic services
O. Palumbo, P. Palumbo, R. Stallone, T. Palladino, L. Zelante, M. Carella;
IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy.
Copy number variations (CNVs) are the most common identifiable causes
of intellectual disability/developmental delay (ID/DD), autism spectrum
disorders (ASDs), or multiple congenital anomalies (MCAs). Chromosomal microarray analysis (CMA), with a 10-20% diagnostic yield, can identify CNVs1 Mb. We report our experience with the use of the Affymetrix
SNP Arrays in 1600 Italian patients during the past 6 years (2008-2013).
We identified CNVs with a high score of pathogenicity in 415 (27%) patients. Among them 143 (34.4%) showed a CNV overlapping with a known
syndrome, 272 (65.6%) a likely pathogenic rearrangement. Of particular
interest, we found some CNVs useful to further delineate the clinical features associated with deletions in 8q12.1q12.3, in 15q25.2, in 17q21.31, in
2q24.1q24.2, in 22q11.2, and duplications in 16p13.3 and in 11p13. Some
CNVs were useful to describe new syndromes such as a 1.7 Mb deletion in
3q13.2q13.31. Also, we have identified a large group of small CNVs (< 1.0
Mb) encompassing, either in whole or in part, functionally related genes to
the phenotypes such as CASK, CNTN6, SNTG2, HIP1, DLG2, NRXN1, MCPH1
and CHL1 genes. Among these small CNVs, we have reported a FOXP1 gene
microdeletion in a boy with autism and speech delay, and a de novo interstitial deletion of 0.122 Mb at 2q24.2 region harboring only TBR1 gene in a boy
with moderate to severe intellectual disability. Variants of uncertain significance (VOUS) because unreported, containing genes of uncertain clinical
significance or non-genic but potentially regulating nearby gene expression,
were identify in 128 individuals (8%).
P08.20-M
The Cohen syndrome-associated protein COH1 functions as Golgi
matrix protein required for Golgi integrity
152
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Intellectual disablity (IQ<70) affects up to 3% of the general population. Until recently, the underlying cause was unclear in about half of the affected
individuals. The introduction of whole exome sequencing (WES) techniques
enables elucidating the genetic background of ID.
We performed WES in a cohort of 250 individuals with unexplained ID and
identified heterozygous de novo CTNNB1 mutations in five unrelated individuals (three frameshift, one stop, and one splice mutation). All five patients have severe motor delay, profound speech impairment, hypotonia of
the trunk and hypertonia of the legs. The craniofacial phenotype comprises
microcephaly (4/5) and some consistent facial features - a broad nasal tip,
small alae nasi, long philtrum and thin upper lip vermillion.
Beta-catenin is a key downstream component of the canonical Wnt signaling
pathway, and acts as a negative regulator of centrosome cohesion. Whereas
somatic gain-of-function mutations have already been found in various tumor types, germline loss-of-function mutations were suspected in animal
models to influence neuronal development and maturation. This was supported by the finding of dominant inactivating CTNNB1 mutations as a cause of ID in 3/865 patients (0.35%, deLigt etal., 2012). Their phenotype was
additionally characterized by absent/limited speech, microcephaly and spasticity with severely impaired walking.
Our finding of five individuals in our cohort of 250 (2.0%) suggests that CTNNB1 loss-of-function mutations might be a more frequent cause of ID than
estimated from the data of deLigt and colleagues. Our data further emphasize the importance of Wnt signalling in human brain development and/or
function.
P08.22-M
Deletions limited to CTNND2 cause mild intellectual disability
The 15q overgrowth syndrome has been associated with the very distal
15q duplication. At least 26 cases with trisomy of 15q25-26qter have been
published, of which approximately 70% have presented with overgrowth
and about 65% have been the result of a parental balanced translocation.
Overgrowth has been associated with the dosage effect of IGF1R (insulin-like growth factor 1 receptor) gene located on 15q26. However, about 50% of
ABSTRACTS POSTERS
patients with larger duplications of distal 15q (15q21-24qter, including the
IGF1R gene) on the contrary presented with growth retardation, although a
few authors have reported overgrowth as well.
We report 12 and 8 years old brothers with identical 0.77 Mb microduplications in 15q26.3 region (chr15: 101,031,538-101,802,565, Hg19). The
elder sib presents with postnatal overgrowth - at the age 12 his height is
175 cm (+4 SD), but his birth length was 52 cm (0 SD). The younger sib
has normal growth (+1 SD). In addition, they both present with expressive
speech disorder, some facial and hand microanomalies, and poor fine motor
skills. Their father has overgrowth (+2.5 SD), prominent facial features and
presented with dysarthria in childhood. Therefore, the duplication is with
high probability of a paternal origin (analysis in work). This is the smallest
pure 15q26 duplication reported so far. Interestingly, the IGF1R gene is not
duplicated in our patients. Furthermore, they demonstrate variable clinical
phenotype. Therefore, we give further evidence that a more complex pathogenesis for the development of somatic overgrowth should exist in case of
distal 15q duplication.
P08.24-M
6p21.33 microdeletion associated with EHMT2 haploinsufficiency
and intellectual disability
K. Kurosawa1, I. Ohashi1, Y. Kuroda1, T. Naruto1, T. Saito1, M. Masuno2;
1
Kanagawa Childrens Medical Center, Yokohama, Japan, 2Kawasaki University of
Medical Welfare, Kurashiki, Japan.
Microdeletion of 6p21 is a rare condition that has been described in patients with multiple congenital malformation and intellectual disability. We
report on a patient with a 0.4 Mb microdeletion in 6p21.33 and intellectual disability. The genomic loss contained 30 RefSeq genes ncluding EHMT2
which is a primary enzyme for mono- and dimethylation at Lys 9 of histone
H3 (H3K9me1/2), and plays critical roles in various biological processes.
The 6-year-old boy was referred for evaluation of intellectual disability and
macrocephaly. He was born at 37 weeks of gestation to a 32-year-old G1 P1
mother and 34-year-old father after uneventful pregnancy. The birth weight
was 2418 g, length 45 cm, and head circumference 33 cm. His psychomotor
development was delayed. He achieved head control at 4 months, sitting at
10 months, and walking alone at 2 years. He spoke his first word at 2.5 years.
He never had seizures and his hearing was normal. His height was 106 cm
(-1.7 SD), weight 20.3 kg (-0.3 SD), and head circumference 53.5 cm (+1.4
SD) respectively. Brain MRI at age of 4 years showed incomplete rotation
of the bilateral hippocampus. EHMT2 is a human homologue of mouse G9a
that exists predominantly as a G9a-GLP heteromeric complex. The human
homologue of the GLP is EHMT1, which is a causative gene responsible for
Kleefstra syndrome with characteristic facial dysmorphism and severe intellectual disability. This case provides insight into the etiological mechanism of histone modification and human development.
P08.25-S
EPHA1 as a new candidate gene for autosomal recessive nonsyndromic intellectual disability
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Virtually all clinical testing for FXS revolves around the determination of the
length of the CGG repeat tract in the 5UTR of the Fragile X Mental Retardation (FMR1) gene. Repeat lengths generally fall into four categories, where
<45 CGG repeats is accepted as normal, 45-54 repeats is the intermediate
range (gray zone), 55-200 repeats is considered the premutation range, and
>200 repeats is a full mutation. Diagnostic testing for FXS has traditionally
employed both standard PCR and Southern blot techniques since they provide distinct, yet overlapping, levels of resolution and complementary pieces
of information. However, newer PCR protocols such as TRP-PCR have the
potential to allow amplification of full mutations, thereby providing a method of screening for expansions at the FMR1 locus. The molecular diagnostic laboratory at the Greenwood Genetic Center has offered FXS testing for
nearly 25 years. In 2010, we implemented a validated, lab-developed FMR1
PCR assay utilizing commercially available TRP-PCR reagents (Abbott Molecular) into our routine diagnostic workflow. Here we summarize our experience with the TRP-PCR assay, discuss how the implementation of this assay
has changed our diagnostic workflow, and compare the hands-on time, cost,
and turn-around time for the samples tested over the past four years to these statistics for samples tested prior to the implementation of the TRP-PCR
assay. In conclusion, the implementation of the TRP-PCR assay has allowed
us to drastically reduce the hands-on time, turn-around time, and number
of Southern blots performed for samples submitted to our laboratory for
diagnostic FXS testing.
P08.28-M
A pilot study for prenatal and preconceptional Fragile-X syndrome
screening in the Balearic Islands
153
ABSTRACTS POSTERS
causes of hereditary intellectual disability. The estimated incidence of FXS
in males in the Spanish population is 1 in 2500 with full mutation and about
1 in 250 with premutation (Fernandez-Carvajal et al. 2009). In women,
although the prevalence in Spain of premutation has not been established,
estimates in other western countries range from approximately 1/150 to
1/250. Given the severity of the disease, its high incidence in the general population, the exclusively maternal expansion, the familial and social impact
of the FXS, and the high level of detection of current techniques (99%), we
think that screening for FXS in women of reproductive age is a reliable and
desirable option. Therefore, we have initiated a pilot study in the Balearic
Islands to determine the feasibility and acceptability of prenatal and/or preconceptional screening in women of childbearing age. The results obtained
so far, in a total of 3118 women (252 preconceptional and 2866 prenatal)
indicate a high acceptability of testing both, in women that are referred for
prenatal or preconceptional consultation. Surprisingly, the incidence of carriers of a premutation (55-200 repeats) is (to date) very high: 1 in 97, which
may indicate a higher prevalence than previously thought. We will present
updated results based on a total of approximately 3500 women.
P08.29-S
GPM6A is duplicated in a patient with learning disability and
influences cholesterol response and long-term memory in Drosophila
melanogaster
154
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studies of patients have reported variants within the GABP2 binding domain
p.Gly876Ser in an individual with autism spectrum disorder (Piton et al.,
2011) and p.Ala864Thr in a patient with mental retardation (Tarpey et al.,
2010). The mutation was confirmed by Sanger sequencing in both patients
and their mother. Extended family studies are ongoing.
P08.31-S
HDAC8 duplication in a patient with de Lange -like phenotype
K.K., female, was referred to our attention with a previous clinical diagnosis
of Silver Russel syndrome. Prenatal ultrasound were normal. No prenatal
cytogenetic examination was performed. She was born at 40+5 week of
gestational age through cesarean section; birth weight was 2,320 ( < 3rd
pc), length was 45 cm (< 3rd pc), head circumference was 30 cm (< 3rd pc).
The karyotype was normal (46,XX). Major malformations were excluded.
We evaluated her at 6 years and a half; she showed prominent eyes, long
thick eyelashes, posteriorly rotated ears, high nasal bridge; short and mild
micrognathia and hirsutism. Her weight was 18,900 Kg (10th pc), length
110,5 cm (3-10th pc) and head circumference was 46,5 cm (<<3rd pc); she
also presented severe mental retardation. Hands and feet X-rays showed hypoplasia of the 5th meacarpal and the 5th metatarsal. A CGH-array showed
duplication of a part of HDAC8 gene (exon 5 and 6) from nt 71,630,467 to nt
71,697,179. HDAC8 gene was recently found implicated in Cornelia de Lange syndrome with variable clinical expressivity. Up now only complete deletion or mutation of the gene have been described. No reports of duplication
of this gene are available. Our patient shows some clinical features related
to de Lange syndrome also if her phenotype is not classic at all. Expression
study are in progress in order to define the biological consequences of this
finding and the relationships with the phenotype.
P08.32-M
Clinical characterization of a patient with a complex rearrangement
involving duplication and deletion of 9p and 9q
ABSTRACTS POSTERS
The 1q21.1 CNV is associated with a variable clinical phenotype and normal
to delayed neurodevelopment. We performed whole genome transcription
and exome sequencing analysis in 5 subjects from 2 families with 1q21.1 deletion (3 subjects) and duplication (2 subjects), in search for genetic changes that could affect the phenotype. The subjects had variable learning difficulties. A pathogenic variant in the ATF6 gene from 1q22-23, which plays a
role in endoplasmatic reticulum stress response, was detected in the mildly
affected father and his more severely affected child with 1q21.1 duplication.
It was validated by Sanger sequencing and associated with reduced ATF6
RNA and protein expression in patient lymphoblast cell lines. However, the
ER stress response, as measured by induction of known ER stress genes
(GPR78, Dnajb9, Sdf2l1) in response to tunicamycin was not altered in patient vs control cell lines. No candidate pathogenic mutation that could be
linked to altered gene expression status and/or phenotype was found in the
3 subjects with the 1q21.1 deletion, either in the CNV region or genomewide. Interestingly, for all 5 subjects with 1q21.1 CNV a larger number of the
107 genes implicated in ER stress response showed altered expression in
comparison to controls on the whole genome expression array (15-28% in
subjects in comparison to 0-6% in controls). Perturbed ER stress response
caused either by dysfunction of genes from 1q21.1, genome wide, or both,
could play a role in the observed phenotypic variability and result in more
severe developmental abnormalities in unfavourable environmental circumstances.
P08.34-M
Contribution of copy number variants (CNVs) in congenital
unexplained intellectual and developmental disabilities in 149
Lebanese patients
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old girl, with pre- and postnatal growth retardation, severe psychomotor
retardation, spastic quadriparesis, dysmorphic features, cleft palate, bilateral congenital cataract, severe epilepsy with status epilepticus episodes.
aCGH on an 105K Agilent platform was done according to manufacturers
recommendations; the results were validated by FISH. In the two siblings,
aCGH revealed the same genetic abnormality: arr 4p16.3p16.1(72,4478,373,151)x3,10q26.3(130,977,858-135,434,178)x1 pat. The balanced
t(4;10)(p16.1;q26.3) translocation was detected by FISH in the genome
of the patients father. In the second case, aCGH showed another genetic
anomaly involving 4p16.3 region: arr 4p16.3p16.1(72,447-9,371,067)
x1,8p23.3p23.1(176,452-8,094,773)x3. The genetic investigations of the
parents were normal. The 4p region includes over 140 genes, many included in OMIM Database as involved in various pathologies. The 10q deleted
region consists of over 60 genes, many also described in the OMIM Database. The 8p duplicated region has a size of 8 Mb and includes 116 genes. The
complexity of the phenotype, both for deletion and duplication of 4p16.3
region, of these cases can be explained by the association of other genetic
anomalies (10q deletion and 8q duplication, respectively). Acknowledgements: project PN 09.33.02.03.
P08.36-M
Recurrent CNVs in 15q11.2-q12 in Bulgarian patients with
generalized epilepsy and intellectual disability
Copy number variants are frequent in autism spectrum disorders and generalized epilepsy. In this study, we performed comparative genomic hybridization assay (aCGH) using Agilent Microarray Kit, 4x180K in a preselected
sample of 36 Bulgarian patients with epilepsy and intellectual disability (ID).
The region most often engaged in copy number changes included 15q11.215q12. In 7 patients (19.4%) CNVs were located in 15q12. Five of them harbored microduplications in GABRG3 gene, 1 patient showed microduplication in GABRB3 gene and in 1 patient both rearrangements were present.
Aberrations in 15q11.2 region were observed in 2 patients. One of them was
a 0.161 Mb deletion, including SNURF/SNRPN upstream reading frame. In
the other patient, a 2.608 Mb duplication covering 36 genes was revealed.
Genomic region on 15q11-13 is involved in many clinically important rearrangements. These include aberrations of various sizes which can affect
the neuronal differentiation by disrupting normal epigenetic control and
gene expression. All Bulgarian patients with CNVs harboring 15q12 shared
a common clinical phenotype of severe ID, speech impairment or complete
lack of speech, different types of seizures, facial dysmorphisms, microcephaly and behavior abnormalities of the autistic spectrum. In contrast, the two
patients with 15q11.2-rearrangements displayed different clinical characteristics and milder forms of mental retardation. Our results are in line with
the central role of GABAergic systems in ID and epilepsy. Further studies are
needed to investigate the parental origin and elucidate the effect of the CNVs
and the phenotype-genotype correlations. Acknowledgement: the study was
supported by DTK02/67/2009, DUNK01-2/2009 funded by NSF.
P08.37-S
Disruption of the Methyltransferase-Like 23 Gene METTL23 Causes
Mild Autosomal Recessive Intellectual Disability
We describe the characterization of a gene for mild non-syndromic autosomal recessive intellectual disability (ID) in two unrelated families, one from
Austria, the other from Pakistan. Genome-wide single nucleotide polymorphism (SNP) microarray analysis enabled us to define a region of homozygo-
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ABSTRACTS POSTERS
sity-by-descent (HBD) on chromosome 17q25.1. Whole exome sequencing
and analysis of this region in an affected individual from the Austrian family
identified a 5bp frameshifting deletion in the METTL23 gene. By means of
Sanger sequencing of METTL23, a nonsense mutation was detected in a consanguineous ID family from Pakistan for which homozygosity-by-descent
mapping had identified a region on 17q25. Both changes lead to truncation of the METTL23 protein, which disrupts the predicted catalytic domain
and alters the cellular localization. 3D-modelling of the protein indicates
that METTL23 is strongly predicted to function as a S-adenosyl-methionine
(SAM)-dependent methyltransferase. Expression analysis of METTL23 indicated a strong association with heat shock proteins, which suggests that
these may act as a putative substrate for methylation by METTL23. A number of methyltransferases have been described recently in association with
intellectual disability. Disruption of METTL23 presented here supports the
importance of methylation processes for brain function and development.
P08.38-M
Targeted next generation sequencing in a new cohort of 996
individuals with Intellectual Disability
We screened DNA from 996 individuals with Intellectual Disability (ID) for
variants in known genes and candidate genes for ID in order to identify the
cause of disease. Overall, we investigated the coding sequence of 565 candidate genes in individual cases affected with moderate to severe ID.
We observed 8225 non-synonymous variants passing quality control criteria and frequency filter (<1% in internal and population controls). The rate
of loss of function (LoF) variants (frameshift, nonsense or canonical splice
site) was 0.43 per person. Overall, we have identified pathogenic LoF mutations in 11% of the cases (110 out of 996) affecting known genes previously
reported to harbour mutations causing ID. LoF mutations in ATRX, CC2D2A,
ARID1B and CUL4B were the most commonly observed (frequencies varied
from 0.4% to 0.6%). The interpretation of the vast number of missense mutations in known genes proved to be more challenging, as rarity of variant
is insufficient to assign pathogenicity in such a genetically heterogeneous
phenotype.
Although the cohort consisted of predominantly non-syndromic ID cases,
the yield of mutations in genes associated with a syndromic phenotype was
high, for example, ATRX, CC2D2A, CHD7 and ARID1B. This suggests that the
distinction between syndromic and non-syndromic ID is an increasingly artificial concept in a genotype-driven diagnostic era.
P08.39-S
Discovery of new mutations using exome sequencing in adult patients
with intellectual disability and psychiatric disorders
N. Baena1, M. Vias1, R. Rabionet2, X. Estivill2, E. Gabau1, S. Esteba3, N. Ribas3, R. Novell3,
M. Guitart1;
1
Corporaci Sanitria Parc Taul, Sabadell, Spain, 2Center for Genomic Regulation,
Barcelona, Spain, 3Specialized Service in Health Mental and Intellectual Disability, Parc
Hospitalari Mart i Juli, Salt (Girona), Spain.
Next Generation Sequencing offers the opportunity to identify new mutations and increasing the proportion of patients with ID receiving a genetic
diagnosis. We have analyzed a cohort of 102 adult patients affected by ID,
psychiatric diseases and minor dysmorphic features, identifying a genetic
cause of ID in 28 (27,5%). We recruited 9 trio cases negative for fragile X
syndrome, chromosomal and subtelomeric rearrengements and pathogenic
copy number variants. The exome was captured with Agilent sure select
technology, multiplexed, and sequenced in an Illumina HISeq2000 lane. Potential damaging variants detected were selected and filtered (condel score,
EVS, 1000genomes, dbSNP). Variants were finally confirmed by Sanger sequencing. We detected an average of 8624 non-synonymous variants in the
cases, of which an average of 23 were de novo. Frequency and functionality
based filtering reduced the number of potential candidate ID genes harbouring de novo variants to 1-10 per case. In 5 cases, potential disease-causing
variants were identified in genes previously implicated in ID syndromes:
One case was compound heterozygous for two RPGRIP1L rare missense
variants, two cases carried a de novo mutation in (an in-frame deletion in
UBE3A and a missense variant in MLL), one familial case was carrying a rare
variant in TCF4 and in the last case a frameshift deletion was identified in
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SATB2. Two cases were compound heterozygous for two very rare or novel
variants in two different potential ID candidate genes, and in the remaining
two cases, no candidates were identified. Work supported by FIS grants:
PI080778 and PI10/01710.
P08.40-M
The degree of Intellectual Disability is significantly associated with an
excess of Runs of Homozygosity (ROH)
I. Gandin1,2, F. Faletra2, M. Carella3, V. Pecile2, G. Ferrero4, E. Belligni4, P. Palumbo3, O.
Palumbo3, P. Bosco5, C. Romano5, C. Belcaro1, D. Vozzi2, A. P. dAdamo1,2;
1
University of Trieste, Trieste, Italy, 2IRCCS Burlo Garofolo, Trieste, Italy, 3IRCCS Casa
Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy, 4AO citta della salute e della
scienza, Torino, Italy, 5IRCCS Oasi Maria SS, Troina(EN), Italy.
Varying degrees of developmental delay or intellectual disabilities and dysmorphic features appear to be strongly associated with deletions involving
5p. Most of these deletions are associated with cri du chat syndrome. These
deletions can be terminal, interstitial, or associated with complex chromosomal rearrangements. To establish the genotype-phenotype of 5p deletions
many efforts had be done in the last years in dissecting the phenotype and
three different critical regions had been determined. But there had been
controversaries about the relationship between MR and deletions of MRIII encompassing bands 5p13.2p14.3. Deletions limited to this region were
reported to show no phenotype, but to aggravate the phenotype in case of
accompanying aberrations (Zang X., et al. 2005). We now observed a novel
3- generation family with a interstitial deletion del(5)(p13.2p14.3) limited
to MRIII without additional rare CNVs in microarray testing. The index patient, a 15 years old girl, showed learning disability (IQ 75), microcephaly,
high pitched voice and a subtle facial phenotype. The patients mother and
grandmother had the same deletion and likewise the learning disabilities,
the high pitched voice and subtle facial features, but no microcephaly. In
contrast to the current literature we therefore propose that deletions limited to the MRIII region have a mild, but distinct phenotype with particular
emphasis on the high pitched voice.
P08.42-M
Inverted triplication of 7q11.22 embedded within the 7q11.21q11.23
duplication segment in a child with stigmata dysplastica,
developmental and speech delay
ABSTRACTS POSTERS
mental disorder with mild craniofacial anomalies and increased incidence
of congenital anomalies. Williams-Beuren syndrome (WBS; OMIM#194050)
is caused by ~ 1.8-Mb hemizygous deletion on chromosome 7q11.23. WBS
triplication syndrome with similar but more severe clinical features has
been also described.
Case presentation: We present a 3-years-old boy with developmental and
severe speech delay, dysmorphic signs, bilateral cryptorhidism and hypoplasic corpus callosum. Cytogenetic and molecular analysis revealed a complex de novo chromosomal rearrangement which we characterize as inverted triplication of 7q11.22 segment embedded within larger 7q11.21q11.23
duplication.
Methods and results: Molecular characterization by array-CGH revealed a
1.93-Mb duplication of segment 7q11.21, 5.27-Mb triplication of segment
7q11.21q11.22 and 1.33-Mb duplication of segment 7q11.23. Further molecular cytogenetic investigation was performed with multiple combinations
of specific FISH probes. Metaphase and interphase FISH confirmed the location of triplication 7q11.22 segment within the 7q11.21q11.23 duplication.
Additional FISH analysis revealed that triplicated 7q11.22 segment on derivative chromosome 7 was inverted. Parental cytogenetic analysis demonstrated de novo origin of this complex chromosomal rearrangement.
Conclusions: Although chromosomal imbalances are the major cause of developmental delay, large de novo CNVs are relatively rare events. We have
identified a novel complex rearrangement on chromosome 7, duplication
with embedded inverted triplication also known as DUP-TRP/INV-DUP
structure. Triplication of segment 7q11.22 is probably generated by long
range inverted repeats which still need to be identified.
P08.43-S
Identification of novel variants in PIGQ, PGAP3 and PIGY further
implicate the GPI pathway in the pathogenesis of neurodevelopmental
abnormalities
A. T. Pagnamenta1, H. Martin1, M. F. Howard1, A. Goriely1, Y. Murakami2, K. Hudspith1,
C. Anzilotti1, S. J. L. Knight1, H. Stewart3, E. Blair3, T. Kinoshita2, P. Donnelly1, J. C. Taylor1,
U. Kini3;
1
University of Oxford, Oxford, United Kingdom, 2Osaka University, Osaka, Japan, 3Oxford
University Hospitals NHS Trust, Oxford, United Kingdom.
Around 30 genes are required for glycosylphosphatidylinositol (GPI) biosynthesis. Mutations in many of these are reported to cause a spectrum of
neurodevelopmental abnormalities. Here we describe novel mutations in
three members of this pathway.
Whole genome sequencing was applied to a sporadic patient with severe
early-onset epilepsy. This approach identified a homozygous PIGQ splice
mutation, resulting in exon skipping and defective GPI biosynthesis. Additionally, autozygosity mapping and exome sequencing were combined to
investigate two consanguineous Pakistani families where multiple affected
individuals presented with intellectual disability and microcephaly. In the
first family, seizures were present in 2/3 affected individuals. A homozygous
p.G92D mutation was detected in PGAP3 that co-segregated with disease.
Affected individuals had elevated alkaline phosphatase, a marker for defective GPI biosynthesis. Functional studies using CHO cells confirmed the
mutation influenced GPI-anchor remodelling. For the second family, the concurrent release of ENCODE data prompted us to examine UTR variants and
a c.-540G>A variant was detected in PIGY which co-segregated with disease.
Disruption of an SP1 binding motif suggested that the variant might influence gene expression. IonTorrent sequencing confirmed that in heterozygote carriers ~9% of transcripts were expressed from the mutant allele.
Our work strengthens the role of the GPI pathway in the pathogenesis of
neurodevelopmental abnormalities. Although PIGQ and PIGY both encode
subunits of N-acetylglucosaminyltransferase, the range of phenotypes seen
in the families described recapitulates the pleiotropic effects of defective
GPI biosynthesis as a whole. This study also highlights the importance of
assessing UTR variants during analysis of exome data.
P08.44-M
Careful consideration is necessary in MECP2 testing and allele drop
out
Allele drop out (ADO) leads to the preferential amplification of one of two
allele due a series of reasons among which the occurrence of sequence
mismatch within a primer-binding site or a complex DNA motif. The drop
out can cause false results during PCR amplification and be responsible of
potential pitfall in diagnosis. It is known that genomic DNA sequences can
assume different complex secondary structures which can prevent DNA replication and transcription and, if altered by mutations, may be responsible of ADO events in PCR amplification. An example of these genomic DNA
Back to index
Xq28 duplications encompassing MECP2 have been described in male patients with a severe neurodevelopmental disorder associated with hypotonia
and spasticity, severe learning disability, stereotyped movements and recurrent pulmonary infections. We report on standardized brain magnetic resonance imaging (MRI) data of 30 affected patients, including 5 symptomatic
females, carrying a MECP2 duplication, the size of which varied between
228 kb and 11.7 Mb. The aim of this study was to compare brain MRI results to seek recurrent malformations and attempt to determine whether
variations in imaging features could be explained by differences in the size
of the duplications. We showed that 90% of patients had brain MRI abnormalities, and that they shared certain non-specific brain malformations
such as corpus callosum abnormalities (n=20), ventricular dilatation (n=9),
reduced volume of the white matter (WM) (n=12), increased T2 signals in
posterior periventricular WM (n=6), and vermis hypoplasia (n=5). The occipito-frontal circumference could be highly variable since it was >+2DS in 5
patient and <-2DS for 4 patients. Among the 9 patients with dilatation of the
lateral ventricles, 6 (67%) had a duplication involving L1CAM. The only patient harbouring bilateral posterior subependymal nodular heterotopia also
carried a FLNA gene duplication. We could not demonstrate a link between
periventricular WM hypersignals / delayed myelinisation and duplication of
IKBKG. These results show that patients with MECP2 duplications share certain common but non-specific brain abnormalities. These imaging features,
therefore, do not constitute a diagnostic clue. We did not clearly demonstrate a genotype-imaging phenotype correlation.
P08.46-M
Clinical relevance of the genotype-phenotype correlation in a patient
with a de novo monosomy 9pter-p24.1 and duplication Xq28-qter
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the first year of life with severe neonatal encephalopathy. Since birth, the
clinical conditions have been worsening during time. Now he shows severe
mental retardation, dysmorphic features, extreme generalized hypotonia,
white matter leucomalacia, drug-resistant epilepsy, blindness, genitourinary and gastrointestinal abnormalities, generalized joint laxity and recurrent
respiratory tract infections. High resolution karyotype was normal. Subtelomeric FISH probes identified a derivative 9 of an unbalanced translocation
t (9p;Xq) with monosomy 9p and Xq disomy. FISH studies of the parents revealed that the alterations were de novo in the patient. Array CGH was performed and identified two rearrangements: a 6Mb deletion on chromosome
9 from p24.3 to p24.1 and a 5Mb duplication on chromosome X from q28 to
qter. This double rearrangement has never been described in literature. Our
patient presents urogenital birth defects, facial dysmorphism (possibly due
to the 9p deletion) and developmental regression, axial hypotonia, Hirschsprung disease, feeding difficulties, recurrent infections, epilepsy, absence
of language, white matter disease (typically related to the Xq28 duplication).
Several genes, included in the deleted (KANK1, DMRT1, SLC1A) and in the
duplicated region (MeCP2, L1CAM), are known to have an important role
in the Central Nervous system development. This report allows to compare the phenomics of our patient to the other cases described in literature,
to contribute to the knowledge about genotype-phenotype correlation and
to provide new informations for future studies about the 9p24.3-pter and
Xq28-qter regions and the genes included.
P08.47-S
Exome sequencing identifies candidate gene for MEHMO syndrome
The patient is a 3,5 years young boy suffering from severe psychomotor delay, microcephaly, epilepsy, and multiple endocrine disorders (i.e. obesity,
diabetes, hypogenitalism, and partial deficiency of pituitary hormones).
There are no other living male members in the mothers family, as mothers
brother and grandmothers brother died in the first months of life. The clinical picture and family history indicated towards the MEHMO syndrome, an
X-linked disease which has been described in three families only.
Exome sequencing of probands DNA revealed 23 previously unreported variants on X chromosome as confirmed also by Sanger sequencing. Haplotype
analyses showed only 3 of them to be shared with probands mother and his
mothers mother. Only one of them, a variant in EIF2S3 gene, is in the region
of X-chromosome previously described to be associated with MEHMO syndrome. EIF2S3 encodes a subunit of eukaryotic translation initiation factor
2 (eIF2) that is responsible for transporting the initiator Met-tRNAiMet to
the 40S ribosomal subunit. Point mutations in this gene were previously described in two families with intellectual disability. The variant found in the
patient is a frame-shift mutation with premature stop codon influencing 8
last amino acids of the protein. In-sillico analyses evaluate this change as
disease causing.
Our results support the role of EIF2S3 as a candidate gene, disruption of
which might significantly contribute to this severe clinical symptomatology.
Supported by APVV 0187-12, KCMM (ITMS 26240220071)
P08.48-M
New insights on cognitive and structural brain imaging phenotype in
primary microcephaly due to ASPM mutations
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areas. Unlike most previous data that linked microcephaly to mental retardation, these findings suggest i) that these microcephalic ASPM-related patients are able to learn despite their cognitive disabilities and ii) that other
master genes than ASPM are necessary for the development of hippocampus formation and function.
P08.49-S
Microduplication 17q12 in a patient with microcephaly and moderate
psychomotor delay
ABSTRACTS POSTERS
Germany, 3CeGaT, Center for Genomics and Transcriptomics, Tbingen, Germany,
4
genteQ, Hamburg, Germany.
Back to index
Noonan syndrome (NS OMIM 163950) is a multisystemic dominant disorder with a prevalence of 1/1000-1/2500 live births. It is clinically and
genetically heterogeneous, with mutations in several genes of the RAS/
MAPK pathway detectable in up to 75% of cases. Pathogenesis of central
nervous system (CNS) developmental anomalies has not been thus far fully
enlightened. We have applied the multiple hit hypothesis to study in deep the
pathogenic mechanisms implicated in NS related CNS anomalies. In this oligogenic model, point mutations and genomic rearrangements cooperate in
an additive manner in the pathologic development of CNS. Using array CGH
technology, we analyzed 15 samples of selected patients with molecularly
confirmed diagnosis of NS and a severe impairment of CNS. We found 37
rare CNVs (one/several per patient) most of them inherited from an healthy
parent. According to the Database of Genomic Variants 12/37 were never
reported and 25/37 were reported in very few cases. Based on the function
of the genes mapping in the identified CNVs, 10/37 (27%) were probably involved in the pathogenesis of CNS anomalies observed in our patients.
We provided first data supporting the hypothesis that CNS involvement in
NS does not depend exclusively on single gene mutations but also on the
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concurrent presence of other genomic penetrant variants. These results represent an initial assumption for the application of the multi-hit hypothesis
in the dissection of the NS pathogenesis. Further studies on larger cohorts
are deserved to better define the meaning and the clinical implications of
these findings.
P08.56-M
NPAS3-related copy number variants: a role in developmental delay?
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Introduction:Autosomal recessive primary microcephaly (MCPH) is a congenital disorder caused by impaired neurogenic mitosis lead to defect in brain
development. This disorder is heterogeneous genetically and characterized
by reduced head circumference (-2 SD or more) under age and sex-based
mean and mild to severe intellectual disability. So far, 12 genetic loci (MCPH112) with twelve corresponding genes (MCPH1, WDR62, CDK5RAP2,CASC5,
ASPM, CENPJ, STIL,CEP135, CEP152, ZNF335,PHC1and CDK6) have been
identified for this disease.Using whole-exome sequencing (WES) as a new
pioneer technology and diagnostic tool, we can provide an opportunity for
identifying the causal mutations with more efficient and cost-effectiveness
in Iranian patients with autosomal recessive primary microcephaly. Case
presentation:We report an Iranian family with two affected individuals with
moderate intellectual disability, large toes and primary microcephaly with
consanguineous marriage. We performed WES for one affected and focused
ABSTRACTS POSTERS
on genes associated with microcephaly and homozygous variants. We identified a homozygous novel frameshift mutation in CDK5RAP2 in the affected
individual. Sanger sequencing confirmed the presence of the homozygous
mutation in the other affected and heterozygous state for parents and normal siblings. Discussion:WES led to cost-effectiveness and rapid identification of novel frameshift deletion in CDK5RAP2 in Iranian family with primary
microcephaly. We can facilitate genetic counseling for this family. To date,
only five different mutations have been reported for CDK5RAP2 gene which
most of them were from Pakistan. Moreover, this study implies that WES is
a suitable diagnostic tool for identifying mutations responsible for MCPH
families.
P08.61-S
New case of biallelic TRMT10A deficiency identified by exome
sequencing confirms the associated phenotype of primary
microcephaly with intellectual disability and short stature
Recently a mutation in the TRMT10A gene was identified in a large consanguineous family of Moroccan origin defining a new syndrome of young onset diabetes, short stature and microcephaly with intellectual disability. By
linkage analysis and exome sequencing a homozygous nonsense mutation
was identified in all three affected siblings. The protein encoded by TRMT10A (also RG9MTD2), which was proposed to have tRNA methyltransferase
activity, was shown to be ubiquitously expressed with enriched levels in the
affected tissues brain and pancreatic islets and to be absent in lymphoblasts
from the affected siblings. We now report a new case of biallelic TRMT10A
deficiency in a girl born to apparently non-consanguineous parents of Kosovo origin. By exome sequencing in our patient we identified a homozygous
nonsense mutation (c.379C>T) in the TRMT10A gene. Of note, this is the
same mutation as recently reported, introducing a premature stop codon
at position 127 of the protein. Our patient presented with primary microcephaly, intrauterine onset borderline growth, mild intellectual disability
and fine motor problems, a high palate with uvula bifida and minor facial
features such as long narrow face with narrow palpebral fissures, long thin
nose and small mouth. At age 4 years a seizure disorder started. Notably,
at age 8 years, our patient did not yet manifest diabetes, which was of adolescent onset in the previously described family. In conclusion, our report of
a novel patient confirms the phenotype of the novel syndrome associated
with biallelic TRMT10A deficiency including short stature and microcephaly
with intellectual disability.
P08.62-M
Analysis of MECP2, CDKL5, and FOXG1 genes in Czech patients with
Rett syndrome and Rett-like features
Background: Rett syndrome is a severe X-linked dominant neurodevelopmental disorder primarily caused by de novo MECP2 mutations. Clinical
features include developmental regression at the age of 6-18 months, acquired microcephaly, autistic behavior, loss or severe impairment of speech
and purposeful hand use, stereotypic hand movements, gait apraxia, and
seizures. CDKL5 mutations have been identified in early-onset seizure variant and FOXG1 mutations in congenital variant of Rett syndrome. We report
the results of mutation analysis of these genes in Czech patients with Rett
syndrome and mental retardation with Rett-like features. Materials and
methods: MECP2 was analyzed in 416 patients, CDKL5 was analyzed in 59
patients, and FOXG1 was analyzed in 20 patients. MECP2 and CDKL5 were
analyzed by high-resolution melting analysis and DNA sequencing. FOXG1
was analyzed by DNA sequencing. Large deletions and duplications were
analyzed by MLPA analysis (MRC-Holland). Results: Pathogenic mutations
in the MECP2 gene in were identified in 45 patients with classic Rett syndrome, 7 patients with atypical Rett syndrome, 11 patients with Rett-like
features, and 1 patient with autism. CDKL5 mutations were found in 2 patients with early-onset seizures and Rett-like phenotype. No FOXG1 mutation
was detected in this study. Conclusions: MECP2 mutations are common in
classic Rett syndrome patients, but they are less frequent in atypical or Rettlike phenotypes. However, analysis of MECP2 in these patients should not be
discouraged. More patients should be examined to determine frequencies
of CDKL5 and FOXG1 mutations in Czech Republic. Supported by grants NT
13120-4/2012, UNCE 204011/2012, MZCR RVO-VFN64165/2012.
Back to index
P08.63-S
Ageing in Rett Syndrome: Characteristics of long term survivors
reported through the British Isles Rett Syndrome Survey (BIRSS)
We report what is known about the health and wellbeing of thirty women of
at least 40 years with Rett syndrome (RTT). The study is based on longitudinal data from the British Isles Rett Syndrome Survey (BIRSS). These 30 women have a clinical diagnosis of RTT: 24 women of 40-49 years, five women
of 50-59 years and one older woman of 64 years. Tweny nine women were
diagnosed with classic RTT and one with atypical RTT (AR).
MECP2 mutations were identified in 14 of the 18 women tested. There were
six missense mutations; one early truncating; two late truncating; three
C-terminal deletions and two large deletions. A simplified Smeets severity
score was calculated for every decade of the womens lives. Little increase in
severity was observed and, for most, the severity rating was mild.
Factors contributing to disease severity have been assessed. Findings include: (1) severity of phenotype is milder amomg older women, indicating survival advantage; (2) depression among middle-age RTT may be a substantial
but under-recognised problem; (3) menopause does not seem to occur earlier than in other women; (4) nutrition standards from the general population will often be inapplicable; (5) multiple opportunities exist to prevent
functional decline through detailed attention to the quality of the medical
and social care.
There is a particular need to increase awareness of RTT amongst staff caring
for older adults with disabilities so that they can identify and meet the needs
of their adult patients with RTT.
P08.64-M
Italian brother and sister with familial Xp22.12 microduplication
including RPS6KA3 gene and phenotype description
A. Fabretto, P. Costa, F. Faletra, P. Gasparini, V. Pecile;
Institute of Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy.
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ning processes, commonly impaired in schizophrenia. Based on this rationale, we analyzed three common genetic variants of Adducins, ADD1 G1532T
(rs4961), ADD2 C1797T (rs4984) and ADD3 IVS11+386A>G (rs3731566) in
a sample of 342 patients with schizophrenia, assessed with a broad battery
evaluating core cognitive domains that are typically impaired in schizophrenia. The analysis showed significant effects of ADD1 genotype on executive
function (p<.025)and of ADD2 genotype on several domains: working memory (p<.003), verbal fluency (p<.018), verbal memory (p<.001), abstract
reasoning (p<.009), cognitive flexibility (p<.02570) and sustained attention
(p<.010). Moreover an interaction effect of ADD1 and ADD3 genotypes was
observed on symbol coding (p<.029) and verbal memory (p<.027). Our findings suggest that genes involved in synaptic building and plasticity, such as
Adducins, may have a key role in cognitive impairment in schizophrenia and
may help us to better understand the ethiopathogenesis of the disease.
P08.66-M
The power of Next Generation Sequencing in identifying mutations in
non-specific ASD-ID phenotypes: the example of SHANK3
H. Poquet1, M. Willems2, J. Thevenon1, C. Redin3, M. Lefebvre1, C. Redin1, A. MoscaBoidron1, C. Thauvin-Robinet1, Y. Duffourd1, S. Lumbroso2, B. Gerard3, J. Mandel3, J.
Rivire1, A. Piton3, L. Olivier-Faivre1;
1
CHU, Dijon, France, 2CHU, Montpellier, France, 3IGBMC, Strasbourg, France.
Autism Spectrum Disorders (ASDs) comprise a range of early onset neurodevelopmental conditions of varying severity, with or without Intellectual
Disability (ID), characterized by impairments in relatedness and communication, accompanied by restricted interests and repetitive stereotyped behaviors. SHANK3 haploinsufficiency implicated in Phelan-McDermid 22q13
microdeletion is one of the more prevalent monogenic causes of ASD, explaining at least 0,5% of cases, but the indication of SHANK3 sequencing in
patients with ASD and ID remain difficult. Here we report on two patients
with de novo SHANK3 mutations identified by next generation sequencing
(NGS). Patient 1, aged 15, was part of a cohort of 40 patients screened by
exome sequencing for undiagnosed severe ID, and a truncating mutation
was identified in SHANK3 (c.4381C>T ; p.Gln1461*). Patient 2, aged 10, was
part of a cohort of 106 patients with ID screened by targeted NGS of 220 ID
genes, and a causative heterozygous truncating mutation of SHANK3 was
identified (c.2955_2970dup;p.Pro992Argfs*325). They both had normal
measurements, severe ID, developmental and speech delay with acquisition
of a few words and secondary regression with absence of speech, attention
deficit and behavioral disorders necessitating treatment, autistic traits, insomnia and tantrum. Patient 1 had eating and digestive difficulties, patient
2 had distal spasticity, epilepsy from age 5, and facial dysmorphic features.
These two clinical presentations appear nonspecific within the ASD-ID spectrum. After reviewed the other patients with SHANK3 mutations, we argue
that NGS will be helpful to determine patients with SHANK3 mutations in
the absence of clear distinctive clinical features.
P08.68-M
Interpretation of TCF4 Variants Requires mRNA Splicing Analysis in
Patients with Pitt-Hopkins Syndrome
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P08.69-S
A novel X-linked trichothiodystrophy associated with a nonsense
mutation in RNF113A
UBE3A gene maps on 15q11q13 chromosome and encodes for the protein E6AP, an Ubiquitine-ligase involved in protein degradation process.
The gene is maternally imprinted in central nervous system and different
studies show that loss of expressed copy causes Angelman Sydrome (AS)
while the duplication of 15q11q13 represents the genetic cause in 1-3% of
autistic patients (dup15 Autism). Using 2 cohorts of AS and dup15 autism
patients, we applied induced Pluripotent Stem cells (iPSCs) technology and
neuronal differentiation to identify molecular targets of a dys-regulated
dosage of UBE3A. To generate iPSCs we used 4 canonical factors identified
by Yamanaka (c-Myc, Klf-4, Oct3/4 e Sox2) through retroviral or lentiviral
vectors. We performed 14 attempts and in 13 we generated cells that we
defined as partially reprogrammed because they lost their typical fibroblasts morphology, but died after passaging. In the last experiment we used
a commercial lentiviral vector with the same 4 pluripotency factors making
a dup15 hiPSCs cell line positive to pluripotent expression markers; further
characterizations are still ongoing. Once we have hiPSCs we will proceed
with neuronal differentiantion. With our experiments we confirm that the
hiPSCs genesis is a stochastic process with a successful rate of 0.1-2% and
that no standard protocol exists. In the other hand this new challenging and
arduous technology give to the scientists the possibility to understand the
pathogenetic mechanisms in tissues that for obviously reasons are difficult
to study.
P08.71-S
Maternal uniparental isodisomy of chromosome 4 in a subject with
mild intellectual disability
P. Palumbo, O. Palumbo, M. Leone, R. Stallone, T. Palladino, L. Zelante, M. Carella;
Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy.
Uniparental Isodisomy (iUPD) is a rare condition characterized by the inheritance of two homologous chromosome from one parent and the absence
of the homologous chromosome from the other parent. Several mechanisms
of UPD formation have been previously decribed, including trisomy rescue,
monosomy rescue and gamete complementation. Problems associated to
ABSTRACTS POSTERS
UPD are homozigosity of autosomal recessive inherited mutations and aberrant genomic imprinting. iUPD of chromosome 4 is a rare conditions. To date
only few cases are reported, with heterogeneous phenotype, and all were
maternal. We describe a patient with mild intellectual disability and slight
speech delay, harboring maternal iUPD of chromosome 4 detected by high
resolution SNP-array and confirmed by microsatellite analysis. Our patient
did not show any dysmorphic feature and did not present any other anomaly. To the best of our knowledge, this is the second patient carrying maternal
chromosome 4 iUPD that show a behavioral phenotype. Chromosome 4 maternal iUPD is a sporadic and rare event, that may present little or not distinguishable phenotype. Thus, it seems unlikely that important maternally
imprinted genes are on this chromosome. Because iUPD is associate with a
complete unmasking of recessive mutations, it is possible that such mutation could be responsible for the intellectual disability in our patient, and this
hypothesis could justify the phenotypic variability among other reported
cases. It will be useful to study further cases with chromosome 4 paternal
iUPD, in order to compare clinical phenotype and to exclude or confirm the
presence of paternally imprinted genes on chromosome 4.
P08.72-M
X- Chromosome imbalances by array-CGH - from single gene to
chromosomal regions imbalances
We report on two unrelated and sporadic patients presenting a novel syndromic XLID featuring macrocephaly, severe elocution disability with open
bite, high arched palate, malar hypoplasia, a thin nasal bridge with deviated
nasal septum, slender build, and scoliosis. Brain MRI showed a thick corpus callosum. High-throughput sequencing identified two distinct mutations (c.1131G>A and c.1394dup, p.Asn466Lysfs*13) in the P54NRB/NONO
X-linked gene. P54NRB/NONO belongs to the DBHS (Drosophila Behaviour
Human Splicing) protein family. In mammals three members belong to this
family: P54NRB/NONO, PSPC1, and PSF/SFPQ. DBHS proteins are multifunctional nuclear proteins implicated in multiple aspects of RNA production and processing. Beyond RNA production, they also play a role in RNA
surveillance (binding and retaining hyper edited RNA in the subnuclear bodies named paraspeckles), as well as in the control of mammalian circadian
rhythms. Finally, PSF and P54NRB have also been implicated in dendritic
RNA transport.
We demonstrated that both mutations lead to complete absence of the
Back to index
P54NRB/NONO protein and overexpression of the two other DBHS proteins. In patients cells, whole transcriptome analysis revealed severe gene expression deregulation, and reporter assays showed reduced circadian clock
amplitude. Finally, CT scans analysis of P54nrb/Nono-deficient mice demonstrate a dramatic flattened nose phenotype that may mimic the severe malar
hypoplasia observed in patients.
These findings demonstrate the existence of a recognizable XLID syndrome
due to mutations in P54NRB/NONO, and highlight the crucial role of DBHS
proteins in brain development and function.
P08.74-M
The KIAA2022 duplication found by high-resolution custom-designed
array CGH in Polish patient with X-linked intellectual disability
Genomic instability is a feature of the human Xp22.31 region: deletions, duplications, triplications and other complex rearrangements were identified
163
ABSTRACTS POSTERS
at this locus. Submicroscopic duplication of Xp22.31 has been reported as
either a possible cause of neurobehavioral phenotypes or a benign variant.
Recently, two large cohorts of patients with the microduplication at Xp22.31
were reported. The size of the Xp22.31 duplication varied between 149 kb
and 1.9 Mb and mostly included the steroid sulfatase (STS) gene. Patients
with Xp22.31 recurrent duplications generally presented with a neurocognitive and behavioral phenotype, including developmental delay. The STS
gene could be a candidate gene contributing to the abnormal phenotype in
Xp22.31 duplication.
Here we report 2 boys with the microduplication of Xp22.31 found by MLPA
analysis. The duplication was minimum 246,2 kb in size and included STS
and HDHD1A genes. Both of them presented with mild to moderate intellectual disability/developmental delay mainly affecting speak ability, behavioural abnormalities and minor facial dysmorphisms. Proband B also had
a hypotonia. The mother of proband A, carrying the same duplication, had a
mild intellectual disability.
Our cases overlap with those previously described in most clinical features.
Although there is no clear evidence to support pathogenicity of the Xp22.31
duplication, there is still enough evidence to consider the Xp22.31 duplication as a risk or modifier factor for intellectual disability and behavioural problems. We hope that the description of these two cases will contribute to the
phenotype delineation and elucidation of the role of Xp22.31 duplication.
P08.77-S
Xq12 and Xq13 submicroscopic duplications in a single patient:
confirming the existence of a new X-linked disorder and narrowing
the critical region.
In 2012 Kaya et al. reported three related patients with a recurrent duplication Xq12-q13.3 with mental retardation, autism, seizures and dysmorphic features. The duplicated region (9.4 Mb) contains numerous genes and
the authors pointed out the possible role of an increased dosage of specific
genes focusing on their presumed role in developmental delay and autism.
Subsequent reports (Prontera et al. 2012; Wentz et al. 2013) described 4
more patients with a variable association of severe global developmental
delay, microcephaly, dysmorphism and autism spectrum disorder, all carrying a duplication in the Xq12-q13.3 region, supporting the existence of
a clinically recognizable X-linked recessive disorder. We describe a further
patient affected by global developmental delay, epilepsy, autistic traits and
carrying two non-contiguous duplications detected by array CGH, involving
Xq12 and Xq13 regions. Detailed clinical description is provided and compared to previous reported cases. Due to the narrowed size of the duplicated
regions (123 Kb and 509 Kb, respectively), this case helps to redefine the
region of interest for this condition. The two duplications in our patients
involve 13 genes, and 7 of them are mainly expressed in the nervous system (OPHN1, SNX12, MED12, NLGN3, GJB1, ZMYM3, TAF1) and all, except
for SNX12, are involved in the pathogenesis of a known human disease. Our
observation provides further evidence of the existence of a new genomic
condition, helping to define the corresponding clinical phenotype, and to
identify a more definite critical region.
P08.78-M
Xq28 duplication detected by SNP-array in two girls with
microcephaly, intellectual disability and neurological features
164
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the ventricular system and evidence of small brain. Our second patient is the
second daughter of an apparently healthy, non-consanguineous couple born
at 39 weeks by cesarean section for small gestational age. The auxological
parameters were all below the 3rd percentile. At birth, brain MRI showed
mild hypoplasia of corpus callosum and cerebellum. She had generalized hypotonia, hyporeactivity, sucking deficit, seizures, and congenital heart disease. Physical examination showed microcephaly, dysmorphic features of the
face and extremities. GDI1 has been associated with ID and non-syndromic
microcephaly in a high percentage of males. Our patients and the revision of
cases from literature and DECIPHER database strengthen the GDI1 involvement in intellectual disability and microcephaly not only in male but also in
female subjects. Of note, neurological and neuroimaging abnormalities, such
as ventricular system enlargements, cerebellar or corpus callosum hypoplasia are recurrent clinical features associated with this rare microduplication
syndrome.
P08.79-S
Mutations in YY1 cause intellectual disability and a distinct facial
appearance
Microdeletions of 1q43q44 were described in patients with intellectual disability (ID) with limited or no expressive speech, microcephaly, dysmorphic
features, abnormalities of the corpus callosum, hand and foot abnormalities, and seizures. Different genes were predicted as candidate genes for the
microcephaly (AKT3), abnormalities of the corpus callosum (ZBTB18), and
seizure (FAM36A, C1orf199, HNRNPU). Recently, a mutation in the ZBTB18
gene was identified in a patient with ID and further signs of the 1q43q44
microdeletion syndrome. Knock-out mice with loss of Znf238 die at birth
with neocortical defects but a brain-specific knock-out of this gene in mice
causes microcephaly, reduced thickness of the cortex and agenesis of the
corpus callosum. The ZBTB18 gene acts as a transcriptional repressor of key
neurogenic genes such as NeuroG2 and NeuroD1 or ID2 and ID3. Here, we
describe two patients carrying de novo mutations in ZBTB18, one patient
with a missense mutation (p.R495G) and a further patient with a nonsense
mutation (p.E286X). The first patient, a 23 years old woman, demonstrated
a non-syndromic ID with mild facial dysmorphic signs. The other patient, a
7 years old boy, showed ID, facial abnormalities, significant speech and motor developmental delay. In both patients normal karyotypes (550 bands),
array-CGH results and normal FMR1 gene analyses were shown. Our results
indicated that ZBTB18 is responsible for neurogenic development and mutations within ZBTB18 cause ID with variable phenotypic abnormalities.
ABSTRACTS POSTERS
P08.81-S
Klinefelter syndrome: 48, XXXY aneuploidy in a patient with mild
mental retardation and psychotic personality traits
M. F. Hernandez-Amaris1, H. Pachajoa1,2;
1
Universidad Icesi, Cali, Colombia, 2Fundacion Clinica Valle del Lili, Cali, Colombia.
Klinefelter syndrome is the most common aneuploidy in males with a prevalence of 0.1-0.2% in the general population, which rises up to 3% in males
with fertility issues, although only 35% of cases are diagnosed. The affected males tend to be tall, have narrow shoulders, wide hips, sparse body
hair, gynecomastia and small testis; they present androgen deficiency and
azoospermia. Besides the previous physical characteristics, there have been
reports on the expression of behavioral and cognitive traits that tend to be
very variable, possibly according to the type of aneuploidy, with an established association between the number of extra X chromosomes and cognitive deficit, and a not so clear association of different chromosomal variants
of Klinefelter and psychotic behavior, with some authors proposing the origin of the extra chromosome as a determinant of different behavioral traits.
Here is presented the case of a 13 years old male with karyotype 48,XXXY,
who besides presenting the classical physical features, is under psychiatric
treatment because of presenting trouble at home and at school for being
aggressive and impulsive. A review on the literature is made, concluding on
the importance of an opportune starting of hormonal therapy and a multidisciplinary approach including endocrinology, pediatrics, genetics, neuropsychology and psychiatry when needed.
P09.001-S
4H (Hypomyelination, Hypodontia and Hypogonadotropic
Hypogonadism) syndrome caused by POLR3B mutations
Leukodystrophies are a heterogeneous group of inherited neurodegenerative disorders characterised by alterations of Central Nervous System white
matter. Several clinical forms have been described and genes involved in
generating the hypomyelinating leukodystrophy, but recently, mutations in
POLR3A andPOLR3Bgenes have been reported to cause a specific syndrome named 4H characterized by the association of the hypomyelination with
hypodontia and hypogonadotropic hypogonadism. We visited a 27-year-old
female born from a non consanguineous mating with a leukodystrophy and
an unlikely clinical / endocrinological diagnosis of Pelizaus - Merzbacher
syndrome. Her phenotype was characterized by teeth alterations (neonatal tooth and permanence of deciduous dentition), thin hair, high myopia,
hypogonadotropic hypogonadism, and minor dysmorphic features. Moreover, a gradual, but consistent motor and cognitive deterioration was noted.
Putting together all the clinical, radiological and phenotipic characteristics
of the proband a clinical diagnosis of 4H syndrome has been made. Subsequently, a Sanger sequence of the entire coding region and the flanking
exon/intron boundaries of POLR3A and POLR3B has been performed. The
analysis revealed the two mutations p.V523E and p.G695fs*5 in the POLR3B
gene. This case further expands the knowledge about the role of the POLR3B
gene in generating the 4H syndrome.
P09.002-M
A familial 9q22.1q22.31 deletion of 3.8 Mb, challenging interpretation
of large, inherited copy-number variation
A. C. tabet1, L. perrin1, S. Toujani1, J. Leger1, V. Vantalon1, C. dupont1, L. bouffard1, E.
pipiras2, A. Delahaye2, B. Benzacken2, A. Verloes1;
1
Robert Debre Hospital APHP, paris, France, 2Jean Verdier Hospital APHP, Bondy, France.
Chromosomal microarray analysis are now the first-line diagnostic test for
patients presented with intellectual disability, autism, or multiple congenital anomalies. An ongoing challenge for the clinician and biologist is the
interpretation of copy number variation. Many tools have been proposed
in order to facilitate their interpretation including linking data to several
genome browsers, gene contains and prioritization, inheritance of the copynumber variation (CNV) and the size of the variant. Indeed, large CNV (>500
kb) are supposed to be strongly associated with morbid consequences. We
report on a large 9q22.1q22.31 deletion of 3.8 Mb identified by whole genome SNP array (HumanCytoSNP-12, Illumina) in a 6 year old girl presented with growth retardation and Attention-Deficit/Hyperactivity Disorder
(ADHD). Neuropsychological evaluation excluded a mental retardation. The
deletion encompassed 24 genes, was inherited from her apparently normal father. Nevertheless, the familial history was marked by hyperactivity
and dyslexia in the fathers childhood. The older sister presented also with
ADHD and carried the same deletion. Grandparents will be further analyzed.
We discuss the implication of this CNV in the abnormal phenotype of the
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Array comparative genomic hybridization (aCGH) is a quick method to analyze the whole genome for imbalances at a higher resolution. In conventional
diagnostic settings aCGH is used as a first-tier diagnostic test in the group
of patients with unexplained developmental delay (DD) and/or idiopathic
intellectual disability and/or dysmorphic features and/or multiple congenital anomalies.
AIM: Our aim was to evaluate the diagnostic yield/clinical utility of aCGH
in the group of children with neurological disorders at Clinical institute of
Medical Genetics Ljubljana, tested in 2012-2013.
PATIENTS AND METHODS: We included 244 children, 91 with DD and/or
dysmorphic features, 44 with epilepsy, 34 with autism, 45 with developmental abnormalities of the CNS, and 30 with abnormal muscle tone. DNA
was hybridized to Agilent 180K or 60K Human CGH microarrays. Discovered genomic imbalances were interpreted according to the data in Internet
databases (ISCA, DECIPHER, ECARUCA) and scientific publications cited in
PubMed.
RESULTS: Altogether, 37 pathogenic copy number variations (CNVs) were
found that could explain the phenotype of patients (Table 1). In addition,
26 variants of unknown significance (VUS) were detected, most of them de
novo. Eleven patients (4,5%) had complex CNVs.
CONCLUSION: We found out that pathogenic CNVs were causative in 15%
of cases with neurological disorders with/or without dysmorphic features
or CNS developmental abnormalities. Our data demonstrate that aCGH is an
important diagnostic tool in neuropediatrics.
Phenotype
DD with or without
dysmorphic features
Developmental abnormalities
of the CNS
Abnormal muscle tone
Epilepsy
Autism
Number of tested
patients
91
45
30
44
34
Pathogenic
VUS
CNV
22
10
2
6
2
4
3
3
Diagnostic
Yield
(pathogenic
CNVs)
24.2%
11.1%
6.7%
13.6%
5.9%
P09.004-M
Genetic analysis of amyotrophic lateral sclerosis in the Slovenian
population
Background: Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease characterised by progressive degeneration and loss of upper and lower motor neurons in the cerebral cortex, brainstem and spinal
cord leading to death due to respiratory failure within 2-5 years from onset. The most common genes involved in the disease process are SOD1, FUS,
TARDBP and C9ORF72.
Patients and methods: Blood samples from 72 Slovenian ALS patients were
collected and the exons of genes of interest were PCR amplified followed by
Sanger sequencing on ABI310 Genetic Analyzer. In case of C9ORF72 repeatprimed PCR was performed followed by fragment length analysis on ABI310
Genetic Analyzer. Results were analyzed using Gene Scan software.
Results: Genotyping of genes SOD1, FUS, TARDBP and C9ORF72 revealed
some changes in the DNA sequence. In SOD1 gene two mutations (V15M and
G94C) were detected. Sequencing of FUS and TARDBP genes did not reveal
any amino acid changes. Although two substitutions were detected, R522R
(G>A) in FUS and L330L (A>G) in TARDBP. The latter was not previously
described. In 2 ALS patients we detected expansion of repeats in the first
intron of C9ORF72.
Conclusions: This study represents first genetic analysis of Slovenian ALS
patients. Our findings are in concordance with other studies provided on
European ALS patients. Of these four analyzed genes repeat expansion in
165
ABSTRACTS POSTERS
C9ORF72 remains the most common cause of ALS although for the majority
of cases the main causes are yet to be revealed.
P09.005-S
Aging-related genes modulate the early altered expression in young
3xTg-AD mice hippocampi: a whole transcriptome approach
M. DAurora1,2, S. Sperduti1,2, L. DOnofrio1,2, A. Granzotto2, S. L. Sensi1,2, L. Stuppia3,2, V.
Gatta3,2;
1
Department of Neuroscience and Imaging, G. dAnnunzio University, Chieti, Italy,
2
Center of Excellence on Aging (Ce.S.I.), Chieti, Italy, 3Department of Psychological,
Humanities and Territorial Sciences, School of Medicine and Health Sciences, G.
dAnnunzio University, Chieti, Italy.
Alzheimers disease (AD) is a multifactorial neurological condition associated with a genetic profile that is still not completely understood. In this
study, using a whole gene microarray approach, we investigated age-dependent gene expression profile changes occurring in the hippocampus of
young and old transgenic AD (3xTg-AD) and wild type (WT) mice. The aim
of the study was to assess similarities between aging- and AD-related modifications of gene expression and investigate possible interactions between
the two processes.
Global gene expression profiles of hippocampal tissue obtained from 3xTgAD and WT mice at 3 and 12 months of age (m.o.a.) were analyzed by hierarchical clustering. Interaction among transcripts was then studied with the
Ingenuity Pathway Analysis (IPA) software, a tool that discloses functional
networks and/or pathways associated with sets of specific genes of interest.
Cluster analysis revealed the selective presence of hundreds of upregulated
and downregulated transcripts. Functional analysis showed transcript involvement mainly in neuronal death and autophagy, mitochondrial functioning, intracellular calcium homeostasis, inflammatory response, dendritic
spine formation, modulation of synaptic functioning, and cognitive decline.
Thus, over-expression of AD-related genes (such as mutant APP, PS1, and
tau, the three genes that characterize our model) appears to favor modifications of additional genes that are involved in AD development and progression.
The study also showed overlapping changes in 3xTg-AD at 3 m.o.a. and WT
mice at 12 m.o.a., thereby suggesting altered expression of aging-related genes that occurs earlier in 3xTg-AD mice.
P09.006-M
The role of p35/CDK5 regulation by miR-15/107 family in
Alzheimers disease
166
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Post-zygotic mosaic aneuploidy is common in the normal and diseased human brain. Somatic chromosomal mosaicism affecting the brain is, probably,
the result of disturbances of adult neurogenesis/gliogenesis during the early
ontogeny. It was proposed that aneuploidy of the brain is somehow involved
in pathogenesis of neurodegenerative brain disorders including Alzheimers
disease (AD). Here, we have analyzed X chromosome aneuploidy (a feature
of aged cell populations) in the brain tissues of females with/without AD.
Molecular cytogenetic analyses were performed by multiprobe/quantitative
FISH and interphase chromosome-specific multicolor banding (ICS-MCB) in
10 AD and 10 age/sex matched control samples, scoring 160,000 cells per
each sample set. In AD, the mean rate of aneuploidy (2.79%, 95% CI 1.883.69) was two times higher than in control (1.32%, 95% CI 0.92- 1.71%); P
=0.013 (Mann-Whitney U-test). An AD sample demonstrated mosaic aneuploidy (X chromosome loss) confined to the hippocampus in about 10% of
cells. More than 75% of cells with X chromosome aneuploidy were NeuNnegative (non-neuronal cells; glia and, probably microglia). These preliminary data indicate that somatic (post-zygotic) chromosome instability causes large-scale genomic variation in a significant proportion of hippocampal
cells in AD. In context of a causal relationship between brain aging and AD
pathogenesis, our findings suggest that X chromosome aneuploidy can contribute to both brain aging and neurodegeneration in females with AD. We
speculate that mosaic aneuploidy in the brain is a new non-heritable genetic
factor predisposing to AD. Supported by BLR 11/002, the Russian Federation President Grant (MD-4401.2013.7), and RFBR 12-04-0021.
P09.009-S
Genetic analysis of Angiogenin gene in Italian patients with
Alzheimers disease
ABSTRACTS POSTERS
(ALS) and Parkinsons disease (PD). A decreased level of ANG protein was
found in AD patients serum [Kim 2012]. Objectives. to investigate the role
of the ANG gene in AD. Methods. Genetic analysis of ANG gene was done in
a cohort of 509 AD patients and 417 healthy volunteers over 65 years of
age using Sanger sequencing of the coding regions. Results. Genetic analysis showed the presence of a nonsynonymous mutation in heterozygosis in
position g.2162012 causing the change of a lysine to a Stop codon (K73X).
This new mutation was found in two AD patients (0.48% of the whole AD
cohort): patient 1 is a man, 69 years old, with family history of dementia;
patient 2 is a woman, 74 years old, with no familiarity for AD or dementia.
No mutations were found in control subjects. Discussion and conclusion.
This study suggests that ANG gene mutations may be associated with rare
cases of AD. Interestingly, the K73X mutation may be specific for AD, since
it has not been found in the numerous genetic studies on ALS and PD so far
reported, proposing an important specificity mutation-disease.
P09.010-M
Dog as an animal model for idiopathic epilepsy: identification of
common risk variants in the ADAM23 gene
Introduction: Microcephaly can be either isolated with no additional anomalies, or it may coexist with other neurological entities and/or multiple
congenital anomalies, known as syndromic microcephaly. Although many
syndromic cases can be classified based on the characteristic phenotype,
some others require further investigation. Aim: The present study describes
the application of high resolution array-comparative genomic hybridization
(array-CGH), as a diagnostic tool for the study of patients with syndromic
microcephaly in order to identify clinically relevant copy number variations (CNVs). We evaluate the MRI findings with patients clinical profile and
the underlying molecular findings. Material and Methods: From a cohort
of 210 unrelated patients, who were referred for genetic evaluation due to
syndromic microcephaly of unknown etiology, accompanied by at least one
further pathological manifestation, 50 undiagnosed cases underwent arrayCGH analysis. In all cases previous standard karyotype as well as other genetic tests were negative. High resolution 4x180K and 1x244K Agilent arrays
(>170.000 and >236.000 probes respectively, average resolution 8.9Kb)
were used in this study. Results: In 32 out of 50 patients (64%) featuring
microcephaly among other phenotypic anomalies, array-CGH revealed si-
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ARSACS is the second most frequent form of hereditary spastic ataxia after
Friedreichs ataxia. ARSACS is caused by mutations in the SACS gene encoding for sacsin which is partially localized to the mitochondria. The function
of sacsin is still poorly understood. Recently, the mitochondrial network abnormal was observed in immortalized fibroblasts of two Quebecs patients
(Girard, 2012). We searched for point mutations and gene dosage anomalies
in SACS using direct sequencing and customized CGH array in 325 patients
presenting with progressive spastic ataxia and age at onset < 45 years recruited through the SPATAX network. We obtained primary cultures from
skin fibroblasts from ten ARSACS patients. On these fibroblasts we performed a labeling of the mitochondrion using the Mitotracker Green. We identified 42 different mutations in a total of 32 patients carrying two mutations
167
ABSTRACTS POSTERS
in SACS. Customized CGH array on the two only patients carrying one heterozygous truncating mutation were revealed no anomalies. The diagnosis of
ARSACS was confirmed in 13% of our cohort, the largest series of ARSACS
patients reported. Mean age at onset was 4 years but late-onset > 25 years
was observed. Although demyelinating neuropathy and cerebellar atrophy
were frequently associated with spastic ataxia, the clinical spectrum appeared larger than previously. Analysis of the mitochondrial network in all
patients fibroblasts revealed altered mitochondrial shape associated with a
significant decrease of the mitochondrial mass. These alterations observed
in the mitochondrial network of ARSACS patients bring a real interest to
study the role of sacsin regarding the mitochondrion.
P09.014-M
The 901bis pedigree of ataxia is linked to SCA37 locus
Autosomal dominant spinocerebellar ataxias (SCAs) are a clinically, genetically and pathologically heterogeneous group of movement disorders
defined by variable degrees of cerebellar ataxia and often accompanied
by additional cerebellar and non-cerebellar symptoms. The clinical symptoms are triggered by neurodegeneration of the cerebellum and its relay
connections. Recently we report the clinical and genetic findings from Spanish kindred (901 family) presenting a cerebellar phenotype characterised
by ataxia, atypical early-altered vertical eye movements, variable severity,
no evidence of anticipation, age at onset ranging from 35 to 64 years and
Cranial CT or MRI showing cerebellar atrophy in several patients without
brainstem involvement. We have found significant linkage with the highest
two-point LOD score Zmax = 3.831 at theta = 0.00 (P < 0.001), between the
locus trait and D1S2742, assuming an age-dependent penetrance model
with twelve liability classes. Multipoint analysis and haplotype reconstruction traced this novel SCA locus to a 0.66-cM interval flanked by D1S200
and D1S2742 (multipoint Zmax = 6.052). The Human Genome Nomenclature
Committee (HGNC) has assigned it as SCA37. We presented here another
ataxia pedigree (901bis family) with 5 affected members over a total of 17
individuals, which show a multipoint lod score of 3.0 to respect the same
genetic markers used in the 901 family. This family comes from the same
region than the previous 901 and probably has a common founder ancestor.
The linkage results from this pedigree reinforces the hypotheses of linkage
from the same ataxia locus trait localized in 1p and named SCA37.
P09.015-S
Reduction of neurological symptoms in Ataxia Teleangiectasia
patients by Intra-Erythrocyte infusion of Dexamethasone (EryDex)
L. Chessa, M. Piane, IEDAT-01 trial group;
University La Sapienza, Roma, Italy.
168
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P09.016-M
Eight new ATM gene alterations in Polish patients with ataxiatelangiectasia
Ataxia-telangiectasia (AT, MIM#208900) is neurodegenerative and immunodeficiency disorder, is inherited in an autosomal recessive manner. AT
results from mutations in the ataxia telangiectasia mutated (ATM) gene.
We studied the entire ATM gene coding sequence for mutations using PCRHD, PCR-SSCP and MLPA techniques. 38 changes in ATM sequence were
detected in 25 AT families. The mutation types are diverse, including 18
nonsense (58,1%), 10 splicing (32.3%), 2 missense alterations (6,4%) and
1 large genomic deletion (3,2%). Only 2 mutations have been found in homozygous state ( [c.4007_4008insA; c.4007_4008insA], [c.9021_9022insA;
c.9021_9022insA]). Most frequent mutations among our AT patients are:
c.6095G>A (7 times), c.7630-2A>C (6), c.5932G>T (5), c.7010_7011delGT
(2). Several families had newly diagnosed DNA alterations in Polish
population (8/38; 21,1%). New detected variants were: c.8441delC,
c.6145T>G, c.434T>G, c.6754_6754delAfsX5, c.4007_4008insA, c.7606G>A,
c.3402+30_3402+32delATC, deletion of 62 and 63 exons of gene. The effects
of two missense changes were predicted to be pathogenic by in silico analysis. The c.3402+30_3402+32delATC was identified in 3 AT families (12%)
and none of control samples. According to data from the NHLBI Exome
Sequencing Project the ATC deletion allele has a frequency of 0.11% total
alleles studied and is not observed in the homozygous state. In this study,
we confirmed status of recurrent mutations, but also detected new changes
in ATM sequence. Most of the Polish patients are compound heterozygotes
and none of them having the same combination of mutations, what makes
molecular diagnostic more difficult.
Supported by grant NN401098240 from the Ministry of Science and Higher
Education in Poland.
P09.017-S
Finding patients with Parkinsons disease (PD) which are
heterozygotes for Atp7b gene in Russia
ABSTRACTS POSTERS
Introduction Attention-deficit hyperactivity disorder (ADHD) is one of the
most common neurobehavioral disorders of childhood, characterized by
age-inappropriate levels of inattention, hyperactivity and impulsivity. Despite its high heritability (76%), the results of association studies have been
inconsistent and poor replicated. The aim of this study was to replicate in a
Spanish cohort, the association of previously reported gene variants. Patients and Methods 324 children and adolescents (6-17 years old) diagnosed
with ADHD according to DSM-IV-TR, and 344 controls were recruited. Patients and controls were genotyped for 23 genetic variants: DAT1: rs2550948,
rs261759, rs2652511, VNTR-3UTR, VNTR-Intron8; DRD2: rs1800497;
DRD4: rs3758653, VNTR-Exon-3, VNTR-Promotor, SLC6A4: VNTR-Promotor,
VNTR-Intron-2; HTR2A: rs7322347; NET1: rs28386840, r5569; ADRA2A:
rs553668, rs1800544; LPHN3: rs1397458, rs2305339, rs6551655; COMT:
rs4680; FADS2: rs498793; SNAP25: rs3746544; DDC :rs6592961. Pearsons
X2 test, and Hardy-Weinberg equilibrium were used to assess genetic association. Results The most significant associations found in this study
were: NET1-rs28386840 A/T genotype was significantly increased in both
ADHD patients (OR:1.54, p: 0.027) and combined-ADHD subtype (OR:1.72,
p:0.0043). DAT1-5/5 genotype resulted increased in inattentive-ADHD patients (OR:2.13, p:0.052) Discussion and Conclusion The difficulties found
identifying risk ADHD associated gene variants could be explained by the
heterogeneity and complexity of this disorder. Further studies designed to
examine the contribution of gene-gene or gene-environment interaction are
needed. In addition, the use of endophenotypes instead of DSM-IV diagnoses
could improve the detection of genetic effects.
P09.019-S
Variation in the phenotype of Machado-Joseph disease (MJD/SCA3):
the role of mitochondrial DNA (mtDNA) haplogroups
Mitochondrial dysfunction has been implicated in the pathogenesis of several neurodegenerative disorders, such as Machado-Joseph disease (MJD),
a late onset poly-Q ataxia that results from an unstable expansion of a CAG
tract in the ATXN3 gene. The CAG expansion size is incompletely correlated
with the age at onset, highlighting the existence of genetic modifiers. Although the way by which mitochondria is involved in the neurodegenerative
cascade is still unknown, mitochondrial DNA (mtDNA) haplogroup-specific
polymorphisms have been associated to other poly-Q disorders. To evaluate whether mtDNA variation contributes to MJD phenotype, namely to age
at onset (AO), we determined the mtDNA haplogroups in 113 MJD patients
with Azorean ancestry, by sequencing the mtDNA hypervariable region I.
The frequency of mtDNA haplogroups in unrelated patients (n=68) was similar to the one previously described for the general Azorean population.
MJD patients classified as haplogroup J present a significantly earlier onset (mean AO of 33 years). Haplogroup W seems to have a protective effect, causing a delay in onset (mean AO of 51 years). Although haplogroup
J has already been implicated in other neurodegenerative disorders, there
are no previous reports of an association between haplogroup W and disease. In conclusion, our results suggest that mitochondrial single nucleotide
polymorphisms defining these haplogroups could modify AO in MJD. The
complete mitochondrial genome of MJD patients classified as J and W haplogroup were further sequenced in order to better understand the impact of
the haplogroup-defining variants on mitochondrial function and their interaction in MJD phenotype.
P09.020-M
Transcriptional profile of Machado-Joseph Disease (MJD): mTOR
signaling pathway is dysregulated in blood cells of patients
Machado-Joseph disease (MJD; MIM #109150; ORPHA98757), or spinocerebellar ataxia type 3 (SCA3) is a protein misfolding-associated disease,
being the worldwide most prevalent autosomal dominant ataxia as well as
the second more common polyglutamine (polyQ) disorder. Abnormal conformation of mutated ataxin-3, promotes a gain of a toxic function compro-
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ABSTRACTS POSTERS
Bench software applications. Results: We present a family based study on
the validity of CNV detection in ASD using microarray analysis. The clinical
utility of this screening is evaluated for different parameters (e.g. sporadic
versus familial ASD, IQ, comorbidity). In some cases, we found a better diagnostic performance of the higher resolution platforms. Conclusion: We
contribute to the diagnostic utility and indication of rare CNV detection in
ASD families with respect to clinical presentation and family history.
P09.023-S
Genetic evidence that hyperactive Th2 and NK immune pathways
contribute to the pathogenesis of Autism Spectrum Disorder
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Band-like brain calcification (BLC) or pseudo TORCH syndrome is a rare autosomal recessive with distinctive clinical and neuroimaging. Severe microcephaly, early onset seizures, profound developmental delay together with
band-like calcification in brain, simplified gyral pattern and polymicorgyria
are the hallmark of the syndrome. In 2010 ODriscoll et al. attributed BLC
to homozygous mutation in occludin (OCLN) gene through a description of
5 families from a worldwide series including an Egyptian one. Since then, a
single family was reported associated with extracranial phenotype namely
renal involvement due to cortical calcification. Herein we describe eight patients derived from six Egyptian Families with BLC. All presented at early life
with severe microcephaly, failure to acquired developmental skills, growth
arrest and mixed seizures types with predominance of the myoclonic. Patients showed a unique calcification pattern; subcortical band, basal ganglia,
dentate nucleus and pons. Molecular analysis revealed that two families
were linked to known genetic mutations while four families had novel mutations in OCLN gene. Three of our patients deceased at the end of the first
year of life. Interestingly, the single patient with novel mutation in exon 5
had non-neurological manifestations including high imperforate anus and
genital anomalies. Further, he had the best life performance with fairly controlled seizures and achieved few skills although no comparable neuroimaging findings were present. This study is considered the largest series with
BLC from same ethnic group. Assigning more families will allow delineation
of the phenotype-genotype spectrum of BLC.
P09.026-M
Association study of 240,000 rare coding variants in bipolar patients
and controls from Germany and Norway
S. Herms1,2, P. Hoffmann1,2,3, T. W. Muehleisen3, S. Djurovic4, F. D. Degenhardt 2,5, A.
Forstner2,5, M. Rietschel6, M. M. Noethen5,2, O. Andreassen4, S. Cichon1,3,7;
1
Genomics Research Group, Department of Biomedicine, University of Basel, Basel,
Switzerland, 2Department of Genomics, Life & Brain Center, University of Bonn, Bonn,
Germany, 3Institute of Neuroscience and Medicine (INM-1), Research Center Juelich,
Juelich, Germany, 4KG Jebsen Centre for Psychosis Research, Division of Mental Health
and Addiction, Oslo University Hospital & Institute of Clinical Medicine, University of
Oslo, Oslo, Norway, 5Institute of Human Genetics, University of Bonn, Bonn, Germany,
6
Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health
Mannheim, Univ. of Heidelberg, Mannheim, Germany, 7Division of Medical Genetics,
University Hospital Basel, Basel, Switzerland.
Genome-wide association studies (GWAS) of bipolar disorder (BD), a highly heritable disorder of mood with a lifetime prevalence of approximately
0,5-1% in all populations world-wide, have identified several common genetic risk factors. It is currently unclear, to what extent low-frequency and rare
variants contribute to disease development. A valid hypothesis seems to be
that low-frequency and rare variants in coding gene regions have a higher
probability to have a functional (deleterious) effect. This subset of human
genetic variation might therefore be enriched for disease-relevant variants.
The Illumina HumanExome arrays make particularly this window of genetic
variation accessible for association studies. This array contains 240000 rare
markers derived from exome sequencing. That array was used to test 1314
bipolar patients (895 from Germany, 419 from Norway) and 2700 controls
(2366 / 339). Genotypes were exproted from a single GenomeStudio project
to minimize clustering artifacts. Statistical analysis was performed using
Cochrane-Mantel-Hansel test statistics. Clusters for all associated variants
were checked by zCall and manually. The two variants with the strongest
association to bipolar disorder were found in the gene SYNE2. Interestingly,
common variants in another member of the same gene family (SYNE1) had
shown genome-wide significant association in the discovery step of the first
mega-analysis of bipolar disorder performed by the international Psychiatric Genomics Consortium (PGC; Sklar et al. 2011). We are currently aiming
to follow-up this and other strong findings of our analysis in independent
samples of bipolar disorder genotyped on the HumanExome array.
P09.027-S
Clinical profile of paediatric Brown-Vialetto-VanLaere syndrome
ABSTRACTS POSTERS
with BVVLS. Our cohort consisted of patients diagnosed at the Molecular
Diagnostic Laboratory at Guys and St. Thomas Hospital and by our collaborators in Germany and Iceland, as well as molecularly-confirmed reports in
the literature. After excluding patients with insufficient clinical data available, we identified a total of 28 SLC52A2 and 15 SLC52A3 patients for analysis.
We identified differences in the clinical presentation and progress of these
two patient groups. In particular, while hearing loss, muscle weakness and
bulbar palsy were common clinical features in both patient groups, amongst
SLC52A2 mutation carriers, ataxia and optic atrophy were also surprisingly
common. These findings suggest that BVVLS may have a wider clinical phenotype than presently appreciated. This is an important observation given
emerging evidence that it can be ameliorated using riboflavin supplementation.
P09.028-M
Effects of sapropterin on endothelium-dependent vasodilation in
patients with CADASIL: a randomized controlled trial
Back to index
ly. Fibroblasts were grown in DMEM with 10% FBS, Glutamine, Streptomycin-Penicillin. At 80% of confluence, cells were washed in TBS and fixed on a
slide with paraformaldehyde.
The slides were treated with a mouse anti Human Notch3 antibody that recognizes the extracellular domain ECD and a rabbit antibody that recognizes
the intracellular domain NICD. Anti-mouse and anti-rabbit secondary antibodies revealed the primary antibodies.
Different patterns of localization were detected. In cells with mutation R61W
an immunoreactive predominance of the intranuclear NOTCH3 NICD, and in
cells from patients with R332C mutation an immunoreactive predominance
of the N3ECD extracellular NOTCH3 domain was observed when compared
both with cells from normal subject, and with the remaining CADASIL cells.
The obtained results show that in CADASIL fibroblasts the mutation type
is associated with a relative differential localization of Notch3. This likely
might be due to alterations in the protein processing.
P09.030-M
aCGH reevaluation reveals clinically relevant CNVs, in patients with
previously unexplained developmental delay / ASD, candidate for
exome sequencing. Presentation of selected cases.
Cerebellar and brainstem congenital defects (CBCD) affect about 1 in 30004000 live births. Typical clinical signs include hypotonia, ataxia, abnormal
ocular movements and psychomotor delay/intellectual disability, but phenotypic variability is wide with possible multiorgan involvement.
With the exception of few autosomal recessive or X-linked conditions, CBCD
are frequently sporadic, suggesting that de novo mutations or genomic rearrangements may represent common pathogenetic mechanisms.
We analyzed by genomic microarray platforms 83 sporadic CBCD patients,
enrolled in a multicentric project focused on studying the genetic causes of
CBCD. Eight pathogenic or potentially pathogenic CNVs were detected in 7
patients (8.4%).
Further 11 patients, investigated in our Cytogenetic diagnostic lab for intellectual disability and/or congenital anomalies, have been subsequently
included in the CBCD project, since they showed CNVs overlapping to deletions/duplications detected in CBCD patients and/or were affected by CBCD.
Of note, we detected eight deletions that defined three recurrent imbalances. Three deletions were partially overlapping in 3q22.3q26.1, three in
6q25.1q27 and two in 10q26.2q26.3. These three chromosomal regions
have been already associated to CBCD, although literature and DECIPHER
database revision suggest incomplete penetrance. In addition we report
six patients with non-recurrent CNVs: del(2q36.3), dup(6p22.3), inv/
dupdel(8p), del(8p21.3p21.2), dup(13q34), dup(Xq28).
These data confirm the crucial role of microarray analysis as a tool to identify
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ABSTRACTS POSTERS
pathogenic CNVs in patients with congenital defects. Our data confirm that
CBCD are characterized by high genetic heterogeneity and incomplete penetrance, suggesting the involvement of further mechanisms (modifier genes,
environmental factors) in cerebellar and brainstem mal-development.
P09.032-M
The structure and functions of the transcription factor PHOX2B:
new insights in the molecular pathogenesis of Congenital Central
Hypoventilation Syndrome
Congenital Central Hypoventilation Syndrome (CCHS) is a very rare neonatal neurological disorder characterized by abnormal ventilatory response to
hypoxia and hypercapnia, owing to failure of autonomic respiratory control:
affected children hypoventilate during sleep, with possible very severe neurological damages.
Frameshift mutations (5%) and poly-alanine triplet expansions (95%) have
been detected in the coding region of the homeobox gene PHOX2B in about
90% of CCHS patients. A correlation between length of the expansion and
severity of the respiratory phenotype has been reported. Since some of the
mutations alter the sub-cellular localisation, the DNA-binding affinity and
the transcriptional activity of the protein, and that mutated PHOX2B proteins can interfere with the activity of the wild-type protein by sequestering it into aggregates, one crucial question concerns the identification of
the functional domains of the protein, the role of the poly-alanine tract and
the effects of its expansion on the general architecture and function of the
protein. We have performed a deletion analysis of PHOX2B and identified
two nuclear localization signals in the homeodomain, both required for the
complete import of the protein in the nucleus, corresponding to residues
necessary for the binding to DNA, and partially blocked by the expanded
poly-alanine tract. By using mammalian two-hybrid system we have also
demonstrated that PHOX2B can form homo-dimers and we are currently investigating if the mutations alter the dimerization properties of the protein
and the potential contribution to the range of phenotypes and pathogenesis
in CCHS patients.
P09.033-S
Transcriptional dysregulation and impairment of PHOX2B autoregulatory mechanism in the pathogenesis of Congenital Central
Hypoventilation Syndrome
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Mutations in the CLCN2 gene encoding the brain chloride channel ClC-2
have been recently associated with a rare autosomal recessive leukoencephalopathy, characterized by variable age at onset and clinical features,
with specific brain MRI findings caused by chronic white matter edema. We
sequenced CLCN2 in 6 patients from 5 families presenting characteristic
MRI white matter alterations involving middle cerebellar peduncles, cerebral peduncles, and posterior limb of the internal capsules. We identified
ABSTRACTS POSTERS
3 homozygous CLCN2 mutations in 5 patients (4F/1M) from 4 families,
while one male patient tested negative. One novel homozygous splice-site
mutation was identified in a young asymptomatic woman who underwent
MRI because of headache. A previously described nonsense mutation was
identified in 3 women from 2 apparently unrelated families from Southern
Italy. One patient presented at 58y with dystonic posture of the neck, mild
postural tremor of the hands, and head tremor. Of the two sisters, one presented at 26y with migraine without aura followed, at 46y, by progressive
postural imbalance, hypoacusia and tinnitus, and degenerative retinopathy;
her sister, aged 60y, suffers from cluster headhache since 30y with negative
neurological examination. Repeated MRI in both sisters show stability of the
leukodystrophic pattern. Finally, a novel homozygous missense mutation
was found in a male patient presenting asymptomatic leukoencephalopathy
discovered during assessment for infertility and azoospermia. This study
shows that CLCN2 mutations can be associated with a wide, but relatively mild, phenotypic spectrum and suggests that a second, yet unidentified,
gene can be associated with this peculiar white matter disease.
P09.037-S
A mutation in SBF2 deleting the plekstrin homology domain affects
carriers
R. Parvari1, E. Wajsbrot1, E. Muhammad1, D. Landau2,1, Y. Sadaka2,1, I. Noyman2,1, Z.
Shorer2,1;
1
Ben Gurion University of the Negev, Beer Sheva, Israel, 2Soroka Medical Center, Beer
Sheva, Israel.
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ABSTRACTS POSTERS
pathy and aneurisms of intracranial vessels. In family LEU-3-TO two male
brothers presented an overlapping clinical picture with history of recurrent
haemorragic strokes in the first two decades and a severe leukoencephalopathy, brain microbleeds and ischemic lacunae at the neuroimages. We
identified two novel missense mutations in COL4A1 (c.1249 G>C, p.G417R
in LEU-1-TO; c.2662 G>A, p.G888R in LEU-2-TO) by sequence analysis. Both
are predicted pathogenic and hit highly conserved Gly residues of the GlyX-Y repeat in the collagen triple helical domain. As reported for COL4A1
missense changes in the first third of the gene, mutation p.G417R seems
associated with a variant phenotype in which porencephalic lesions are not
the major characteristic. Patients in family LEU-3-TO, negative for COL4A1/
COL4A2, suggest a genetic heterogeneity for this disease.
P09.041-S
The role copy number variations (CNVs) in cryptogenic Cerebral Palsy
R. Segel1,2, H. Ben-Pazi1,2, S. Zeligson1, A. Fatal-Valevski3,4, A. Aran1,2, V. Gross-Tzur1,2, D.
Shmueli5, N. Shneibaum3,4, D. Lev6, S. Perlberg1,7, L. Deutsch8, E. Levy-Lahad1,2;
1
Shaare Zedek Medical Center, Jerusalem, Israel, 2Hadassah School of Medicine, Hebrew
University, Jerusalem, Israel, 3Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 4Tel
Aviv University, Tel Aviv, Israel, 5Child Development Center, Clalit, Jerusalem, Israel,
6
Metabolic-Neurogenetic Clinic, Wolfson Medical Center, Holon, Israel, 7Hebrew
University, Jerusalem, Israel, 8Biostatistical Consulting, BioStats, Jerusalem, Israel.
Background: Cerebral palsy (CP) is an umbrella term for congenital, nonprogressive motor disability, reflecting multifactorial insults to the developing brain. Etiologic factors include perinatal asphyxia, infection, inflammation, and stroke. However, in many individuals with CP the pathogenesis is
unknown. Copy number variations (CNVs) cause various neurodevelopmental conditions. We investigated the prevalence and characteristics of CNVs in
individuals with cryptogenic CP.
Methods:52 participants with non-progressive pyramidal or extra-pyramidal signs since infancy and no identified etiology were enrolled. Individuals
with acquired cause (i.e. stroke) were excluded. Analysis was performed
using the Affymetrix HD array. CNVs were classified as: pathogenic CNVs,
likely pathogenic, or likely benign. Main outcome measures were: clinically
significant (pathogenic and likely pathogenic CNVs) or not-significant (likely
benign and no CNVs) findings.
Results:40 CNVs were found in 26/52 (51%) participants. Most CNVs were
considered clinically significant (11/26 pathogenic and 5/26 likely pathogenic vs. 10/26 likely benign) and were not previously reported to cause
motor disability (12/16). Most CNVs were de-novo. Compared to individuals
without clinically insignificant CNVs, individuals with clinically significant
CNVs were more likely to have multiple CNVs (p<0.001), dysmorphic features (p=.01) and non- motor comorbidities (p=0.03). In 2/16 participants
with clinically significant CNVs the phenotype was characteristic for the genomic findings (SPAT and KANK1 deletions).
Conclusions: Clinically significant CNVs were found in 16/52 (31%) of individuals with cryptogenic CP and were often multiple and de-novo. We recommend that this useful test be performed in individuals with cryptogenic
CP especially in individuals with dysmorphic features and comorbidities.
P09.042-M
Behavioral phenotype in Costello Syndrome with atypical mutation: a
case report
P. Alfieri1, C. Caciolo1, G. Piccini1, L. DElia1, G. Valeri1, M. Tartaglia2, M. Digilio1, B.
Dallapiccola1, S. Vicari1;
1
Bambino Gesu` Childrens Hospital, IRCCS, Roma, Italy, 2Istituto Superiore di Sanit,
Roma, Italy.
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Mutations in PRNP that encodes the prion protein are causative for inherited Creutzfeldt-Jakob disease (CJD). The E200K substitution is the most
frequent mutation. Earlier age at death in successive generations has
been reported in two clusters, from Israel (Rosenmann et al. Neurology
1999;53(6):1328-9) and Italy (Pocchiari et al. PLoS One 2013;8(4):e60376).
No molecular or environmental explanations have been found. The aim was
to analyze a possible observational bias in E200K CJD patients from families living in France. Ages at death from 42 parent-offspring pairs from 19
families were collected and compared: parents 63.5 years 13.9 (range 43
to 90) offspring 59.8 years 10.4 (25 to 80), indicating a significantly earlier
age at death in offspring (p=.015 paired t-test). Including ages at last followup or at death of an unaffected but obligate carrier-parent, the difference
increased to - 9.8 years anticipation p=.004. Considering the large range of
ages at death and the intra-sib-ship variability, the most obvious bias lies in
the fact that sibs could develop disease later in life. When analyzing pairs
with at least 50% affected with known ages at death, the anticipation was
lost: parents 68.1 19.9 years and offspring 62.8 7.9 years p=.957, n=12.
This result could indicate a possible observation bias. Another hypothesis
could be the loss of modifying genes outside inbred clusters. The search for
genetic modifiers in familial CJD is of great importance.
P09.044-M
A new autosomal recessive syndrome: Severe developmental delay
and dysmorphic features causing by a missence mutation in FTO gene
ABSTRACTS POSTERS
targeting 42 dystonia genes. A total of 310kb is enriched and sequenced by
Illumina MiSeq (2x 150 bp paired-end). A first batch analysis in 27 dystonia
patients showed that HaloPlex enrichment provided enrichment efficiency
superior to standard whole exome procedures (>95% covered >20 reads;
90-95% of reads on target; mean coverage >500 reads per base).
We identified presumed disease-causing mutations in 2 out of 27 patients
including one patient with a DYT6 dystonia and one patient carrying a mutation in SLC2A1. In 7 patients, we identifed so far unknown probable disease-causing variants including mutations in ATP7B, PLA2G6, PARK2, FBCO7,
VPS13A and GCDH. This technology enabled the identification of a genetic
cause in approximately 7 % of patients in an unselected cohort of dystonia
patients in whom the most common genetic form (DYT1) was excluded. Targeted NGS may be a useful and cost-effective method to screen for mutations
in multiple genes associated with dystonia.
P09.047-S
Using exome-sequencing for the diagnosis of rare disorders: two
siblings affected by a congenital encephalopathy with microcephalia,
polymicrogyria and dystonia
We report a 15 year-old girl presenting with generalized epilepsy and intellectual disability. After normal development in the first two and a half years
of life, delayed language development became apparent. At age 3 drop attacks occurred. Later on the patient developed clonic and clonic-tonic seizures, predominantly at night time. Most remarkably, photosensitivity and a
special EEG phenomenon occured. Under the treatment with antiepileptic
drugs the patient remained nearly seizure-free. Anti-convulsive treatment
was stopped at the age of 13, leading to aggravation of the EEG only. Astonishingly, two years later the EEG showed normalization of the paroxysomal activity triggered by eye opening. We performed next generation sequencing (NGS) for a panel of genes known or suspected to be causative
in the pathogenesis of seizures. We identified a heterozygous stop mutation (c.348C>A, p.Y116*) in SYNGAP1 which was validated using Sanger sequencing. Analysis of the parents suggests that the mutation arose de novo.
The gene SYNGAP1 encodes an excitatory synapse-specific Ras GTPase and
is a major component of the postsynaptic density. It is thought to regulate
synaptic strength e.g. by suppressing signaling pathways linked to NMDARmediated synaptic plasticity. Mutations in SYNGAP1 are known to cause
autosomal dominant mental retardation type 5 (MRD5). More recently, the
publication of SYNGAP1 mutations in patients with seizures suggested a role
Back to index
in idiopathic generalized epilepsy combined with a variable degree of intellectual disability including severe speech impairment. Our patient shows
additional symptoms, expanding the spectrum of phenotypes for patients
with SYNGAP1 mutations.
P09.049-S
Linkage and subsequent exome sequencing analyses in a
consanguineous family reveal two novel genes associated with
pattern-sensitive idiopathic generalized epilepsy
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ABSTRACTS POSTERS
P09.051-S
Exome sequencing reveals mutations of a solute carrier gene in an
autosomal recessive form of epileptic encephalopathy of the first days
of life
Epileptic encephalopathy (EE) refers to a clinically and genetically heterogeneous group of devastating disorders characterized by seizures combined
with abnormal inter-critic encephalography Age of onset can be a key diagnostic feature for epileptic syndrome definition, treatment and prognosis.
We ascertained two multiplex families (including one consanguineous family) consistent with an autosomal recessive inheritance pattern of EE. All seven affected individuals developed seizures in the first day of life, resistant
to treatment and leading to repetitive Etat de mal. Evolution was marked
by severe EE with major delay in motor acquisitions and one patient died
at 11 years of age. No facial dysmorphism was noted, but oligodontia. Given
the similarity in clinical presentation in the two families, we hypothesized
that the observed phenotype was due to mutations in the same gene, and
performed exome sequencing in three affected individuals. Analysis of rare
variants in genes consistent with an autosomal recessive mode of inheritance led to identification of mutations in a solute carrier gene. Causality
was confirmed by co-segregation analysis in additional family members.
To assess the frequency of alterations of this gene in early onset EE, coding
exons were screened for mutations in a cohort of 70 unrelated affected individuals by targeted sequencing on a MiSeq instrument (Illumina). This experiment led to identification of compound heterozygous pathogenic variants
in a simplex case with a similar clinical presentation as the other affected
patients. These results highlight the value of careful clinical characterization
for genetic studies in heterogeneous diseases such as EE.
P09.052-M
Exome sequencing identifies novel SCN8A mutation associated with
neonatal epileptic encephalopathy, multiple congenital anomalies
and movement disorder
Epileptic encephalopathies represent clinically and genetically heterogeneous group of disorders of which the majority are of unknown aetiology.
It is hypothesized that novel variants may contribute to most of these devastating group of epilepsies. Within recent years the role of SCN8A in human disease has become apparent from next generation sequencing studies.
Whole-exome sequencing of a parent-offspring trio was used to identify the
genetic cause of severe early infantile epileptic encephalopathy in a boy,
where previous chromosomal, gene and metabolic investigations revealed
no abnormalities. The patient had neonatal seizures, movement disorder,
multiple congenital anomalies. He died at the age of 17 months due to respiratory illness. We identified a de novo heterozygous missense mutation
(c.3979A>G; p.Ile1327Val) in SCN8A (voltage-gated sodium-channel type
VIII alpha subunit) gene. The variant was confirmed in the proband with
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ABSTRACTS POSTERS
FOXG1 point mutations or submicroscopic 14q12 deletion, which involve FOXG1 gene. It is now known as FOXG1 syndrome which clinically causes postnatal microcephaly, severe mental retardation, absent language,
dyskinesia, and dysgenesis of the corpus callosum. More than 30 cases of
FOXG1 syndrome have been published. Cases of a submicroscopic 14q12
deletion, involving regulatory elements of FOXG1, with the coding region of
FOXG1 being unaffected, are described very seldom. A cis-acting regulatory sequence, acting as a silencer, is deleted more than 0.6 Mb distally from
FOXG1 in these cases. We report a new case with clinical features of FOXG1
syndrome and 14q12microdeletion. She was born at term with birth weight
3636g, length 51 cm and head circumference 33.5 cm. Since the birth developmental delay was noticed. At 11 months she has only weak head control, microcephaly (-3 SD), focal epilepsy, deep set and almond shape eyes,
protruding tongue, increased muscle tonus and brisk tendon reflexes. Brain
MRI showed hypogenesis of the corpus callosum, hypomyelinisation, arachnoid cyst in the left temporal region and subtle pachygyria. Chromosomal
microarray analysis revealed 1.5-Mb size 14q12 microdeletion (arr[hg19]
14q12(27,584,943-29,170,974)x1) appeared to be de novo. The end of this
deletion is 65-kb proximal from FOXG1 leaving the gene itself intact. There
are no known protein-coding genes located in the deleted area. Therefore,
we can hypothesize that there is some regulatory element of FOXG1 located
proximal to the gene which deletions can also cause FOXG1 syndrome.
P09.056-M
Cy5 Analysis System in molecular diagnosis of Fragile-X Syndrome
and Huntington disease
Expansion of DNA repeats causes hereditary disorders in humans; our genetic diagnosis center is studying (CGG)n repeat in the FMR1 gene in Fragile-X
Syndrome and (CAG)n repeat in the HTT gene in Huntingtons disease (HD)
by a high performing systems, Cy5-labeled Fragile-X Syndrome The critical
issues in FMR1 analysis usually are:
- discriminate between fully mutated females from normal homozygotes.
- determine the proper CGG repeat size between 110 and 200 repeats
- determine methylation status
We routinely use a combination of two PCR-systems (CE-IVD) to determine the proper CGG repeat size until 200 repeats and alternative methods to
Southern Blot (MS-MLPA, High Resolution Melting ) to determine methylation status.
Huntington Disease In HTT gene the critical issue is define the exact CAG
repeat size considering the small range that exists between premutation (36
repeats) and mutation status (40 repeats). The PCR-system (CE-IVD) we introduced in 2013 in our laboratory includes a positive control that helps to
estimate the proper CAG repeat size in sample.
Our results refer to 400 Fragile-X Syndrome suspected cases analyzed from
2010 to 2013 and to 30 Huntingtons Disease suspected cases analyzed in
2013. Use of these systems (CE-IVD), high performing, fast and easy, has
allowed us to reach good diagnostic results using instruments already supplied in our department.
P09.057-S
Comparison of Friedreich Ataxia Patients with Trinucleotide Repeat
Expansions and Point Mutations in FXN-Encoded Frataxin
R. Gavrilova, R. Dhamija, J. Hesemann, C. Teigen, J. Johnson, M. Ackerman, G. Isaya, M.
Patterson, P. Tebben, D. Oglesbee;
Mayo Clinic, Rochester, MN, United States.
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ABSTRACTS POSTERS
sylceramidase (GCase). It promotes rearrangement of the organization of lipids in lysosomal membranes and provides GCase greater accessibility to GC
substrate. Rare functional deficiency in Sap C results in a rare variant form
of Gaucher disease (GD). Sap C has six conserved cysteine residues involved
in three disulphide bonds making the protein structure remarkably stable
to acid environment and degradation. Five different mutations (i.e., p.C315S,
p.342_348FDKMCSKdel, p.L349P, p.C382G and p.C382F), four of them involving a cysteine residue, are known. Here we report on the functional and
biological behaviour of disease-associated Sap C proteins. Lipid-binding
properties and activating efficiency on GCase of Sap C mutants, analyzed
by surface plasmon resonance and biochemical assays, were comparable to
those of wild type Sap C. On the contrary, mass spectrometry analyses of
protease-treated mutants revealed a rapid degradation of those carrying a
mutation involving a cysteine residue. Our data provide evidence that mutant activator protein instability is the underlying pathogenetic mechanism
of Sap C deficiency.
P09.061-S
Genetic analysis of glucocerebrosidase (GBA) gene in Italian patients
with Parkinson disease and Lewy Body Dementia
Mutations in ATP1A3 have been reported in rapid-onset dystonia-parkinsonism (RDP). Dystonia in RDP has a characteristic sudden onset, typically
in adolescence in response to physical or mental stress. Dystonic symptoms
usually involve the bulbar region and are accompanied by symptoms of parkinsonism. More recently, mutations in ATP1A3 have been linked to alternating hemiplegia of childhood (AHC) and CAPOS syndrome, respectively.
We investigated a family with dystonia from New Zealand with ten affected members. Interestingly, only females were affected. After exclusion of
mutations in TOR1A and THAP1, we performed genome sequencing in two
affected cousins. For filtering, we used the KnomeDiscovery Data Filtering
Software. Since re-sequencing of 20 candidate variants did not elucidate the
genetic cause, we performed exome sequencing in another affected individual. Analysis of the raw data using an in-house bioinformatics pipeline revealed a previously unreported three base-pair deletion (c.443_445delGAG,
p.148_149delSer) in ATP1A3 in all three genome/exome sequenced patients. Segregation analysis showed the mutation in all patients and in one
unaffected male. The mutation was not found in 200 controls. Subsequent
clinical re-examination, revealed sudden onset of dystonic symptoms after
stressful events. None of the patients reported any history of AHC or CAPOS
syndrome.
In conclusion, our study identifies a novel mutation in ATP1A3 as cause of
RDP and highlights two important challenges when using next generation
sequencing: 1) The importance of detailed clinical information. In our family,
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ABSTRACTS POSTERS
Diseases, Department of Molecular Biology, Khartoum, Sudan.
Objective: To obtain a proof-of-concept for an anaplerotic therapy in Huntington disease (HD) using a validated functional biomarker of brain energy
metabolism.
Background: Energy deficit has been greatly implicated in the pathophysiology of HD. Our previous work has indicated a need to refill the Krebs cycle
which can be achieved using anaplerotic therapy.
Methods: 31P brain magnetic resonance spectroscopy (MRS) was coupled
with the activation of the occipital cortex to measure the levels of PCr and Pi
before (rest), during (activation) and after (recovery) a visual stimulus. At
visit 1, we performed 31P brain MRS in 10 patients at the early stage of HD
and 10 controls. HD patients were then treated at home for one month with
triheptanoin and came back for a second visit during which we performed
31P brain MRS.
Results: At visit 1, we confirmed a significant increase in Pi/PCr ratio
(p=0.022) during brain activation in controls - reflecting increased ATP synthesis - followed by a significant return to baseline levels during recovery
(p=0.008). In HD patients, we confirmed an abnormal brain energy profile
with decreased Pi/PCr ratio before treatment. After one month of triheptanoin therapy, the MRS profile was greatly improved in HD patients with
increased Pi/PCr ratio during visual stimulation (p=0.004).
Conclusion: This study suggests that triheptanoin is able to correct the bioenergetic profile in HD patients brain at an early stage of the disease. The
administration of triheptanoin over a longer period of time is now required
to assess its clinical benefit.
P09.067-S
PMCA, SERCA and VEGF as potential patogenetic factors in
Huntingtons disease and as biomarkers of onset and progression
Back to index
Kufs disease (KD) is the rare adult form of neuronal ceroid lipofuscinosis
(NCL). Mutations in the cathepsin F gene (CTSF) (MIM 603539) have recently been discovered in autosomal recessive Type B KD families characterized by movement and behavioral abnormalities and dementia. We present
a family in which pseudo-dominant transmission of Type B KD was explained by a novel homozygous splice site mutation in CTSF leading to the lost
of exon 1.
Three affected individuals showed similar neurological pictures characterized by generalized seizures, cerebellar dysarthria and cognitive decline,
evolving into frank dementia. The presence of three additional relatives with
a neurological syndrome compatible with KD and two instances of parent-
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ABSTRACTS POSTERS
to-child transmission proposed an initial hypothesis of a dominant form
of dementia representing a confounding factor for genetic testing. A more
detailed clinical assessment of the patients, meticulous collection of family history and recognition of the high degree of inbreeding in the isolated
community where this family lives, were crucial in disclosing an autosomal
recessive pattern of inheritance, prompting investigation of CTSF.
In vitro experiments in cultured skin cells from the proposita and her aunt
demonstrated a dysregulated autophagy and aggresome-like structures,
confirmed by ultrastructural studies in skin biopsies. These data put forward the hypothesis of a cytoplasmic toxicity of pathological cathepsin F in
KD type B.
P09.070-M
Lamin B1 expression is affected by EBV infection in lymphoblasts of
patients with Autosomal Dominant Leukodystrophy through mir-23
deregulation
180
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Mutations in LRRK2 are recognized as the most common genetic determinant of sporadic and familial Parkinsons disease (PD). Recently, several
studies of exonic variants in large populations were published; however,
none of them was focused in the 3UTR region. Our aim was to screening
the 3UTR of LRRK2 looking for risk variants, modifying factors, disrupted
targets for microRNA binding and a possible effect of this region in mRNA
expression.
Our cohort consisted of 743 PD patients (6710 years; 52% male) and 523
healthy controls (6712 years; 50% male). The human post-mortem tissues were obtained from three different brain regions of 9 PD and 5 healthy
donors.
Patients were genotyped for the LRRK2 mutations G2019S and R1441G/
C/H resulting in 16 G2019S carriers and 15 R1441G carriers. None of the
tissue donors was mutation carrier. We identified a total of 12 variants; 2 of
them new (c.*130_131del and c.*1382C>A) in the 3UTR region. We found
rs66737902 T>C as a possible variant related with PD risk being the C allele overrepresented in patients; p= 0.01 OR=1.37 CI=1.07-1.74. To tested
if rs66737902 had an effect in the gene expression through the binding of
miRNAs, we study miR-138-2* as candidate miRNA (TargetScan, microRNA.org) and miR-205 as control miRNA. We did not find any effect of miR138-2*. In the mRNA expression study we found significant differences
between TT and TC rs66737902 genotypes in the SN of PDs, with a minor
expression level in the TC group (p=0.011). No differences between patients
and control were found.
P09.074-M
Variation in the promoter of the autophagic beclin-1 gene (BECN1)
and its impact on expression levels: a study in Machado-Joseph
disease (MJD/SCA3) patients
ABSTRACTS POSTERS
Porto, Portugal, 3Laboratorio Nacional de Genmica para la Biodiversidad (LANGEBIO),
Unidad de Genmica Avanzada, CINVESTAV-IPN, Irapuato, Mexico.
Background and Objectives: Autophagy, as a process of intracellular components degradation, is especially important in disorders where accumulation
of the mutant protein is a hallmark, such as MJD, a late onset polyglutamine
ataxia. We documented the variation in the promoter of the BECN1 gene
whose overexpression has been reported to exert neuroprotective effects in
MJD. The relation between BECN1 promoter variation and expression levels
were studied, as well as its impact on disease onset.
Methods: The BECN1 promoter was sequenced in 95 MJD patients and 120
controls. In silico analysis (PROMO) were performed to detect differences in
putative transcription factor binding sites (TFBS). BECN1 expression level
was quantified by Real-time PCR in 29 MJD patients and 27 controls, for
which cDNA from peripheral blood was available.
Results: Two previously described variants (rs60221525 and rs116943570)
were found in MJD patients and controls. In silico analysis predicted the
existence of less putative TFBS for rs60221525 and of more TFBS for
rs116943570. BECN1 expression levels were in agreement with the in silico
predictions, showing decreased and increased expression for rs60221525
and rs116943570, respectively. CAG number explained 60.5% of the variance in onset; when rs60221525 and rs116943570 were added to the
multiple regression model, there was a tendency for the increase in the explanation.
Conclusions: Variation found in the BECN1 promoter modulates expression
and has a potential to modify onset of MJD. The analysis of further patients
should increase the power of the study and confirm the role of BCN1 as modifier of MJD.
P09.075-S
Study of the genetic architecture behind mood disorders by whole
exome sequencing on a large Italian pedigree
Major depressive disorders (MDD) and bipolar disorder (BD) are mood disorders with a lifetime prevalence in the adult population of approximately
16% and 4%, respectively. Estimated heritability is about 37% for MDD and
75% for BD and the two disorders have a genetic correlation of about 43%.
As of today their genetic architecture remains unclear and no major genetic risk factors or causative genes have been identified yet. With the aim of
identifying susceptibility loci responsible for the MDD/BD phenotype, we
studied a IV generation Italian pedigree composed of 20 subjects, 7 of whom
are affected by mood disorders (5 MDD, 2 BD). Linkage analysis identified 5
regions that were inherited by all the affected members, extending for 222
Mb overall. Exome sequencing performed on the 7 affected subjects and 2
unaffected relatives identified ~1930 genetic variants within linkage regions, 222 of which are functional variants (missense, LoF, splicing) shared by
all affected subjects. These latter variants affect several genes already associated to MDD/BD and other relevant biological pathways, such as synaptic
development and neurodevelopment. We also identified 28 rare variants
(MAF<1 % in dbSNP, 1000G and ESP6500), 8 in the linkage regions, present
only in the affected subjects. Among the genes affected by these variants, we
selected 4 candidates that could differentiate affected from unaffected subjects in the family. In conclusion, our data suggest that MDD/BD arise from
the combination of a shared mutational burden on risk genes together with
a few rare variants triggering the observed phenotype.
P09.076-M
Spectrum of Mutations in a New Cohort of Pakistani Families with
Primary Microcephaly
Autosomal Recessive Primary Microcephaly (MCPH) is a neurodevelopmental disorder resulting in diminution of brain growth in utero. The noticeable
features are reduced head circumference at birth, and varying degree of intellectual impairment without other neurologic findings. To date, twelve genes
have been reported to be associated with MCPH, including MCPH1, WDR62,
CDK5RAP2, CASC5, ASPM, CENPJ, STIL, CEP135, CEP152, ZNF335, PHC1, and
CDK6. Among these, ASPM has been found to be most frequently mutated in
the Pakistani population. In the current study, 30 new families were ascertained from different regions of Pakistan. We performed SNP array-based
Back to index
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ABSTRACTS POSTERS
4
Kabuki syndrome (KS; OMIM# 147920) is a rare congenital disorder, characterized by typical facial features including: long palpebral fissures, eversion of the lateral third of lower eyelids, arched and broad eyebrows with
lateral sparseness, short columella and prominent ears. Point mutations and
large intragenic deletions and duplications of the histone methyl transferase
MLL2 gene are the main causes of KS. MLL2 encodes a large protein that
belongs to the SET1 family of human SET-domain protein methyltransferase
superfamily. Recently, de novo partial or complete deletions and point mutations of KDM6A gene, have been identified as additional causes of KS. Case
report: We report the molecular and phenotype studies of a Spanish patient
who have typical features of KS. The patient is a 9 y.o. girl with mental retardation, a peculiar facies characterized by long palpebral fissures, a broad
and depressed nasal tip, large prominent earlobes, a cleft palate, scoliosis,
radiographic abnormalities of the vertebrae, hands, and hip joints, and
recurrent otitis media. Genomic DNA was extracted from the patient and
parents and mutation screening of coding exons and intron-exon junctions
of the MLL2 gene was performed by direct sequence analysis using a 3130XL
Genetic Analyzer. All found changes were in silico analyzed (Polyphen 2.0) to
estimate a possible damaging effect in the protein. We identified a deletion
in exon 32 that expands to the first portion of exon 33 (c.20711-c.21014
NG_027827). This deletion is absent in both parents and it has not been previously described.
Back to index
In 1977, Sourander and Wlinder described a case of hereditary multi-infarct dementia (MID) in a Swedish family. Later their disease was suggested
to be cerebral autosomal dominant arteriopathy with subcortical infarcts
and leukoencephalopathy (CADASIL). The clinical picture of recurrent strokes resembles CADASIL, but we did not detect any pathogenic mutations
in the entire 8091 bp reading frame of NOTCH3 nor found evidence for the
NOTCH3 gene linkage. Neither did we find any evidence of the CADASILtypical granular osmiophilic material (GOM) in skin biopsies. These multiple approaches suggest that in this Swedish family the hereditary MID
suspected to be CADASIL is a different disorder with dissimilar pathological
features, and belongs to the growing group of genetically uncharacterized
familial small vessel diseases (SVDs).
In order to search for the pathogenic mutation behind the disease, we did
a whole-exome sequencing for two healthy and three affected family members. The exon targeting was done with NimbleGens sequence capture
(Roche Inc. USA) and the sequencing with Illuminas sequencing platform
(Illumina Inc. USA).
After sequencing the results were filtered against public SNP databases in
order to identify the sequence variants that have not been published previously as polymorphisms. Subsequently, we concentrated on the variants
that are shared by all the three patients and excluded the variants that are
also shared by the healthy controls. Since the disease is dominant we narrowed our search by excluding the variants that are homozygous in all patients. Detailed results of our studies will be presented at the congress.
P09.085-S
Multiple sclerosis risk variant at the ch12q14.1b locus affect the
expression of the CYP27B1 gene in activated monocytes
P09.083-S
SGK223, a novel candidate gene for an autosomal recessive form of
dHMN
E. Gregianin1, A. Vettori1, E. Tognon1, D. Tessarolo2, M. Mostacciuolo1, G. Vazza1;
1
University of Padova, Padova, Italy, 2San Camillo Forlanini Hospital, Roma, Italy.
Distal hereditary motor neuropathies (dHMN) are a subgroup of Hereditary Motor Sensory Neuropathies characterized by a predominant motor involvement of the peripheral nervous system. In the present study a family
affected by an autosomal recessive form of dHMN was examined by using a
combined strategy of linkage analysis and whole-exome sequencing (WES)
in two patients. WES variants in the known disease-related genes associated
with similar phenotypes, and copy-number variants shared by the affected
subjects were excluded. Considering the presence of two consanguineous
marriages in the family, the homozygosity mapping approach was performed and a candidate autozygous region shared exclusively by the affected
subjects was highlighted on chromosome 8p23.1-p22. The candidate region
contains 120 genes and is characterized by a high number of pseudogenes
and paralogs which led to many false positive calls due to multiple-mapping
reads. No evident mutations were identified in the also poorly-covered exons
of the linkage region but the prioritization analysis of the about 230 WES va-
Background: The region ch12q14.1 was shown to be associated with several autoimmune diseases in different Genome Wide Association Studies. We
have determined that a variant which alters the enhancer activity of a regulatory element explains the association of the locus with multiple sclerosis
(MS). This region contains the CYP27B1 gene encoding the enzyme 1-alphahydroxylase which catalyses the conversion of 25(OH)D to 1,25(OH)2D, the
active form of vitamin D. Since the vitamin D is a key regulator of the immune response and plays a role in MS susceptibility, we want to determine the
effect of the variant on the expression of the CYP27B1 gene in specific population of immune cells. Methods : The monocytes CD14 cells were purified
from 111 healthy individuals. They were activated with Interferon-gamma
(IFN) and lipopolysaccharide (LPS). The CYP27B1 expression was then
measured by quantitative real time PCR and correlated with the genotype
of the variant. Results: We observed that the MS risk allele of the polymorphism rs10877013 was significantly associated with a low expression of the
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ABSTRACTS POSTERS
CYP27B1 gene (p=0,007) in activated monocytes. Conclusion: In this work
we demonstrate that the causal variant of MS association at the ch12q14.1
locus, alters the transcription of CYP27B1 gene suggesting that the intracellular vitamin D levels could be affected. Our data point to lower production
of 1,25(OH)2D is the functional cause of the genetic association with MS.
P09.086-M
Alterations of gene expression at the peak level in the experimental
allergic encephalomyelitis
A. Sen, G. Celik, O. Ozgun, S. Arslan;
Pamukkale University, Denizli, Turkey.
Multiple Sclerosis (MS) is a multifactorial autoimmune demyelinating disease of the CNS. Several large international association studies detected over
100 MS loci. However, for the majority of them the causal variant is not yet
identified and, generally, only common variants have been studied. The aim
of this study was to follow-up the MS loci in the Italian population, searching
for the primarily associated variants, focusing also on rare variants. After
an association study in 1750 Italian MS cases and 2272 matched controls
(Illumina 660-Q, and Immunochip), we selected for target resequencing 32
MS associated regions showing a significant association in the Italian population. The selected regions (1.9 Mb), either including the whole genomic
segment (N=17 regions, 45 genes) or only the coding sequences (further 48
genes), were captured (Agilent SureSelect) and sequenced (GAIIx Illumina)
in 600 Italian MS patients and 400 matched controls pooled in groups of 12
individuals.We used the variant caller CRISP to call the variants, ANNOVAR to
annotate them, and custom R-scripts to calculate allele frequency. Validation
of a subset of variants by individual genotyping demonstrate a high correlation with allele frequency in the pools.After QC, results in the MS patients
showed that among the 23065 detected variants, 67% were absent in public
databases, including 1061 variants with a predicted functional consequence
on the gene product (missense, nonsense, splice variants), thus potentially
directly involved in the MS susceptibility. The comparison with the data of
the controls and the replication in independent cohorts is ongoing
P09.088-M
Modulation of Protein Kinase C Alpha (PRKCA) mRNA expression
and alternative splicing by functional polymorphisms contribute to
multiple sclerosis susceptibility
E. M. Paraboschi1, V. Rimoldi1, C. DallOsso1, E. Saba1, T. Tabaglio1, L. Clario1, M. D.
Benedetti2, S. DAlfonso3, M. Leone4, D. Gemmati5, S. Duga1, G. Sold1, R. Asselta1;
1
Department of Medical Biotechnologies and Translational Medicine, University of
Milan, Milan, Italy, 2Department of Neurosciences, University Hospital, Verona, Italy,
Back to index
Congenital myotonias are a group of hereditary muscle disorders that present in infancy and childhood, characterized by impaired muscle relaxation
following a voluntary forceful contraction. The core symptoms either relate
to myotonia or to periodic weakness. These disorders are caused by mutations in genes encoding the skeletal muscle chlorides, sodium and calcium
channels.
We report a 7-years old boy with a severe early onset unique phenotype of
myotonia permanents. He had neonatal respiratory failure and as well as
severe recurrent episodes of laryngospasm during infancy. Massive general
muscle hypertrophy, eye lid myotonia and prolonged hand grip relaxation,
were present since early childhood. An unusual complication of tongue hypertrophy was noted during infancy and reoccurred again after surgical resection.
A novel V717G mutation in SCN4A was identified in the proband, but not in
his parents.
This is the first case of myotonia permanens reporting recurrent tongue
hypertrophy as a major clinical feature in this case. Tongue hypertrophy
resulted most probably from continued tongue contraction caused by his
permanent myotonia .
The novel mutation found in SCN4A in this boy, has not been found in his
parents. It has been suggested to be pathological by a number of in silico
prediction programs. It is located in a well conserved area of the gene in
the transmembrane helical part of the Sodium channel protein subunit. This
mutation has not been describes previously in SCN4A related disorders.
This case further expands the genetic and clinical spectrum of myotonia
permanents.
P09.090-M
PANK2 and C19orf12 mutations in neurodegeneration with brain iron
accumulation (NBIA)
G. Annesi1, P. Tarantino1, M. Gagliardi1,2, G. Lesca3,4, E. Broussolle5,6, G. Iannello1,7, A.
Quattrone1,8;
1
Institute of Molecular Bioimaging and Physiology, Section of Neuroimaging, National
Research Council, Germaneto (CZ), Italy, 2University of Magna Graecia, Germaneto (cz),
Italy, 3Departement of Medical Genetics, Hospice Civiles de Lyon and ClaudeBernard
Lyon, University Lyon France, Lyon, France, 4CRNL, CNRS UMR 5292,INSERM U1028,
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ABSTRACTS POSTERS
Back to index
P09.093-S
Familial case of NBIA
P09.091-S
Identification of the genetic factors involved in neural tube
development and defects
Neural tube is the embryonic precursor of brain and spinal cord, and forms
as a consequence of closure of the developing neuroepithelium. If closure
events fail, the embryo will manifest a neural tube defect (NTD: spina bifida
or exencephaly). Despite significative advances in the field, the elucidation
of genetic factors associated to NTD has remained elusive and NTDs are
the second most common congenital defects affecting human pregnancies.
This project aims at investigating two different aspect of NTDs: on one side
the analysis of the transcription factor Sax-1, that was found to be downregulated in a microarray screening in murine NTDs model (Zic2Ku). Sax-1
expression correlates remarkably closely with the progression of posterior
closure of the neuroepithelium, overlapping with Zic2 expression. Functional studies of Sax1 and Zic2 in zebrafish will be flanked to mouse embryos
and in vitro analyses. On the other side, this project investigates the role of
miRNAs in neural tube development on human samples. From a database of
534 fetal autopsies, we selected 9 fetuses with NTDs (7 myelomeningocele, 2 anencephaly). Using a combination of bioinformatics tools (MirWalk,
CO-ME-TA, DAVID), we have identified 4 candidate miRNAs whose predicted
targets are significantly enriched for functional pathways related to neurulation, that are now under functional evaluation.
P09.092-M
Mutations in RNA kinase CLP1 cause neurodegeneration
V. R. C. Eggens, J. L. Cazemier, P. R. Kasher, M. A. J. Weterman, F. Baas;
Academic Medical Centre Amsterdam, Amsterdam, Netherlands.
Cleavage and polyadenylation factor I subunit (Clp1) is an important kinase in RNA metabolism. It is involved in processes as tRNA maturation and
mRNA 3 end processing. Recently, a missense mutation in the CLP1 gene has
been identified in patients with atrophy of the cerebellum, pons and corpus
callosum. We have isolated an ENU induced zebrafish mutant, harbouring
a p.R44X nonsense mutation in the Clp1 gene. Using in situ hybridization
and morpholino knockdown of p53 we examined the phenotype of homozygous p.R44X zebrafish embryos. We show that Clp1 knockout zebrafish
do not survive beyond 4 days post fertilisation (dpf), have a reduced head
size and show an S-curved body. At 2dpf, Clp1 knockout fish show reduced
expression of midbrain marker otx2, increased cell death in the brain and a
disturbed organisation of motor neurons. The phenotype can be partly rescued by injecting human wild type, but not by mutant p.R140H CLP1 mRNA.
Morpholino knock down of p53 partially rescues the phenotype as well. We
show that Clp1 is an essential gene in both the central and peripheral nervous system. Similar to the human situation, clp1 mutations cause neurodegeneration and motor neuron problems in zebrafish. We show that the
neurodegeneration is mediated by the p53 apoptosis pathway. Our data supports the hypothesis that amongst other RNA processing genes, CLP1 plays a
crucial role in neurodevelopment.
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P09.094-M
A cell reprogramming-based approach to study 7q11.23 gene dosage
imbalances in Williams Beuren syndrome and autism spectrum
disorder
Symmetrical gene dosage imbalances at 7q11.23 cause two neurodevelopmental diseases, Williams Beuren Syndrome (WBS) and the 7q11.23
microduplication associated to autistic spectrum disorder (7dup-ASD). Besides intellectual disability and craniofacial dysmorphisms, WBS patients
display hypersociality and comparatively well-preserved language skills
while 7dup-ASD is associated with impairments of varying severity across
the autistic spectrum. The striking symmetry in genotype and phenotype
between the two conditions points to the 7q11.23 cluster as a surprisingly
small subset of dosage-sensitive genes affecting social behavior and cognition. The molecular phenotypes of these syndromes in disease-relevant celltypes remain to be elucidated however, due to scarce availability of primary
diseased tissues. Convergent evidence both from human studies and mouse
models, points to transcriptional dysregulation as a critical aspect of both
conditions, consistent with the presence of several transcription factors in
the 7q11.23 interval.
Here we present the first analysis of transcriptional dysregulation in human
physiopathologically relevant cell types carrying 7q11.23 dosage imbalances. We selected a large and unique panel of WBS and 7dup-ASD patients
and derived induced pluripotent stem cells (iPSC) with the most advanced
reprogramming technology based on synthetic mRNAs. These were then
differentiated into relevant lineages to establish experimentally tractable
models of these conditions. Next, we integrated high-throughput sequencing for transcriptional and chromatin analysis and present here a functional dissection of these symmetric conditions that uncovers the principles of
dosage-dependent transcriptional dysregulation in disease-relevant human
cells types.
P09.095-S
Next-generation sequencing in the diagnosis of neurodevelopmental
disorders
ABSTRACTS POSTERS
Department of Medical Genetics,Osaka Medical Center and Research Institute
for Maternal and Child Health, Osaka, Japan, 2Laboratory for Medical Science
Mathematics, Center for Integrative Medical Sciences, RIKEN, Yokohama, Japan,
3
Department of Pediatrics, Yamagata University Faculty of Medicine, Yamagata,
Japan, 4Department of Pediatrics and Neonatology, Nagoya City University Graduate
School of Medical Sciences, Nagaoya, Japan, 5Department of Pediatric Neurosurgery,
Takatsuki General Hospital, Osaka, Japan, 6Division of Regenerative Medicine, Institute
for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka,
Japan, 7Department of Neurosurgery, Osaka National Hospital, National Hospital
Organization, Osaka, Japan, 8Center for Medical Genetics, Keio University School of
Medicine, Tokyo, Japan.
1
Introduction
Recently, genetic bases of neurodevelopmental diseases including CNS
anomalies, intellectual disability (ID), and epilepsy have been significantly
elucidated, but are still largely unknown. We have developed a target and
whole exome sequencing (WES) based mutation screening strategy. The target sequencing system enabled us to screen 284 genes associated with neurodevelopmental disorders. Unresolved patients were studied by probandparent trio approach using WES.
Materials and Methods
Forty seven patients were included in the study. Fourteen patients were studied by WES. They were analyzed under the approval by our institutional
ethics committee. To capture the exonic DNAs, we used SureSelectXT Custom capture library for neuronal genes capture. We performed sequencing
using Illumina HiSeq 2000 sequencer and obtained paired-end sequence
reads. We excluded known variants found in dbSNP, 1000 Genomes Project,
ESP6500 and our control samples, and narrowed the candidates to missense, nonsense SNVs and frameshift indels.
Results
Fourteen mutations including FOXG1, AHI1, PTPN11, DYRK1A, ACTB, CASK,
GABRD were identified by target screening and conventional sequencing.
WES revealed de novo mutations in CREBBP, KIF1A, GRIN2A and ALDH18A1.
HNRNPH2 and AMMECR1 mutations were noted in X-linked pattern. None
of these mutations were recorded in database. Some other mutations have
unknown pathogenic significance.
Discussion
NGS method could clarify a consistent percentage of patients with neurodevelopmental disorders. NGS method would be an alternative to current
technologies for identifying the multiple genetic causes of neurodevelopmental disorders.
P09.096-M
Seven novel neurofibromatosis 1 (NF1) gene mutations identified in
Spanish pediatric patients
Introduction: Neurofibromatosis type 1 (NF1) is one of the most common human autosomal dominant disorder with an estimated incidence of
1:3500. This is a neurodermal dysplasia characterized by caf-au-lait spots,
axillary freckling, dermal neurofibromas, and Lisch nodules of the iris. The
condition is fully penetrant and has a highly variable expression. It is caused
by mutations in the neurofibromin gene (NF1) located on chromosome
17q11.2. Subjects and Methods: Seven children suspected of having NF1
were tested for mutations in the NF1 gene. Some of them only have cafau-lait spots and two presented other clinical features. Single and multiexon deletion/duplication were tested by MLPA assay. PCR and sequencing
analysis in RNA was performed. Segregation of the mutations was checked
in 3 cases. Results: Seven pathogenic not previously reported mutations in
NF1 gene were identified in heterozygous (Table 1). Only 1 sample showed
a deletion of the exon 23 by MLPA. In one case the mutation was considered
to be de novo. Discussion: Mutational NF1 analysis is considered to be a difficult task owing to the size of the gene, the absence of clear mutational hot
spots and the presence of pseudogenes. RNA and MLPA-based techniques
should be used in conjunction to provided reliable and accurate molecular
genetic testing.
Table 1
Case
1
2
3
4
5
6
7
Mutation
c.4009C>T, p.R1337W
c.2894T>G, p.I965R
c.3639_3640insA
c. 3935C>T, p.S1312F
c.23505_3508insAAGT
c. 36086_6087insG
c.3611G>T, p.R1204L
Familiar study
yes
yes
yes (de novo)
ND
ND
ND
ND
Back to index
P09.097-S
Genetic diagnosis of neurological diseases using NGS: first year of
experience
Background
Neuronal migration disorders are an important cause of severe psychomotor retardation and seizures, frequently resistant to antiepileptic medication. Based on cerebral MR imaging (cMRI) neuronal migration disorders
(NMD) can be subdivided into various radiological forms including classic
and cobblestone lissencephaly.
Method
Genetic testing was performed for 1034 index patients either (1) individually after in-house cMRI reevaluation (n=214) or (2) as assigned by the referring doctor (n=820). Individual testing strategies included MLPA analysis,
Sanger sequencing and massive parallel sequencing. In an ongoing study we
assess long-term development and genotype dependent response to antiepileptic and supportive therapies.
Results
119 NMD patients of study arm 1 were genetically analyzed leading to the
identification of the underlying genetic alterations in 35.3% of the analyzed samples incl. classic lissencephaly 65.4%, subcortical band heterotopia
83.3%, polymicrogyria 10.0%, cobblestone lissencephaly 14.3%, periventricular nodular heterotopia 37.5% and complex cortical malformations
20.0%. In comparison, the overall mutation detection rate in study arm 2
without in-house cMRI reevaluation was only 18.5% (p<0.0005).
Overall, pathogenic mutations were identified in 18.9% of independent patients incl. LIS1 (34), DCX (47), ARX (10), TUBA1A (4), TUBB2B (4), GPR56
(10), FLNA (30), POMT1 (22), POMGnT1 (15), FKTN (3), FKRP (9), ISPD (1),
LARGE (2) and DAG (1). Our preliminary data suggest a genotype dependant
antiepileptic response in LIS1 associated classic lissencephaly.
Conclusion
Genome wide genetic testing strategies will further improve the number of
identified genes and genetic alterations, but continue to require their critical interpretation in the context of clinical findings and cerebral imaging.
185
ABSTRACTS POSTERS
P09.099-S
In a consanguineous family with two patients showing a novel
autosomal recessive inherited syndrome another patient with an
unlinked autosomal nonsyndromic form of mental retardation was
identified
186
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probably not accidental, because intron 27b of the the NF1 gene contains
OMGP gene (OMIM * 164 345 ), which acts in myelination processes and
seems to be involved in immunopathogenesis of MS. Moreover, NF-1 and
MS may share a common influence from genes on chromosome 17 affecting cell proliferation and inflammation processes. We report our 12 years
experience in the follow up of NF1 adult patients. The cohort is composed
by 301 patients, with an age range of 18-72 years, and Males/Females ratio
of 111/190. A multidisciplinary equipe evaluates patients yearly, thus allowing a precise knowledge about the natural history of the condition. Over
the years, MS has been diagnosed in four of our patients (3 women and 1
man). This observational study enables us to define for the first time MS
prevalence in a large cohort of NF1 adult patients (1.3%). Detailed clinical
features of each MS-NF1 patient, together with review of literature and current aetiopathogenetic hypothesis will be provided.
P09.102-M
An NGS gene panel for the genetic diagnosis of rare autosomal
recessive cerebellar ataxias
Autosomal recessive cerebellar ataxias (ARCA) are a genetically highly heterogeneous group of neurological disorders involving both central and peripheral nervous system. One of the goal of the E-Rare EUROSCAR project is
to establish an NGS-based targeted genotyping for the diagnosis of known
ARCA genes. One-hundred-forty-two ataxic patients referred from different
European partners were analyzed using a HaloPlex-based gene panel targeting the coding regions of 127 genes involved in recessive and dominant ataxia. Patient inclusion criteria for analysis were: progressive ataxia, exclusion
of nongenetic causes, family history suggestive of autosomal recessive ataxia (AR) or sporadic (S) patients with onset before age 40. We contributed
with 34 Italian patients (14AR and 20S) previoulsy tested for FRDA, AVED,
or APTX, as appropriate. This approach allowed us to identify homozygous
or compound heterozygous pathogenic mutations in ADCK3 and 2 challenging genes (SYNE1 and SACS) in 6 patients (4S and 2AR). Moreover, different missense variants of uncertain pathogenicity were identified in genes
responsible for dominant ataxia (PDYN, SPTBN2, CACNA1A). Finally, several
heterozygous mutations were identified in recessive genes. For 6 patients,
analysis revealed no candidate variants in screened genes. All high-quality
variants were confirmed by Sanger sequencing indicating reliability of this
approach. Further analyses are required for the validation of uncertain variants and the evaluation of large in/del mutations in recessive genes in which
heterozygous mutations have been identified. In conclusion, NGS gene panel
represents a necessary tool for genetic diagnosis of highly heterogeneous
diseases such as ataxia. (E-Rare grant to MK, PB, and FT)
P09.103-S
Complete recovery from psychosis upon miglustat treatment in a
juvenile Niemann-Pick C patient
Niemann-Pick disease type C (NPC) is a rare recessive lipid trafficking disorder characterized by the accumulation of unesterified cholesterol and
glycosphingolipids in the brain and viscera. In the juvenile form, patients
in their early years are symptom free, but present with neurodegeneration
later in their lives. These include clumsiness, seizures, ataxia, and motor and
intellectual decline. Psychiatric manifestations (schizophrenia, presenile
dementia, psychosis or depression) may occur at any stage of the disease.
Recently, miglustat was approved for the therapy of NPC. We present a case
of a patient with juvenile NPC disease whose psychosis was reversed completely by miglustat treatment. At age 14 of the patient, slurred speech and
coordination problems, frequent falls and loss of balance were observed.
Later, dyslexia and dysgraphia developed. Fine motor skills started to decline more rapidly at age 17, and dysphagia developed. Vertical supranuclear
gaze palsy when looking downwards, a hallmark of NPC was observed. Laboratory diagnosis of NPC was established by sequencing of the NPC1 gene.
Genotype of the patient was [c.3019C>G] + [c.3182T>C] . Both pathogenic
mutations were described previously in the literature. Brain MRI was performed at age 17, 20.5, and 23. At age 17 no signs of atrophy could be detected. Moderate degree atrophy developed between ages 17 and 20.5 years.
Miglustat therapy was introduced at age 20.5. Between ages 20.5 and 23,
atrophy showed no progress. In conclusion, the introduction of miglustat in
NPC patients even in the most advanced cases, with respect to psychiatric
ABSTRACTS POSTERS
illness might be beneficial.
P09.104-M
VEGF-mediated sphingosine kinase activity decreases in NiemannPick Type C neurons and contributes to pathology in NP-C mice
H. Jin1, J. Bae2,3;
1
College of Veterinary Medicine, Kyungpook National University, Daegu, Korea, Republic
of, 2School of Medicine, Kyungpook National University, Daegu, Korea, Republic of, 3BK21
Plus KNU Biomedical Convergence Program, Daegu, Korea, Republic of.
Cortical tremor (CT) is characterized by fast, small-amplitude, focal or multifocal jerks. It is commonly induced by movement or somatosensory stimulation. CT may have different etiologies: metabolic abnormalities, various
neurodegenerative disorders and posthypoxic myoclonus. CT may also be
present in familial adult myoclonic epilepsy (FAME) defined by an autosomal dominant inheritance, but despite the locus has been mapped to chromosomes 8, 2 and 5, no causative mutations have been identified to date. A
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The NPC1 gene spans more than 47 kb, located at locus 18q11, and encodes
for an 1278-amino-acid protein located primarily into late endosomes. Mutations in this gene are associated to Niemann-Pick type C (NP-C) disease,
which is a fatal autosomal recessive lysosomal disorder characterized by
storage of unesterified glycolipids and cholesterol that leads to progressive
neurodegeneration. The aim of this work was to define pathogenic variations in patients with clinical suspicion of NP-C. In total, 244 DNA samples
were included. DNA was isolated from peripheral blood by standard methods. The whole coding region of the gene was amplified by PCR and sequenced by Sanger sequencing followed by electrophoresis in the genetic
analyzer ABI3130xl. Sequence variations were compared to data in the NP-C
database (http://npc.fzk.de/) and in silico analyses were performed when
necessary. Fourteen new variations were identified in 20 DNA samples that
were classified as pathogenic. From those, 4 (28,57%) mutations (p.P434S,
p.S667L, p.G911S, p.V1115F) are responsible for change in amino acid polarity, and two of those are located within transmembrane regions. Additional changes were observed in other functional domains, such as in disulfide
bond (p.C238R), and in cholesterol binding and transfer (p.A183T) regions.
Same of them (p.C238R, p.P434S, p.R615H, p.S667L) were shown to be loca-
187
ABSTRACTS POSTERS
ted in conserved regions based on gene alignment among different species.
We can predict that alterations reported here can affect protein conformation structure and/or protein function. These findings can contribute to the
understanding of important functional domains in the human NPC1 gene.
P09.109-S
PARK2 deletions in patients with Autism Spectrum Disorder (ASD)
and other neurodevelopmental pathologies
188
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P09.111-S
Polymorphisms and mutations in a specific population of Central
Europe (South-Eastern Moravia, Czech Republic) with autosomaldominant Parkinsonism
Background: In an epidemiological study carried out in an isolated population of South-Eastern Moravia in the Czech Republic, a surprisingly high
prevalence of parkinsonism was found, differing from the published prevalence rates in other European countries. Objective: To determine the type of
mutation in families with autosomal-dominant parkinsonism with dementia. Methods: On the basis of a detailed genealogical examination of all the
individuals with confirmed parkinsonism, the pedigrees were compiled and
a DNA analysis of probands from each pedigree was subsequently initiated.
A massive parallel sequencing method using Ion Torrent technology was
used; the DNA sequence analysis was focused on the gene loci in which the
causal mutations related to Parkinsons disease (PD) have been described.
Results: Three large pedigrees with an autosomal-dominant inheritance
pattern with reduced penetration of parkinsonism were identified. None
of the previously described pathogenic mutations associated with PD were
found; rare variants or yet-undescribed mutations were also found. In 5 of
10 examined probands, a novel missense Q230H mutation of the MAPT gene
was detected. Polyphene and SIFT in silico predictors indicate this mutation as probably damaging. Conclusion: Confirmation sequencing using an
independent method is underway to examine the targeted MAPT mutation
in other individuals from all three pedigrees and to compare it with healthy
controls. Supported by grants: IGA MZ R NT - 14407-3/2013 and IGA LFUP
2013-024
P09.112-M
HOMER1 promoter analysis in Parkinsons disease: association with
levo-dopa dosage
ABSTRACTS POSTERS
velopmental delay. Segregation analysis is complemented with an in silico
analysis of mutation effects on the protein structure and function. Using
sequence information, we compared different computational prediction
methods. We also used homology modelling to build structural models of
two PCDH19 EC-domains containing the novel missense mutations. Wildtype and mutant models were compared to identify the differences in the
presence and type of residue interactions or in the biochemical properties
(conservation, electrostatic charge, hydrophobicity) of the model surfaces.
This analysis revealed that these novel mutations exert their pathogenic role
using different molecular mechanisms. Two of them interfere with or alter
functional residues predicted to mediate ligand or protein binding, another
alters the EC-domain folding stability, and the frame-shift mutation produces a truncated protein lacking the intracellular domain.
We also observed that the two girls with severe neurological impairment
carry mutations predicted to have the worst effects on the protein structure.
P09.115-S
Molecular genetic background of patients with PEHO-like features
PEHO syndrome (Progressive encephalopathy with Edema, Hypsarrhythmia and Optic atrophy; MIM 260565) is an autosomal recessive inherited
progressive infantile encephalopathy. The main features of PEHO syndrome
are hypotonia, infantile spasms and/or hypsarrhythmia, psychomotor retardation, absence or early loss of visual fixation, edema of the face and limbs,
and typical dysmorphic features. Brain atrophy is progressive and most prominent in the cerebellum, where the molecular layer is strongly reduced,
Purkinje cells are deformed and misaligned, and the cells of the granule cell
layer are significantly reduced in number. In addition, the optic nerves show
varying degrees of loss of myelinated axons and gliosis, and the retinal nerve fiber and ganglion cell layers are atrophic. A number of patients present
with many of these clinical features, but lack typical neuroradiological and
neuropathological findings or progression of brain atrophy and do not have
the PEHO founder mutation. These patients are classified as having PEHOlike syndrome. This group is clinically heterogeneous and therefore it is likely that there are multiple underlying genes. To characterize the genetic
background of patients showing PEHO-like features, we performed exome
sequencing of 33 Finnish patients, and parents of six of them. We identified
likely pathogenic mutations in known disease genes (CDKL5, ABAT, SPTAN1,
SCN2A, MT-CYB, WDR45 and KCNQ2) in nine individuals. Analysis of mutations in novel disease genes is ongoing. Our preliminary findings imply that
patients with PEHO-like features are genetically highly heterogeneous and
that the entity overlaps with early-infantile epileptic encephalopathies.
P09.116-M
De novo mutations in periventricular heterotopia: an exome
sequencing study
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direct binding between calmodulin and filaminA. Although the exact pathogenic status of this variant has yet to be corroborated by a second individual,
this illustrative case demonstrates that functional genetic relationships can
contribute to the implication of extremely heterogeneous disorders such as
PH.
P09.117-S
PLA2G6-associated neurodegeneration (PLAN): long-term followup, genotype-phenotype correlates and identification of a founder
mutation in a large North-African cohort
Mutations in the PLA2G6 gene are causative of PLAN, a spectrum of neurodegenerative conditions including infantile, childhood and adult onset forms.
Seventeen North African patients with a clinical suspicion of infantile-onset
PLAN underwent clinical and instrumental examinations and PLA2G6 sequencing. Haplotype analysis was performed to date the identified founder
mutation. All patients carried pathogenic biallelic mutations in PLA2G6. Sixteen children had the commonest form of infantile-onset PLAN, with early
onset of psychomotor regression, hypotonia, cerebellar signs and abnormal
ocular movements. The phenotype was highly homogeneous, with rapid development of severe spastic tetraparesis, cognitive impairment, optic atrophy, and cerebellar atrophy at brain MRI. Motor neuropathy and EEG fast
rhythms were also frequent. Nine patients from six families shared the same
founder mutation (p.V691del) which likely arose in the late seventeenth
century. Only one patient fits the diagnosis of the much rarer childhoodonset PLAN. Despite the early onset (18 months), progression of cerebellar
and pyramidal signs was slower, with behavioral disturbances and dystonia.
Typical features of infantile-onset PLAN such as hypotonia, nystagmus/strabismus, optic atrophy, EEG fast rhythms and motor neuropathy were absent.
Besides cerebellar atrophy, MRI showed iron deposition in the basal ganglia.
This patient carried a missense mutation predicted to be less deleterious.
P09.118-M
Identification and functional characterisation of a new KCNJ2
mutation
189
ABSTRACTS POSTERS
gene in a patient presenting with symptoms characteristic of HCS, but without cystinuria.
Immediately after birth, the patient presented with severe muscular hypotonia and feeding problems, followed by growth hormone deficiency and
hypergonadotropic hypogonadism. Initial molecular analyses excluded a
number of syndromes, including Prader-Willi (PWS). The homozygous deletion in PREPL identified by array analysis corresponded to heterozygous
deletions in each of the nonconsanguineous parents. qPCR analysis confirmed these findings and pinpointed the extent of the deletion to exons 5-10.
An isolated homozygous microdeletion involving only PREPL has not been
previously described, however, Rgal et al., (in press) recently identified a
patient carrying a nonsense mutation in PREPL combined with a microdeletion involving SLC3A1 and PREPL, resulting in a similar phenotype to the
case presented here. These findings may contribute to further delineate the
normal function of PREPL, as well as support the notion that homozygous
PREPL inactivation should be considered in the diagnosis of patients with a
PWS phenotype, but no PWS genotype (Maartens et al., Biol Chem 387;879883,2006).
P09.121-S
Compound heterozygous mutations in two known MCPH genes in
autosomal recssesive primary microcephaly
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Introduction: Since ,mutations in MECP2 gene were described as causing Rett syndrome (RTT, OMIM#312750) in 1999, other genes have also
appeared to be responsible of RTT: X-linked cyclin-dependent kinase-like
5 (CDKL5) lying in Xp22 and forkhead box G1 (FOXG1) in 14q12. We present our experience in clinical and molecular diagnosis of congenital form
of RTT.
Materials and methods: We have studied the FOXG1 gene by direct sequencing and MLPA (Probemix-P075, MC-Holland) in patients RTT-without
ABSTRACTS POSTERS
MECP2 mutation and in patients with mental retardation and Rett-like clinical features.
Results: We analyzed 211 patients with clinical presentation likely to have
mutations in FOXG1 gene: 143 RTT (classic and congenital form MECP2negative), 39 male patients with severe mental retardation and hypotonia
and 29 patients with RTT like features. We detected 9 mutations: 5/23 girl
patients with congenital form and 4/39 male patients with severe mental
retardation.
None of classical form or girls with RTT-like clinical features carried mutation in FOXG1.
Conclusions: Genetic etiologies of variant Rett syndrome are heterogeneous; screening the FOXG1 gene should be done not only in females, but
also in male patients with severe hypotonia and acquired microcephaly since the first months of life.
FOXG1 is not an X-linked gene and therefore there can be a higher incidence
of mutation detection in RTT-like males than in MECP2 and CDKL5 genes.
P09.126-M
Modifiers of age at onset in spinocerebellar ataxia type 2: a
preliminary study in a Brazilian population
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Spinocerebellar ataxia type 8 is a dominantly inherited ataxia, mainly occurring in adulthood, caused by a CTG.CAG tract expansion in the ATXN8OS
gene, an untranslated antisense RNA partially overlapping the KLHL1 gene,
and the complementary CAG repeat in the ATXN8 gene.
We report a father (59 years) and his daughter (10 years) carrying a genomic deletion (800 kb) completely overlapping ATXN8OS gene and part of
KLHL1 (from 70,345,271 to 71,196,665 - NCBI Build 37/hg19 - Array-CGH
60K) and a 4 Mb duplication in 8q13.33q21.11 (72,268,801-76,311,849)
segregating in an unaffected brother. The girl was evaluated for dysarthria,
difficulties in sentence structuring, short attention span and mild intellectual deficit. No other neurological signs were reported. Brain MRI was normal.
Neurological examination of the father revealed short attention span; coordination, sensitivity, reflexes and cranial nerves were normal; no abnormal
gait or dysarthria. Brain MRI revealed several areas of gliosis in the frontal
white matter.
Databases mining showed a partially overlapping 357 kb deletion involving
only KLHL1 and ATXN8OS (chr 13:70,486,026-70,843,210) in a patient with
mild speech delay (Decipher 278559), inherited from a healthy mother.
In contrast with the KLHL1/ATX8OS knockout mouse model, our data suggest that heterozygous deletion of KLHL1/ATX8OS is not associated with
ataxia/cerebellar involvement in humans.
P09.128-M
Genetics of Schizophrenia: preliminary results of an Italian
multicenter study
Schizophrenia (SCZD) is a major psychiatric disease causing severe disability and with a prevalence of 1% worldwide. Genetic factors play a key role
in the etiology of schizophrenia but its genetic bases are complex and not
yet clarified. In the last few years, innovative technologies have been widely
applied to the genetic study of schizophrenia. This multicenter study aims to
apply aCGH and NGS technologies to investigate genetic risk factors for specific SCZD-related endophenotypes that greatly affect real-life functioning of
people with schizophrenia or variables strongly associated with it. To investigate the contribution of de novo CNVs to schizophrenia vulnerability, we
have used the Enhancer Chip (PLoS One 2012;7(12):e52264), a customized
aCGH recently developed by our group, which is able to examine CNVs in
the whole human genome as well as testing over 1,250 enhancers for their
potential pathogenic role. Clinical research units have provided us DNAs
from 60 sporadic SCZD patients and their parents (family trios). To date,
we have identified two de novo partially-overlapping deletions at 7q31.2 in
unrelated SCZD patients. Interestingly, both CNVs include MET. This protooncogene has been previously associated to schizophrenia (Am J Psychiatry
2010;167(4):436). Another SCZD patient presented a de novo duplication
at 19p13.3. A similar rearrangement is reported in Decipher, possibly associated to intellectual disability. De novo protein-altering mutations will be
also investigated by NGS-based exome sequencing of all family trios recruited (now in progress). These preliminary data may help to strengthen the
role of de novo variants in the pathogenesis of schizophrenia.
P09.129-S
Genome-wide methylation profiling of schizophrenia
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participate in apoptosis, synaptic transmission and nervous system development (GABRA2, LIN7B, CASP3) were discovered. Methylation profiles differed between the genders. Among females the most important genes participate in apoptosis and synaptic transmission (XIAP, GABRD, OXT, KRT7).
Among males DHX37, MAP2K2, FNDC4, GIPC1 genes represent the most significant candidate-genes.
Conclusion: Our data revealed substantial differences in methylation profiles between schizophrenia patients and controls and between male and
female patients. Dysregulated activity of these candidate genes could play a
role in schizophrenia pathogenesis.
P09.130-M
De novo mutations identified in sporadic cases of Childhood Onset
Schizophrenia
Childhood Onset Schizophrenia (COS) is a severe neurodevelopmental disorder, for which the genetic etiology remains largely unknown. Recently,
whole exome sequencing studies have identified severe rare de novo variants in sporadic cases of adult onset schizophrenia and autism. In this study,
we performed exome sequencing of 17 COS trios with the aim of identifying
de novo variants in the COS affected children. We identified 20 de novo mutations in 17 probands, which is consistent with the de novo mutation rate
thus far identified in adult form of schizophrenia. Our findings also suggest
that de novo missense variants identified in our study have a high likelihood
for pathogenicity. In fact, the pool of genes we found to present de novo variants appears to be enriched for genes that are less likely to tolerate such
alterations. Among the genes found to be disrupted in our study, there is
evidence suggesting that some of them are involved in neuronal functions.
Moreover some of these genes were previously reported to be associated
with neurodevelopmental disorders. Hence this list of genes should be considered to represent genuine COS candidate genes. We believe that insights
from the identification of COS genes may help to develop diagnostic tools,
which in turn may help to intervene before the onset of symptoms. Eventually, knowing the genetic etiology may lead to the identification of therapeutic avenues for better treatment.
P09.131-S
Systematic association analysis of human microRNAs with
schizophrenia
Schizophrenia (SCZ) is a severe neuropsychiatric disorder. SCZ genomewide association studies (GWAS) have identified common single nucleotide
polymorphisms (SNPs) and a large polygenic contribution to illness risk,
but biological mechanisms remain unclear. The known role of microRNAs
(miRNAs) as potent disease modifiers in neuropsychiatric disorders raises
the question of whether genetic variation in miRNAs plays a critical role in
SCZ etiology.
Therefore, we implemented a systematic set-based test for all miRNAs defined in the miRBase based on test statistics from GWAS data. Results from
the largest SCZ meta-analyses to date [Ripke et al., 2013] provided the basis
for this analysis. Alike the popular gene set testing tool VEGAS [Liu et al.,
2010], SNPs in within miRNAs were grouped together, corrected for linkage
disequilibrium and controlled for the number of SNPs within each miRNA to
calculate a miRNA test statistic.
From all analyzed miRNAs, 2.76% were significantly associated with SCZ
after correction for multiple testing; further 18.90% were nominal significant. As expected from the GWAS results, the strongest association was
found for hsa-mir-137. Moreover, miRNAs involved in neural and synapse
development such as mir-9 and let-7 as well as in miRNAs with yet unknown
function were identified. Further evaluation of targets from significantly associated miRNAs as well as their presence in brain QTLs will be presented.
Overall, our results give the first unbiased screening of miRNA association
based on large SCZ GWAS data and might lead to the discovery of key players
suitable for further functional studies.
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P09.132-M
Whole exome sequencing of schizophrenia patients with high level of
autozygosity
C. Magri1, E. Giacopuzzi1, P. Valsecchi2,3, M. Traversa1, R. Gardella1, G. Borsani1, S.
Barlati1, E. Sacchetti2,3, M. Gennarelli1,4;
1
Department of Molecular and Translational Medicine, University of Brescia, Brescia,
Italy, 2Department of Clinical and Experimental Sciences, University of Brescia, Brescia,
Italy, 3Department of Mental Health, Brescia Spedali Civili, Brescia, Italy, 4Genetic Unit,
IRCCS San Giovanni di Dio, Fatebenefratelli, Brescia, Italy.
Schizophrenia (SZ) has a high heritability (about 80%), but the genetic architecture of the disease is still unclear. The numerous genome wide association studies and the recent next generation sequencing analyses indicated
that about 32% of the genetic variability of SZ is due to the additive effect
of a high number of susceptibility alleles with a modest effect size and/or
to the presence of rare deleterious copy number variants (CNVs) or coding
single nucleotide variants (SNVs). To evaluate the contribution of rare recessive homozygous SNVs in SZ, we initially identified, with SNP-array analysis
of 180 unrelated SZ patients, the offspring of mates that are closely related
(inbreeding) and therefore have an increased prior probability to carry potentially deleterious recessive mutations; subsequently, we performed whole exome sequencing analysis of these patients.
An average of 269 (min-max: 196-384) functional (frameshift, missense and
nonsense) homozygous SNVs has been observed in the autozygous regions
of the 7 sequenced patients. About 8 SNVs per individual are novel (not in
dbSNP, 1000G and dbESP6500) and have a high phyloP conservation score.
Among these, we identified, in three patients, three different SNVs representing good candidate SZ mutations, since they are predicted to be damaging
and map in genes mainly expressed in brain and previously associated with
SZ.
Our data suggest that rare homozygous SNVs could be involved in the clinical phenotype of SZ, at least in patients with high level of autozygosity.
P09.133-S
Copy number variants analysis and targeted resequencing of the
schizophrenia candidate gene RB1CC1
Schizophrenia is a severe neuropsychiatric disorder with heritability estimates of ~80%. Xu et al. (2011) published the first exome-sequencing study
focusing on de novo mutations in patients with schizophrenia. To provide
additional genetic evidence for any of the genes suggested by the exomesequencing study, we performed a follow-up study focusing on copy number
variants (CNVs). We screened 1,637 patients and 1,627 controls for CNVs in
any of the genes suggested by the exome-sequencing study. Duplications in
RB1CC1 on chromosome 8 were overrepresented in patients. The duplications were followed-up in independent European samples. In the combined
analysis, comprising of 8,461 patients and 112,871 controls, duplications
in RB1CC1 were associated with schizophrenia (P = 1.29 x 10-5; odds ratio
= 8.58). The aim of the present study was to further explore RB1CC1 as a
candidate gene for schizophrenia. The gene consists of 24 exons. We focused
our targeted resequencing on exon 15: (i) it contains >30% of the genes
total protein-coding sequence, and (ii) Xu et al. (2011) identified a de novo
frameshift deletion in this exon. After quality control, the data from 1740
patients were available. Among 22 patients, a total of 17 different variants
were identified and verified by sequencing the complementary strand. Of
these, 10 were neither detected in the 1000 Genomes Project nor the Exome Variant Server. Currently, we are analyzing whether these variants cosegregate with a psychiatric diagnosis within the families of the affected
probands. Furthermore, detailed phenotypic description of the mutation
carriers are being assembled.
P09.134-M
Combination of whole-genome and whole-exome sequencing, to
identify rare and de-novo variation in cases of schizophrenia and
bipolar disorder from the Faroe Islands
ABSTRACTS POSTERS
Kingdom, 3Genetic Biobank of the Faroes, Faroe Islands, Denmark, 4Mental Health Centre
Amager, Capital Region of Denmark, Denmark, 5Aarhus University Hospital, Risskov,
Denmark, 6BGI - Beijing Genomics Institute, Beijing, China.
Isolated populations represent an advantage to identify rare disease variants that may appear at higher frequencies compared to outbred populations. In this work, we use the Faroese population to test this hypothesis,
and combine low-depth (6X) whole-genome (WGS) and high-depth (35X)
whole exome (WES) sequencing approaches to describe genomic variation,
de-novo mutations and perform association mapping in patients with schizophrenia (SZ) and bipolar disorder (BP).
Our sample consists of 106 SZ cases, 28 BP and 214 controls (344 total):
together with unrelated individuals, it includes 54 complete trios.
A total of 17,345,307 and 259,904 variants have been called in WGS and
WES respectively. We discovered 9,130 de-novo mutations in WGS and 417
in WES. Clear differences emerge between WGS and WES in identifying different types of variants. A specificity of this work is the combination of WGS
and WES in the discovery of de-novo mutations: both approaches discover
the same number of de-novo loss-of-function mutations but high-depth
WES is clearly more powerful in calling coding variants, mostly because of
depth filtering criteria. In the association mapping we identified 6 genomewide significant loci, which are currently being replicated in 3,300 BP and
controls from UCL. The results of this analysis, the concordance between
WES and WGS, and their perspectives will be presented and discussed.
All Variants
De Novo Variants
P09.135-S
Identification of a de novo 15q13.1-13.3 deletion in a reading and
language impaired cohort
Dyslexia and specific language impairment (SLI) are developmental disorders presenting deficits of written or spoken language respectively in individuals with normal intelligence and education, and without overt neurological abnormalities. Copy number variations (CNVs) have been implicated in
neurodevelopmental and psychiatric conditions, such as autism and schizophrenia, but it is not clear to what extent they might contribute to reading
and language abilities. Using data from a longitudinal study investigating
the development of children between 3 and 7 years of age, we performed
CNV analysis (n=94 children; 65% family risk of dyslexia, 23% language impaired, 12% typically developing) and identified a large deletion on chromosome 15q13.1-13.3 in an individual with an early age SLI diagnosis. This
single copy deletion spanning BP3-BP5 (~3.2Mb) was validated by qPCR,
and shown to be de novo. Both parents and a sibling are non-carriers of this
deletion and do not display any reading or language deficits, suggesting this
deletion may be directly involved in the phenotype of the proband. This is
the first report of a BP3-BP5 deletion to be identified in an SLI cohort, in the
absence of intellectual disability. The identification of a de novo chromosome
15q CNV in this cohort provides further support for the implication of this
locus in contributing to a wide range of neurodevelopmental phenotypes.
P09.136-M
Gain-of-function Nav1.7 and Nav1.8 mutations in patients with
idiopathic small fiber neuropathy
Small fiber neuropathy (SFN) is a relatively common disorder of thinly myelinated and unmyelinated nerve fibers and is clinically characterized by burning pain and autonomic complaints. We have recently described the presence of gain-of-function variants in Nav1.7 and Nav1.8 (encoded by SCN9A
Back to index
Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are
both motor neuron disorders. Several studies have tried to establish a link
between the two diseases but the subject is still under debate. It has been
reported that large expansions of the hexanucleotide GGGGCC in intron 1 of
the C9ORF72 gene are often responsible for familial and sporadic ALS cases.
On the other side, mutations in the SMN1 gene cause SMA and its highly homologous copy, SMN2, is a phenotypic modifier. Objective: We investigated
whether the number of the hexanucleotide repeats in C9ORF72 was associated with the phenotype and the number of SMN2 copies in a group of 162
SMA patients. Methods: Conventional PCR was used to determine values
within the normal range (less than 30 repeats), while Repeat Primed-PCR
(RP-PCR) and Southern blot methods were used to exclude the existence of
large expansions that are out of the range to be amplified by conventional
methodology. Results: No pathological (> 30 repeats) or premutated alleles
(20-30 repeats) were found. The allelic distribution of the C9ORF72 gene in
SMA patients overlapped with the data obtained in our control population
discarding putative repeats that may be associated with the disease. No association was either observed with the SMA phenotype or the number of
SMN2 copies. Conclusion: The involvement of C9ORF72 as a genetic marker
in SMA is unlikely. Current investigation of modifier genes in SMA should
consider other possible candidates (Supported by FIS 11-2606).
193
ABSTRACTS POSTERS
P09.139-S
Spinocerenellar ataxia type 6(SCA6): clinical pilot trial with medicinal
herbs
T. Okabe;
University of Tokyo Hospital, Bunnkyo-ku, Japan.
Spinocerebellar ataxia type 2 (SCA2), an autosomal dominant neurodegenerative disease, is caused by the expansion of a CAG triplet repeat located in
the N-terminal coding region of the ATXN2 gene. Alleles of the ATXN2 gene
that carry 13-31 CAG-trinucleotide repeats are present in normal individuals. Contrariwise, alleles with a CAG triplet repeat number of >31 and up to
approximately 200 are present in patients with SCA2. Although the detail
mechanism of pathogenesis is yet to be defined, neurotoxin, especially reactive oxygen species (ROS), released from aggregated mutant proteins, may
play a role in the pathogenic process. In this study, the lymphoblastoid cell
lines (LCLs) isolated from SCA2 patients were utilized to compare with the
wild-type lymphoblastoid cells. We investigated the crucial relationship between the expression of p-ERK1/2 and autophagy. Results found the endogenous p-ERK1/2 and autophagy marker protein, Atg8 (LC3 class II) were
higher in SCA2 patients. Electron micrographs showed that only the cells
expressing expanded Ataxin-2 contained aggregated protein and autophagic
vacuoles. Based on the above observations we hypothesized that the aggregated mutant Ataxin-2 proteins may generate ROS in mitochondria, which
subsequently up-regulate Atg8 expression levels and ultimately lead to autophagy and cell death.
P09.142-M
Intragenic deletions affecting two alternative IMMP2L transcripts in
patients with Tourette syndrome
194
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ABSTRACTS POSTERS
have a first postzygotic mutation in neuroectoderm or neural crest, explaining the negative mutational studies in leukocytes. They may present with 3
clinical recognizable phenotypes: a)possible TSC, b)TSC without tubers and
SENs and c)TSC with tubers and without SENS.
P09.145-S
Novel compound heterozygous UBE3B mutations in two sisters with a
craniofacial-intellectual disability syndrome
Ubiquitination is a fundamental post-translational modification pathway involved in a wide range of cellular activities. The substrate specificity of the
Ubiquitination is mostly dependent on the E3 ubiquitin-protein ligase family. Homozygous and compound heterozygous mutations in the E3 ubiquitinprotein ligase UBE3B were found to cause Kaufman Oculocerebrofacial Syndrome (OMIM 244450) or Blepharophimosis-Ptosis-Intellectual Disability
Syndrome (OMIM 615057) in six patients from five families.
We describe two affected sisters, carrying a compound heterozygous mutation in trans in UBE3B. They exhibited cranio-facial anomalies including
microcephaly, microphthalmia, blepharophimosis, upslanting palpebral
fissures, high-arched and interrupted eyebrows, and myopia. In addition,
they had laryngomalacia, abnormal genitalia, severe intellectual disability,
delayed motor development, hypotonia, failure to thrive, and growth delay.
The younger sister had small kidneys and impaired hearing, and the older
sister had corpus callosum hypoplasia. The facial dysmorphisms including
arched and interrupted eyebrows and long eyelashes overlap with Blepharophimosis Ptosis and Epicanthus inversus Syndrome and Kabuki Syndrome.
By exome sequencing, followed by Sanger sequencing, we found in both sisters a UBE3B missense mutation in exon 1 (p.Met1Val) and a 1bp frameshift
deletion (p.Phe591fs) in exon 17. The mutations were predicted to result in
a truncated UBE3B protein and cause the disease.
We compared the findings in our patients with the previously described
patients. Loss of UBE3B seems to result in a relatively homogeneous phenotype, recognizable by the distinct facial dysmorphisms of the ocular region
in particular, and the severe psychomotor developmental delay.
P09.146-M
Exome Sequencing reveals a novel WDR45 Frameshift mutation and
POLR3A compound heterozygous variants in a female with complex
phenotype and mixed brain MRI findings
M. Khalifa , L. Naffaa ;
1
Akron Childrens Hospital, Akron, OH, United States, 2Northeast Ohio Medical University,
Rootstown, OH, United States.
1,2
WDR45 and POLR3A are newly recognized genes; each is associated with a
distinct neurodegenerative disease. WDR45 is an X-linked gene associated
with a dominant form of Neurodegeneration with Brain Iron Accumulation
(NBIA), manifested by progressive disabilities, dystonia, cognitive decline,
spastic paraplegia, neuropsychiatric abnormalities and iron deposition in
the basal ganglia on brain imaging. POLR3A on the other hand is an autosomal gene and its mutations cause a recessive form of a hypomyelination
with Leukodystrophy disease, also known as 4H syndrome, characterized by
congenital Hypomyelination with thinning of corpus callosum, Hypodontia
and Hypogonadotropic Hypogonadism. We report a female child with severe intellectual disability, aphasia, short stature, ataxia, failure to thrive and
structural brain abnormalities. MRI of the brain obtained in late infancy showed hypomylelation involving the central periventricular white matter and
corpus callosum with no evidence of iron accumulation. MRIs of the brain
obtained in childhood showed stable hypomyelination, with progressive
iron accumulation in the basal ganglia in particular in the globus pallidus
and substania nigra. Whole Exome Sequencing (WES) identified a novel
WDR45 frameshift deleterious mutation in Exon 9 (c.587-588del). WES also
revealed POLR3A 3 missence heterozygous variants. The first is a novel missence variant in exon 4 which is maternally inherited (c.346A>G). Exon13
carried 2 heterozygous missence variants; a maternally inherited variant
(c.1724A>T) and a paternally inherited variant (1745G>A). These variants
are considered likely damaging. The patients complex clinical phenotype
and mixed brain MRI findings might be attributed to the confounding effects
of the expression of these 2 genes.
Back to index
P09.147-S
Severe presentation of WDR62 mutation: is there a role for modifying
genetic factors?
195
ABSTRACTS POSTERS
protein (Rho-GAP) which is mutated in a syndromic form of intellectual
disability associated with cerebellar hypoplasia. In vitro and in vivo studies
on hippocampal neurons of ophn1 deficient mice have shown defects in synaptic morphology and function. The administration of Y-27632, an inhibitor
of the ROCK signaling pathway, specifically hyperactivated in ophn1 loss of
function, fully rescues the cellular and biochemical phenotype. We have developed a cellular model based on human induced pluripotent stem cells
(iPSCs) technology and in vitro neurogenesis to analyze the morphological
and biochemical properties of OPHN1 loss of function in patients cells. The
neurogenic potential of the OPHN1-defective iPS cells has been assessed
and compared with those of control iPS cells following the protocols for
differentiation into different neuronal cell lineages. The results obtained
show that human OPHN1-defective neurons have altered morphology, with
shorter neurites and decreased branching level, when compared with control neurons. We also confirmed the hyperactivation of the ROCK signaling
pathway in OPHN1-defective iPSCs through Western blot and immunofluorescence assays of the Myosin Phosphatase Target Subunit 1 (MYPT1) and
its phosphorylated state on Thr-853. We treated control iPS cells with ROCK
inhibitors and observed a phenotypic rescue in terms of ROCK activity and
neuronal morphology. We are currently exploring the molecular mechanisms underlying these processes and analyzing in detail the phenotype of
OPHN1 defective cells at different stages of neuronal differentiation, before
and after treatment.
P09.150-M
Rett syndrom:current situation in Algria
Autism spectrum disorders encompass a group of childhood neurodevelopmental and neuropsychiatric disorders characterized by deficits in verbal
communication and impairment of social interaction. Numerous researches
have pointed out that strong genetics components are involved in susceptibility to autism. Genome wide association studies have revealed out strong
association signal for CDH10 gene with SNP rs4307059 and rs4327572.
This gene had not been investigated for its association with Autism in South
African population. Aim: In this study we aimed to investigate the association of two SNPs (rs4307059 and rs4327572) of CDH10 gene of autism in
the South African (SA) population. For SNPS rs4307059, the present study
group was comprised of typed cases (188, unrelated autistic children) and
typed controls (212, unrelated healthy children) where the respective figures for rs4327572 were 72 and 209 respectively. The Taqman Real-Time
PCR and genotyping assay was utilized to determine the genotypes. Results:
There was no significant association of SNP rs4307059 and4327572 with
autism in the South African (SA) population. Conclusion: There might be a
possible role of CDH10 in autism but we need to validate it with a larger
sample number. The present study represents the first report on study of
genetic association of CDH10 gene in SA population.
P09.152-M
The Haplotype analysis to test for the association of three SNPs with
autism in a South African Population
Z. Arieff1, J. R. Sharma1, M. Davids1, M. Kaur2;
1
Department of Biotechnology, University of Western Cape, Bellville, South Africa,
2
Computational Bioscience Research Center, King Abdullah University of Science and
Technology, Thuwal, Saudi Arabia.
196
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of this study was to investigate the association of SNPs from genes ABHD11
(rs2293484 and rs10279013) and ABHD13 (rs17060) with haplotype analysis in South African autistic population. A total of 435 individuals were
recruited including 197 autistic and 213 control subjects. The Taqman Real-Time PCR and genotyping assay was used to determine the genotypes. A
significant association of SNP rs10279013 with autism in the South African
population is observed. Haplotype analysis with three SNPs revealed that
the frequency of CGC, TGT and TCT haplotypes is significantly higher in controls than cases (p<0.05). The haplotypes TGC and TCC presented a significantly higher frequency in cases and the odds documented nearly 3-3.47
fold higher risk to the disease in carriers of the haplotypes [OR=3.0 (1.167.79), OR=3.47 (0.95-12.63), respectively]. In conclusion there is a role of
various haplotypes of these SNPs in increasing susceptibility to autism for
South African populations. The present study represents the first report on
haplotype analysis on these SNPs in a SA population.
P09.153-S
Mutations in XRCC4 associated with a complex ataxic syndrome
We studied two monozygotic twins (II-1 and II-2), born to first cousin
parents (I-1 and I-2), affected by a multisystem disease. Since birth they
presented with cryptorchidism, dysmorphism (short arms, hypotelorism)
and short stature. In the adulthood they suffered of an ataxic syndrome with
cognitive decline, pyramidal signs and depression; dilatative cardiomyopathy was also observed. We performed an exome-sequencing analysis on one
proband (II-1) and his unaffected sister (II-3). First we filtered out common
variants (>0.1% in public databases); then we selected non-synonymous
variants that were homozygous only in II-1, based on a hypothesized recessive trait and on the known consanguinity of the parents. The remaining
28 gene variants were prioritized according to the predicted deleterious
outcome of the corresponding amino acid substitutions. The most severe
variant was the nucleotide change c.673C>T in the gene XRCC4, predicted
to create a stop codon (p.R225*) causing the synthesis of a truncated protein; this variant segregated within the family. Quantitative PCR on cDNA
extracted from patients fibroblasts revealed that the XRCC4 transcript level
was strongly reduced, probably due to mRNA decay. Functional assays after
gamma-ray irradiation demonstrated a reduction in DNA repair efficiency
in mutant cells. These are the first patients reported with XRCC4 mutations.
XRCC4 protein has a role in double-strand DNA repair processes; mutations
affecting other proteins with a role on DNA repair (SCAN1, AOA1, ATM) have
been associated with peculiar forms of ataxia, suggesting an important role
for DNA repair in the nervous system, in particular in the cerebellum.
P09.154-M
Screening for C9ORF72 repeat expansion in amyotrophic lateral
sclerosis
L. Mosca, C. Tarlarini, C. Lunetta, V. Sansone, S. Penco;
Niguarda Ca Granda Hospital, MIlano, Italy.
ABSTRACTS POSTERS
P10.01-S
Analysis of the c9orf72 GC- rich low complexity sequence in ALS cases
carrying GGGGCC expansion
L. Corrado1, A. Bagarotti1, N. Barizzone1, L. Mazzini2, S. DAlfonso1;
1
University A.Avogadro, Dept of Health Sciences, Novara, Italy, 2Department of
Neurology, A. Avogadro University and Maggiore della Carita` Hospital, Novara, Italy.
Recessive mutations in ANO5 gene have been reported in patients suffering from limb girdle muscular dystrophy (LGMD) 2L, distal myopathy resembling Miyoshi myopathy and patients manifesting with mialgia and high
CK levels.
ANO5 gene, located on chromosome 11p14, is composed of 22 exons. To
date, forty different mutations have been described in the literature, being
c.191dupA the most frequent mutation. Interestingly, previous studies show
considerable geographical differences regarding the prevalence of ANO5
mutations. LGMD2L is the second most common form of LGMD in Northern
Europe, in contrast to the low prevalence (2%) that has been reported in the
Italian cohort of LGMD patients.
We present data from an ANO5 gene molecular study in 27 Spanish patients
manifesting the three different phenotypes: 1) LGMD 2L, 2) distal myopathy
resembling Miyoshi myopathy, 3) mialgia and high CK levels. We identified
six different mutations, two of them not previously described, in ten individuals (8 males and 2 females). Six patients presented 2 mutations and 4 only
1, suggesting symptomatic carrier status.
Among patients presenting two mutations, six of them carried the c.191dupA:
three in homozygous state, 2 in compound heterozygous state and one as a
single mutation. In our cohort of patients, c.191dupA is, as in the majority of
populations, the most frequent ANO5 mutation.
Our mutational screening reveals a higher prevalence of ANO5 mutations
in Spanish population compared to the Italy cohort of patients previously
reported.
P10.03-S
Deletions involving the 17-22 repeats in the rod domain of dystrophin
gene result in a late-onset Becker Dystrophinopathy
Dystrophin, the protein product of DMD gene is a rod-shaped protein consisting of 3684 amino acids composed by four domains: actin binding domain;
central rod domain; cysteine-rich domain and carboxy-terminal domain.
Mutations in DMD gene cause phenotypes ranging from severe (DMD) to
mild (BMD) myopathic form or X-linked cardiomyopathy (XLCM). Mutations
causing premature translation termination result in a total absence of the
protein that leads to DMD, while mutations derived from in-frame deletions
result in a reduced protein expression leading to BMD or XLCM.
Back to index
We investigated the percentage of patients sharing mutations in the rod domain in a cohort of 160 BMD patients with gene deletions, followed at the
Cardiomyology and Medical Genetics of Naples Second University. We found
that 20 of them (12.5%) shared a deletion involving exons from 45 to 57, at
the 17-22 repeats level.
From a clinical point of view, based on muscular, cardiac and respiratory assessment, 8 (40%) were asymptomatic, 11 (55%) had a mild phenotype and
only 1 (5%) had dilated cardiomyopathy, as the first manifestation.
Compared with data reported by Kaspar et al. (2009), the percentage of
asymptomatic patients in our cohort was higher (40 vs 8%) while that of
patients with mild phenotype was lower (89 vs 11%). The difference probably lies in a younger mean age of our patients in both groups (30.5 vs 20.2
and 24.3 vs 30.5, respectively).
Data here reported confirm that mutations involving repeats 17-22 of the
rod domain result in a late-onset mild Becker Dystrophinopathy.
P10.04-M
Mutations in the Charcot-Marie-Tooth diseaseassociated GDAP1 gene
leads to calcium homeostasis dysfunction and endoplasmic reticulum
stress
D. Pla-Martin, E. L. Calpena, F. Palau;
Centro de Investigacin Prncipe Felipe and CIBERER, Valencia, Spain.
Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder and is characterized by large locus heterogeneity. Mutations in the GDAP1 gene show phenotypic and Mendelian heterogeneity in
CMT patients. GDAP1 is a mitochondrial outer membrane protein related
to mitochondrial dynamics network and different effects on the fission and
fusion pathways have been reported for recessive and dominant mutations,
respectively. Recently we have described the role of GDAP1 in the regulation of store-operated calcium entry (SOCE), a complex process needed to
fill endoplasmic reticulum (ER) after Ca2+ release. Dysregulation of calcium
processes and ER stress represent a common pathway in neurodegenerative diseases. Here we present how missense mutations of GDAP1 expressed
in the human neuroblastoma SH-SY5Y cells also affect calcium homeostasis
depending of their mode of inheritance and its relative position in the protein. Recessive mutations within the protein interaction domain of GDAP1
blocks Ca2+ influx during SOCE avoiding ER-Ca2+ refilling, while dominant
mutations show an exacerbated Ca2+ influx with high resting Ca2+ levels.
These mechanisms may affect the proper function of ER, which produce ER
stress that eventually could lead to neurodegeneration.
P10.05-S
Diagnostic time trend and genetic analysis of Charcot-Marie-Tooth
Disease in Denmark
S. Vaeth1, H. Andersen2, R. Christensen1, M. Duno3, U. B. Jensen1;
1
Dept. Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark, 2Dept. of
Neurology, Aarhus University Hospital, Aarhus, Denmark, 3Dept. Clinical Genetics,
Copenhagen University Hospital, Copenhagen, Denmark.
Aim: To classify Danish patients diagnosed with CMT in the period 19772012 according to genetic analysis, sex, and age at diagnosis.
Background: Charcot-Marie-Tooth Disease (CMT) is the most common inherited neurological disease. More than 70 genes associate with a CMT phenotype, but mutations in only four genes accounts for the vast majority of
cases. Little is known about the epidemiology of CMT in Denmark.
Material and Methods: Records with diagnostic codes ICD8 33009 (atrophia mm. neuropathica, Charcot-Marie-Tooth) and ICD10 G60.0 (hereditary
motor sensory neuropathy) from 1977 to 2012 were retrieved from The Danish National Patient Register (DNPR). These data were linked with data on
genetic analysis for CMT between 1990 and 2012 at Department of Clinical
Genetics, Aarhus University Hospital and Copenhagen University Hospital.
Results: A total of 2084 patients with a CMT diagnosis were identified in
DNPR. Of these 712 patients (34%) had genetic testing, a genetically confirmed CMT diagnosis was obtained in 50%. In total, 17% of the patients
clinically diagnosed with CMT since 1977 had a genetically confirmed CMT
diagnosis. This percentage was largest in the 0-9 years age group (35%). In
the 2008-12 cohort, 573 patients received a CMT diagnosis, 246(43%) had
a genetic analysis for CMT, of which 124 (50%) confirmed the diagnosis.
The majority of confirmed CMT cases where caused by duplication of the
PMP22 gene (58%).
Conclusion: Only half of the newly clinically diagnosed CMT patients have
been genetically tested. Fifty percent of the tested patients had a genetically
confirmed diagnosis.
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P10.06-M
Combined 12q24.3del and 17p12dup in a patient with truncal
hypotonia and psychomotor delay
The use of substantially improves the diagnosis of chromosomal abnormalities that are not evident by conventional karyotype. We report the clinical
and molecular findings of a 2 year old boy presented with truncal hypotonia,
absent deep tendon reflexes and psychomotor delay, but no major malformations or dysmorphic features. Cytogenetic analysis was normal and DNA
analysis for SMA revealed a triplication of SMN1. Subsequently an array
CGH analysis with high resolution 4X180K Agilent arrays (> 236.000 probes, average resolution of 8.9 Kb) showed a combination of the following:
1) 12q24.33 deletion [del12q24.33, 1.5 Mb; 132,302,325-133,733,528;
hg19] containing the genes FBRSL1, P2RX2, POLE, PXMP2, PGAM5, ANKLE2,
GOLGA3, CHFR, ZNF605, ZNF26, ZNF84, ZNF140, ZNF10, ZNF268 2) 17p12
duplication[dup 17p12,1.3Mb; 14,111,772-15,379,208; hg19] involving
the Charcot Marie Tooth syndrome type 1A (CMT1A) critical region within
which PMP22 gene is contained. To our knowledge this is the first report
of combined 12q24.3 deletion/ 17p12 duplication in a patient whose phenotype is not typical of either the described 12q24.3 deletion or CMT1A,
perhaps the combination of dosage sensitive OMIM genes in the aberrant
regions contribute to the specific phenotype. Niyazov et al., 2007; Kehrer et
al., 2013; Choi et al., 2011;
P10.07-S
Whole exome sequencing in patients with congenital myopathies
198
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lyses of quadriceps muscle biopsies were performed. Western Blotting revealed elevated desmin level in the patients muscles. The increased expression was confirmed using immunostaining. Abnormal localization of desmin
with aggregates within the fibers was also observed. Additional staining for
M-cadherin, -actinin and MHCs confirmed severe disruption of myofibrillar organization. Abnormalities were more prominent for the Q348P muscle
which additionally displays numerous centrally localized nuclei in the aberrant muscle fibers.
Based on the in silico and ex vivo analyses, Q348P and A357_E359del mutations may be assumed pathogenic. It could be speculated that abnormal
structure of mutated desmin results in aberrant folding and aggregation,
triggering myofibril disruption.
P10.09-S
Haplotype analysis and age of mutation in DMPK gene in Yakutia.
M. Swarovskaia1,2, S. Stepanova3, A. Marusin1, A. Suhomiasova3, N. Maksimova3, V.
Stepanov1;
1
Institut of Medical Genetics, Tomsk, Russian Federation, 2Siberian State Medical
University, Tomsk, Russian Federation, 3Yakut Scientific Center complex medical
problems, Yakutsk, Russian Federation.
ABSTRACTS POSTERS
Dipartimento di Scienze della Vita, Universit degli Studi di Modena e Reggio Emilia,
Modena, Italy, 2Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda
Universit degli Studi di Napoli, Napoli, Italy, 3Telethon Institute of Genetics and
Medicine, Napoli, Italy, 4Dipartmento di Medicina Clinica e Sperimentale, Universit di
Pisa, Pisa, Italy, 5Dipartimento di Scienze Neurologiche, Neuropsicologiche Morfologiche
e del Movimento, Universit di Verona, Verona, Italy, 6S.S. Malattie Neuromuscolari,
Universit degli Studi di Torino, Torino, Italy, 7Unit Neuromuscolare, Ospedale
Maggiore Policlinico, Universit degli Studi di Milano, Milano, Italy, 8Dipartimento
di Scienze Neurologiche, Universit Federico II, Napoli, Italy, 9Neurologia Clinica,
Universit degli Studi di Brescia, Spedali Civili, Brescia, Italy, 10Dipartimento di
Neuroscienze, Universit degli Studi di Messina, Messina, Italy, 11Dipartimento di
Neuroscienze, Istituto Besta, Milano, Italy, 12Dipartimento di Neuroscienze, Universit
di Padova, Padova, Italy, 13Centro per le malattie neuromuscolari, Universit G.
dAnnunzio, Chieti, Italy, 14Unit di Neuropsichiatria Infantile, IRCCS C.Mondino
Foundation, Pavia, Italy, 15IRCCS Medea, Bosisio Parini (Lecco), Italy, 16Dipartimento
Ospedale Binaghi Centro Sclerosi Multipla, Cagliari, Italy.
1
Facioscapulohumeral muscular dystrophy has been associated with reduction of the number of D4Z4 repetitive elements at 4q35. FSHD is characterized by great clinical variability within families in which D4Z4 reduced allele
(DRA) segregates. An increasing number of cases are sporadic with no other
affected relatives and several findings suggest that additional factors (genetic modifiers) might modulate FSHD expression. Thus molecular diagnosis,
prognosis and genetic counseling have become more challenging. To gain
additional information on the complexity of FSHD, we tested 40 FSHD patients belonging to families with reduced penetrance by testing, a broad core
panel of 93 genes involved in myopathies (Motorplex). This Next Generation
Sequencing-based workflow permit the analysis of 2,544 exons. We studied
40 samples from FSHD patients belonging to families in which other DRA
carries are healthy. In all subjects we found putative pathogenic variations
in genes causing different myopathies. In addition to DRA, all patients carried at least one damaging variations in other disease genes. These variants,
if they had been detected alone in the context of a single gene testing, would
have been considered as causative. The high number of damaging mutations identified in each sample support the hypothesis of multiple factors
leading to the FSHD phenotype. In conclusion, the use of a reliable, sensitive
and specific method has been able to identify putative pathogenic mutations
that can explain the variable penetrance of DRA. Importantly these large set
of mutations observed in FSHD patients highlight the genetic complexity
that might contribute to the disease expression.
P10.12-M
Clinical and molecular characterization of FSHD cases with 1-3 DRA
:data from the Italian National Registry of FSHD
Back to index
P10.13-S
HPSE2, mutated in human urofacial syndrome (UFS), directs
neuromuscular differentiation
Introduction. We try to modeling gene-gene interaction between 5 mutations in 4 genes, involved in folat/homocysteine metabolism (FHMG) and
eNOS- play a pivotal role in vascular homeostasis and endothelial function,
that may be alter the severity (in our case on the age at wheelchair dependency- 9 or 12 years) of this genetically simple disease.
Methods: A retrospective single institution long-term follow-up study was
carried out in 148 corticosteroids-free DMD patients. The genotyping of
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ABSTRACTS POSTERS
dystrophin gene were performed by the MPCR to detect the deletion in
DMD gene and the PCR-RFLP to identify the MTHFRC677Tand A1298C,
MTRA2756G, MTRRA66G, eNOS polymorphisms. Gene-gene interactions
were analyzed using entropy-based multifactor dimensionality reduction
(MDR).
Results: We evaluated single-site allelic and genotypic associations, genotype equilibrium and multilocus genotype associations, using MDR, which
failed to show a genetic model of severity of myopathic process. Have been
adopted the MDR method to explore the synergistic effects of the studied
polymorphisms on modifying to myopathic process. We selected the best
model, which included the. MTHFRC677T and A1298C, MTRA2756G, eNOS
polymorphisms, cross-validation consistency is 9/10 (2 =54.22, p<0.0001)
for case of wheelchair up to 12yrs). Since the selected polymorphisms were
not associated with DMD there is evidence for the existence of epistasis between the two polymorphisms MTHFRC677T and eNOS (CVC10/10, 2=5.3,
p=0.02) in case of wheelchair up to 9yrs.
Conclusion: Our results indicate that the MTHFR, MTR and eNOS genes are
modifying loci and presence of the high-risk alleles may associate with an
increase in the severity of DMD.
P10.16-M
Limb girdle muscular dystrophy in the Czech Republic
About one third of autosomal recessive limb-girdle muscular dystrophy patients show no mutations in the ARLGMD genes so far identified. The possible explanations are that several genetic cause of LGMD cannot be discovered
by traditional DNA sequencing tests, or that the genetic heterogeneity is
greater than expected with many other genes that should be analyzed. However, the present genetic testing is long, expensive and ineffective to cope
with this second possibility. New powerful approaches for DNA analysis, like
next-generation sequencing (NGS) and array CGH are revolutionizing the
field, with the entire human genome that can be properly analyzed.
We combined SNP array-based linkage analysis and NGS technology to discover orphan LGMD mutations.
The patients to be studied were selected according to the following criteria:
a) clinical diagnosis of LGMD; b) inconclusive molecular testing after the
complete study of the known LGMD genes c) severity of disease.
A number of mutations were identified. In particular in a LGMD family with
an autosomal recessive inheritance, we have recently identified a new homozygous mutation in the ACADVL gene, Acyl-CoA Dehydrogenase- Very Long
Chain, that is shared by all the affected family members and by another
patient from the same town. In other two family a heterozygous compound
mutations and homozygous mutation in the ACADVL gene was also found.
This demonstrates that specific amino acid changes in the catalytic domain
of the ACADVL Very gene may be associated with an LGMD-like phenotype
and this should be considered in the differential diagnosis.
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P10.18-M
Exome sequencing identifies mutations in a gene coding for the LIMdomain protein N-RAP in a BAG3 myofibrillar myopathy-affected
patient
F. DAvila1, M. Barcella1, M. Meregalli1, S. Lupoli1, C. Sitzia1, S. Erratico2, D. Cusi1, C.
Barlassina1, Y. Torrente1;
1
University of Milan, Milan, Italy, 2YStem s.r.l., Milan, Italy.
In myopathic patients molecular diagnosis is hampered by the genetic heterogeneity and the clinical interpretation of molecular findings are further
hindered by the presence of low penetrant variations and by the effect of
modifier genes. To gain a comprehensive view of all sequence variants in
patients, we developed a broad core panel of 93 genes involved in myopathies. To analyze their 2,544 exons, we have developed a Next Generation
Sequencing-based workflow, based on a Haloplex custom enrichment strategy (Motorplex). We studied 200 samples from myopathic patients with
prevalent limb-girdle muscle involvement and 150 samples with congenital
myopathies. In about 20% of patients we found typical causative mutations,
while in an additional 30% other putative pathogenic variations were detected. In addition to the causes of monogenic disorders, we also discovered
more than 35% of patients showing damaging variations in other disease
genes. These variants, if they had been detected alone in the context of a single gene testing, would have been considered as causative. The high number
of damaging mutations identified in each sample support the hypothesis of
multiple troubles leading to a such complex phenotype. In conclusion, in a
large cohort of patients with non-specific symptoms, our reliable, sensitive
and specific method has been able to identify pathogenic mutations, despite of the heterogeneous conditions, improving significantly the diagnostic
yield. Finally, intrafamilial and interfamilial phenotypic variability of patients sharing the same pathogenic mutations must be reevaluated considering the comprehensive view of all sequence variants.
ABSTRACTS POSTERS
P10.20-M
Childhood onset tubular aggregate myopathy associated with de novo
STIM1 mutations.
We investigated on three unrelated patients with tubular-aggregate myopathy and slowly progressive muscle weakness manifesting in the first years of
life. All patients showed type 1 muscle fiber predominance and hypotrophy
of type 2 fibers. Tubular aggregates of the sarcoplasmatic reticulum were
abundant. In all three patients, de novo heterozygous mutations were identified in the Stromal Interaction Molecule 1 gene, STIM1. In one of the patients, the mutation was identified by exome analysis, coupled to a hypothesis-driven filtering strategy. Two patients harbored the previously described
c.326A>G p.His109Arg change while the third patient carried a previously
unreported mutation (c.343A>T, p.Ile115Phe). All mutations affected the
EF-hand motif, which is required for Ca2+ ions binding. Besides, the tubular
aggregates, autophagic debris and myophosphorylase deficiency were documented in patients muscle cells. Consistent with previous findings, the
c.326A>G change represent a recurrent mutation specifically related to early onset muscle weakness.
P10.21-S
Myotonia congenital type Becker in an endemic Bulgarian region
Back to index
Myotonic dystrophy type 1 (DM1), the most common adult muscular dystrophy, is caused by expansion of a CTG repeat in the 3UTR of the DMPK
gene. Repeat size is correlated positively with phenotypic severity and negatively with age-of-onset, with affected individuals classified as mild (50-150
CTGs), classic (100-1000 CTGs), or congenital (>1000 CTGs) DM1. Southern
analysis reliably detects all expansions, but requires micrograms of DNA,
yields approximate allele sizing, and is labor-intensive, time-consuming,
and costly. PCR across the repeat accurately sizes all normal and also expanded alleles, although the upper limit of detection is unknown. Newer tripletprimed PCR (TP-PCR) assays will detect all normal and expanded alleles,
but product analysis by capillary electrophoresis (CE) is still costly when
applied in high throughput screening situations where a majority of tested
samples may be screen-negative. We have now adapted the 5 and 3 TP-PCR
assays for single-step detection of DM1 expansions by melting curve analysis (MCA), wherein melt peak profiles from normal samples were distinctly
different from samples carrying an expansion. In a blinded validation of 60
clinical samples enriched for affected individuals, a 48-repeat plasmid clone
was used to establish a cut-off temperature separating normal from affected
samples, and 100% concordance with their known genotypes was achieved.
We conclude that TP-PCR MCA is an accurate yet simple, rapid and inexpensive screen useful for identifying DM1 expansion mutations. Follow-up
confirmation and sizing can be accomplished by capillary electrophoresis
following a quick 5-cycle extension-labeling of the positive TP-PCR product.
P10.24-M
Elevated miRNA levels in serum of myotonic dystrophy patients relate
to disease progress
A. Koutsoulidou1, T. Kyriakides2, Y. Christou1, E. Zamba Papanicolaou1, L. Phylactou1;
1
The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus, 2Yale Center for
Analytical Sciences at Yale School of Public Health, University of Yale, New Haven, CT,
United States.
201
ABSTRACTS POSTERS
P10.25-S
Large copy number variations in NEB are frequent in nemaline
myopathy patients
Recently, new large mutations have been identified in the nebulin gene
(NEB) causing nemaline myopathy (NM). NM constitutes a heterogeneous
group of disorders among the congenital myopathies, and mutations in NEB
are a main cause of the recessively inherited form. NEB consists of 183 exons
and it includes a 32 kb triplicate region (TRI) where eight exons are repeated three times. We have designed a custom NM-CGH microarray to detect
copy number variations in the currently known nine NM genes and one unpublished gene. To date, 230 samples from 170 families have been run with
the NM-CGH microarray and we have identified NEB TRI variation in approximately 14% of the NM families in this study cohort. The results suggest
that the adjacent intronic repeat elements may predispose to the recurrent
TRI variations. The possible pathogenicity of the TRI variations warrants
further studies and elucidation of the exact breakpoints in each family. Moreover, we have identified seven different, novel, large disease-causing aberrations in NEB in seven different families. The size of the aberrations varies
greatly, covering from only a part of one exon (0.9 kb) to more than half of
the gene (133 kb). The NM-CGH microarray method is currently available
for mutation analysis in our laboratory. Additionally we have analyzed ten
samples with exome sequencing and identified causative mutations for half
of the families. We believe that the combination of the NM-CGH microarray
followed by exome sequencing will accelerate mutation detection and improve the diagnostics of NM and related disorders.
P10.26-M
A novel homozygous deletion in SGCB identified by a targeted next
generation sequencing approach and the Motor Chip
202
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Myopathies are a clinically and genetically heterogeneous group of disorders, most of which are genetic and many cause progressive weakness and
atrophy of muscles. Mutations in more than 200 different genes are known
to cause the disorders and several genes may have to be sequenced in order
to identify the molecular defect in a patient. The large number of possible candidate genes, overlapping phenotypes as well as an enormous size of
some of the genes e.g. DMD, TTN and NEB bring difficult challenges to diagnostics. Molecular characterization is nevertheless important for an accurate diagnosis and management of the diseases. Targeted next-generation
sequencing (NGS) is an efficient and cost-effective method to sequence several genes simultaneously. Our first targeted NGS custom panel was designed
for the coding exons and UTRs of 180 myopathy related genes with a total
size of 1.3 Mb. For DNA capture a custom NimbleGen SeqCap EZ Choice Library was designed. Sequencing was performed at Institute for Molecular
Medicine Finland (FIMM) using Illumina MiSeq with sequencing depth of
100X, and files were processed with their Variant Calling Pipeline. In the pilot study DNA samples of 20 patients were sequenced, of which four served
as mutation controls with previously reported mutations. We were able to
detect all the mutations in the control samples, except for one heterozygous
11-bp insertion/deletion mutation which was missed in the variant calling.
Disease-causing mutations were detected in seven of the 16 patients, with
three of them having previously reported mutations and four showing novel
mutations.
P10.29-S
Highly efficient genetic diagnostic testing in patients with
neuromuscular disorders using Panel-based Next Generation
Sequencing
J. Mohr, N. Gloeckle, T. Grau, F. Battke, S. Biskup, K. Hoertnagel;
CeGaT GmbH, Tbingen, Germany.
ABSTRACTS POSTERS
P10.30-M
Genetic testing of inherited muscular dystrophies and myopathies
using Sequence capture and targeted resequencing
The inherited muscular dystrophies and myopathies comprise a heterogeneous group of muscle diseases that share similar clinical features as progressive skeletal muscle weakness and wasting. Mutations in several genes
that encode proteins of extracellular matrix, endoplasmic reticulum, nuclear
envelope, sarcolemmal proteins and glycosyltransferases are known to be
responsible for muscular dystrophies and myopathies. The large overlap of
phenotypic manifestations resulting from different gene mutations poses
a challenge for determination exact type muscular disease. Targeted resequencing using next-generation sequencing technology is a cost-effective
strategy to accelerate patient diagnosis. We designed capture library to target the coding and all flanking intron regions of 42 genes associated with
group of disease as muscular dystrophies, congenital muscular dystrophies,
congenital myopathies, distal myopathies and other myopathies. We performed targeted capture combined with next-generation sequencing using
NimbleGen SeqCap EZ Choice library and Roche 454 sequencing platform
in 33 Czech probands. Mutations associated with muscular dystrophies or
myopathies were identified in 16 of them. Mutations were detected in ACTA1, CAPN3, COL6A1, COL6A3, DNM2, DYSF, LAMA2, RYR1, SGCB and SEPN1
gene. This work was supported by grants IGA MZ CR NT14574-3.
P10.31-S
Swiss Cheese localization and function in the nervous system of
Drosophila melanogaster third instar larvae
Neuropathy target esterase (NTE) is the 6th member of 9-proteins of patatinlike phospholipase domain-containing proteins, PNPLA1-9. Mutation in the
catalytic domen of NTE (PNPLA6) can lead to the slowly developed diseases,
known as organophosphorus compound-induced delayed neuropathy
(OPIDN) and hereditary spastic paraplegia called NTE-related motor neuron disorder (NTE-MND). The Swiss Cheese (SWS) protein is an ortholog
of NTE in Drosophila and shares 39% sequence identify with human NTE.
The Drosophila sws mutants are characterized by progressive degeneration of adult nervous system, glial hyperwrapping, and neuronal apoptosis.
Drosophila sws mutant thus provides an experimental model for functional
studies of the SWS/NTE role in maintaining neural integrity. There is an evidence of SWS role in the age-dependent brain changes. However, possible
disturbance caused by point mutations in sws gene in Drosophila larvae remained unknown.
The sws-point mutants were used to investigate SWS localization and function in the nervous system of D.melanogaster larvae. We were shown SWS
localization mainly in glial tissue of larvae brain as well as in glial cells,
wrapped axons in all investigated lines. Presence of SWS was also observed in the postsynaptic membranes of the larval NMJ. SWS protein in small
amount was also detected in larvae neurons. Quantitative and qualitative
parameters of NMJ in all investigated sws-mutants also were changed comparing with wild type flies.
Thus, SWS dysfunction in earlier stages of Drosophila ontogenesis can be
important for nervous system development and aging in D.melanogaster
adults.
P10.32-M
Central bradypnoe and hypotonia as diagnostic feature for male Rett
syndrome
A. Baumer1, E. Wey1, D. Gubler2, A. Rauch1;
1
Institute of Medical Genetics, Schlieren, Switzerland, 2Childrens Hospital, Zurich,
Switzerland.
Mutations in the MECP2 gene causing the X-linked dominant Rett syndrome are a relatively common cause of intellectual disability in girls. Although
MECP2 mutations in males are usually considered lethal, leading to the prenatal loss of the fetuses, few observations of boys with severe congenital
encephalopathy were reported. We present here a novel case of a newborn
boy with Rett syndrome caused by the de novo MECP2 frameshift mutation
c.806delG. The clinical suspicion of male Rett syndrome was triggered by
the rare combination of neonatal hypotonia and central bradypnoe.
At the age of four days the newborn was transferred to the neonatal unit of
a tertiary childrens hospital because of poor suck, dysregulation of muscle
tone and a pathological breathing pattern. On admission, the neonate was
Back to index
Spinal and bulbar muscular atrophy (SBMA) is caused by a pathological expansion over 38 of a CAG repeat in the first exon of the androgen receptor
(AR) gene on chromosome X, coding for a polyQ tract (La Spada et al, 1991).
SBMA is an androgen-dependent disorder, with males with full disease manifestations, and females showing only mild symptoms even if homozygous
for the mutation. While a correlation between expansion size of polyQ tract
and disease severity has been reported, patients with the same number of
CAG repeats have different age at onset and disease progression even if relatives. Human AR exon 1 encodes further short aminoacidic stretches. The
effect of these sequences on SBMA phenotype has not been studied yet. In
order to further characterize the effect of AR coding repeated sequences on
SBMA phenotype, we genotyped AR exon1 polymorphisms in 132 molecularly defined SBMA patients, referring to the Motor Neuron Clinic of the
University of Padua and to Fondazione IRCCS Istituto Neurologico Carlo
Besta, Milan. Among AR exon-1 trinucleotide stretches, a polymorphic GGN
sequence coding polyG showed a tendency to be higher in patients with
an earlier onset. Our study confirms that the length of SBMA-causing polyQ tract does not fully explain the disease phenotype and point to a polyG
strech within AR exon 1 that is a potential disease modifier in SBMA:
REFERENCES:
La Spada AR, Wilson EM, Lubahn DB, Harding AE, Fischbeck KH. Androgen
receptor gene mutations in X-linked spinal and bulbar muscular atrophy.
Nature. 1991;352:77-9.
P10.34-M
Mutation analysis of SCN1A gene in Slovak patients with various types
of childhood epilepsy
M. Surovy1, A. Soltysova1,2, M. Kolnikova3, P. Sykora3, D. Ilencikova4, A. Ficek1, L. Kadasi1,2;
1
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University,
Bratislava, Slovakia, 2Institute of Molecular Physiology and Genetics, Slovak Academy
of Sciences, Bratislava, Slovakia, 3First Department of Pediatrics, Department of Child
Neurology, Department of Clinical Biochemistry, Comenius University Childrens
Hospital, Bratislava, Slovakia, 42nd Pediatric Department of the Comenius University,
Medical School and University Childrens Hospital, Bratislava, Slovakia.
Mutations in SCN1A gene are by far the most frequent cause of Dravet syndrome (DS) worldwide, with mutation prevalence approximately between
70 and 80%. One of the main features of DS is the fact that many patients do
not respond well to the treatment with anticonvulsive drugs. Furthermore,
there have been observations that some drugs, which function as sodium
channel blockers, may induce aggravation of seizures in patient, thus worsening their symptoms. As most of the DS SCN1A mutation result in truncation of the protein, the use of antiepileptic drugs would further decrease the
inhibitory function in central nervous system. The aim of this study was to
identify mutations in SCN1A gene in a broad testing group of patients with
phenotypes raging from DS to milder phenotypes such as Genetic Epilepsy
with Febrile Seizures Plus (GEFS+). Completing mutation screening in 37
unrelated patients, 4 causative mutations located in the coding region were
identified. All mutations were found in DS patients and in accordance with
previously identified disease variants, found mutations, of which three are
novel, result in creation of a premature stop codon, either by substitution or
an insertion and thus to a possible loss of function of the altered protein. In
summary, our findings contribute to the broadening of the mutation spectrum of the SCN1A gene and enrich the clinical data of DS. Genetic testing
may prevent the need for unnecessary clinical examinations and provide
genetic counseling for families.
203
ABSTRACTS POSTERS
P10.35-S
De-novo deletion or uniparental disomy as SMA determining cause: a
case report
We report a 3-months old female with type I spinal muscular atrophy (SMA)
born to a young and non-consanguineous couple. Molecular analysis confirmed the diagnosis of SMA with an homozygous deletion of SMN1. Parents
analysis revealed that only the mother was a carrier of the SMA causative
deletion. It is important to find out if the father, that carries two SMN1 copies, has a CIS-duplication of the gene on one chromosome bearing to a 2/0
genotype or the deletion occurred de-novo during gametogenesis. Indeed,
the risk for future pregnancies could be reduced from 25%, usually estimated for SMA couple, to residual risk arising from recurrent de-novo mutation
(2% of the affected individual). Alternatively, the uniparental disomy (UPD)
of the maternal-deleted-chromosome, with an estimated incidence of about
1:3500 live birth, must be taken into account. Gene dosage analysis of the
fathers relatives does not support a paternal 2/0 genotype. To discriminate
between a paternal de-novo mutation and a maternal isodisomy we analyzed in the affected infant and parents a total of nine microsatellite markers
in a region spanning 3.0 Mb at the SMN locus. Five microsatellites at 5/3ends of that region demonstrate a maternal and paternal inheritance but
could not definitively exclude the maternal segmental UPD. Finally, using a
NGS approach we identify three paternal and one maternal-inherited variants in the SMN2 genes of the infant. All together our data support for a
de-novo mutation of paternal chromosome 5 as could result by unequal
crossing-over during gametogenesis.
P10.37-S
Prevalence of SMN1 gene duplication in different ethnic groups:
implication for carrier testing
P. Rimessi, C. Trabanelli, M. Fabris, A. Venturoli, B. Dolcini, M. Taddei Masieri, R.
Selvatici, A. Balboni, L. Melchiorri, A. Ravani, F. Gualandi, A. Ferlini;
U.O. Medical Genetics, Azienda Ospedaliero Universitaria, Ferrara, Italy.
Spinal muscular atrophy (SMA) is an autosomal recessive disorder, characterized by symmetrical muscular weakness and atrophy. The incidence is
variable from 1 in 6000 to 1 in 10000 live births in different geographic
areas. A homozygous deletion involving exon 7 in SMN1 gene is present in
more than 95% of the cases. Carrier testing in parents and relatives of SMA
patients is complicated by the occurrence of SMN1 gene duplication (4-5
%).
Here we report on the SMN1 genetic test results obtained in our lab in the
last two years. The most frequent clinical indication is represented by positive family history for SMN1 gene deletion or SMA, partner heterozygous for
the deletion and consanguinity with the partner.
We calculated the frequency of different SMN1 genotypes in 326 tested subjects. We found a percentage of SMN1 gene duplications (subjects with 3
copies of SMN1) a bit higher than expected, ranging from 6 to 6.5%. The
55% of the subjects with the duplication is represented by North and West
Africans that have been tested because of consanguinity with the partner,
the remaining are Italians, 25% with SMA positive family history and 20%
with partners heterozygous for the deletion.
Our results highlight once again the importance of taking into account the
occurrence of SMN1 gene duplication when performing carrier testing and
suggest that the prevalence varies in different ethnic groups, therefore affecting the recurrence risk assessment .
P10.38-M
Methylation level of SLC23A2, NCOR2 and CDK2AP1 genes regulatory
regions correlates with spinal muscular atrophy severity
G. Y. Zheleznyakova1,2,3, M. A. Maretina1,4, A. V. Kiselev3, E. Nilsson2, V. S. Baranov3, H. B.
Schith2;
1
Saint-Petersburg State University, Saint-Petersburg, Russian Federation, 2Uppsala
University, Uppsala, Sweden, 3D.O. Ott Research Institute of Obstetrics and Gynecology,
Saint-Petersburg, Russian Federation, 4D.O. Ott Research Institute of Obstetrics and
Gynecology, Saint-Petersburg, Austria.
204
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SMA patients. The methylation level of CpG sites situated in regulatory regions of ARHGAP22, CDK2AP1, CHML, NCOR2, SLC23A2, RPL9 genes were
analyzed in 24 I type, 43 II type and 29 III-IV SMA patients using bisulfite
sequencing. The methylation level of target CpG site and one nearby CpG site
belonged to 5UTR of SLC23A2 was significantly lower by 11-15% in III-IV
type comparing to I type SMA patients. Moreover, a significantly increased
allele A frequency of polymorphism rs1279683 (g.G>A) situated in target
CpG site was found in III-IV type SMA patients. III-IV type comparing to I
type SMA patients demonstrated decreased methylation level by 9-16% of
target CpG site and nearest CpG site belonged to 5UTR of NCOR2. Significant difference in the methylation level between different types SMA patients was revealed for three CpG sites located in the region 1735-1398 bp of
TSS of CDK2AP1. Thus this study confirms that DNA methylation changes of
SLC23A2, NCOR2, and CDK2AP1 might be associated with spinal muscular
atrophy severity.
P10.39-S
Molecular analysis of common mutation associated with SMA n
Romanian population
We report on a patient who presented with clinical features of Ullrich congenital muscular dystrophy. The patient is a girl, four years of age, the first
child of healthy non-consanguineous Lithuanian parents. The proband was
born at 40 weeks gestation in normal delivery. Hip dysplasia was noted
from birth. The girl has chronic constipation from 2 months of age. Her intelligence is in normal range, but motor development is delayed. She could
sit at age of 6 months, stand up with support at age of 2 years, can walk
only with support. Clinical examination at the age of 4 years revealed the
contractures in elbows and knees but striking distal phalanx hyperlaxity.
The consentration of CK was mildly elevated (315 U/L). Spine CT and MRT
investigations, cardioechoskopy were normal. The clinical diagnosis of Ullrich congenital muscular dystrophy was suspected and molecular genetic
testing of COL6A1, COL6A2 and COL6A3 genes was performed. The mutation
c.6210+5G>A, IVS16+5G>A, in intron 16 of the COL6A3 gene in a heterozygous state was detected. The analysis of parents confirmed de novo origin of
the mutation. The protein immunohistochemistry analysis of the muscular
biopsy concluded that the finding is compatible with dominant in frame deletion in exon 16.
The mutation c.6210+5G>A, IVS16+5G>A, has already been described as
causative for a dominant form of Ulrich congenital muscular dystrophy. The
molecular and clinical characterization of our patient provides additional
information for genotype-phenotype correlation and confirms dominant acting mutation in collagen VI gene as a course of Ullrich disease.
ABSTRACTS POSTERS
P10.41-S
Paternal germline mosaicism in COLVI related myopathies: a case
report
Mutations in the COL6A1, COL6A2 and COL6A3 genes cause collagen VI related disorders, characterized by muscle wasting, weakness, joint contractures, distal laxity, serious respiratory dysfunction and cutaneous alterations.
The severe Ullrich congenital muscular dystrophy (UCMD) can be due to
autosomal recessive mutations in one of the three genes of collagen VI with
a related 25% recurrence risk. In the majority of UCMD cases nevertheless,
the underlying mutation is thought to arise de novo and the recurrence risk
is considered as low.
Here we report a family with recurrence of UCMD in two sibs only by
fathers site. In both, the molecular analysis revealed heterozygosity for the
c.896G>A mutation in COL6A1 exon 10 (Gly299Glu) and for the COL6A1
c.1823-8G>A variation within COL6A1 intron 29. The Gly299Glu mutation,
despite not previously reported, is likely to be pathogenic, leading to the
disruption of the Gly-Xaa-Yaa motif in the triple-helix domain of collagen VI
alfa(1)-chain. The intronic variation was inherited from the father and RNA
analysis in skin fibroblasts allowed to exclude its role in affecting COL6A1
transcript processing. The Gly299Glu mutation occurred apparently de novo
in the two sibs.
The described mutational segregation strongly suggests the occurrence of
paternal germline mosaicism , to be confirmed by mutational analysis of the
sperm. The reported family represents the first observation of gonadal mosaicism in collagen-VI related myopathies and, similarly to other collagen
related diseases as osteogenesis imperfecta, this possibility deserve to be
considered in genetic counseling and recurrence risk estimation.
P10.42-M
Diagnosis of spinal muscular atrophy in Algeria
We described a young man, the third son of healthy and non consanguineous
parents. He was born small for gestational age. He had delayed height growth and psychomotor development. Karyotype was 46,XY. At the age of 6,
he developed precocious puberty At the age of 9 years, brain MRI showed
partially empty saddle. At the age of 15, we evaluated him for microsomia,
delayed psycomotor development, dysmorphisms. On physical examination,
height and weight were below 3 centile, there was microcephaly, triangular face, frizzy hair, laterally sparse eyebrows, strabismus, hypotelorims,
hypoplasia of midface, wide nasal bridge, short broad nose, large and low
set ears, pointed chin, micrognatia, arched palate, scoliosis, kyphosis, long
hands with tapering fingers, hallux valgus bilaterally, clubfeet. An aCGH showed 14q32.214q32.31 de novo microdeletion of 1.106 Mbp. At the age of
20, he had enlargment of the thyroid gland, exophtalmos, weight loss, tachy-
Back to index
We describe two cases with opposite rearrangements (deletion and duplication) in 15q region including Insulin-like Growth factor I receptor (IGF1R)
gene. The IGF1R is involved in growth, insulin-related phenotypes, and longevity and is expressed equally from the maternal and paternal alleles.
Case 1: Girl of 11 years with unexplained intrauterine growth retardation
(IUGR), birth weight <-1.5 SD and persistent short stature (<-2.0 SD). She
displayed dysmorphic features suggestive of Silver-Russell Syndrome, with
unspecific multiple hyperpigmented lesions on the body. She had a normal
methylation study of the 11p15.5 region. Significant increases in IGF-I levels
were observe during two IGF-I generation tests. SNP-array analysis identified a heterozygous de novo 4,92 Mb deletion in15q26.2 including IGF1R
gene.
Case 2: proband was first evaluated at the age of 4 months for overgrowth,
hypotonia and some dysmorphic features (a prominent nasal bridge, deep
set eyes, epicanthic folds and micrognathia). He delivered by C-section at
39 weeks of gestation for breech presentation, with a birth weight of 3600
g (50th centile), a birth length of 53 cm (75th centile) and an OFC of 35 cm
(+1.7 SD). He had presented craniosynostosis, corrected at 3 months of age.
Array-CGH analysis showed a 17,3 Mb de novo duplication from 15q25.2 to
26.3 encompassing the IGF1R gene.
Our two cases again demonstrate that overgrowth, craniosynostosis, macrocephaly, and mild developmental delay in patients with trisomy 15q26.1
result from dosage increase of IGF1R, whereas gene dosage decrease of IGF1R can cause IGF1-resistance and underline IUGR with subsequent short
stature.
P11.003-S
Two children with triplication of 16p11.2 associated with global
developmental delay and dysmorphic features
205
ABSTRACTS POSTERS
Republican Medical Centre Mother and Child, Minsk, Belarus.
206
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Here we describe a case of a 5 year old boy who came to our genetic service due to small stature in association with hypospadias and psychomotor retardation. Convenctional karyotype was normal, while a microarrayCGH detected a 2.8Mb deletion spanning from 3q27.1 to 3q27.3: 46,XY.arr
3q27.1q27.3(185852379-188697676)x1[Hg18]. Recently a new 3q27.3 microdeletion syndrome has been described, overlapping molecular features
with our case, but only partially concordant regarding clinical phenotype.
Our patient exhibit a genitourinary malformation previously unreported
and a borderline IQ, opposed to reported severe mental impairment. IUGR
and subsequent small stature and peculiar dysmorphisms are also seen. It is
remarkable that all cases described before are adults, and as we concern this
is the first case described at young age. This case expands the phenotype
and clinical features of the 3q27.3 microdeletion syndrome and offers new
insights about genotype-phenotype correlation.
P11.009-S
A New Syndrome? Severe growth and develop mental delay associated
with Bilateral Polycystic kidney Disease.
S. Sakazume1, Y. Kido1, H. Numabe2;
1
Dokkyo Medical University Koshigaya Hospital, Koshigaya, Saitama, Japan,
2
Ochanomizu Womens University, Tokyo, Japan.
In this report, we describe two girls with severe growth and developmental
delay with polycystic kidney disease. Long arm of chromosome 4was consistently deleted in almost same posisions. The common locus in the chromosome is 4q21-22. One patient had a simple 4q contiguous gene deletion,
whereas the other patient had a complicated chromosomal rearrangement.
In the first patient, a smaller part of the 4q was inserted to 3p. In both patients, abdominal ultrasonography revealed renal cysts. In the deletions
PKD2, which causes autosomal dominant polycystic kidney disease (MIM
613095) was mapped. , The common 52 genes deleted in both patients. Typical phenotypes, were severe growth and developmental retardations, and
a characteristic facial appearance consisting of frontal bossing, thin broad
ABSTRACTS POSTERS
eyebrows, epicanthus fold, missing, missing teeth. And mild hand and foot
anomalies. Although these patients carry different chromosomal aberration,
the common deletion causes the quite similar phenotype, suggesting a new
syndrome due to 4q21-q22 deletion.
P11.010-M
9p deletion syndrome-like in a girl with a 9p insertion on
chromosome 2 without 9p deletion
Partial deletions of 9p have been reported as a clinically recognizable syndrome characterised by the variable association of intellectual disability,
speech delay, hypotonia, cardiac anomalies and dysmorphisms.
We report a 3 years old girl with midface hypoplasia, downslanting palpebral fissures, teeth agenesis and teeth fusions. Language was not acquired.
At birth, severe neonatal hypotonia and patent ductus arteriosus were present. Phenotype was consistent with the clinical diagnosis of 9p deletion
syndrome (OMIM#158170), with uncommon features such as bilateral cataract (diagnosed at 5 months), chronic constipation, high pain threshold
and poor temperature control.
Karyotype analysis showed an apparent 9p deletion. Array-CGH (60K
Agilent) demonstrated a 2q microdeletion (600 Kb, from 166,993,501 to
167,602,904, NCBI Build 37/hg19) with normal dosage of chromosome 9.
Subsequent FISH analysis revealed a 9p22.1p24.3 insertion in chromosome
2q [46,XX,ins(2;9)(q24.3;p22.1p24.3)dn]. The 2q microdeletion involved
only two genes: SCN9A and SCN7A.
SCN9A recessive mutations cause congenital insensitivity to pain. SCN7A is
not associated with human disease, and in mouse is likely to be a sodiumlevel sensor of body fluids in the brain. SCN9A haploinsufficiency may support the clinical observation of the patient high pain threshold and the poor
temperature control. We are verifying the presence of a second mutation on
the remaining allele. Assuming that the 2q deletion cannot account for the
whole phenotype, we speculate that a critical gene/regulatory region key
for the 9p deletion syndrome is located in the 9p breakpoint(s). This patient deserves future studies that may help to refine 9p critical region.
P11.011-S
Identification of de novo 2,62Mb deletion in chromosome 15q26.1 in
a boy with stigmata dysplastica, developmental delay, short stature,
microcrania, nasal polyposis, hypermetropia
S. Bertok1, U. Groelj1, M. Jekovec-Vrhovek1, L. Lovrei2, V. Marija2;
1
University Childrens Hospital, 1000 Ljubljana, Slovenia, 2Clinical Institute of Medical
Genetics, 1000 Ljubljana, Slovenia.
Back to index
Triple A (Achalasia-Addisonianism-Alacrima, Allgrove) syndrome is an inherited condition characterized by three specific features: achalasia, Addison disease, and alacrima. Allgrove syndrome is inherited in an autosomal
recessive pattern and is caused by mutations in the AAAS gene localized at
12q13.13. The gene encodes a protein called ALADIN. It localizes to nuclear
pore complexes, large multiprotein assemblies that are the sole sites of nucleocytoplasmic transport. Achalasia-Addisonianism-Alacrima syndrome is
a rare condition, incidence is unknown, and only scattered family and case
reports are noted in the literature. Here we report a female patient with
Triple A syndrome born to non-consanguineous parents from Bulgarian origin. She was diagnosed as Triple A at the age of 17. The patient express a
typical phenotype: achalasia, Addison disease, and alacrima. The affected
patient also has muscle weakness, peripheral neuropathy, skin hyperpigmentation, optic atrophy and papillae atrophy. Many of the features of triple
A syndrome are caused by dysfunction of the autonomic nervous system
and the neurological symptoms have worsen over time. Molecular genetic
testing of the patient showed 2 heterozygous mutations in the AAAS-gene:
an already reported nonsense mutation c.1024C>T, p.(Arg342*) and a novel
splice site mutation c.1331+2dupT. The one basepair duplication, located 2
basepairs upstream exon 14, disrupts the donor splice site of intron 14 of
the AAAS-gene. Based on Alamut software prediction, it is very likely that
the mutation leads to skipping of exon 14.
P11.013-S
Acrocallosal syndrome caused by novel mutations in the KIF7 gene_a
report on a Polish family and review on KIF7 syndromology
P. Chiurazzi1, S. Swagemakers2,3,4, B. C. van der Eerden5, F. Pirozzi1,6, M. SchreudersKoedam5, D. Heijsman2, M. Moscarda1, G. Zampino7, C. Carrozza8, P. Belli9, P. Caradonna10,
L. Padua11,12, E. Tacconelli13, A. Kremer2, J. Goos2,14, D. Pirolli15, M. De Rosa15, G. Neri1, J. P. T.
van Leeuwen5, P. J. van der Spek2;
1
Institute of Medical Genetics, Catholic University, Rome, Italy, 2Department of
Bioinformatics, Erasmus University MC, Rotterdam, Netherlands, 3Department of
Genetics, Erasmus University MC, Rotterdam, Netherlands, 4Cancer Genomics Centre,
Rotterdam, Netherlands, 5Department of Internal Medicine, Erasmus University MC,
Rotterdam, Netherlands, 6Department of Genetics and Genome Sciences, Case Western
Reserve University, Cleveland, OH, United States, 7Department of Pediatrics, Catholic
University, Rome, Italy, 8Institute of Biochemistry and Clinical Biochemistry, Catholic
University, Rome, Italy, 98Department of Bio-Imaging and Radiological Sciences, Catholic
University, Rome, Italy, 10Department of Internal Medicine, Catholic University, Rome,
Italy, 11Institute of Neurology, Catholic University, Rome, Italy, 12Fondazione Don Carlo
207
ABSTRACTS POSTERS
Gnocchi, Milan, Italy, 13Department of Infectious Diseases, Catholic University, Rome,
Italy, 14Department of Plastic and reconstructive Surgery and Handsurgery, Erasmus
University MC, Rotterdam, Netherlands, 15Istituto di Chimica del Riconoscimento
Molecolare, Consiglio Nazionale delle Ricerche, Rome, Italy.
The index patient, a boy, presented neonatally with conjugated hyperbilirubinemia which resolved in 2-3 months. He was born five days post term
(birthweight 2840 g, length 50 cm, occipitofrontal head circumference 34
cm) after an uneventful pregnancy. His healthy non-consanguineous parents
are of Norwegian descent. Of note is that his father was icteric for the first
2-4 months of life. In the non-dysmorphic index patient, hepatic ultrasound
and scintigraphy were normal neonatally. Bile duct paucity, giant cell transformation and intracanalicular cholestasis was evident histologically. Serum cholesterol was <5mmol/L. His proteinuria improved over the first 12
months. Additional findings included posterior embryotoxon and bilateral
mild peripheral pulmonary artery stenosis. Butterfly vertebrae were not demonstrated. No sequence or copy number variants were detected in JAG1.
Sequencing of ABCB4 and CFTR revealed no pathogenic variant. Sequencing
208
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Karyotype analysis has been the standard procedure for prenatal cytogenetic diagnosis since the 1970s. However, the major limitation remains
requirement for cell culture, resulting in a delay of as much as 14 days to
get the test results. FISH and QF-PCR do not provide a genome screen for
unexpected imbalances. CGH array technology has proven to be useful in
postnatal diagnosis of mental retardation, development delay, and congenital malformation syndrome. We evaluated the use of array comparative
genomic hybridization (aCGH) for prenatal diagnosis including assessment
of variants of uncertain significance, and the ability to detect abnormalities
not detected by karyotype. Women undergoing amniocentesis or chorionic villus sampling (CVS) for karyotype were offered aCGH analysis using a
targeted cytochip ISCA 4x44K v1.0 oligonucleotide array. Parental samples
were obtained at the same time to exclude maternal cell contamination and
determine if copy number variants (CNVs) were de novo, or inherited.We
analyzed 163 samples, most were CVS (75.5%) and amniotic fluid (19.6%).
The most common indication were advanced maternal age (N=74), abnormal ultrasound finding (N=46) and a previous child with multiple congenital abnormalities (N=40).We detected 27 CNVs (16.7%). Of these 16 (9.8%)
were interpreted as likely benign, 9(5.5%) were of defined pathological significance, while 2(1.2%) were of uncertain significance. We concluded that
array CGH identified clinically significant abnormalities in approximately
5.5% of fetuses with ultrasonographic abnormalities and normal conventional karyotype. This study demonstrate the potential for array CGH to replace
conventional cytogenetic in the great majority of prenatal diagnosis cases.
ABSTRACTS POSTERS
P11.019-S
Deletion of 9q22.3-33.1 in a child with corpus callosum hypoplasia
and thymus asymmetry
M. Czak, B. Duga, K. Hadzsiev, K. M. Komlsi, P. Kisfali, G. Kosztolnyi, B. Melegh;
University of Pcs, Dept. of Medical Genetics, Pcs, Hungary.
Back to index
The fathers karyotype was normal 46,XY while the mothers karyotype was
46,XX,t(5;14)(p13;q22) identical with her sons rearrangement. The performed array-CGH of the patient identified a heterozygous deletion in 5p12
chromosome with 489 kb size, including only one mapped gene: FGF10
(fibroblast growth factor 10) gene. Mutations in FGF10 gene may underlie autosomal dominant conditions such as lacrimo-auriculo-dento-digital
(LADD) syndrome and aplasia of lacrimal and salivary glands (ALSG). The
main features of lacrimo-auriculo-dento-digital (LADD) syndrome are abnormal tear production, malformed ears with hearing loss, decreased saliva production, small teeth, and hand deformities. The haploinsufficiency of
FGF10 gene caused by heterozygous loss of on gene copy may explain the
phenotype in our patient. This rare case of a cytogenetically balanced familial translocation causing abnormal phenotype demonstrates the diagnostic
importance of genome analysis in patients with chromosomal rearrangements detected on routine karyotype.
P11.022-M
Whole exome sequencing identifies a vertically transmitted novel
ACTB mutation in a mother-son pair presenting with atypical
Baraitser-Winter syndrome
Background: Bardet-Biedl syndrome (BBS) (OMIM 209900) is a rare autosomal recessive, pleiotropic ciliopathy. To date, at least fifteen genes causing
BBS have been reported.
Case: A 26-year-old Korean male from non-consanguineous Korean parents
presented with rod-cone dystrophy, truncal obesity, mental retardation and
end stage of renal disease. A 28-year old his brother showed rod-cone dystrophy, truncal obesity, mental retardation and deep vein thrombosis. To
rule out the BBS, genomic DNAs were obtained from the peripheral blood
leukocytes of all the family members. All the exons and intron flanking regions of the fifteen BBS genes were analyzed by direct sequencing. A compound heterozygous mutation of BBS7 gene (NM_176824) was identified
in both brothers; one was a novel acceptor splice site c.103-1G>A mutation (IVS2-1G>A) altering the splicing recognition site at the intron 2 and
exon 3 boundary of BBS7 gene. The other was a novel missense mutation
of c.728G>A changing codon 243 from cysteine to tyrosine. The father was
a heterozygous carrier for c.103-1G>A, and the mother was a heterozygous
carrier for c.728G>A. Array comparative genomic hybridization analysis revealed a normal hybridization pattern with no evidence of significant chromosome imbalance.
Conclusion: This is the first BBS case in Korean family genetically confirmed
that had a compound heterozygous mutation in BBS 7 gene. Considering
that the BBS is genetically heterogeneous and the prevalence differs among
ethnic group, our clinical experience would be helpful to diagnose these patients accurately and understand the genetic events in BBS.
209
ABSTRACTS POSTERS
P11.024-M
Genotype-Phenotype correlation for BBS1 gene in Bardet-Biedl
Syndrome patients
S. Castro-Snchez, M. Alvarez-Satta, D. Valverde;
University of Vigo, Vigo, Spain.
Bardet-Biedl syndrome (BBS, #209900) is a rare genetic disorder characterized by highly variable phenotype and genetic heterogeneity. BBS belongs
to a group of diseases known as ciliopathies, which show partial overlapping
phenotypes that further complicates the molecular diagnosis. BBS1 gene accounts for 25% of total mutations described in BBS, of which p.M390R is
the most frequent mutation in European population. Many efforts have been
done to establish a genotype-phenotype correlation for BBS genes in order
to facilitate diagnosis confirmation. In this sense, the objective of this study
was to correlate different BBS1 mutations with some clinical features. We
selected 36 patients from 22 families for whom a BBS1 mutation had been
identified. Clinical features of these patients were analysed by SPSS v.19.
p.M390R mutation was the predominant mutation identified in 33 patients
(92%), 20 of them harboured this mutation in homozygous state (56%) and
13 were compound heterozygous (36%). Only 3 patients (8%) had two mutated alleles with BBS1 mutations different from p.M390R. By comparing
all clinical features of these three groups of patients, we found statistically
significant differences between these groups: while homozygous p.M390R
showed more frequently high blood pressure, compound heterozygous
manifested more secondary features, especially psychomotor impairment/
retardation, hearing loss and worse visual defects. In conclusion, we confirm the important role of p.M390R mutation in the diagnosis of BBS, and
it seems that homozygous patients have a milder phenotype. This will be
important for genetic counselling purposes in these families.
P11.025-S
Potential impact on splicing of BBS12 variations found in Bardet-Biedl
syndrome Spanish patients
M. Alvarez-Satta, S. Castro-Snchez, D. Valverde;
University of Vigo, Vigo, Spain.
Back to index
P11.026-M
Cognitive, behavioural, and adaptive functioning of patients affected
with Bardet-Biedl syndrome (BBS)
P11.028-M
A novel IGF2/H19 domain triplication in the 11p15.5 imprinting
region - new insights into the pathogenesis of Beckwith-Wiedemann
and Silver-Russell syndromes
Bardet-Biedl syndrome is a rare genetically heterogeneous multisystem disorder characterized by retinal degeneration, genital and kidney malformation/function, and other features. Variability in cognitive, social, and emotional impairment is reported; however, studies were small and completed
prior to availability of molecular characterization. Our aim is to define neuropsychological function in a group of patients with molecular diagnosis of
BBS to better understand phenotypic variability.
Methods: Eighteen patients (12 females; mean age: 20.95 years; range 6.438 years) completed standardized measures of cognition. Magnification was
available. Standardized informant-proxy questionnaires were completed to
assess behavioural and adaptive functioning. Results were compared to nor-
The imprinted 11p15.5 region contains two domains (IGF2/H19 and KCNQ1OT1/CDKN1C), each of them under control of its own imprinting control
region. Defects of 11p15 imprinting result in two growth disorders with opposite phenotypes: Beckwith-Wiedemann (BWS) and Silver-Russell (SRS)
syndromes. Various 11p15.5 genetic and epigenetic aberrations have been
revealed, among them microduplications and microdeletions.
210
ABSTRACTS POSTERS
We report a novel IGF2/H19 domain triplication in the 11p15.5 region identified in a girl with BWS and her father with symptoms of SRS. Methylation
specific multiplex ligation-dependent probe amplification (MS-MLPA) performed in the patients DNA revealed triplication of IGF2/H19 domain as
well as increased methylation of H19 region. The same genetic change in the
fathers DNA was associated with reduced methylation of H19 region. The
presence of triplication was confirmed by aCGH.
This is the first report of IGF2/H19 domain triplication associated with BWS
or SRS. A few BWS patients with IGF2/H19 domain duplication of paternal
copy have been described so far. Duplications of maternal copy of this domain have been reported in three individuals and were not associated with
an abnormal phenotype (i.e. Silver-Russell syndrome). The clinical outcome
of 11p15.5 copy number variations depends on their size, localization and
the parental inheritance. These aberrations may influence chromatin organization affecting the regulation of imprinted genes. Our findings bring new
insights into the regulation of genomic imprinting at 11p5.5 region and underline difficulties of genetic counseling in patients with 11p15 defects.
The study was financed by National Science Centre, project 1149/B/
P01/2011/40 (NN407114940) and EU Structural Funds, POIG.02.01.00-14
-059/09.
P11.029-S
Uniparental disomy in Beckwith-Wiedemann syndrome: new insights
from genotype-phenotype correlations
F. Cerrato1, A. Freschi1, A. De Crescenzo1, A. Sparago2, P. Palumbo3, O. Palumbo3, M.
Carella3, A. Riccio1,2;
1
DISTABIF, Second University of Napoli, Caserta, Italy, 2Istituto di Genetica e Biofisica
Adriano Buzzati Traverso CNR, Napoli, Italy, 33IRCCS Casa Sollievo della Sofferenza,
Unit di Genetica Medica, San Giovanni Rotondo, Italy.
Back to index
Aim: The identification of de novo disease causing mutations in three Caucasian patients with sporadic Caudal Regression Syndrome (CRS). CRS is a rare
and diverse congenital disorder which is characterised by different degrees
of agenesis of the caudal spine. Known genetic mutations are only able to
explain a fraction of cases and are not accounting for sporadic occurrences
or the diversity of the disorder. Methods: Exome sequencing assay was conducted of the three sporadic cases and their biological parents. We targeted rare genetic variants as the underlying cause of CRS as well as de novo
mutations. Further we investigated de novo indels, copy number variations
(CNV) and compound heterozygosity. Identified mutations were ranked and
filtered based on genomic, genetic and statistical features. Results: Sanger
sequencing confirmed two different de novo mutations in two cases (detailed results will be presented). In addition, our analysis revealed several
potentially causal compound heterozygous mutations which are also under investigation. Conclusion: CRS may be caused by de novo or compound
heterozy mutations thus, i) the diversity of the disorder is mirrored in the
underlying genetic architecture and its mutations; ii) ranking of compound
heterozygous mutations enables identification of candidate genes.
P11.032-M
A de novo microdeletion of chromosome 18q11.2q12.2 causes a new
distinct clinical phenotype with coarctation of aorta and intellectual
disability.
Cardiac abnomalities are diagnosed in 25-30% of the patients with 18q syndrome and the most common heart abnormalities are reported in patients
with terminal 18q deletion (18q22): atrial septal defect, ventricular septal
defect, polmonary valve anomalies and total anomalous polmonary venus
return. We report on one girl with coarctation of the aorta, moderate development delay, behavioral problems, with an interstitial de novo deletion
within chromosomal band 18q11.2 of 13.4Mb. The propositus was born at
37 weeks via caesarian section after a pregnancy complicated by gestosis.
Her birth weight was 2400g (<10th centile), birth length was 45cm (3-10th
centile) and occipital frontal circumference (OFC) 34 cm (5thcentile). Family history revealed no abnormalities or congenital heart diseases. At the age
of one month, cardiac echo revealed tight istmic coarctation of the aorta and
the patient underwent surgery. Clinical evaluation showed a distinctive craniofacial apparence characterized by brachicephaly, plagiocephaly, telecanthus, strabismus, low nasal bridge, smooth philtrum, thin lips, high arched
palate, low set ears with hypoplastic lobule, proximally placed thumbs. The
development milestones were delayed and the propositus developed behavioral problems: poor concentration, hyperactivity and distractibility. CGH
analysis showed an interstitial proximal microdeletion of chromosome 18
of 13,4Mb [arr 18q11.2(22,032,122-35,430,900)x1]. In this region some genes are present that are involved in development of the heart, DSC2 ,DSG2,
TTR, DTNA, FHOD3. This is the first report of aortic coarctation mapping to a
proximal microdeletion involving the chromosome 18q11.2q12.2 segment.
The report also expands the spectrum of clinical phenotype associated with
18q11.2q12.2 deletion.
P11.033-S
A further case of pure 15q deletion: Clinical and molecular
cytogenetic findings
211
ABSTRACTS POSTERS
Back to index
on is rarely described. Here we report an 11 year-old girl, from non-consanguineous parents, was referred to the Pediatric Genetics Department with
growth retardation and multiple congenital abnormalities. In her medical
history, she was born at-term after an uncomplicated pregnancy. Her birth
weight was 2200 g. She had a cleft palate, hip dislocation and crossed renal ectopia. On physical examination, her weight, height and head circumference were below the 3rd percentile. Dysmorphological evaluation revealed a triangular face, low-set ears, fissured cleft tongue, micrognathia,
proximally placed hypoplasic thumbs, genu valgus, 2-3 toe skin syndactyly,
clinodactyly and nail hypoplasia. Speech problems were also noticed. The
complete blood count, basic biochemical parameters and hormones profile were within the normal range. The karyotype was normal. Subtelomeric
fluorescene in-situ hybridisation (FISH) analysis showed a de novo terminal
deletion of chromosome 15. BAC FISH analysis of the patient indicated that
the deletion breakpoint was at 15q26.3 and the deletion comprised 700870 kb. The deleted region includes the CHSY1 gene that is responsible for
Temtamy preaxial brachydactyly syndrome which shares clinical features
with 15qter deletion syndrome. To the best of our knowledge, this deletion
is the smallest among reported cases. It is considered that the case presented here significant contribution to phenotype-genotype correlation in 15q
deletion patients.
P11.036-M
Haploinsufficiency of MEIS2 is associated with orofacial clefting and
learning disability
P11.034-M
Association between HLA-G and non-syndromic Oral Cleft
Non-syndromic oral clefts (nsOCs) aect 1 per 1,000 live births worldwide.
There is some evidence suggesting that embryo-maternal interaction can
play a relevant role in the etiology of nsOCs. HLA-G has a protective function
at the maternal-embryo interface, protecting the embryo from destruction
by mothers immune system.
In this study we investigated the association between a functional variant in
HLAG gene and the risk of nsOCs. A 14 nt insertion in the 3UTR of HLAG was
genotyped in a group of 222 Italian nsOC trios, including affected children
and their parents.
The analysis of transmission disequilibrium resulted in no evidence of association in nsCL/P (p=0.16) nor in nsCP trios (p=1.00). However, when considering the length of pregnancy, a significant overtransmission of the insertion was observed in the trios with pregnancy length >38 weeks (p=0.005).
In trios with affected children born after 38 weeks or less, the transmission
of minor allele was reverse (p=0.031), so that in the effect of the asymmetric segregation was canceled when the two groups were pooled. In children
born after more than 38 weeks of pregnancy the ins/ins genotype resulted
specifically associated with a three-fold increased risk of the more severe
phenotype (cleft lip and palate, CLP) (p<0.0001).
This results suggests the existence of a link between HLA-G genotype, length
of pregnancy and chance of developing CLP. We hypothesize that HLA-G
could be involved in prenatal loss of abnormal embryos (teratothanasia)
and drive selection against those which fail to fuse lip and palate.
P11.035-S
Association between chromosome 8q24.21 and susceptibility for
nonsyndromic cleft lip with or without cleft palate in Iraqi population
F. Mattioli1, F. Abenavoli2, A. Franchella3, F. Pacelli2, C. Baluardo1, V. Aiello1, G. Astolfi1, D.
Balestra1, F. Conte1, A. Edris1, E. Bonomo Roversi1, P. Franceschelli1, M. Rubini1;
1
University of Ferrara, Ferrara, Italy, 2Emergenza Sorrisi ONG, Roma, Italy, 3Pediatric
Surgery Unit, S. Anna Hospital, Ferrara, Italy.
Cleft lip without or without cleft palate (CL/P) are the most common craniofacial congenital malformations. Most of CL/P cases are non-syndromic
(nsCL/P), with a multifactorial etiology. Recent genome-wide association
studies (GWAS) have identified susceptibility loci for nsCL/P, among which
one of the most relevant is located in 8q24.21. In particular, the polymorphism rs987525 has been shown to be highly associated with nsCL/P in
population of European origin, while yielding only marginal significance in
Oriental populations.
We present the result of case-control study of a sample of Iraqi population from Nasiryiah: 418 affected children, and 273 unaffected controls. The
rs987525 C>A variant was genotyped using TaqMan assay. The frequency of
minor allele (A) resulted significantly higher among cases compared to controls. Odd Ratio (OR) for carriers of heterozygous (C/A) genotype was 1.69
(95% C.I. 1.24-2.31), while OR for homozygotes (A/A) was 1.95 (95% C.I.
1.16-3.29). In the studied Iraqi sample the rs987525 variant acts in a dose
dependent fashion, in agreement to what resulted from association studies
in population of European origin, suggesting that 8q24.21 locus confers risk
for nsCL/P also in Middle-East population.
212
MEIS2 is a homeodomain-containing transcription factor of the TALE superfamily that has been proven important for development. We confirm and
extend a recent single case report stating that deletions in MEIS2 can cause
cleft palate (Crowley et al, Am J Med Genet 2010, 152A:1326-7). Here we report five additional cases with 15q14 deletions of sizes 0.6, 0.6, 1.0, 1.9 and
4.8 Mb, respectively, all involving MEIS2. In addition, we present a family
with four affected individuals and an intragenic 58 kb direct duplication disrupting MEIS2. In total, 7/9 cases had clefting, from mild (submucous cleft
palate) to severe (cleft lip and palate), and 3/9 cases had ventricular septal
defects. All cases had delayed motor development and most had learning
disability, at worst in the mild intellectual disability range. Our results show
that MEIS2 clearly is a gene needed for palate closure. In syndromic cases of
cleft palate, MEIS2 should be considered among the candidate genes, e.g. in
cases without 22q11.2 deletions.
P11.037-S
Familial Beckwith-Wiedemann Syndrome: Follow up
CLOVES syndrome (congenital lipomatous overgrowth, vascular malformations, epidermal nevi, and skeletal abnormalities (MIM 612918) is a nonhereditary regional overgrowth disorder distinct from Proteus syndrome.
Gain-of-function mutations in PIK3CA have been identified in affected tissues demonstrating somatic mosaicism of varying degrees. Activating PIK3CA
somatic mutations have also been shown in further regional overgrowth
conditions predominantly affecting the limbs like in isolated macrodactyly
and fibrolipomatous gigantism. In the current study, hot spot mutations of
PIK3CA were identified in six patients presenting with CLOVES syndrome.
Analysed tissues included epidermal nevi, skin, fatty tissues, and connective
tissue. Blood was available from five patients. Molecular analysis was performed for all six patients by Sanger sequencing. Mutant allele ratios of 30-
ABSTRACTS POSTERS
50% were observed in scrapings from epidermal nevi or affected fatty tissue samples. In none of the blood samples PIK3CA mutations were detected.
Because detection levels and quantification of mutant alleles were limited
to 10-15% by Sanger sequencing, fragment analysis and amplicon-deep sequencing on a GS Junior platform (Roche) were applied. This increased the
mutant allele detection to 1-5%. In blood samples mutant allele ratios were
2% or less. Our data confirm that cells from affected tissue are essential for
mutation analysis in CLOVES syndrome whereas blood is an inappropriate
source. Improved detection methods may be required for other tissues with
low level somatic mosaicism.
P11.039-S
Endocrinological study in a new case of Coffin Siris syndrome due to
ARID1B gene deletion
Cohen syndrome (CS) is a rare autosomal recessive disorder with multisytemic clinical features due to mutations in the VPS13B gene, which has recently been described encoding a mandatory membrane protein involved
in Golgi integrity. As the Golgi complex is the place where glycosylation of
newly synthesized proteins occurs, we hypothesized that VPS13B deficiency, responsible of Golgi apparatus disturbance, could lead to glycosylation defects and/or mysfunction of this organelle, and thus be a cause of
the main clinical manifestations of CS. The glycosylation status of CS serum
proteins showed a very unusual pattern of glycosylation characterized by a
significant accumulation of agalactosylated fucosylated structures as well
as asialylated fucosylated structures demonstrating a major defect of glycan
maturation in CS. However, CS transferrin and 1-AT profiles, two liver derived proteins, were normal. We also showed that ICAM-1 and LAMP-2, two
highly glycosylated cellular proteins, presented an altered migration profile
on SDS-polyacrylamide gels in peripheral blood mononuclear cells (PBMCs)
Back to index
from CS patients. RNA interference against VPS13B confirmed these glycosylation defects. Experiments with Brefeldin A demonstrated that intracellular retrograde cell trafficking was normal in CS fibroblasts. Furthermore,
early endosomes were almost absent in these cells and lysosomes were
abnormally enlarged, suggesting a crucial role of VPS13B in endosomallysosomal trafficking. Our work provides evidence that CS is associated to a
tissue-specific major defect of glycosylation and endosomal-lysosomal trafficking defect, suggesting that this could be a new key element to decipher
the mechanisms of CS physiopathology.
P11.041-S
A Novel Frameshift mutation in the PIEZO2 gene in a Turkish
pediatric patient with Distal Arthrogryposis 5A
The distal arthrogryposis (DA) are characterized by congenital contractures of two or more different body areas without a main neurological and/
or muscle disease. Currently, DAs are subdivided into 10 types, depending
on the number and nature of additional features. DA5 is unique among
DAs because, in addition to contractures, affected individuals have ocular
abnormalities. These abnormalities include ptosis, ophtalmoplegia, and/or
strabismus. Recently, it has been shown that a subtype of DA5 that includes
restrictive pulmonary disease is caused by gain-of-function mutations in the
mechanically activated cation channel PIEZO2. We report 14 years old boy
with DA5 who initially presented with multiple contractures at the age of
2 months. The main clinical findings included pitosis, ophtalmoplegia, micrognathia, clinodactyly, pectus excavatum, recurrant pulmonary infections,
operated inguinal hernia/midpenil hypospadias, elbow extansion/bilateraly wrist supination restriction, knees lightly in flexion moode and pes valgus. Sequencing of the PIEZO2 gene in the proband revealed a de novo one
base-pair deletion in Exon 52 of PIEZO2, which results in a frameshift mutation (c.8208delA). The mutation leads to a p.Y2737Ifs*7 change within the C
terminal domain of PIEZO2. Recently electrophysiologic studies of E2727del
variant showed that mechanically activated current inactivation were clearlly slower and E2727del channels spend about two fold less time in an inactivated state following mechanical stimulation than in wild channels, thus can
be reactivated quicker. Therefore because our pathologic variant is at the
same domain as E2727del, it can be speculated that increased response to
mechanical force may explain the phenotype of our patient also.
P11.042-M
The contribution of discrepant DNA variations in discordant
monozygotic twins with Congenital Diaphragmatic Hernia (CDH) or
Esophageal Atresia/ Tracheoesophageal Fistula (EA/TEF).
213
ABSTRACTS POSTERS
tegies to determine if we could distinguish false positive differences from
actual ones. Currently, we are evaluating with whether these remaining discrepancies are true differences.
P11.043-S
Associated noncardiac congenital anomalies among infants with
congenital heart defects
C. Stoll, Y. Alembik, B. Dott, M. Roth;
Genetique Medicale, Strasbourg, France.
214
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ABSTRACTS POSTERS
di Milano, Milano, Italy, 5Laboratory of Medical Cytogenetics and Molecular Genetics,
IRCCS, Istituto Auxologico Italiano, Milano, Italy.
Score
1,5
2
1
1
3,5
0
1,5
The probability of a mild MR increases with the reducing final score less than
2, the probability of a severe MR increases with the increasing final score more
than 3.
This prognostic index allows to define, precociously in the life of a baby, the
probability of a better or worse intellectual outcome in CdLS patients.
P11.048-M
SNP arrays in the diagnostic strategy of corpus callosum agenesis
associated with intellectual disability (an update)
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215
ABSTRACTS POSTERS
genes in the clinical manifestations and will provide insight into the underlying biological mechanisms.
P11.052-M
Non-mosaic 4p16.3 deletion concomitant with low-level mosaicism
for deletion at 21q11.1q21.2
O. S. Kurinnaia1,2,3, S. G. Vorsanova1,2,3, V. Y. Voinova1,2,3, T. Liehr4, A. D. Kolotii1,2, Y. B.
Yurov1,2,3, I. Y. Iourov1,2,5;
1
Institute of Paediatrics and Paediatric Surgery, Ministry of Health, Moscow, Russian
Federation, 2Mental Health Research Center, Russian Academy of Sciences, Moscow,
Russian Federation, 3Moscow City University of Psychology and Education, Moscow,
Russian Federation, 4Institute of Human Genetics, Jena, Germany, 5Department of
Medical Genetics, Russian Medical Academy of Postgraduate Education, Ministry of
Health, Moscow, Russian Federation.
Juvenile polyposis can be associated to hereditary hemorrhagic telangiectasia (HHT) due to SMAD4 gene mutations. We describe the first case of juvenile polyposis and HHT in a patient with SMAD4 gene loss due to a chromosomal deletion. The male patient presents moderated intellectual disability,
limited verbal language repertoire, attention-deficit/hyperactivity disorder,
corpus callosum agenesis and brain arteriovenous malformation. Colonoscopy revealed inflammatory intestinal polyposis, with typical juvenile polyp
histology, fulfilling juvenile polyposis syndrome clinical criteria. Three small
haemangiomas in scalp and bilateral telangiectasias in Kiesselbach area were
found. The patient presents a 46,XY,t(6;18)(q13;q21)dn karyotype. Chromosomal microarray detected two non-continuous de novo microdeletions
encompassing 2.7 Mb and 0,4 Mb as follows: arr 18q21.1q21.2(47,553,46850,257,792)1,18q21.2(50,644,595-51,052,896)1. FISH with BAC probes
confirmed both deletions and the presence of a segment between them. This
is the first case of juvenile polyposis and HHT in a patient with a chromosomal rearrangement that resulted in interstitial deletions of 18q and loss
of the SMADA4 gene, among others. The loss of a copy of the entire SMAD4
gene, added to the fact that the patient had intestinal polyps at a young age,
prompted us to look for telangiectasia. Thus, we showed the importance of
the detailed screening for these phenotypes in patients in whom cytomolecular studies indicate deletion of SMAD4 gene. Additionally, we demonstrate
the relevance of cytogenomic investigation in patients with juvenile polyposis, dysmorphisms and intellectual disability. Financial support: FAPESP,
Brazil.
P11.054-M
Microdeletion 19p13.12 in a fetus with severe microcephaly and
paraventricular cysts: case report and review of the literature
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ABSTRACTS POSTERS
P11.056-M
A novel micro-deletion 7p14.3 associated with complex
neurocognitive phenotypes and distinct Cardiac malformations
We report a 20 year old male with global developmental delay and a novel
microarray finding. His past medical history included a diagnosis of supravalvular mitral ring moderate endocardial fibroelastosis at age two, following recurrent pneumonia and congestive heart failure. His neurocognitive history, in addition to global delay, also included a diagnosis of autism,
attention deficit disorder and bipolar disorder with episodes of psychoses
that required admission. Other medical issues included moderate bilateral
hearing loss, eosinophilic esophagitis and sleep apnea. He had minor dysmorphic craniofacial features, a narrow palate and a bifid uvula, as well
as hypoplastic nails. Psoriasis and striae were noted on skin exam. Chromosomal microarray analysis revealed a de novo deletion at 7p14.3p14.2
(chr7:33,453,828-36,924,450), confirmed by fluorescence in-situ hybridization. The genes in the deleted segment include BBS9, BMPER, NPSR1,
DPY19L1, TBX20, HERPUD2, SEPT7, EEPD1, KIAA0895, ANLN, AOAH and
ELMO1. BMPER mutations are thought to be associated with craniofacial
dysmorphism. TBX20 is a transcription factor involved in the formation of
cardiac chambers and valves. Other components within this 7p14.3 deletion
with features of cardiac malformations and learning disabilities may also be
implicated and will be discussed. By sharing these novel findings we hope
to aid other healthcare providers in the care and management of similar
patients.
P11.057-S
A futher case of de novo 10p14-pter deletion detected by multiplex
ligation dependent probe amplification (MLPA) assay in a newborn
Pure distal monosomy of the long arm of chromosome 10 is a rare cytogenetic abnormality; the location and size of the deletions described in this
region are variable. The associated phenotype associated with this deletion
is also variable but reported features include developmental delay/learning
disability/mild to moderate intellectual disability, speech and language delay, poor attention, strabismus and distinctive facial features. We report two
siblings; a male aged 15 and a female aged 21 years, with intellectual disability, microcephaly, motor impairment and ataxia. MRIs showed mild dilatation of the lateral ventricles and a prominent cisterna magna. Karyotype
was normal. We performed whole-exome sequencing (WES) and they were
found to have a 5.5 Mb terminal chromosome 10q26.3 deletion using the
program FishingCNV. The deletion was confirmed by FISH and the mother
was found to carry a pericentric inversion. A third child, age 6 years, presented in early childhood with global developmental delay, poor coordination
and ataxia. MRI showed no abnormality of the posterior fossa. A high-resolution (105 K) comparative genomic hybridization microarray identified a
deletion of the terminal 4.6 Mb of the long arm of chromosome 10, within
cytogenetic band 10q26.3. This finding was confirmed by FISH analysis. The
parents were tested and this is a de novo change. Our findings add three new
cases of 10q26.3 monosomy to the literature; we summarize the previous
cases and highlight the features of this emerging cytogenetic syndrome.
Back to index
P11.059-S
Oxidative stress a Phenotypic Hallmark of Fanconi anemia and Down
syndrome: The effect of antioxidants
H. T. El-Bassyouni1, H. H. Afifi1, M. M. Eid1, R. M. Kamal2, H. H. El-Gebali2, G. S. M. ElSaeed1, M. M. Thomas1, S. A. Abdel Maksoud1;
1
National Research Center, Guiza, Egypt, 2Institute of Postgraduate Childhood Studies,
Ain Shams University, Cairo, Egypt.
Oxidative stress plays a major role in the pathogenesis of leukemia-prone diseases such as Fanconi anemia and Down syndrome. Objectives: To explore
the oxidative stress state in children with Down syndrome and Fanconi anemia by estimating the levels of antioxidants (e.g., malondialdehyde, total antioxidant capacity and superoxide dismutase [SOD] activity) and DNA damage, and to evaluate of the effect of antioxidant treatment on these patients.
Methods: The study included 32 children clinically diagnosed with Down
syndrome (15 patients) and Fanconi anemia (17 patients) in addition to 17
controls matched for age and sex. Malondialdehyde, total antioxidant capacity, superoxide dismutase (SOD) activity and DNA damage were measured.
Antioxidants including vitamin A, E and C were given to the patients according to the recommended daily allowance (RDA) for 6 months. Clinical follow-up and re-evaluation were conducted for all patients. Laboratory tests
including complete blood count, karyotyping, DNA damage and oxidative
stress were re-evaluated. Results: Children with Fanconi Anemia and Down
syndrome had elevated levels of oxidative stress and more DNA damage than
controls. Oxidative stress parameters and DNA damage improved in Fanconi
anemia and Down syndrome patients after antioxidant administration. Conclusions: Early administration of antioxidants to Fanconi anemia and Down
syndrome patients is recommended for slowing of the disease course with
symptoms amelioration and improvement of general health.
P11.060-M
Report of the first case of robertsonian translocation in Down-Turner
mosaicism (mos 45, X / 46,XX, + 21, rob (21;21)(q10;q10))
M. D. F. Carvalho1, E. D. F. Carvalho2, M. Montenegro3, K. M. Carvalho4;
1
UECE/Unichristus/APAE-Fortaleza CE, Fortaleza, Brazil, 2Unichristus/USP, Fortaleza,
Brazil, 3USP, So Paulo, Brazil, 4UECE, Fortaleza, Brazil.
The short arm of chromosome 16 is rich in segmental duplications rendering this region susceptible to rearrengement through non-allelic homologous recombination. Several syndromes resulting from microdeletions
or microduplications in this region have been reported. The chromosome
16p12.2-p11.2 deletion syndrome, 7.1-to 8.7-Mb [OMIM#613604] is characterized phenotypically by dysmorphic facial features, feeding difficulties,
recurrent ear infections, developmental delay and cognitive impairement.
Reciprocal duplication of 16p12.2-p11.2 has been observed in few patients
with dysmorphic features, short stature, developmental delay and intellectual disability but a specific phenotype hasnt been established and more
cases are needed.
We report two new unrelated cases of chromosome 16p12.2-p11.2 dupli-
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cation analysed by 180K oligonucleotide arrayCGH (Agilent Technologies).
Both children showed a de novo 7.8 Mb duplication extending from 21.5 Mb
to 29.3 Mb, comprising the same region involved in the deletion syndrome.
We discuss the phenotype and molecular findings of our cases with respect
to previous reported ones to further define the syndrome.
P11.062-M
Redefining the contiguous gene syndrome in the era of highthroughput sequencing
We ascertained an Algerian consanguineous family in which two sibs present with psychomotor delay, progressive microcephaly, spasticity, thin corpus callosum, and severe and early onset obesity. Exome sequencing identified two homozygous substitutions cosegregating with the phenotype and
locating 170 kb apart on 7q22.1: a c.1137+1G>T splice mutation in AP4M1
previously described in a Moroccan family and a c.595A>T misense variation in AZGP1 which encodes zinc-alpha2-glycoprotein (ZAG). Haplotyping
analysis indicated that the AP4M1 mutation was a founder mutation shared
between both families, whereas the AZGP1 mutation is secondarily and unique in our family.
Mutations in AP4M1 cause AP4-deficiency syndrome, a condition characterized by severe intellectual disability, progressive microcephaly and spasticity. Notably, none of the 25 previously reported cases with AP4-deficiency
syndrome exhibited obesity. On the other hand, ZAG is an adipokine stimulating lipolysis in adipocytes; ZAG likely regulates body weight since administration of human ZAG to ob/ob mice resulted in progressive weight loss.
We propose that the phenotype of our patients resulted from the additional
effects of the two mutations in AP4M1 and AZGP1 accounting for the neurological signs and the precocious morbid obesity, respectively.
The contiguous gene syndrome was proposed in 1986 to explain the association of multiple and unrelated clinical features due to the deletion of
multiple adjacent genes: the phenotype results from the combination of the
endophenotypes of each contiguous gene sensitive to haploinsufficiency.
Today, high-throughput sequencing allows us to enlarge this concept to describe simultaneous transmission of independent mutations that are genetically linked.
P11.063-S
Insight into genetic heterogeneity of complex diseases by exome
sequencing
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Fanconi anemia (FA) is an inherited disease characterized by congenital malformations, pancitopenia, cancer predisposition, and sensitivity to
cross-linking agents. The molecular diagnosis of FA is relatively complex,
due to several aspects including genetic heterogeneity with mutations in at
least 16 different genes. In this study, we report the mutations identified in
100 unrelated FA probands enrolled into the National Network of the Italian Association of Pediatric Hematology and Oncology. We identified 108
distinct variants of FANCA, FANCG, FANCC, FANCD2, and FANCB genes in 85,
9, 3, 2, and 1 families, respectively. Particularly, in FANCA we found mainly private mutations of all different categories (large intragenic deletions,
nonsense, frameshift, splicing and missense mutations). Expression level of
FANCA protein was studied in 32 lymphoblastoid cell lines of complementation group FA-A and a correlation between the type of mutation and the
expression level of FANCA was observed. In case of nonsense or frame-shift
mutations FANCA is not detectable whereas it is expressed at the same levels as in controls when alleles are hit by missense or in frame mutations.
Since it will be interesting to determine whether there is a correlation between stable expression of mutant FANCA and phenotype, we have been evaluated a series of different aspects at both clinical and cellular levels in order
to provide insights into potential residual activities of altered but expressed
proteins.
P11.065-S
A familial case of Fanconi anemia-related VACTERL-H association
due to a mutation of FANCF gene, identified using a next generation
sequencing approach
ABSTRACTS POSTERS
severe frontofacionasal dysplasia phenotype with extreme microphthalmia
and oblique facial clefts. It is shown that mice homozygous for deficiency in
Cart1, the mice homolog to ALX1 are born with acrania and meroanencephaly. In the limited number of ALX1-related FND patients, neural tube closure
defects have not been reported.
We report a unique FND case, a 20-week-old male fetus terminated due to
anencephaly. Postmortem examination showed severe hypertelorism, clinical anophthalmia, bifid nose and midline upper lip cleft. The parents were
first degree cousins, and their first pregnancy was also terminated due to
anencephaly.
Array-comparative genomic hybridization using the NimbleGen CGX-3 1.4M
DNA oligoarray set revealed normal results. Molecular analysis of ALX1 did
not reveal any mutations. Trio exome sequencing revealed a homozygous
mutation in a candidate gene, linked to a human phenotype for the first time.
While the mouse model for this gene is well described, further studies are
needed to establish its role in human development.
P11.067-S
High throughput analysis in Goldenhar syndrome
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P11.069-S
Unusual presentation of Goltz syndrome with minimal ectodermal
involvement in a 3-year-old Iranian girl
A. Rajaee, A. Rajaee;
employee in genetics center, Tehran, Islamic Republic of Iran.
Hamamy syndrome is a very rare, autosomal recessively inherited malformation syndrome characterized by craniofacial findings, bone fragility,
conductive heart defects and sensorineural hearing impairment. Patients
have common dysmorphic features such as severe telecanthus with hypertelorism, sparse lateral eyebrows, thin upper vermilion border, flat philtrum
and dysplastic, protruding ears. High myopia with retinal changes, absent
or dysfunctional nasolacrimal structures, teeth anomalies, mycrocytic hypochromic anemia and mild intellectual deficit may also be present.
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The syndrome was clinically described as a new syndrome by Hamamy et
al. in 2007, in two brothers born to double first cousin parents of JordanianArabic origin. Identification of two further patients from a Turkish family
led to the localization of the causative gene to 16q12.2-q21, using homozygosity mapping. By locus re-sequencing, two different homozygous missense mutations were identified in the IRX5 gene. A member of the Iroquis
family of transcription factors, IRX5 plays an active role in face, heart, blood,
brain, bone and gonad development.
We report here a clinical and molecular evaluation of three new patients
carrying two novel homozygous mutations in IRX5, along with previously
unreported clinical findings, including acute myeloid leukemia with maturation. The phenotypic and molecular features of the Hamamy syndrome
patients will be reviewed to further delineate the clinical and mutational
spectrum.
P11.072-M
Novel de novo heterozygous FGFR1 mutation in two siblings with
Hartsfield syndrome: suggesting gonadal mosaicism
R. Dhamija, S. Kirmani, X. Wang, D. Babovic;
Mayo Clinic, Rochester, MN, United States.
We describe two siblings with Hartsfield syndrome(association of holoprosencephaly, ectrodactyly, cleft lip and palate) and a novel de novo FGFR1
mutation suggesting gonadal mosaicism. The proband presented at age 6
years for genetic evaluation.He was a product of a non-consanguineous union.Multiple congenital anomalies were detected at birth.His phenotype was
consistent with Hartsfield syndrome (global developmental delay with spastic quadriplegia due to holoprosencephaly, ectrodactyly of bilateral hand
and feet and bilateral cleft lip and palate).Previous genetic evaluation included normal karyotype, oligonucleotide array and single gene testing for
non-syndromic holoprosencephaly (SHH, SIX3, ZIC2, TGIF).At the age of 6
years, exome sequencing was performed on the patient at BCM, Houston,TX
and a de novo novel missense variant was identified in FGFR1 (coding for fibroblast growth factor-1) on chromosome 8p12:c.1880G>C (p.R627T).This
variant affects a highly evolutionarily conserved area of the gene, replacing
arginine with theorine.Online prediction programs suggest this variant is
a deleterious mutation.Subsequently a younger sibling was born with the
same phenotype (holoprosencephaly, ectrodactyly of bilateral hand and feet
and bilateral cleft lip and palate).Sequencing of FGFR1 revealed the identical variant. We report a novel heterozygous FGFR1 mutation in Hartsfield
syndrome in two siblings, making this as the first case of familial recurrence
of this rare syndrome. Both parents were negative for the sequence variant
in FGFR1, thus suggesting gonadal mosaicism. This report also expands the
phenotypic spectrum associated with loss of function mutations in FGFR1
gene to include Hartsfield syndrome and confirms autosomal dominant inheritance of this condition.
P11.073-S
Clinical and molecular dissection of two novel cases of
hemihyperplasia
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ABSTRACTS POSTERS
Pediatra. Hospital Clnico Universitario Virgen de la Arrixaca, Murcia, Spain, 5Unidad
de Medicina Fetal. Servicio de Ginecologa y Obstetricia. Hospital Clnico Universitario
Virgen de la Arrixaca, Murcia, Spain, 6Unidad de Gentica Mdica. Servicio de Pediatra.
Hospital Clnico Universitario Virgen de la Arrixaca. Grupo Clnico Vinculado al
CIBERER-ISCIII (Madrid). Ctedra de Gentica Mdica. Universidad Catlica San Antonio
de Murcia, Murcia, Spain.
Introduction: Holt-Oram syndrome (HOS) is an autosomal dominant disorder characterized by upper-limb malformation in combination with congenital heart defect. More than 70% of patients who meet diagnostic criteria
have an identifiable mutation in TBX5.
Clinical report: 32-year-old woman. Second gestation. Abortion at 20
weeks because of bilateral upper-limb amelia in a male fetus. Normal karyotype. No necropsy.
The patient had a healthy son. Her partner had required surgery to correct
atrial septal defect in childhood. He displayed low-set thumbs with no major
upper-limb anomalies.
In a further gestation, ultrasound detected again bilateral upper-limb amelia
in a male fetus at 12 weeks. Karyotype and heterochromatic repulsion analysis were normal. The patient underwent abortion. Post-mortem examination didnt reveal heart defects. No environmental hazards were identified.
A new pregnancy was followed closely by high-resolution ultrasound, without detecting anomalies in a female fetus. At birth, triphalangeal thumbs
and atrial septal defect were present. TBX5 gene sequencing analysis revealed no mutations. MLPA showed a deletion in exon 9, not previously described. The same deletion was detected in the second affected fetus and the
father, co-segregating with the disease.
Discussion: Upper-extremities abnormalities are variably expressed, even
within families. Although phocomelia has been described in HOS, there are
few reports of frank amelia. TBX5 analysis should be therefore considered
in such finding, even in the absence of heart defect. We report on a new
exonic deletion in TBX5. Upcoming expression and exome analysis will help
elucidate the mechanisms and modifying factors underlying its remarkable
variable expressivity.
P11.077-S
Three TBX5 gene mutations resulting in Holt-Oram syndrome
Holt-Oram syndrome (HOS) is characterized by the congenital malformations of the heart, limbs, often accompanied with variable skeletal abnormalities. HOS-associated limb malformations usually involve absent or hypoplastic radial bone(s) and often abnormal thumb(s), which may be digitalised,
absent, hypoplastic, triphalangeal or bifid. In a subset of patients, the syndrome results from TBX5 mutations. To date, about 60 different TBX5 gene
lesions resulting in HOS have been identified. In this report, we describe
three index cases of Polish ethnicity suspected of HOS, in whom we performed TBX5 Sanger sequencing. In all three probands we identified heterozygous TBX5 causative mutations: c.255-256delCA(p.P85fs94X), c.C524T(p.
S175F), c.C668T(p.T223M). The first two mutations were novel, whereas
the latter one represented previously identified variant. In the first two
probands we observed severe bilateral hypoplasia of the radial bones and
absent thumbs. Both probands presented with complex congenital heart
defect composed of ventricular septal defect and persistent foramen ovale.
The third patient was born with inborn heart defect in the form of atrial
septum defect with patent ductus arteriosus and skin syndactyly of thumbs
with second fingers. Upon MRI of the head, hypoplasia of corpus callosum
was noted. Similar hand anomaly was observed in her father without heart
involvement. Although based on a small sample, our study shows that TBX5
mutations may account for a significant proportion of HOS causative alterations and points to the importance of TBX5 mutational screening in the
patients clinically suspected of this syndrome.
P11.078-M
Diamond-Blackfan anemia and intellectual disability: a new
contiguous gene syndrome at 15q25.2
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We report on a 15-year-old girl presenting with moderate intellectual disability, delays in speech and language acquisition, strabism and severe chronic constipation looking like Hirschsprung disease in spite of the presence
of ganglion cells. Her parents are not consanguineous and in good health
but her paternal half-brother presents speech difficulties and moderate
intellectual disability. Conventional chromosome analysis was considered
as normal, but array CGH showed a 10,2Mb interstitial duplication of the
2q24.3q31.2 region. In situ hybridization of paternal metaphases revealed a
direct intrachromosomal insertion of the long segment of chromosome 2 at
band q32.3 between bands 2q24.3 and 2q31.2. The duplication observed in
the patient results from an abnormal meiotic recombination of the fathers
insertion. Given its risk of recurrence for another child, an abnormality
of the same chromosomal region has now to be searched in the girls half
brother. The function of several duplicated genes can explain the phenotype
of the patient. As it is, to our knowledge, the first 2q24.3q31.2 duplication
reported, additional patients are needed to improve the description of the
phenotype.
P11.080-M
Comprehensive sequencing of all known Joubert genes in a large
Joubert syndrome cohort in search of oligogenicity
Joubert syndrome (JS) is a ciliopathy characterized by a distinctive hindbrain malformation, ataxia and cognitive dysfunction. It is typically inherited in an autosomal recessive manner caused by biallelic mutations in one
of >20 genes, but despite the large number of causal genes, the underlying
cause remains unknown in more than one third of affected individuals. Affected individuals carrying multiple heterozygous rare deleterious variants
(RDVs) in known JS-associated genes raise the possibility of more complex
genetics. To search for evidence of oligogenicity (defined as disease causation through combined effects of two or more heterozygous RDVs) and
genetic modifiers, we used a novel molecular inversion probe-based (MIP)
capture technology to sequence all known JS-associated genes in a large JScohort. Using a recessive model where biallelic RDVs in any of the known
JS-associated genes were considered causal, we were able to determine the
cause in less than two thirds of our subjects. In the remaining subjects, only
a small number carried heterozygous RDVs in two or more JS-associated genes, and this proportion was not significantly different from that observed
in a control population. Moreover, Sanger sequencing identified a second
RDV that had been missed by MIP sequencing in several subjects, thereby
establishing a recessive cause in these individuals. Thus, our data do not
support the hypothesis that a significant proportion of JS cases are due to
oligogenicity involving RDVs in the known JS genes. Current analyses focus
on determining whether genetic burden correlates with disease severity.
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P11.081-S
Multi-gene-panel diagnostics detects MKS1- gene mutations in a boy
with Joubert-Syndrome
Mutations in the MKS1 gene are known to be a major cause for Meckel-Gruber-Syndrome, a genetically heterogeneous condition and the most common
form of syndromic neural tube defect . The MKS1 gene also accounts for a
minor fraction of the total mutational load in Bardet-Biedl-Syndrome.
We report the phenotype of a five year old boy from Austria with episodes
of apnoe during the first 7 months of life, severe hypotonia, psychomotor retardation, congenital nystagmus and a molar tooth sign detected in the MRI
of the brain, which suggested the diagnosis of Joubert syndrome.
Next-Generation sequencing based multi-gene-panel diagnostics from a
blood sample revealed two mutations located in the MKS1 gene: The first
mutation, c.1407-7_1408_35del29, is known to be a major cause for MeckelGruber-Syndrome in homozygous state. The second mutation is a missensemutation that has not yet been reported. Five bioinformatic tools predict an
alteration of protein function caused by the mutation. We therefore assume
that these two mutations in the MKS1 gene in compound heterozygous state
are causative for the phenotype.
Mutations in the MKS1 gene are primarily reported in patients with Meckel-Gruber-Syndrome, a phenotype which denotes the most severe (usually lethal) end of the spectrum of ciliopathies with occipital encephalocele
and other early embryonic malformations. Here we describe a patient with
classical Joubert syndrome which underlines the genetic heterogeneity in
Joubert syndrome and illustrates the pleiotropy of MKS1 mutations.
P11.082-M
Kabuki syndrome: clinical and molecular diagnosis in the first year of
Life
M. Dentici, A. Di Pede, F. Lepri, M. Gnazzo, M. Lombardi, C. Auriti, S. Petrocchi, E.
Pisaneschi, R. Capolino, A. Braguglia, A. Angiorni, A. Dotta, M. Digilio, B. Dallapiccola;
Bambino Ges Pediatric Hospital, IRCCS, Rome, Italy.
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drome were unknown. At present, two causative genes for KS have been
identified, the MLL2 and the KDM6A genes. In this study we analyzed the
prevalence of CHD, cardiac anatomic types and molecular characteristics of
41 patients with KS from a single institution. All patients underwent cardiological evaluation. Analysis of MLL2 and KDM6A genes was performed
through targeted resequencing, using MiSeq sequencing platform. All mutations were validated through Sanger sequencing. MLL2 mutations were
found in 34/41 (83%), including nonsense, frameshift, in-frame duplication, and missense variants. Two/41 (5%) had KDM6A mutations. Additional
chromosome rearrangements were detected in 3. CHDs in MLL2 mutated
patients (23/34=68%) included LVOTOs (bicuspid aortic valve, aortic coarctation, Shone complex) in 11/23 (48%), subaortic ventricular septal defect
in 5/23 (22%), atrial septal defect in 4/23 (17%), abnormal pulmonary venous return (APVR) in 2/23 (9%), double outlet right ventricle (DORV) in
one (4%). The patients with KDM6A mutation had normal heart. In conclusion, the high proportion of left-sided obstructions and the cardiac overlap
with Turner syndrome (LVOTOs and APVR) have been confirmed. No specific hot spots for CHD have been identified, but mutations are preferentially
located in proximal and terminal MLL2. Unusual CHD (DORV) is diagnosed
in association with an additional chromosomal rearrangement.
P11.084-M
Hypermobility in Individuals with Kabuki Syndrome
C. Stumpel, M. Klaassens, P. Staps, N. Schott;
Academic Hospital Maastricht, 6202AZ Maastricht, Netherlands.
Autosomal dominant or de novo KMT2D (MLL2) mutations account for nearly one-third cases of Kabuki syndrome (KS1, MIM 147920). Large deletions and nonsense or frameshift point mutations involving KDM6A (UTX) on
Xp11.3 are another very rare cause of this condition (KS2, MIM 300867).
Here, we describe seven new patients with de novo KDM6A mutations, which
doubles the number of reported cases with point mutations in KS2. We report, for the first time, germline missense and splice-site KDM6A mutations
and confirm their pathogenicity via multiple lines of evidence. Combined
analysis from two centres shows that less than 5% cases of Kabuki syndrome are due to KDM6A mutations.
Our findings enable a detailed review of the clinical features of KS2. We
demonstrate that the developmental delay and learning disability in KS2 is
generally moderate-severe in boys and mild-moderate in girls. Speech and
cognition tend to be more severely affected than motor development. Some
girls with KDM6A mutations may have a normal developmental profile. Similar to the commoner KS1, KS2 patients are characterized by hypotonia
and feeding difficulties during infancy and poor postnatal growth and short
ABSTRACTS POSTERS
stature. Increased susceptibility to infections, join laxity, heart, dental and
ophthalmological anomalies are common. Hypoglycaemia is more common
in KS2 than in KS1. Importantly, diagnosis on facial gestalt alone may be
difficult in many patients because the facial dysmorphism with KDM6A mutations is highly variable. Hypertrichosis, long halluces and large central
incisors may be useful clues to an underlying KDM6A mutation in some patients.
1
P11.086-M
Examining Kabuki syndrome causing mutations in Czech population
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P11.088-M
Familial case of KBG syndrome caused by a novel ANKRD11 gene
mutation
Background: KBG syndrome is an autosomal dominant disease characterized by intellectual disability, seizures, short stature, skeletal anomalies and
distinct craniofacial features. It is caused by mutations in ANKRD11 gene.
Methods: We report the cases of a 19-year-old woman and her 41-year-old
mother presenting with intellectual disability, short stature and short 5th
fingers. While the mother had already lost most of her teeth, we observed
that her daughter had macrodontia of the upper central incisors. The diagnosis of KBG syndrome was suspected. Hence, we proceeded with mutation screen of the coding region of the ANKRD11 gene.
Results: ANKRD11 gene sequencing revealed a heterozygous pathogenic
nonsense mutation, c.1318C>T [p.(Arg440*)], previously not described in
the literature.
Conclusion: Our patients showed clinical features of KBG syndrome. The
molecular analysis of ANKRD11 gene confirmed our clinical hypothesis,
which allows a more precise genetic counselling for our patients and their
family.
P11.089-S
X chromosome-linked copy number variations in Klinefelter
syndrome
M. S. Rocca1, V. Pecile2, R. Selice1, N. Caretta1, C. Foresta1, A. Ferlin1;
1
University of Padova, Padova, Italy, 2IRCCS Burlo Garofolo, Trieste, Italy.
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eyebrows, short fifth finger, and hypotonia in infancy were more frequent
in the KMT2D mutation group than in the KDM6A mutation group. Short
stature and postnatal growth retardation were observed in all individuals
with KDM6A mutations, but in only half of the group with KMT2D mutations.
The genetic basis of the patients who tested mutation-negative (20-45%)
remains elusive. Further studies are necessary to understand the whole picture of the genetic aspects of KS and its genotype-phenotype relationships.
P11.091-S
Large cryptic genomic rearrangements with apparently normal
karyotypes detected by array-CGH
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P11.093-S
Phenotype of two patients with mandibulofacial dysostosis with
microcephaly (MFDM) associated with esophageal atresia and
choanal atresia caused by EFTUD2 mutations
Progeroid features with concomitant cardiac, skeletal, and muscular anomalies, lipodystrophy and insuline resistance define rare genetic diseases
named laminopathies, caused by mutations in genes encoding nuclear proteins.
Here, we report a case of a 13-yr-old girl, second-born of non consanguineous parents, who showed retarded growth, anemia and xerotic skin
at age 1. She showed triangular facies, micrognathia, low-set and small
ears and sensorineural deafness at age 8. Other clinical features included
hypertrichosis, a notable subcutaneous fat loss from her extremities with
accumulation at abdomen level and insuline-resistance. She also presented
generalized hypotonia, severe muscular hypotrophy and joint contractures.
Given that the clinical overlapping with progeroid disorders, genetic analysis of known mutated genes was performed. We identified an in frame single
codon deletion (c.1812_1814delCTC, p.S605del) in POLD1 gene, encoding
DNA polymerase , has been recently associated to a multisystem disorder
named MDP syndrome, characterized by Mandibular hypoplasia, Deafness
and Progeroid features, detected in other four MDP patients.
The case underlines the clinical and genetic heterogeneity of patients with
adipose tissue, skeletal and muscular anomalies, associated to progeroid
features.
P11.095-S
Loss-of-function mutations in MED13L cause a distinctive clinical
phenotype mimicking the 1p36 deletion syndrome
ABSTRACTS POSTERS
the 1p36 deletion syndrome and with normal array-CGH (15kb resolution). By means of WES in two of them, we found two causative mutations:
p.M303K in SYT1 (subject 1) and p.P1255Pfs*3 in MED13L (subject 2). The
missense mutation in SYT1 involves an essential functional domain. By
Sanger sequencing of both genes in the other subjects, we identified another MED13L mutation (p.S203Sfs*32) in one. All mutations were de novo.
Missense mutations involving MED13L have already been identified as responsible for isolated congenital heart defects. One only literature report
deals with complete or partial gene deletion, which were also associated to
intellectual disability (ID). Mutations in SYT1 have never been described in
humans.
Our three patients presented with moderate ID and shared facial features,
including brachycephaly, horizontal eyebrows, high forehead, long eyelashes, depressed nasal bridge, mid-face hypoplasia. Additional clinical signs
were epilepsy (subject 1), cleft palate, clubfoot and conductive hearing loss
(subject 2) and atrial septal defect (subject 3). Patient 2 was diagnosed with
acute lymphoblastic leukemia.
These results suggest that the haploinsufficiency of MED13L causes a typical phenotype that should be considered in the differential diagnosis of the
1p36 deletion syndrome. We tentatively suggest SYT1 as another candidate
gene for the same clinical presentation, but it needs to be further confirmed.
P11.096-M
An interstitial microdeletion of 20q11.21 in a boy with
cheilognathopalatoschisis, anorectal malformation, severe
microcephaly, craniofacial features, feeding difficulty, mild growth
impairment, and mild intellectual disability
Interstitial microdeletions involving 20q11.2 are very rare with only four
reported patients. We
have identified a de novo interstitial 20q11.2 microdeletion in a 7-year-old
boy, clinically showing cheilognathopalatoschisis, anorectal malformation,
severe microcephaly, craniofacial features (triangular face, hypertelorism,
hypoplastic alae nasi, long philtrum, low set ears), feeding difficulties, mild
growth impairment, mild intellectual disability (IQ67), and attention-deficit
hyperactivity disorder. G-banded chromosomes were normal, cytogenomic
microarray (135K Oligo, Roche) revealed a de novo 1.15 ~ Mb microdeletion at 20q11.2 [arr[hg18] 20q11.21(29,297,619-30,447,117)x1 dn]. The
deleted segment encompassed 15 OMIM genes (COX4I2, MYLK2, ASXL1,
etc). The sizes of deleted segments in four reported patients were 2.6Mb,
6.5Mb, 6.6Mb, and 6.8Mb[Iourov et al.2013; Hiraki et al.2011; Callier et
al.2006;Iqbal et al.2007; ]. Clinical features shared by those with > 6Mb deletion included feeding difficulty, facial features (triangular face, hypertelorism, hypoplastic alae nasi, long philtrum, and low set ear), developmental
delay and/or intellectual disability, though the other with 2.6Mb had a milder manifestation. The present patient with the smallest deletion showed
clinical features of previously reported patients with 20q11.2 microdeletion. Although long-term follow-up and collection of additional patients is
needed to delineate the phenotypic spectrum of the condition, we propose
the microdeletions at 20q11.2 to be a clinically recognizable syndrome characterized by craniofacial features, feeding difficulty, growth impairment,
and intellectual disability.
P11.097-S
The clinical phenotype of 3q29 microdeletion and 3q29
microduplication syndrome in three female patients
It has been demonstrated that whole genome scanning technologies (arrayCGH) is especially suited to identify chromosome abnormalities in individuals with unclear or variable presentations. Application of this technology
resulted in the delineation of several previously unrecognized microdeletion/microduplication syndromes such as recently described del3q29 (OMIM
609425; ORPHA65286) and dup3q29 (OMIM 611936; ORPHA251038) syndromes with highly variable clinical phenotypes.
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The most common features of del3q29 phenotype include mild-to-moderate intellectual deficit and slightly dysmorphic facial features: microcephaly,
long and narrow face/asymmetric face, short philtrum, large posteriorly rotated ears, high nasal bridge, crowded/dysplastic teeth, tapered fingers with
occasionally observed autism and gait ataxia. Clinical features of dup3q29,
included micro/macro-cephaly, round face, bulbous nose, short or downslanting palpebral fissures, excessive hand creases, obesity and pes planus.
Herein, we report on two unrelated female patients with different phenotypic
consequences (high interindividual phenotypic variability) due to de novo
deletion of an identical segment [spanning ~2.0Mb (start 195,438,699bp
- 197,404,251bp)] and one female patient with de novo duplication of
3q29 which overlap this region [spanning ~1.58Mb (start 195,740,387bp
- 197,317,074 bp)].
P11.098-M
Molecular analysis of ACTG2 in a cohort of patients with Megacystis
Microcolon Intestinal Hypoperistalsis Syndrome suggests locus
heterogeneity
We report on a 2 year-old boy with mental retardation, muscular hypotonia, deafness with atresia of the external auditory canal and a preauricular
tag on the right side. The boy was referred for global intellectual disability with very limited speech capabilities and autistic traits. The pregnancy
had been uneventful except for an emergency Cesarean section because of
breech position. Birth measurements were normal, however, the baby was
hypotonic and exhibited difficulties in feeding. At physical examination at
the age of 2 years measurements were still normal but we noted discrete
dysmorphic features: long eye lashes, broad mouth with cupid bow lips,
small fingers with broad short endphalanges, clinodactyly of toes V on both
sides, tender translucent skin, fine hair and cryptorchidism on the right
side. On the MRI at the age of 2 years delayed myelinisation was visible.
As the atresia of the external auditory canal is typical for a deletion 18q we
performed a karyotype on lymphocytes which was first reported with no
pathological findings.
The array (2.7 Affymetrix SNP-oligo array platform) analysis revealed a deletion in mosaic form of 20.7 Mb of the long arm of chromosome 18, encompassing 69 genes in total. The mosaic was confirmed with FISH, which also
revealed that the mosaic ratio in this case was 13% in cultivated blood and
81% in buccal swab. Reevaluating the karyotype the deletion was visible.
This case demonstrates the higher sensitivity of SNParrayCMA testing for
mosaic aberrations.
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ABSTRACTS POSTERS
P11.100-M
Molecular diagnostics of rare hereditary diseases using next
generation sequencing
O. Mazal, J. tika, J. Sabov;
Synlab genetics, Prague, Czech Republic.
We report a Caucasian man first seen at age of 25 months, referred with the
diagnosis of Cerebral Palsy due to prematurity. Furthermore, he presented
severe developmental delay, hypotonia, seizures, failure to thrive, dysplasia
of the right optic nerve, and pulmonic stenosis. We also found dolichocephaly, open fontanel, hypertelorism, large ears with posterior pits, wide neck,
mild pectus excavatum, short 4th metacarpals, and minor ridge dysplasia
and radial loops in the indexes and right 3rd finger. Chromosomes and metabolic tests were normal. Because features of a RASopathy, sequence analysis
of the PTPN11, RAF1, SOS1 and KRAS genes was performed, but no mutations were found. At age 21, his older sister was diagnosed with plexiform
neurofibromas, requiring amputation of a leg. CT scan of the proband after
accident unexpectedly showed massive plexiform neurofibromas infiltrating
the spine, pelvis and extremities. Molecular testing showed an NF1 mutation, c.2326-6T>G, resulting in in-frame skipping of exon 20 (r.2326_2409del),
therefore expected to result in a protein lacking 28 amino acids from within
the Cystein-Serine Rich Domain (CSRD) (p.Trp777_Ala804del). This is a
private mutation, not previously reported and observed in 1/6,500 NF1positive unrelated patients (UAB cohort). This was also found in his sister
and father who both developed spinal neurofibromas but no classic skin
findings (cutaneous neurofibromas, CALMs, freckling). This family illustrates the importance of molecular confirmation in patients with minimal or
no pigmentary manifestations and with features of other RASopathies. Our
proband, who was exceptional despite of all his limitations, died at age 26
(January 2014).
P11.102-M
Unexpected karyotypes in patients with non-syndromic phenotype
studied in the postnatal period
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ABSTRACTS POSTERS
important mechanism that underlies persistent activity-induced changes in
the brain function in the process of neuronal plasticity. While the Ptpn11D61Y mutation is usually found to evoke higher levels of phosphorylated
ERK (pERK), we found no differences in the nuclear level of pERK comparing wild type and mutant cells under basal conditions. However, neuronal
activity-driven induction of nuclear translocation of pERK was affected in
the mutant neurons, suggesting a dysregulation of the activity-induced signaling. In line with this finding, we found differences in the size of total
recycling synaptic vesicle pools, which is a subject of regulation during
homeostatic adaptation in neurons.
The lacking response to stimulation found in the Ptpn11D61Y neurons suggest a deficit in cellular signaling underlying usage-dependent neuronal plasticity and might contribute to the intellectual disability found in patients
with NS.
P11.105-S
Cryptorchidism and pulmonary stenosis as the most important
features in Noonan patients with mutation in PTPN11 in Slovak
population
M. Gos , S. Fahiminiya , J. Klapecki , E. Obersztyn , A. Abramowicz , A. KutkowskaKamierczak1, M. Piotrowicz3, J. Pilch4, R. Posmyk5, J. Wierzba6, J. Bal1, J. Majewski2;
1
Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland,
2
Department of Human Genetics, McGill University and Genome Quebec Innovation
Center, Montreal, QC, Canada, 3Department of Genetics, Polish Mothers Memorial
Hospital - Research Institute, d, Poland, 4Department of Pediatric Neurology, Medical
University of Silesia, Katowice, Poland, 5NZOZ Genetics, Center for Clinical Genetics,
Biaystok, Poland, 6Department and Clinic of Pediatrics, Hematooncology, Oncology and
Endocrinology, Department of General Nursing, Medical University of Gdask, Gdask,
Poland.
1
Noonan syndrome (NS) is a rare disease belonging to the group of RASopathies caused by the germline mutations in genes encoding proteins of RAS/
MAPK signaling pathway. The majority (about 50%) of cases is caused by
PTPN11 mutations. Also mutations in RAF1 and SOS1 are quite common (up
to 17 and 13%, respectively). The aim of the study was the identification
of molecular defect responsible for the NS phenotype using whole exome
sequencing (WES). Forty two patients with primary clinical diagnosis of
Noonan syndrome and excluded mutation in PTPN11, SOS1 and RAF1 genes were included in the study. The WES was performed using Illumina
sequencing platform. The WES analysis allowed for the identification of
mutation in RASopathies-related genes in 7/42 (16.7%) NS patients. The
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227
ABSTRACTS POSTERS
thway, respectively. The present observation is in accordance with the hypothesis that OAVS is a genetically heterogeneous condition, underlying the
importance of SNP array analysis in patients with OAVS features.
P11.109-S
Screening of CD96 and ASXL1 in twelve patients affected with Opitz C
or Bohring-Opitz syndromes
Opitz trigonocephaly (or C syndrome, OTCS) and Bohring-Opitz syndrome (or C-like syndrome, BOS) are two rare genetic disorders which show
phenotypic overlap. The genetic bases of these diseases are not well understood. Two genes have previously been associated with OTCS or BOS with a
dominant pattern of inheritance. Whereas the CD96 gene has been related
to OTCS (one case out of 25) and to BOS (one case in 4 patients analyzed),
ASXL1 (Additional Sex Combs-Like 1) has been related to BOS only (around
half of the patients). In this study we analyzed CD96 and ASXL1 in a cohort
of twelve individuals, including two sibs. Eight of them were diagnosed with
OTCS, two displayed a BOS phenotype and another two could not be accurately diagnosed. Exome sequences were available for six patients with OTCS
and three couples of parents, in which CD96 and ASXL1 were inspected
using bioinformatic tools. For the remaining patients, Sanger sequencing of
all exons in these genes was carried out. Detailed scrutiny of the sequences
allowed identification of only one potentially pathogenic mutation in one of
the patients. In this subject, affected with BOS, we identified a de novo mutation in ASXL1 (c.2100dupT). By nature and location within the gene, this
insertion resembles those previously described in other BOS patients and
we conclude that it may be responsible for the disease. Our results indicate
that for eleven out of the twelve patients, the disease (OTCS or BOS) is not
caused by mutations in CD96 or ASXL1.
P11.110-M
A case of Opitz GBBB syndrome: clinical presentation
E. Caramaschi, F. Lami, E. Bigi, A. Guerra, A. Percesepe;
Universit degli studi di Modena e Reggio Emilia, Modena, Italy.
Opitz GBBB syndrome is a rare congenital syndrome characterized by hypertelorism, hypospadias, cleft lip/palate, laryngotracheoesophageal abnormalities, imperforate anus, developmental delay, and cardiac defects. Opitz
GBBB syndrome is genetically heterogeneous, with both X-linked and autosomal dominant forms. X-linked form of Opitz GBBB syndrome is caused by
mutation in the MID1 gene (OMIM 300552).
We here report two male cases with Opitz GBBB syndrome. One of them
was referred with preliminary diagnosis of FND due to hypertelorism at
228
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Overgrowth syndromes (OGS) are a group of disorders in which all parameters of growth and physical development are above the mean for age and
sex. Partial, localized, or regional OGS are those disorders in which excessive
growth is confined to one or a few regions of the body. We evaluated a series
of 270 families from the Spanish Overgrowth Syndrome Registry with no
known overgrowth syndrome. We identified a deletion and three missense
mutations in a new gene in six patients from 4 families with overgrowth,
macrocephaly, intellectual disability, mild hydrocephaly, hypoglycemia and
inflammatory diseases resembling Sjgren syndrome. Our studies of this
gene point to disruption of three important signaling pathways as the putative final effectors.
P11.113-S
An apparently balanced translocation in a boy with multiple
congenital anomalies of the eye
A 2 year old boy product of the 1st pregnancy was referred for karyotype
testing because of multiple congenital anomalies, including bilateral cataracts diagnosed at birth, hypospadias and right cryptorchidia, hypotonia
and failure to thrive Glaucoma was diagnosed at 3 months of age. MRI showed generalized brain atrophy. At one year of age developmental delay was
noted and height, weight, and head circumference had fallen below the 3rd
percentile. There was no family history of similar findings and the parents
are first cousins. Karyotype showed an apparently balanced translocation
46,XY,t(3;6)(p25;q15). Karyotype of both parents was normal. Further testing for genes involved in anterior segment dysgenesis and/or congenital
cataracts including PITX3, FOXE3, and CYP1B1 was done and all were negative for mutations. Sequencing the breakpoints of the translocation did not
find genes at or near the direct breakpoint. Whole exome sequencing ruled
out mutations in known cataract genes, but identified a homozygous mutation in the PAH gene (NM_000277:c.1139C>T, p.(Thr380Met). This mutation
has been reported to cause Hyperphenylalaninemia (HPA) when combined
with another deleterious mutation. Homozygosity for this mutation has not
been reported previously, to our knowledge. We are confirming this result
by Sanger sequencing and analysis of parental samples is ongoing. We are
still investigating the link between HPA/PKU and congenital cataracts (and
ABSTRACTS POSTERS
reviewing variants in other genes in the WES data), but untreated PKU has
been clearly linked to microcephaly and learning disabilities.
P11.114-M
New severe learning difficulty syndrome with skeletal features due to
de novo missense mutation in the polycomb group ring finger protein
2 (PCGF2) gene
P. D. Turnpenny1, M. Wright2, M. Sloman3, D. D. D. Study4;
1
Clinical Genetics, Royal Devon & Exeter Hospital, Exeter, United Kingdom, 2Newcastle
upon Tyne Hospitals, Newcastle, United Kingdom, 3Molecular Genetics, Royal Devon &
Exeter Hospital, Exeter, United Kingdom, 4Wellcome Trust Sanger Institute, Cambridge,
United Kingdom.
Two UK patients, each known to a clinical genetics service (Northern, Peninsula), were independently recruited to the Deciphering Developing Disorders (DDD) study, Cambridge. Patient 1 presented with poor weight gain
and hypotonia in infancy, and subsequently global developmental delay. He
had relative macrocephaly, enlarged cerebral ventricles, and normal myelination on brain MRI at 17 months. At 2 years a skeletal survey revealed
delayed epiphyseal ossification, particularly carpal bones, pseudo-epiphyses of many metacarpals, hypoplasia of L1, and thoraco-lumbar kyphosis. He
has severe intellectual disability, no clear speech, drools heavily, has short
tapering fingers and valgus deformity of the feet, and multiple pigmented
naevi. Patient 2 presented at 6 months of age with poor weight gain. Subsequently her development was delayed with a moderate learning disability.
She has relative microcephaly; an MRI scan performed at 7 years of age was
normal. She developed constipation which was sufficiently severe to require
an ACE procedure aged 12 years. She has fine hair, a long face with high
arched palate and prognathism, long thin hands and fingers and mild generalised joint hypermobility. She does not have spine or foot abnormalities.
Both subjects were found through DDD to have a de novo p.P65L (c.194C>T)
missense variant in exon 4 of the PCGF2 gene (17q12). Previously, PCGF2
had no associated human phenotype. This identical de novo finding in 2 subjects is strong evidence for a new syndrome associated with a novel morbid
gene, despite some divergence of clinical features.
P11.116-M
A nonsense mutation in BMP2 causes a syndromic form of Pierre
Robin sequence
We report on a 6 year boy with body overgrowth at birth, megalencephaly and motor delay, who is a carrier of a de novo heterozygous mutation
in PIK3CA (NM_006218.2):c.335T>A, p.Ile112Asn. The mutation appeared
germline in peripheral blood, buccal swabs and skin fibroblasts (Sanger sequencing); it has not been observed in published PIK3CA-related segmental
overgrowth patients. Phosphatidylinositol-3,4,5-triphosphate immunostaining of patient cells and Epidermal Growth Factor-mediated PI3K-AKTmTOR pathway stimulation proved that p.Ile112Asn leads to increased PI3K
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activity. The p.Ile112Asn change lies adjacent to the p85 (PIK3R2) regulatory binding domain on the p110 (PIK3CA) catalytic subunit of PI3K. Altered
stoichiometry within the p85-p110 complex could underlie the hyperactive
PI3K-AKT-mTOR signaling in this instance. Further experiments to investigate this are currently ongoing.
Interestingly, the phenotype of this child appears unique comparing to
published PIK3CA-related segmental overgrowth patients. The boy did
not show somatic asymmetry, focal overgrowth, capillary malformations,
epidermal nevi, or digital abnormalities. His brain MRI at 6 years showed
a thick corpus callosum, large cerebellar vermis and possible restricted
right posterior persiylvian polymicrogyria but his cerebral cortex otherwise
looked largely normal.
Our observation adds an isolated megalencephaly with mild body overgrowth to the PIK3CA-associated clinical spectrum. The identified novel mutation is among the few known germline PIK3CA mutations. We demonstrate
robust and rapid functional assays to enable interrogation of PI3K-activity
in this context from patient-derived cells. The constitutional distribution of
PIK3CA mutation might be of prognostic advantage regarding somatic overgrowth, cortical malformations and consecutively favorable development.
However, this hypothesis needs further assessment.
P11.118-M
A clinical score system as useful tool in selecting subjects with clinical
presentation in the spectrum of Pitt-Hopkins syndrome
M. Zollino1, S. Ricciardi1, D. Orteschi1, M. Murdolo1, R. Tenconi2, A. Baroncini3, B.
Dallapiccola4, E. Ponzi1, A. Asaro1, E. Mercuri5, G. Marangi1;
1
Institute of Medical Genetics, Catholic University, Roma, Italy, 2University of Padova,
Padova, Italy, 3Medical Genetics Unit, ASL of Imola, Imola, Italy, 4Bambino Ges
Childrens Hospital and Research Institute, Roma, Italy, 5Department of Paediatric
Neurology, Catholic University, Roma, Italy.
We describe the case of a 3 years 6/12 old girl presenting marked developmental delay, hypotonia, and dysmorphisms. She was born at term from
apparently healthy, non consanguineous parents. Pregnancy and delivery
were uncomplicated except for polyhydramnios. Neonatal growth parameters were in the normal range. She showed right choanal stenosis and velopharyngeal incompetence which made feeding difficult. A brain magnetic
resonance imaging at age 14 months showed a retarded myelination in parietal and periventricular areas.
At last evaluation general hypotonia was still present, with global developmental delay and absent speech. Weight and height were in the upper part
of the normal range, while cranial circumference was slightly under the average. She presented facial and skeletal dysmorphisms, including depressed
nasal bridge, alternating esophoria, protruding tongue, pectus excavatum,
hyperextensible ligaments, thoracic kyphosis, bilaterally recessed IV toes,
broad halluxes.
229
ABSTRACTS POSTERS
Array-CGH analysis disclosed a de novo 5.6 Mb microdeletion of Xq22.1q22.3 chromosomal region. The pattern of X-inactivation was markedly
skewed. The microdeleted region includes 52 genes 3 of which are OMIM
morbid: PLP1, RAB40AL and SERPINA7.
There have been only a few reports of whole PLP1 gene deletions. PLP1 mutations have been associated with a continuum of neurologic phenotypes
from severe forms of Pelizaeus-Merzbacher disease (PMD) to spastic paraparesis. Null alleles tend to associate with milder PMD features in hemizygous males but higher rate of neurological manifestations in heterozygous
females. PLP1 haploinsufficiency could explain at least part of the girls neurological phenotype since it encodes for a primary constituent of myelin in
the brain.
P11.120-M
Dissecting the genetic bases of Poland Syndrome by exome
sequencing
Noonan syndrome (NS) is a genetically heterogeneous developmental disorder characterized by reduced growth, dysmorphic facial features, congenital
heart defects, skeletal and hematological anomalies, and variable cognitive
deficits. NS is caused by aberrant signaling through the RAS-MAPK cascade,
and we previously identified PTPN11 as the major gene underlying this condition. PTPN11 encodes SHP2, an SH2 domain-containing protein tyrosine
phosphatase functioning as a signal transducer that positively modulates
RAS function. Mutations in the same gene are implicated in LEOPARD syndrome (LS), a disease clinically related to NS, or contribute to leukemogenesis. Here, we explored further the molecular spectrum of germline PTPN11
230
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lesions and their associated phenotype. Mutation scanning of the entire PTPN11 coding sequence was performed in large NS/LS cohorts collected in
the frame of the NSEuroNet Consortium. Among the 452 mutation-positive
subjects, 68 different variants deemed to be of pathological significance, including 12 novel missense changes and 2 in-frame indels, were identified.
Besides the previously characterized mutations destabilizing SHP2s inactive state or increasing binding to phosphotyrosyl-containing partners, a novel mutation cluster was recognized. Specifically, eleven changes affecting
Leu261, Leu262 and Arg265 were identified in ten unrelated subjects with clinical features fitting NS. Biochemical and structural characterization of these
mutants provided novel insights on disease pathogenesis. Finally, these data
and published records were used to define more accurately the mutational
spectrum of germline and somatic PTPN11 mutations in human disease.
P11.122-M
A paternally inherited interstitial deletion of 15q11.2 causing clinical
features of PWS: refinement of the PWS-IC
T. N. Nelson1, L. Brown1, K. Schlade-Bartusiak1, E. Gagne1, J. Livingstone1, S. Anson2, L.
Armstrong1;
1
University of British Columbia, Vancouver, BC, Canada, 2Childrens & Womens Health
Centre of British Columbia, Vancouver, BC, Canada.
ABSTRACTS POSTERS
13 years old female was referred for genetic counseling because of intellectual disability (ID), short stature, microcephaly, dysmorphic features (low
frontal hairline, left convergent strabismus, wide nose root, abnormal shape of the pinnas, flat philtrum, large mandible, hypodontia, malocclusion
of teeth), club-foot, partial syndactyly of II-III th toes. A sister of proband`s
mother presented with ID, delay of speech, club-foot and additional features
not common to proband: normal stature, obesity, short philtrum, high narrow palate, crowded teeth, IIth toes overlapping thumbs. Mothers second
sister died in infancy because of multiple congenital anomalies. Array-CGH
of proband`s DNA revealed trisomy of the distal bands of chromosome 10
short arm (10pter-->10p15.1) and monosomy of the distal bands of chromosome 10 long arm (10q26.12-->10qter). Subtelomeric FISH analysis confirmed a rearranged chromosome 10 [rec dup(10p] due to a large, maternal
pericentric inversion. A reverse segmental imbalance [rec dup(10q)] was
detected in proband`s aunt by subtelomeric FISH. The high frequency of recombinants in this family and data from literature review suggests a high
recurrence risk in similar cases with large pericentric inversions comprising
almost entire chromosomes. The research leading to these results was funded by the Lithuanian-Swiss cooperation programme to reduce economic
and social disparities within the enlarged European Union under project
agreement No. CH-3-MM-01/04, UNIGENE project.
P11.125-S
Functional characterization of L440Xfs mutation in the thyroid
hormone receptor beta (TR) in an individual with RTH syndrome
Back to index
Schinzel-Giedion syndrome was first described in 1979, and is characterized by typical facial gestalt, severe developmental delay, frequent epilepsy
and various malformations (urinary tract, genitalia, heart and skeletal). The
reported cerebral malformations are ventriculomegaly, thin or absent corpus callosum, cortical atrophy, gyration anomalies. Hypoplastic pons was
found in 2 patients.
In 2010, SETBP1 gene was identified as responsible for Schinzel-Giedion
syndrome, with identification, by exome, of de novo missense mutations,
some recurrent, all localized in the SKI oncogene homologous domain.
We present 5 novel patients with Schinzel-Giedion syndrome (2 girls and 3
boys) with mutation in SETBP1 (proven de novo for 3). Two mutations were
previously reported (c.2602G>A, c.2608G>A) and the 3 others were novel
(c.2607C>A, c.2608G>T, c.2606G>C). All patients had typical facial gestalt,
severe developmental delay, epilepsy, genitalia anomalies. Hydronephrosis
was present in 4 patients, and the 5th patient had coralliform kidney stones
with recurrent infections. Cerebral MRI showed constant corpus callosum
anomalies, and frequent cortical atrophy, ventricular dilatation, septal rupture. Two patients had gyration anomalies. Four patients displayed similar
posterior fossa anomalies with hypoplastic pons and enlarged medulla oblongata, 3 patients had dysplastic cerebellar hemispheres. Atrophic caudate
nuclei was noted in 4 patients.
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ABSTRACTS POSTERS
These novel cases further delineate the cerebral spectrum of malformations in Schinzel-Giedion syndrome, in particular with similar posterior fossa
anomalies.
The 3 novel mutations, as all the previously described mutations, are localized in the known mutational hotspot.
P11.129-S
Towards a better understanding of de novo germline mutations in
SETBP1 in Schinzel-Giedion syndrome
Schinzel-Giedion syndrome (SGS) is a rare and severe developmental disorder characterized by malformations in multiple organ systems. Exome sequencing recently identified the cause of SGS as de novo germline mutations
clustering within a 12-bp exonic region in SETBP1. Mutations in this SETBP1
hotspot are presumed to be gain-of-function mutations which disrupt a degron (a consensus motif for protein degradation), disturbing normal SETBP1 proteolysis. Interestingly, somatic mutations in this hotspot have also
been found in leukemic cells and shown to increase cell proliferation in this
context.
We have collected clinical information and samples from over 25 SGS patients, which to our knowledge constitutes the largest cohort of SGS patients
yet. To better understand germline de novo mutations in SETBP1, we investigated the parental origin of the allele in which these mutations occur in
SGS. Using a combination of long range PCR and single molecule real time
sequencing (Pacific Biosciences), we determined the parent of origin for
four out of five trios analyzed. In three out of four cases, the de novo mutation in SETBP1 occurred in the paternal allele and only in one in the maternal
allele. Additionally, we examined the molecular and cellular consequences
of SETBP1 mutations in the context of SGS. When compared to controls, fibroblast and lymphoblastoid cell lines of SGS patients have alterations in
downstream targets of SETBP1, such as higher levels of SET protein and deregulated gene expression.
Our studies provide insight into the occurrence and molecular consequences
of de novo germline SETBP1 mutations causing SGS.
P11.130-M
5p15.33-31 deletion and unilateral open lip schizencephaly: causal or
casual association?
A. V. Gulino1,2, G. Traficante1, I. Sani3, S. Guarducci3, A. Petracca1, S. Bargiacchi1, C.
Barba4, M. Falchi4, R. Guerrini4, S. R. Giglio1,3;
1
Department of Clinical and Experimental Biomedical Sciences, University of Florence,
Florence, Italy, 2Paediatric Department, Assisi Hospital, Assisi, Italy, 3Medical Genetics
Unit, Meyer Childrens University Hospital;, Florence, Italy, 4Department of Neurology,
Meyer Childrens University Hospital, Florence, Italy.
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Leri-Weill dyschondrosteosis (LWD) is a dominantly inherited genetic disorder characterized by short stature, mesomelia and Madelung wrist deformity. The LWD is caused by haploinsufficiency of the Short Homeobox contaning gene (SHOX), located in the 2.6 Mb pseudoautosomal (PAR1) region. At
the heterozygous level most mutations found are of different size deletions,
point mutations within the coding sequence or mutations on regulatory enhancers downstream of the SHOX gene. We present the clinical and molecular data of a three generation family with LWD. The index patient is a 43 year
old female with disproportionate short stature, reduced arm span to height
ratio (particularly mesomelic shortening), a muscular body habitus and no
evidence of a classic Madelung deformity. We report for the first time a novel 15kb deletion detected by microarray-CGH using a chromosome X exonspecific array (OGT) in the index patient. The deletion resides in the coding
region of the SHOX gene, includes exons 3-6 containing the homeodomain
region. RT-PCR studies confirmed the deletion and revealed the same 15 kb
deletion in her sister, father and son. The youngest male member of the family has a very mild form of LWD, a phenomenon previously observed, that
SHOX deficiency is more pronounced in females than in males and it can
also exhibit inter-familial and intra-familial variable expressivity. Extended
family studies (paternal side) have identified another 5 members to carry
the deletion. Detailed clinical evaluation of these family members is ongoing
which will shed more light in the phenotypic effect of this deletion.
P11.133-S
SKI gene analysis clarifies the diagnosis in cases suspected of
Shprintzen-Goldberg syndrome
T6857
T7178
female, 2y
male, 28y
c.95T>A
(p.Leu32Gln)
+
+
+
+
+
+
+
+
+
+
+
+
+
c.100G>T
(p.Gly34Cys)
n/a
+
+
-
T7386
male,
46y
T7424
male, 29y
none
c.185C>G
(p.Ala62Gly)
n/a
+
+
-
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
n/a
n/a
+
+
ABSTRACTS POSTERS
In conclusion, the molecular analysis of SKI should be considered to differentiate
between marfanoid phenotypes such as MFS, LDS and SGS.
P11.134-M
Submicroscopic genomic alterations detected by array CGH analysis
in a cohort of patients with Silver Russell syndrome found negative to
classical genetic and epigenetic tests
Silver Russell syndrome is characterized by pre- and postnatal growth retardation, variable facial dysmorphisms, clinodactyly of the fifth fingers, and
sometimes asymmetry of face, trunk and extremities. Genetic and epigenetic
aberrations on chromosomes 7 and 11 are commonly found in SRS, leaving
out however, a fraction of up to 50% of cases with unknown genetic aetiology. A cohort of 32 clinically selected SRS patients, without detected genetic
or epigenetic alterations was analyzed by high resolution array CGH analysis
to identify possibly pathogenetic CNVs. Twenty-eight patients (87.5%) were
found to carry one or more rare CNVs, according to the Database of Genomic
Variants and, overall, 55 rare CNVs, 25 gains (45.5%) and 30 losses (54.5%)
were identified. Inheritance, established for 36 of the identified rare CNVs,
showed that 7 occurred de novo (19.5%). Interrogation of public databases
allowed us to pinpoint genomic regions containing genes, either imprinted
or not, that according to their function, appeared plausible candidates for
SRS. Interestingly 4 CNVs span genomic regions already associated with
growth defects, 3 CNVs contain known genes found altered in previously
described SRS patients and 3 CNVs include genes implicated in growth control pathways not yet associated with SRS. These results confirm the genetic
heterogeneity of SRS and the high percentage of potentially causative imbalances, attesting the wide clinical expressivity of patients with a phenotype
strongly suggestive for this syndrome. Genome-wide scan is reconfirmed an
appropriate and powerful tool to achieve a differential diagnosis between
SRS and SRS-like patients.
P11.135-S
Mosaicism of the H19 hypomethylation in a patient with very low
weight and severe insulin resistance
Back to index
P11.136-M
Regulatory element deletion cause a down-regulation of ZDHHC15
gene in a proband with Smith Magenis syndrome phenotype
Here, we describe a boy aged 3 years who showed mild facial minor anomalies such as brachycephaly, square face, thick eyebrows, broad palate, generalized hypotonia, developmental delay, behavioural problems (self injury),
sleep disorders and congenital heart defect.
Based on suspected Smith Magenis syndrome (SMS), chromosomal analysis was performed and showed a 46,XY karyotype. FISH analysis of the SMS
locus at 17p11.2, as well as MLPA, mutational analyses and quantitative expression of the RAI1 gene, gave normal results. High-resolution array-CGH
analysis disclosed two rare maternal deletions at 4q35.2 and Xq13.3, both
yet unreported in healthy subjects according to the Database of Genomic Variants. Whereas it is not possible to assign a pathogenetic role to the 4q35.2
deletion, the Xq13.3 loss of 54 kb involves a predicted conserved insulator
that maps 29 kb far from the 5 end of the ZDHHC15 gene. ZDHHC15 acts as
a palmitoyl-transferase in brain, and its null expression has been reported
in a syndromic patient. We thus investigated in the patients blood the causative role of the identified CNV on ZDHHC15 by RT-qPCR. The ZDHHC15
expression level was significantly reduced in the male proband compared to
controls, whereas the expression level in the mother was within the control
range.Our results suggest the involvement of ZDHHC15 perturbation in the
onset of the probands phenotype and point to this gene, sharing interactors
with RAI1, as a novel candidate gene for SMS.
P11.137-S
A novel NSD1 mutation in Sotos syndrome with constriction of vena
cava
We present a 2 year-old male patient with poor growth, developmental delay and epilepsy. He is the second-born child of healthy non-consanguineous
parents, with an unremarkable family history. He was born at gestational
week 40 after an uncomplicated pregnancy. Seizures appeared at 16 months
and the EEG revealed focal abnormalities in the left frontal and parietal
regions during sleep. The array-CGH analysis identified a de novo 2.32 Mb
233
ABSTRACTS POSTERS
deletion on chromosome 8q23.1q23.2 due to an unbalanced translocation
t(8;17)(q23;q24). The analysis detected two additional anomalies which
were both inherited from the healthy father: a 165.8 Kb duplication involving chromosome 7p15.2 and a 914.6 Kb duplication involving 22q13.2. In
the deleted region, SYBU and KCNV1 appear to be candidate genes for the
patients neurologic and electroclinical phenotype. In fact, SYBU encodes
a protein which is part of a kinesin motor-adaptor complex that is critical
for the anterograde axonal transport and contributes to activity-dependent
presynaptic assembly during neuronal development. KCNV1 encodes a neuronal modulatory subunit of a voltage-gated potassium channel and it is
predominantly expressed in the brain.
P11.140-M
TBC1D7 mutations are associated with intellectual disability,
megalencephaly, patellar dislocation and celiac disease
Mutations in both TSC1 and TSC2 cause the tuberous sclerosis complex
(TSC), a multisystemic disorder characterized by the development of hamartomas or benign tumors in various organs as well as epilepsy, intellectual disability (ID) and autism. Whereas the binding of TBC1D7, the third
constitutive subunit of the TSC1-TSC2 complex, is required to maintain its
integrity, sequencing of TSC patients with no TSC1-TSC2 mutations indicated that TBC1D7 is unlikely to represent a TSC3 gene. Loss of function of
TBC1D7 results in an increase in mTORC1 signaling, and consequently a delay in the induction of autophagy.
Mutations in TBC1D7 were recently reported in a family with ID and macrocrania. Using exome sequencing we identified two sisters homozygote for a
novel TBC1D7 truncating mutation. In addition to the already described macrocephaly and mild ID, they share osteo-articular defects, patella dislocation, behavioral abnormalities, psychosis, learning difficulties, celiac disease,
prognathism, myopia and astigmatism. Consistent with a loss-of-function of
TBC1D7 the probands cell lines show an increase in the phosphorylation of
4EBP1, a direct downstream target of mTORC1 and a delay in the initiation
of the autophagy process.
This second family allows enlarging the phenotypic spectrum associated
with TBC1D7 mutations and defining a TBC1D7 syndrome. Our work reinforces the involvement of TBC1D7 in the regulation of mTORC1 pathways
and suggests an altered control of autophagy as possible cause of this disease.
P11.141-S
Temple syndrome - introducing a new name for a characteristic
chromosome 14 imprinting disorder
Chromosome 14 harbours an imprinted locus at 14q32. Maternal uniparental disomy of chromosome 14, paternal deletions and loss of methylation at
the intergenic differentially methylated region, result in a human phenotype
of low birth weight, hypotonia, early puberty and short stature. This rarely
diagnosed imprinting disorder has considerable overlap with other better
known imprinting conditions such as Russell Silver and Prader Willi syndromes.
We have reviewed the world literature of 51 cases to identify the key diagnostic features to increase awareness, enhance diagnosis and improve
treatment.
Key findings
1) Small for gestational age: The median birth weight standard deviation
score (SDS) was -1.88 and none had a birth weight SDS >0. The median birth
length SDS was -1.64.
2) Hypotonia and motor delay (93% and 83%).
3) Mildly reduced intellectual ability; IQ, 75-95.
4) Small hands and feet (87% and 96%)
5) Early puberty (86%)
6) Short stature in adulthood; the median final height SDS was -2.04
7) Metabolic syndrome; the median final adult weight SDS was -1.07 demonstrating a relatively greater weight for height in adults; median BMI of 27. Of
15 patients over the age of 11 years, three developed non-insulin dependent
diabetes mellitus at the ages of 12 years, 19 years and 20 years.
The facial appearance distinguishes this condition from other imprinting
disorders.
234
Back to index
The use of the name Temple syndrome is not universal, as yet, but rather
than the somewhat cumbersome use of maternal uniparental disomy of
chromosome 14 related conditions we propose this name change.
P11.142-M
Testicular regression and cerebral abnormalities: a new syndrome
We report three male siblings affected by a Multiple Congenital Abnormalities/Intellectual Disability (MCA/ID) syndrome which, to the best of our
knowledge, has not been reported to date. Patient 1 presented with a clinical phenotype including micropenis, anorchia and a very low serum level of
antimullerian hormone, consistent with progressive testicular regression;
severe developmental delay; spasticity; epilepsy; dysmorphic features; deafness; retinopathy; cerebral atrophy, corpus callosum hypoplasia and periventricular cysts. The two subsequent pregnancies were terminated at 34
weeks gestation because of recurrences. The fetuses (patients 2 and 3) showed cerebral cysts (2/2), thin corpus callosum (1/2), very low levels of antimullerian hormone in the amniotic fluid (2/2) associated with ambiguous
genitalia in patient 2. Pathological examination of patient 2 showed a testicular atrophy, micropenis, a thin corpus callosum, white matter abnormalities (gliosis, cysts), and cerebellar focal neuronal migration anomalies. A
large genetic and metabolic assessment performed in patient 1 was normal:
standard karyotype and array CGH (105k, Agilent); molecular analysis of
the genes SRY, SF1, ARX, ATRX, KAT6B (exon 18); analysis of cholesterol precursors, screening for peroxysomal disorders (blood and fibroblasts), CDG
syndromes, creatine deficiencies, adenylosuccinate deficiency, blood lactate
and pyruvate, maternal X inactivation pattern. Magnetic resonance spectroscopy was normal. An exome sequencing analysis has been performed and
we are interested to study other possible patients showing a similar clinical
picture. In conclusion, we report the preliminary results of the clinical and
molecular study of a previously unreported MCA/ID syndrome, characterized by testicular regression and cerebral abnormalities.
P11.143-S
Autosomal recessive POLR1D mutation with decreasing of TCOF1
mRNA is responsible for Treacher Collins syndrome
ABSTRACTS POSTERS
P11.144-M
Molecular and cytogenetic characterization of an unusual case of
partial trisomy 13q mosaicism
The following is the case of a patient born 2 weeks preterm weighing 2.800
kg. She had a multicystic kidney that was removed immediately after birth.
At age 9, due to a significant growth delay, a cytogenetic analysis was made
on peripheral blood revealing a homogeneous 45,X karyotype thus leading
to Turner syndrome. Growth hormone treatment contributed to stature increase but with the development of secondary sex characters and the menstrual cycle, it became necessary to repeat blood cytogenetic analysis that
confirmed the 45,X karyotype. At 30 years, following absence of menstrual
cycle , she realized she was pregnant. She had to undergo a caesarian delivery and was simultaneously operated for ovariectomy. Karyotype obtained
from gonadic tissue was mos 45,X/46,X,der(X). The insufficient amount of
cells however did not help to investigate on marker nature. As the daughter presented the same stature growth delay, at 5 years her constitutional
karyotype was analyzed and was the same one found in her mothers ova-
Back to index
rian culture: 46,X,der(X), in this case present homogeneously. Cytogeneticmolecular characterization by FISH with various X specific probes allowed
to define, beyond doubt, that the X derivative is a dicentric isochromosome
with an inactive centromere and with symmetric point in Xp21: 46,X,idic(X)
(qterp21::p21qter). This data allowed to demonstrate : 1) the presence
of phenotype atypia. 2) ovarian functionality, 3) the short stature of both
mother and daughter due to SHOX gene deletion.
P11.147-S
SNP array revealed maternal UPD16 in a boy with short stature,
craniosynostosis and psychomotor retardation.
We report on a 15 months old boy (gemini after IVF, healthy sister) with
positive prenatal anamnesis (IUGR, oligohydramnion), craniosynostosis,
short stature, hypotonia, hypospadia, cryptorchidia, pulmonary vein stenosis and delayed psychomotor development. Chromosomal examination
revealed normal male karyotype 46, XY. The whole genome genotyping was
performed using SNP array (300K, Illumina) and no pathogenic CNV was
found. However, three extensive blocks with loss of heterozygosity spanning almost one third of the whole chromosome 16 were found. Parental
DNAs were analyzed and the maternal UPD16 was confirmed. Chromosome
16 showed the mixture of isodisomy and heterodisomy with homozygous
haplotype within the centromere. This haplotype arrangement implies the
following origin of the UPD16: meiosis II nondisjunction, recombination
and mitotic loss of the paternal chromosome 16 (trisomy rescue). Data for
UPD16 are inconsistent, prominent phenotype of maternal UPD16 is IUGR
with or without catch-up growth. Other features may include heart defects,
inguinal hernia, hypospadia, pulmonary hypoplasia, although these features
may be partly due to hidden mosaic trisomy 16. Mental development ranges
from normal to severely delayed. We consider a potential impact of two imprinted genes and five predicted imprinted genes on chromosome 16 to the
distinct phenotype of the UPD16.
P11.148-M
Whole exome sequencing identifies novel genetic variants in the
PRDM5 gene in Brittle cornea syndrome and Axenfeld-Rieger
syndrome
Background: Brittle cornea syndrome (BCS) and Axenfeld-Rieger syndrome (ARS) are disorders affecting the anterior segment of the eye, often
leading to secondary glaucoma. BCS is an autosomal recessive disorder associated with mutations in the PRDM5 and ZNF469 genes, while AxenfeldRieger syndrome is inherited in an autosomal dominant fashion that has
been associated with genetic defects in PITX2 and FOXC1. The purpose of
current study is to identify the underlying genetic causes in a family with
autosomal recessive BCS and in a family with autosomal dominant ARS by
whole exome sequencing (WES).
Methods: WES was performed for a single affected individual of both families. Missense, nonsense and splice site variants identified by WES were
prioritized based on pathogenicity prediction scores (PhyloP, Grantham). Segregation analysis of selected variants was performed in family members.
Results: A homozygous splice site variant (c.93+5G>A) was identified in
the PRDM5 gene, which was found to segregate with the disease in the BCS
family. In the ARS family, a novel heterozygous PRDM5 missense variant
(c.877A>G; p.Lys293Glu) was prioritized from the WES data, and was found
to segregate with the disease in an autosomal dominant fashion. Both variants were absent from population-matched controls, the Exome Variant
Server and an in-house exome variant database.
Conclusions: In the current study we identified a homozygous splice site
variant in a BCS family and a heterozygous missense variant in an ARS family
in the PRDM5 gene. This suggests that genetic variants in PRDM5 can lead to
different disorders affecting the anterior segment.
235
ABSTRACTS POSTERS
P11.149-S
WHSC/NSD2 is the major candidate gene for growth delay and facial
dysmorphisms in Wolf-Hirschhorn syndrome: expression analysis
and functional studies on primary fibroblasts and immortalized
peripheral lymphoblasts from three patients.
C. Cafiero1, G. Pani2, D. Samengo2, G. Marangi1, M. Zollino1;
1
Institute of Medical Genetics, Catholic University, Rome, Italy, 2Institute of General
Pathology, Catholic University, Rome, Italy.
Wolf-Hirschhorn Syndrome (WHS, OMIM194190) is a contiguous gene syndrome caused by partial deletion of the short arm of one chromosome 4.
The core WHS phenotype includes growth delay, intellectual disability, distinctive facial appearance and seizures. It maps within the terminal 1.9 Mb
region on 4p16.3, where the critical region, WHSCR-2, was described. With
respect to pathogenic genes falling within WHSCR-2, WHSC1 is the major
candidate gene for both facial characteristics and growth delay. WHSC1 is
expressed mainly in embryonic tissues and presents homology with Drosophila dismorphy genes. Its PWWP, HMG-box, PHD and SET structural domains suggest an histone methyltransferase activity and a role in the epigenetic regulation of morphogenetic transcriptional programmes. However, to
which extent whsc1 expression and activity are reduced in patientscells due
to 4p16 deletion has not been thoroughly investigated. Additionally, while
very little is known about whsc1 regulation, it is intriguing that this gene is
a potential target of hsa-miR948, that also maps to 4p16.3, and whose concomitant deletion may, in a subset of patients, reduce the effect of WHSC1
monosomy by increasing the expression of the residual allele. Based on these consideration, we decided to evaluate at a mRNA and protein level the expression of WHSC1 and of hsa-miR948 in primary and immortalized cell lines from selected patients and healthy individuals as controls. Furthermore,
the expression levels have been correlated with biochemical characteristics
(H3K36 and H4K20 methylation) and cellular phenotypes (DNA damage
response, inflammatory signaling) reportedly related to WHSC1 biological
activity at least in tumor cells.
P11.150-M
Coeliac disease in Williams syndrome
Williams syndrome is a multi-system disorder characterized by distinctive facial features, growth delay, mental retardation with typical neurobehavioral profile, cardiovascular anomalies, endocrine anomalies including
autoimmune pathologies, and hypercalcemia. Coeliac disease is an autoimmune, gastrointestinal disorders characterized by intolerance to the dietary
grain protein gluten. An increased prevalence of Coeliac disease has been
reported in Down syndrome and Turner syndrome, but there has been only
few previous report which respect to the association of Coeliac disease in
Williams syndrome.
The aim of this study was to estimate the prevalence of Coeliac disease in
our 24 molecularly confirmed Williams syndrome patients (12 girls, 12
boys, mean age 9.4 years). All patients were analysed by the dosage of tissue
transglutaminases IgA and IgG HLA genotyping and intestinal biopsy was
performed to the patients with positive serology. Celiac disease symptoms
and gastrointestinal problems were recorded. Biochemical profile (calcium,
urine calcium/creatinin ratio, thyroid functions), IgA tissue transglutaminases (IgA tTG) and IgG tissue transglutaminases (IgG tTG) were assessed in
all cases. Serum total IgA levels assessed only in tTG positive patients. Patients were selected for the haplotypes in the HLA class II region (HLA DQ),
endoscopy and intestinal biopsy on the basis of tTG positivity.
The results show that the prevalence of Coeliac disease in Williams syndrome was higher than in the general population. As our results, Coeliac disease
questioning and serologic screening is recommended at certain intervals in
all cases with Williams syndrome to explain the cause of growth retardation
and gastrointestinal problems.
P11.151-S
A novel ZCD2 intragenic deletion in Wolfram Syndrome 2
236
Back to index
Federico II, Naples, Italy, 6Second University of Naples, Eye Clinic, Multidisciplinary
Department of Medical, Surgical and Dental Sciences, Naples, Italy, 7Department of
Molecular Medicine and Medical Biotechnology, University of Naples Federico II,
Naples, Naples, Italy.
X;Y translocation (tXY) is a rare chromosomal rearrangement typically inherited from a tXY carrier mother. Patients with tXY commonly display 46, X
or Y, der(X),t(X;Y)(p22.3;q11). Women with tXY show a mild phenotype due
to X inactivation and are fertile. Men with tXY show a range of phenotypes
with variable degree of severity according to the size and position of the
deletion of the X chromosome, comprising short stature, chondrodysplasia
punctata, ichthyosis, ocular-albinism, mental retardation, and Kallmann
syndrome (KS). Here we describe a boy with tXY inherited from a carrier
mother. His phenotype was characterized by mesomelic short stature, midface retrusion, ichthyosis, micropenis, bilateral undescended testes, hypoplastic scrotum, agenesis of right kidney and central hypogonadism as assessed by gonadotropin-releasing hormone (GnRH) and human chorionic
gonadotropin (hCG) stimulation tests. Magnetic resonance imaging (MRI)
of the olfactory tracts showed hypoplastic olfactory bulbs bilaterally, aplasia
of the left olfactory sulcus and hypoplasia of the right olfactory sulcus. The
patients mother and sister showed mesomelic short stature and his mother
displayed Madelung deformity. Single nucleotide polymorphism (SNP) array
analysis defined the breakpoint in intron 7 of the KAL1 gene. Conclusion:
For early diagnosis in patients with KS, it would be useful to search for other
symptoms including renal abnormalities (agenesis of right kidney) and to
assess the presence of olfactory tracts by MRI (hypoplastic olfactory bulbs
bilaterally).
P11.153-S
Chromosome Xq21 deletion syndrome - rare cause of deafness and
mental retardation
A. Klmov;
Department of Medical Genetics, st nad Labem, Czech Republic.
Our case is a family, where Chromozome Xq21 deletion syndrome was diagnosed.
Genetic counselling was recommended because of mental retardation, bilateral hearing loss and paleocerebellar syndrome at 5-year boy. He also
had hypotonia, developmental delay and hyperactivity. We started genetic
examination and we found normal karyotype 46,XY,9qh+. Molecular genetic examination excluded X-Fragile syndrome, Prader-Willi-Angelman syndrome and mutations in the GJB2 gene. Microarray comparative genomic
hybridisation identified a 5-Mb deletion of Xq21, which include the POU3F4,
ZNF711 and CHM genes. The boy did not have any symptoms of choroideremia. Multiplex ligation-dependent probe analysis (MLPA) showed that the
unaffected mother also carried the deletion. The boy had no siblings.
ABSTRACTS POSTERS
Next time the family was examinated in the following pregnancy of the mother, but the case was complicated because of twin pregnancy. Prenatal diagnosis was recommended and chorionic villus sampling was performed with
the result one male and one female foetus. Molecular genetic examination of
the Chromozome Xq21 deletion syndrome in the male foetus was performed
and the same familiar deletion was identified. Mother underwent selective
fetocide of the affected male foetus at 16 weeks gestation. Further course
of gravidity is normal.
P11.154-M
17q21.31 microdeletion syndrome: neurocognitive functioning in two
italian young patients
M. Vendemiale1, F. Ortolani2, M. Settembre1, A. Rossiello1, D. Cornacchia1, G. DAniello1, A.
Labbate1, R. Tripoli1, M. Riccardi1, M. Lucarano1, F. Nicastro3, M. Carella4, M. Gentile5, E.
Piccinno6, F. Papadia6, R. Fischetto6;
1
Servizio Psicologia Clinica-Dir.Medica P.O.Giovanni XXIII AOU Policlinico, Bari, Italy,
2
UOC Malattie Metaboliche Genetica Clinica e Diabetologia Ospedale Pediatrico, Bari,
Italy, 3UOC Malattie Metaboliche Genetica Clinica e Diabetologia, Ospedale Pediatrico
Giovanni XXIII, Bari, Italy, 4UOC Genetica Medica IRCCS Casa sollievo sofferenza, San
Giovanni Rotondo, Foggia, Italy, 5UOC Genetica Medica ASL BA4 P.O.Di Venere, Bari,
Italy, 6UOC Malattie Metaboliche Genetica Clinica e Diabetologia Ospedale Pediatrico
Giovanni XXIII, Bari, Italy.
The 17q21.31 microdeletion syndrome causes a wellknown syndrome recently described in the scientific literature. Clinical features are: neonatal
hypotonia, low birth weight, craniofacial dysmorphism, developmental delay, intellectual disability and amiable behaviour disposition. The present
study analyzes neuropsychological assessment in two young italian patients
with a genetically described 17q21.31 microdeletion. Case 1: 18-year-old
male. Case 2: 13-year-old female.
Neuropsychological Assessment Wechsler Adult and Children Intelligence
Scales Revised, Italian Neuropsychological Battery, Peabody, Vineland Adaptive Behavior Scales, Adaptive Behavior Inventory, Parent Stress Index, Brief
Cope, Multidimensional Scale of Perceived Social Support, Child Behavior
Checklist. Case 1: severe mental retardation (Intelligence Quotient, IQ = 32),
immature representative capacity, impairment of receptive and expressive
communication, anxiety and apprehension. The Adaptive functioning is impaired in all areas (communication, skills of daily living, socialization and
motor skills). Mental Age 4y2m. Case 2: moderate to severe mental retardation (IQ<40), expressive and receptive language deficits, decline in all areas
of adaptive functioning (M.A. 5y7m), socially indiscriminate attachment
behavior, hyperactivity. Reading and writing disabilities, semantic memory
deficits, verbal and visuospatial short-term memory deficits, ideational and
motor dyspraxia, deficit in scheduling tasks and spatio-temporal organization were present in both patient. The emotional systems are characterized by trust in peers and adults, kindness, dependence and poor individual
autonomy. The attachment is secure with important people.To our knowledge these observations can be added to preliminary evidences already
present in literature: 17q21.31 microdeletion syndrome correlates to low
intellectual capacities, learning disability, good interpersonal skills and an
approaching behaviour.
P12.001-S
Impact of Aberrant Promoter Hypermethylation on down-regulation
of MTS2 and MTS1 in Childhood ALL
K. MalekZadeh1,2;
1
Molecular Medicine Research Center, Bandar Abbas, Islamic Republic of Iran, 2Moleculr
MEdicine Research Center; Hormozgan University for Medical Science, Bandar Abbas,
Islamic Republic of Iran.
The tumor suppressor genes MTS1 and MTS2 are cycline dependent kinas
inhibitors inactivated in some human neoplasms via several mechanisms
such as hypermethylation. We have investigated the methylation status
of MTS1 and MTS2 in its effect on transcriptional down-regulation in 125
bone marrow aspirate (7 cases T-cell and 118 cases B-cell phenotypes) from
childhood ALL patient and 100 healthy control in north Indian population
by using MSP-PCR ,bisulfite sequencing, SQRT-PCR and RT-PCR. There were
significant differences in pattern of hypermethylation between patients and
healthy controls of MTS2 (p=0.000) and MTS1 (p=0.001) and also when both
genes methylated. Patients with hypermethylated of both genes showed an
increasing risk for 2.33 fold (95%CI=2.33(1.97-2.77), p=0.03). Significant
association of MTS1 hypermethylation was observed only among the male
patients (p=0.004) in contrast hypermethylation of MTS2 was significantly
associated with increasing risk of ALL among both genders. Down-regulation of mRNA expression was found in cases in which MTS1 and MTS2 were
hypermethylated. In conclusion our data also indicate the impact of hypermethylation mediated inactivation of these genes which is associated with
risk of childhood ALL. This abnormality particularly in promoter of MTS2
occurs in leukemogenesis and can be considered as an important factor in
Back to index
The etiology of acute myeloid leukemia (AML) is currently unknown although genetic background and environmental exposure postulated to be a
possible cause of AML development. The CYP2B6 enzyme plays a vital role
in the degradation of many genotoxic compounds, protecting cells from
oxitative damage. CYP2B6 gene is subjected to the A785G germline polymorphism which reduces enzyme activity. Thus, individuals homozygous or
heterozygous for mutant allele (G/G or A/G) present decreased enzymatic
activity. The purpose of this study was to investigate the role of the A785G
polymorphism in the AML susceptibility. Possible associations with specific
AML-chromosomal abnormalities were also investigated. CYP2B6 genotyping was performed in 220 de novo AML patients and 243 healthy donors
by RCR-RFLP and Real-Time PCR assays. Cytogenetic analysis was successful
in 98% of patients. Among them, 72.7% presented an abnormal karyotype.
A significantly higher incidence of the mutant genotypes (A/G and G/G) was
observed in de novo AML patients compared to the controls (p<0.0001). The
mutant allele frequency was similar between the different gender and age
groups. Interestingly, a higher frequency of heterozygotes A/G was observed
in normal karyotypes compared to abnormal (51.7% vs 30.0%, p=0.010).
Furthermore, a significantly higher incidence of G/G genotype was observed in patients with t(8;21) and MLL rearrangements compared to patients
with normal karyotypes (28.5% and 20.0% vs 6.6%, respectively). Our study comprises the first investigation of the A785G CYP2B6 polymorphism in
AML susceptibility. Our results reveal a possible implication of this genetic
variant in AML development and its specific chromosomal aberrations.
P12.003-S
Higher incidence of co-existing Anaplastic Lymphoma Kinase (ALK)
rearrangements and Epidermal Growth Factor Receptor (EGFR)
mutations in Non-Small Cell Lung Cancer (NSCLC) in a South-East
Asian population
T. Lim1, A. Lim1, L. Oon1, T. Lim1, D. Tan2, Y. Ng1, Y. Yeap1, E. Chen1, J. Ho1, S. Tien1;
1
Pathology Department, Singapore General Hospital, Singapore, Singapore, 2Medical
Oncology, National Cancer Centre Sinagpore, Singapore, Singapore.
Unbalanced whole-arm translocations (WATs) of the long arm of chromosome 1 result in complete trisomy 1q. These are rare chromosomal abnorma-
237
ABSTRACTS POSTERS
lities detectable in both solid tumors and hematologic neoplasms including
cases of myelodysplastic syndrome and acute myelogenous leukemia (AML),
in most cases described as dicentric derivative chromosomes. We report on
1q unbalanced WATs detected at diagnosis in 4 patients as resulting from a
t(1;15)(p10;p10) in a case with myelofibrosis (MF) and in one with AML,
t(1;14)(p10;p10), and t(1;22)(p10;p10) in two other AML patients. Conventional cytogenetics was supplemented by FISH analysis using specific probes
for the centromeric alpha-satellite region of chromosome 1 (D1Z7; 1p11.1q11), chromosomes 14/22 (D14Z1/D22Z1; 14p11.1-q11.1/22p11.1-q11.1),
and chromosome 15 (D15Z4; 15p11.1-q11.1). FISH analysis showed that
the signal of chromosome 1 alphoid region D1Z7 was absent on derivative
chromosomes in all patients suggesting that breakpoint on chromosome 1q
is distal to the centromere, while D14Z1 on t(1;14), D15Z4 on t(1;15), and
D22Z1 on t(1;22) were present. Therefore all derivative chromosomes showed unique centromere (monocentric) derived from the acrocentric chromosome rearranged with 1q, then resulting in reclassification of 1q WATs as
der(15)t(1;15) (both cases), der(14)t(1;14) and der(22)t(1;22). Moreover,
during follow up in the MF case cytogenetic analyses revealed progressive
expansion of the clone with der(15) up to homogeneity, thus suggesting a
proliferative advantage for the der(15)-related clone. Although studies on a
large cohort of patients are needed, present results confirm 1q pericentromeric region instability in leukemia, and support trisomy 1q role to favor
leukemogenesis and hematopoietic tissue alterations.
P12.005-S
Involvement of APC gene in Acinar Cell carcinomas of the Pancreas
Survivin, encoded by BIRC5 gene, belongs to the family of inhibitors of apoptosis (IAP) proteins. In healthy organisms it is not expressed in differentiated tissues, while its expression is markedly increased in tumors. BIRC5
polymorphisms have been previously associated with increased expression,
stability and localization of survivin, all of which can affect tumor development.
In this study we investigated the role of BIRC5 polymorphisms in oral and
oropharyngeal squamous cell carcinoma. Genetic testing of 38 patients and
74 healthy controls was conducted using high resolution melting analysis
and Sanger sequencing.
Results showed different significance of individual BIRC5 polymorphisms.
c.-235G>A showed significantly higher frequency in patients compared to
control samples. For c.-644T>C and c.-625G>C, minor homozygous genotypes were significantly associated with higher Broders grade, which was
also observed for c.-235G>A. The minor homozygous genotypes of either
c.9194G>A or c.9809T>C were associated with higher TNM stage. For c.-
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644T>C and c.-625G>C, the presence of minor allele was associated with
higher survivin mRNA expression. Besides eight frequent, six rare polymorphisms were found, c.9349G>C found in 3 UTR previously unpublished.
This was the first study in Croatia which demonstrated correlation between
BIRC5 polymorphisms with the level of survivin expression and the risk of
oral and oropharyngeal squamous cell carcinoma. Since it is known that
increased expression of survivin is associated with increased resistance to
chemotherapy and radiation, as well with poorer survival, BIRC5 polymorphisms could be used as predictive and prognostic biomarkers in the etiology of head and neck squamous cell cancer.
P12.008-M
Bladder cancer risk in relation to DNA repair capacity, gene
expression and epigenomic profiles.
The ability to repair DNA damage is strongly associated with the risk of
cancer and inter-individual variation in DNA repair capacity (DRC) might
account for different susceptibility of developing cancer. DRC represents a
complex marker comprising the sum of several factors such as gene variants, gene expression, stability of gene products, and effect of inhibitors/
stimulators. Individuals with low DRC will tend to accumulate more damage
than those who have a better ability to repair such damage. This variability
is modulated by the genetic background to which SNPs/haplotype combinations in DNA repair genes, as well as epigenetic regulation are likely to
contribute.
Phenotypic assays to evaluate DNA repair activity (in particular NER comet
assay, H2AX phosphorylation and micronucleus assay) were performed
on cryopreserved lymphocytes from 159 bladder cancer cases (collected
before treatment) and 159 controls matched by age and smoking habits,
enrolled in Turin Bladder Cancer Study (TBCS). We aimed at studying the
relationship between DRC (evaluated by comet assay, micronuclei and H2AX
phosphorylation assay) and bladder cancer integrating, gene expression
and epigenetic profile data (methylation levels and microRNA expression).
In particular, we performed an integrated analysis on the genotype/phenotype correlation in a population of bladder cancer cases and controls provided with detailed description of the follow-up for response to therapy/
recurrence/survival. This integrated approach will help in elucidating the
role of DRC (and its determinants) as predictive and prognostic marker and
to identify DNA repair phenotypic assays to be performed in blood cells as
non-invasive predictive method.
P12.009-S
Mutation detection in urines from bladder cancer patients as noninvasive prognostic tool
In Europe, bladder cancer (BC) is the sixth most commonly diagnosed tumour and the second most common cause of death among patients with
genitourinary tract malignancies. Patients with non-muscle-invasive bladder cancer (NMI-BC) have excellent survival; however two-thirds develop
recurrences. Tumour specific mutations can be used to detect recurrences
in urine assays, presenting non-invasive diagnostic procedure respect to cystoscopy. The present study involves male subjects recruited between 1994
and 2012 and diagnosed with BC. Urine samples have been collected for the
first time at the time of diagnosis and exfoliated cells have been extracted. A
subgroup of patients have been followed up for three years, collecting a urine
sample every six months. The mutation spectrum of hTERT, FGFR3, HRAS,
KRAS, NRAS and PIK3CA in 286 patients has been investigated at diagnosis.
Thirty-eight patients have completed six samplings, twenty-five patients have
five samplings and nineteen patients have four samplings, for a total of 454
follow-up samples. Mutations are detected using three different multiplexed SNaPshot assays. The first allows to detect in one reaction the nine most
frequent FGFR3 mutations. The second multiplex screens simultaneously for
nineteen RAS mutations. The last multiplex detects seven mutations, three in
hTERT gene and four in PIK3CA gene. Preliminary analyses show that mutations in hTERT gene promoter and in FGFR3 gene are the most frequent
somatic mutations in tumours of the urinary bladder. Mutational profiler will
be correlated with recurrences and survival in order to verify the usefulness
of urinary exfoliated cells to follow-up BC progression.
ABSTRACTS POSTERS
P12.010-M
SEPT9/SYHR1, novel fusion gene identified in bladder cancer by RNAseq.
Background: BRCA1/BRCA2 mutations are the most common cause of hereditary breast and ovarian cancer (HBOC). The HBOC-Consortium was
created for unified data collection on Israeli BRCA carriers.
Methods: A uniform computerized-database was used by 12 cancer-centers
Results: Data were collected on 2145 BRCA1, 1131 BRCA2, and 22 doublemutation carriers.
BRCA1 vs. BRCA2 mutation carriers had more breast cancer (BC) (40.0%
vs. 36.3%, p=0.06), ovarian cancer (OC) (17.1% vs. 10.6%, p<0.001), were
younger at cancer diagnosis (BC 45yrs. vs. 49 yrs., p<0.001, OC 53yrs. vs.
62yrs., p<0.001), had more familial BC (64.5% vs. 59.0%, p<0.001), and familial OC (36.7% vs. 27.3% p<0.001).
BRCA1 carriers of 185delAG vs. 5382insC had less BC (38.6% vs. 46.4%,
p=0.02), at older age (46yrs. vs. 40yrs., p=0.023), but more OC (17.6% vs.
12.9%, p=0.04).
Risk-reducing-bilateral-salpingoooporectomy (RR-BSO) rates were assessed
in carriers 38-80yrs. BC patients had higher rates than unaffected carriers
(92.6% vs. 78.1%, p<0.001), but underwent RR-BSO later (51yrs. vs. 48yrs.,
p<0.001). Carriers with familial OC had higher rates than carriers without
(83.0% vs. 74.7%, p=0.05). Carriers with familial-cancer had RR-BSO earlier
than those without (48 yrs. vs. 50yrs., p=0.037), especially BRCA2 carriers
with familial OC (47 yrs. vs. 51yrs., p=0.018).
18/497(3.6%) male carriers had BC. 1/3 were BRCA1 carriers, and were
younger at cancer diagnosis compared to BRCA2 mutation carriers (50yrs.
vs. 57yrs., NS).
Conclusions: RR-BSO rates in Israeli carriers are very high, particularly in
women affected with BC and family history of OC. Consortium data are consistent with lower cancer rates for BRCA2 mutations.
P12.013-S
Setting up the basis for translating omic data for BRCA1/2 into
clinical practice
Back to index
Double heterozygosity for BRCA1 and BRCA2 mutations is a very rare finding, particularly in non-Ashkenazi individuals, and only a few cases have
been reported to date. Here we describe genetic and clinical data of two female double heterozygotes for both BRCA1 and BRCA2 mutations found in
a cohort of 201 mutated Italian breast/ovarian cancer families out of 942
cases analyzed. The first one is a female patient affected by bilateral breast
cancer at 47 and 49 years of age, carrying both a BRCA2 nonsense mutations (c.7408A>T - p.Arg2394X) and a BRCA1 proven splicing defect (IVS512A>G or c.331_332ins11 - p.Arg71SerfsX21). The second one is a female
patient affected by ductal breast cancer at 42 years of age, carrying both
a BRCA1 nonsense mutations (c.3726C>T - p.Arg1203X) and a BRCA2 frameshift mutation (c.3036_3039delACAA - p.Ala938ProfsX21). Although this
event is rare (2/201: 1% in our clinical records, consistent with literature
239
ABSTRACTS POSTERS
data) and the phenotype is not worse than carriers of a single mutation, it
has to be considered in the assessment of the biological effect of variants of
uncertain biological effect. Furthermore the presence of a second mutation
has important consequences for genetic counselling of relatives. We suggest
that mutation analysis of index cases should always be extended in order to
avoid missing a second BRCA mutation.
P12.016-M
BRCA mutation testing in all newly diagnosed patients with breast- or
ovarian cancer: The DNA BONus study
C. Bjorvatn1,2, T. Aas3, B. E. Fiane4, K. Woie5, H. Espelid6, T. Rusken7, H. P. Eikesdal8,2, M. T.
Henanger1, W. Listl1, P. M. Knappskog1,2, V. M. Steen1,2, N. Hoogerbrugge9, H. H. Vetti1;
1
Western Norway Familial Cancer Center, Haukeland University Hospital, Bergen,
Norway, 2Department of Clinical Science, University of Bergen, Bergen, Norway,
3
Department of Surgery, Haukeland University Hospital, Bergen, Norway, 4Department
of Gynaecology and Obstetrics, Stavanger University Hospital, Stavanger, Norway,
5
Department of Gynecology and Obstetrics, Haukeland University Hospital, Bergen,
Norway, 6Department of Surgery, Haugesund Hospital, Haugesund, Norway,
7
Department of Surgery, Frde Central Hospital, Frde, Norway, 8Department of
Oncology, Haukeland University Hospital, Bergen, Norway, 9Department of Human
Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands.
C. Tessereau1, M. Buisson1, M. Imbert1, L. Barjhoux1, F. Damiola1, M. Dondon2, S. EonMarchais2, GENESIS investigators, F. Lesueur2, D. Stoppa-Lyonnet2, N. Andrieu2, O.
Sinilnikova1,3, S. Mazoyer1;
1
Cancer Research Centre of Lyon, Lyon, France, 2Institut Curie, Paris, France, 3Unit Mixte
de Gntique Constitutionnelle des Cancers Frquents HCL/CLB, Lyon, France.
The RNU2 macrosatellite is a tandem array of a 6.1 kb repeat unit containing the 190 bp-long gene coding for snRNA U2, RNU2-1, and 3.8 kb of interspersed repetitive DNA. Although located 124 kb telomeric to the breast
and ovarian cancer susceptibility gene BRCA1, the RNU2 locus is absent
from the human genome assembly due to the inherent difficulty to assemble
repetitive sequences. Thus, the influence that these large tandem repeat arrays might exert on neighbouring gene expression, stability or architecture
and their contribution to the genetic basis of common human diseases remains largely unexplored. We have recently characterized in more details
the RNU2 locus and tested the association between RNU2 copy number and
breast cancer risk. Indeed, the proximity of this macrosatellite to BRCA1
combined with its high degree of polymorphism raises the interesting possibility that it could be involved in breast cancer susceptibility, especially
as no mutation is identified in BRCA1/2 in 80% of the families tested in a
diagnostic setting. We conducted a case-control study by genotyping the
RNU2 macrosatellite with a qPCR assay in 1452 familial breast cancer cases, 1457 affected and unaffected relatives and 1241 unrelated controls of
the French study GENESIS. We found that mean RNU2 global copy number
(GCN) is higher in cases (52 copies) than in controls (50 copies). In addition, we found more very high RNU2 GCN (>100) in affected women. We are
presently investigating the functional impact of extremely large RNU2 GCN
on the BRCA1 locus.
240
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P12.018-M
Investigating the management of symptomatic and pre-symptomatic
BRCA gene mutation carriers in an Irish Tertiary referral centre
T. P. McVeigh1, R. Irwin1, N. Cody2, K. J. Sweeney1, A. J. Green2, N. Miller1, M. J. Kerin1;
1
National University of Ireland Galway, Galway, Ireland, 2National Centre for Medical
Genetics, Dublin, Ireland.
Aims: Germline mutations in BRCA1 and 2 confer a high risk of breast cancer. The aim of our study was to outline the disease phenotype and management of BRCA gene mutation carriers in the West of Ireland.
Methods: A longitudinal cohort study was undertaken. The study group included patients proven to carry a single gene mutation in BRCA1 or BRCA2
between 2000 and 2013. Clinicopathological information was obtained by
chart review.
Results: Fourteen pathogenic mutations were identified in BRCA1 in 45 individuals from 24 families. The most common mutations were large genomic
rearrangements, with deletion of exons 1-23 in 5 families, deletion exons 1420 in 3 families and deletion exons 21-24 in 2 families. Fourteen pathogenic
mutations were identified in BRCA2, in 32 individuals from 19 families. The
most common was frameshift mutation 8525delC, in 5 families. Of forty-one
patients affected with breast cancer, 17 carried mutations in BRCA1 and 24
mutations in BRCA2. Median age of onset of breast cancer in BRCA1 mutation carriers was 40years(25-67), and 45years(35-64) in BRCA2 mutation
carriers (p=0.02, Mann-Whitney). Eight patients developed bilateral breast
cancers including 6 BRCA1 mutation carriers. Five of six patients with ovarian cancer carried BRCA1 mutations. Ten of 51 pre-symptomatic carriers
underwent surgical prophylaxis, including 9 prophylactic mastectomies and
5 oophorectomies.
Conclusions: BRCA gene mutations account for a small proportion of inherited predisposition to breast cancer. Carriers of these mutations require
intensive surveillance or surgical prophylaxis. Counselling and testing of
pre-symptomatic family members can facilitate intervention and modify
disease phenotype.
P12.019-S
Characteristics of Greek patients with breast cancer rearrangements
in BRCA1 gene
Breast cancer is the most fatal disease for women in Western countries, despite mammography screening and adjuvant therapy with tamoxifen and
polychemotherapy. Knowing the molecular mechanisms underlying this
ABSTRACTS POSTERS
disease may contribute to the identification of new targets for future therapies. Tp53, Tp63 and Tp73 tumor suppressor family members encode for
transcription factors which control genome integrity. They take part in cell
response and in tumor suppression. Wild-type p53 protein is a growth modulator and its inactivation is a critical event in malignant transformation
of breast cancer stem cells (CSCs). Otx1 is an homeobox gene involved in
central nervous system development, and when deregulated, plays a role in
tumorigenesis. We showed that Otx1 is over-expressed in ductal and lobular
invasive breast cancers and that is involved in adult mammary gland development. We demonstrated that p53 directly regulates Otx1 gene expression
binding to the 3p53 responsive element (RE) on its promoter, and that this
pathway regulates the LA7 breast CSCs differentiation. Here we will show
that the TAp73 isoform of p73 is able to bind the 5p53RE on the Otx1 promoter, leading to the LA7 cell differentiation and the asymmetric division
of breast CSCs. Furthermore we will demonstrate that Otx1 and TAp73 are
over-expressed in ductal and lobular invasive breast carcinoma. Finally we
will show the functions of the TAp73/Otx1 pathway in differentiation of
mammospheres obtained from wild-type and c-ErbB2 transgenic mice, and
the response to cisplatin treatment in MCF7 and MBA-MB-231 breast cancer
cells.
P12.021-S
One in three Greek patients with early onset or familial breast cancer
carries a loss of function mutation in a known or candidate breast
cancer gene
Heterozygotes for mutations in CHEK2 and NBN genes were found to be associated with increased risk of developing cancers, including breast cancer
(BC). The aim of our study was to determine the frequency of three common
mutations in CHEK2 gene (1100del, I157T and IVS2+1G>A) and two mutations in NBN gene (R215W and 657del5) among Macedonian BC patients
and controls from the general population. For this purpose we have designed
a multiplex PCR followed by SNaPshot analysis. A total of 299 BC patients, of
whom 112 with a cancer family history and 283 controls were included in
the study. Mutations were more frequent among BC patients (n=13, 4.3%)
than among controls (n=5, 1.8%), although without statistical significance.
Twelve patients were heterozygous for one of the analyzed mutations, while
one patient had two mutations (NBN R215W and CHEK2 I157T). The most
frequent mutation was CHEK2 I157T, found in 10 BC patients and 4 controls.
The frequency of this mutation was statistically higher among BC patients
with a cancer family history when compared to the controls (p=0.028). NBN
R215W was found in one BC patient and one control, while CHEK2 1100delC
and NBN 657del5 were found each in one BC patient and no control. CHEK2
IVS2+1G>A has not been found in our study. In conclusion, our study sug-
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gests that mutations in CHEK2 and NBN genes might play a role in the development of breast cancer among Macedonian patients. A study including
larger number of patients and controls are needed to confirm these results.
P12.023-S
BRCA1 and BRCA2 mutation detection by a Next Generation
Sequencing approach: an epidemiological study conducted in
Southern Italy
Hereditary breast and ovarian cancer (HBOC) accounts for about 10% of
all breast cancers and BRCA1 and BRCA2 are the most prevalent genes causing this pathology. BRCA1/BRCA2 germline mutations escalate the risk of
developing HBOCs by up to 20 fold. Testing for BRCA gene mutations is important to improve the clinical management of the high-risk patients and of
their mutation carriers family members.
A NGS screening for BRCA1/2 germline mutations of 300 patients, with
early-onset breast cancer (under forty) and/or with positive family history, is reported in order to identify mutation carriers. BRCA1/BRCA2 coding
regions were amplified using the BRCA MASTR v2.1 Assay (Multiplicom).
Sequencing reactions were performed with the 454 GS FLX System (Roche)
and the downstream data analysis was carried out through the SeqNext tool
(JSI Medical Systems).
More than 15% of the analyzed patients, including a few men, carried a
causative mutation. Several novel variants were identified: 1 splice variant,
causing the loss of a canonic donor splice site at position +2 in the intron 21
of BRCA1 gene, and 1 missense variant falling in the BRCA2-DNA binding
region; one double mutation including 1 missense mutation on BRCA1 gene
causing a premature stop codon; 2 synonymous variants and 1missence variant predicted without clinical significance were found. Functional in vitro
evaluations are ongoing to assess their pathogenicity.
Subsequent analysis of the mutation carriers families, allowed the identification of at higher risk subjects, including men healthy carriers, that have
been involved in surveillance program of preventing health care.
P12.024-M
Rare key functional domain missense substitutions in MRE11A,
RAD50, and NBN contribute to breast cancer susceptibility
The MRE11A-RAD50-Nibrin (MRN) complex plays several critical roles related to repair of DNA double-strand breaks. Inherited biallelic mutations in
any one of the three components predispose to genetic instability disorders.
Inherited heterozygous mutations in the MRN genes have been implicated
in breast cancer (BC) susceptibility, but the underlying data are not entirely
convincing. In order to verify if rare MRN variants are intermediate-risk BC
susceptibility alleles, we mutation screened the coding exons and proximal
splice junctions of the MRN genes in 1,313 early onset BC cases and 1,123
population controls.
Considering the extremely low frequency of likely pathogenic variants in
these genes in the general population, we have decided to evaluate the genes
as if they constitute one relatively large gene (though still smaller than ATM,
another gene in the same pathway).
Limiting our analyses to variants with MAF<0.1% and combining protein
truncating variants, likely spliceogenic variants, and key functional domain
rare missense substitutions, we found significant evidence that the MRN
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ABSTRACTS POSTERS
genes are indeed intermediate-risk BC susceptibility genes (OR= 2.88, P=
0.0090). In addition we found that key domain missense substitutions were
more frequent (24 vs 12 observations) than the truncating variants and
conferred a slightly higher OR (3.07 vs 2.61) with a lower P-value (0.029
vs 0.14). Thus, the spectrum of pathogenic variants in MRN genes includes,
as for ATM and CHEK2, a relatively high proportion of missense, and differs notably from the BRCA1/2 pattern where most susceptibility alleles are
protein-truncating variants.
P12.025-S
Search for recessive cancer predisposition genes: the advantage of
multiple primary cancer cases vs. family history-positive patients
Virtually all known tumor predisposing genes have been identified via the
analysis of familial cancer cases. Here we argue that this approach is likely to
miss recessively acting cancer genes and suggest the analysis of family history-negative patients with multiple primary malignancies for identifying homozygous at-risk genotypes. We performed calculations showing that in cases of transmission of a recessive cancer predisposing allele (a), which has a
frequency of 0.1 and a penetrance of 100%, only a minority of patients with
the aa genotype (19%) will have one or both parents with the same genotype
and hence report a family history of cancer. Therefore, while the focus on
familial cancer clustering is a powerful tool for identifying dominant mutations, the cases of disease caused by the homozygous recessive at-risk alleles
may be easily missed. We further revealed that the c.2515_2519delAAGTT
homozygous mutation in a Holliday junction resolvase, GEN1, was overrepresented in women with bilateral breast cancer (BC) as compared to
healthy controls [11/360 (3.1 %) vs. 18/1305 (1.4 %); OR = 2.25 (1.02 4.75); p = 0.031], although this trend was not pronounced in unilateral BC
patients. Noticeably, the presence of biallelic c.2515_2519delAAGTT mutation was associated with the absence of BC in mothers of both bilateral and
unilateral BC patients [7/239 (3.0 %) vs. 0/41 (0 %) and 21/1,558 (1.3 %)
vs. 0/215 (0 %), respectively; p = 0.041]. This study confirms that patients
with multiple cancers may be particularly fruitful for the identification of
recessive determinants of cancer predisposition.
P12.026-M
High-throughput genetic analysis in 100 hereditary breast cancer
patients
Objective: Here, we report our first results from our HBOC-NGS-panel that
includes 56 genes associated with breast and ovarian cancer. So far, 100
samples were analyzed in a diagnostic setting.
Methods: A custom NGS-panel (HaloPlex, Agilent) was used to target 56 genes. Depending on the patients family history, a set of diagnostic genes
(mostly BRCA1/2, RAD51C/D) as well as screening genes were defined.
DNA was isolated from all samples, enriched and sequenced on the Illumina
MiSeq (2x 150 bp paired-end) following standard protocols. For diagnostic
genes, regions with low depth (< 20x) were complemented by Sanger sequencing as well as MLPA. All mutations were confirmed by conventional
Sanger sequencing.
Results: So far, we have sequenced 100 hereditary breast cancer patients.
Overall 91-99 % of all targeted exons were represented with a diagnostic
average depth of > 20x. Roughly 70 SNVs were identified per sample and
stringent filtering resulted in less than seven variants for validation.
We identified 15 mutations that are known to cause HBOC as well as mutations likely to cause HBOC. Further experiments and segregation analysis
is required to determine the pathogenicity in the latter. In addition heterozygous mutations were found in genes relevant for different autosomal
recessive cancer syndromes.
Conclusion: Taken together, we demonstrate that NGS is a fast and cost efficient genetic screening tool to analyze for variants in genes associated with
the development of hereditary breast cancer. By applying this approach we
were able to uncover both known and novel sequence variants
P12.027-S
BRCA1 and BRCA2 germline mutational spectrum among Macedonian
women with breast cancer detected by next generation sequencing
D. Plaseska-Karanfilska1, I. Maleva1, M. Jakimovska1, K. Popovska-Jankovic1, K.
Kubelka2, M. Karagjozov2, L. Stojanovska3, A. Arsovski3;
242
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ABSTRACTS POSTERS
Oncology, Istituto Europeo di Oncologia, Cogentech, Milano, Italy, 8Division of Cancer
Prevention and Genetics, Istituto Europeo di Oncologia, Milano, Italy, 9Unit of Medical
Genetics, Department of Preventive and Predictive Medicine, Fondazione IRCCS Istituto
Nazionale dei Tumori, Milano, Italy.
Germline deleterious mutations of PALB2 are associated with breast cancer risk and have been reported in several populations. In our initial survey, truncating PALB2 mutations were detected in 12/575 (2.1%) familial
breast cancer cases negative for BRCA gene mutations (BRCAX), recruited
at two large cancer centres in Milan (Istituto Nazionale Tumori and Istituto Europeo di Oncologia). One mutation (c.1027C>T; p.Gln343X) recurred
in three cases, two of whom originally from the province of Bergamo. The
genotyping of the c.1027C>T in 113 BRCAX cases ascertained at Azienda
Ospedaliera HPG23 of Bergamo, detected a total of 6 carriers (5.3%), while
only 2 carriers were observed among 477 female blood donors recruited in
the same area (0.4%). The estimated age-adjusted odds ratio of these frequencies was 13.4 (95% confidence interval: 2.767.4). This value is similar
to those previously observed for a few PALB2 mutations recurrent in other
countries and suggests that the c.1027C>T is associated with a relatively
high breast cancer risk. Of note, we also found that among breast/ovarian
cancer families recruited in Bergamo the mutational spectra or both BRCA1
and BRCA2 were much less heterogeneous than in families ascertained in
Milan. In particular, one BRCA1 founder mutation was observed in >8% of
Bergamo families (Caleca et al., 2014). Further analyses are required to verify whether genotyping for specific recurrent mutations can be proposed as
a cost-effective strategy for the rapid identification of individuals genetically
predisposed to breast/ovarian cancer in the Bergamo area.
P12.030-M
RNA-Sequencing in MCF-7 cells: identification of a new transcript of
SEMA3F and its expression in breast cancer
Breast cancer is the most common tumor in women, and the second leading
cause of death. Tumor cells invasiveness is mainly due to an alteration of
cell-cell and cell-matrix connections. Thus, an altered expression of adhesion molecules and their receptors is a crucial event in this process. Among
them, semaphorins, a large family of transmembrane or secreted molecules
that regulate cell migration and adhesion, are of peculiar interest. A growing
number of studies - and our recent work on SEMA6B in breast cancer among
these - has demonstrated their involvement in cancer progression, often
with divergent functions.
In order to simultaneously investigate gene expression and alternative
splicing for all the genes encoding adhesion molecules, and particularly for
semaphorins, their receptors and co-receptors, we performed RNA-Sequencing experiment on MCF-7 cells, a well-established and widely used cellular
model of breast cancer. Interesting preliminary results were obtained, particularly for SEMA3F gene. Indeed, we identified a novel transcript generated
by alternative splicing, predicted to encode for a truncated semaphorin. Moreover, through semiquantitative PCR and quantitative Real-Time assay we
measured SEMA3F expression on a panel of breast cancer tissues compared
to their healthy counterpart. The analysis revealed a strong and significant
up-regulation of SEMA3F in breast cancer and, intriguingly, the sole presence - in the healthy tissues - of the newly identified transcript. These evidences suggest SEMA3F to be a potential biomarker for onset/progression
of breast cancer.
P12.031-S
Investigating the importance of variants at 12p11, 12q24 and 21q21
in breast cancer in the west of Ireland
U. M. McVeigh, T. P. McVeigh, K. J. Sweeney, M. J. Kerin, N. Miller;
National University of Ireland Galway, Galway, Ireland.
Back to index
prising 1191 cases and 448 controls. The minor allele at locus 12p11 was
found to confer a significant protective effect, with a per allele odds ratio of
0.7 (0.5-0.9, p=0.002, X2). The minor allele at 12q24 had a slight protective
effect (per allele OR =0.9 (0.8-1.1, p =0.29, X2)). The minor allele 21q21 did
not confer a protective effect, and was in disequilibrium as per test of HardyWeinberg.
Conclusion: All three genetic variants were detected in the population in the
west of Ireland. The G allele at 12p11 was associated with reduced breast
cancer risk. Population-specific genome wide association studies are required to identify susceptibility loci specific to the Irish subgroup
P12.032-M
DNA-diagnostics for inherited breast and/or ovarian cancer: standard
approaches and novel technologies
L. N. Lyubchenko1, I. S. Abramov2, E. I. Bateneva1, A. S. Tyulyandina1, O. V. Krokhina1, I. K.
Vorotnikov1, V. A. Sobolevsky1, S. M. Portnoy1, T. V. Nasedkina2;
1
N.N.Blokhin Russian Cancer Research Center, Russian Academy of Medical Sciences,
Moscow, Russian Federation, 2Engelhardt Institute of Molecular Biology, Russian
Academy of Sciences, Moscow, Russian Federation.
Modern molecular techniques allow revealing the most characteristic genetic changes responsible for hereditary breast and/or ovarian cancer (hBC/
OC), calculating the risk of neoplasia development, defining treatment and
prevention. In routine diagnostics of hBC/OC two methods: biochip-based
approach and real-time PCR were used for the analysis of founder mutations in Russian Federation, namely, 185delAG, 300T>G, 4153delA, 4158A>G,
5382insC, 6174delT, 1100delC. However, only 15% of patients were found
to carry the BRCA1/2 or CHEK2 mutation. Other genes, like TP53, PALB2,
ATM, BRIP1, RAD50, BLM and so on, are widely tested in patients with
hBC/OC susceptibility. The aim was to study an impact of TP53 germline
mutations and polymorphisms into predisposition to bilateral BC and/or
secondary-primary multiple neoplasia (SPMN). Genomic DNA was isolated
and exons 4-11 of 53 gene were amplified with commercial primer set
SeqPlateTP53. Next-generation sequencing was performed using a platform
GS Junior (454/Roche). The results were validated by Sanger sequencing.
Exons 4-11 of TP53 gene in 22 patients with SPMN including BC or bilateral
BC and without founder mutations in BRCA genes (wtBRCA) were analyzed. The findings included several rare variants with population frequency
less than 0.001%. Previously found nonsense mutation 2637/T, leading to
stop-codon instead of arginine (codon 306) in patient with bilateral BC was
confirmed by NGS. A heterozygous germline missense mutation in codon
241 (.722>A) was diagnosed in patient with SPMN (Li-Fraumeni syndrome). The mutation was confirmed by Sanger sequencing. The data demonstrated the clinical utility of TP53 testing in selected patients with SPMN
and/or bilateral BC.
P12.033-S
The experiences and views of health care professionals and
researchers regarding the feedback of results in the context of next
generation sequencing in oncology
243
ABSTRACTS POSTERS
P12.034-M
Whole-genome profiling of cervical carcinomas patients with
CGH+SNP microarrays: correlations with clinical outcome
Cholangiocarcinoma (CCA) presents significant diagnostic challenges, resulting in late patient diagnosis and poor survival rates. Primary Sclerosing
Cholangitis (PSC) patients pose a particularly difficult clinical dilemma,
since they harbor chronic biliary strictures that are difficult to distinguish
from CCA. MicroRNAs (miRs) have recently emerged as a valuable class of
diagnostic markers; however, thus far, neither extracellular vesicles (EVs)
nor miRs within EVs have been investigated in human bile. We aimed to
comprehensively characterize human biliary EVs, including their miR content. Conclusion: We have established the presence of extracellular vesicles
in human bile. In addition, we have demonstrated that human biliary EVs
contain abundant miR species, which are stable and therefore amenable to
the development of disease marker panels. Furthermore, we have characterized the protein content, size, numbers and size distribution of human
biliary EVs. Utilizing Multivariate Organization of Combinatorial Alterations (MOCA), we defined a novel biliary vesicle miR-based panel for CCA
diagnosis which demonstrated a sensitivity of 67% and specificity of 96%.
Importantly, our control group contained 13 PSC patients, 16 patients with
biliary obstruction of varying etiologies (including benign biliary stricture,
papillary stenosis, choledocholithiasis, extrinsic compression from pancreatic cysts, and cholangitis), and 3 patients with bile leak syndromes. Clinically, these types of patients present with a biliary obstructive clinical picture
that could be confused with CCA. These findings establish the importance
of using extracellular vesicles, rather than whole bile, for developing miRbased disease markers in bile.
P12.036-M
Functional studies on post-transcriptional regulation of FAS/FASL in
chordoma
Chordoma, originating from notochord remnants, is characterized by chemoresistance. Since the apoptotic Fas/Fasl pathway is involved in notochordal
cell apoptosis, we investigated its possible role in chordoma. Accordingly
we detected the lack of FASL mRNA and the presence of both FAS anti- and
pro-apoptotic isoforms and the inactive Caspases 8 and 3 forms in most of
the tumors analyzed, suggesting the Fas/Fasl pathway inactivation in chordoma. The enhancement of apoptosis in U-CH1 chordoma cells, expressing
244
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Fasl and Fas anti- and pro-apoptotic isoforms after Soluble Fasl treatment,
indicates that this pathway can be re-activated. These findings lead us to hypothesize that Fas receptor binds Soluble Fasl that, undermining Fas antiapoptotic isoform, activates apoptosis. Soluble Fas isoform could have a key
role as antiapoptotic factor as the alternative splicing underlying the expression regulation of the two Fas isoforms. Knowing that HuR is one of the splicing factors leading to Fas transmembrane isoform, we are performing functional studies aimed at modulating the reciprocal amount of Fas isoforms
by interfering with the expression of HuR. U-CH1 apoptosis, cellular vitality
and migration will be evaluated after HuR iperexpression and silencing to
assess the role of Fas antiapoptotic isoform in chordoma apoptosis regulation. We are also silencing miR-21 targeting FASL mRNA to verify whether the
increase of endogenous Fasl may activate apoptosis. This study, providing
findings on the involvement of Fas/Fasl pathway in chordoma could help to
elucidate the role of apoptosis in chordoma tumorigenesis, addressing the
identification of new potential pharmacological targets.
P12.037-S
Transcriptome analysis of cancer cells in chronic myeloid leukemia
The aim of this study was to explore the association between polymorphisms of three DNA repair genes (XPD, XRCC1 and XRCC4) and clinical parameters in patients with Philadelphia-positive (Ph+) Chronic Myeloid Leukemia
(CML) treated with imatinib. Sixty-two patients with CML and 70 healthy
controls were examined for XPD (A-751G), XRCC1 (A399G), XRCC4 (intron
3 VNTR and G-1394T) polymorphisms. DNA was extracted from peripheral blood samples using salting out procedure. All polymorphisms were
genotyped by PCR and/or PCR-RFLP and analyzed. All data were analyzed
using de Finetti program and SPSS version 14.0 for Windows. No significant differences were detected between CML group and healthy controls
with respect to the distributions and numbers of genotypes and alleles in
XPD, XRCC1 and XRCC4 (-1394). However, intron 3 VNTR polymorphism in
ABSTRACTS POSTERS
XRCC4 gene was showed an association with CML patients. The distribution
of DD, DI and II genotypes for the gene was 3.2%, 51.6% and 45.2% in CML
compared with 14.3%, 60% and 25.7% in the controls. In the univariate
analyses of clinical parameters, there was no significant prognostic factor
found to influence overall survival. The four factors that were predicted for
better event-free survival from univariate analysis were younger age (< 60)
(p=0.020), absence of splenomegaly (p=0.011), lower Sokal risk score at
diagnosis (p=0.0148) and XRCC1 GG genotype (p=0.033). Our results suggest that XRCC1 GG genotype may be a useful marker for CML prognosis and
II genotype of intron 3 VNTR polymorphism in XRCC4 may be associated
with susceptibility.
P12.039-S
Polymorphism in genes CYP2C9 and ODC1 involved in aspirin
handling influence colorectal cancer risk
Long term regular use of aspirin is associated with a reduced risk of colorectal cancer (CRC) of up to 60%. However, not all aspirin users receive
the beneficial chemopreventive effect. This is potentially attributable to the
presence of single nucleotide polymorphisms (SNPs) in genes involved in
aspirins pharmacokinetic and pharmacodynamic pathways. We investigated the impact of 32 SNPs within 9 genes and 2 SNPs in intergenic regions
on CRC risk using the Leeds Colorectal Cancer Panel which contains 1910
cases and 1276 controls; these SNPs were chosen for their potentially functional effect. Sixteen out of 34 SNPs were removed from analysis as they
had frequency of <3%. All remaining SNPs were in Hardy-Weinberg equilibrium. P values for the tests of association were adjusted for sex and age.
The presence of rs11694911 T allele (OR=0.79, 95% CI=0.63-1.0, p=0.04)
and rs2302615 T allele (OR=0.81, 95% CI=0.67-0.99, p=0.024) at ODC1 locus were associated with a reduced CRC risk. When the data was stratified
(colon versus rectum), CYP2C9 rs1799853 T allele (OR=0.73, 95% CI=0.600.90, p=0.003) and ODC1 rs2302615 T allele (OR=0.76, 95% CI=0.61-0.93,
p=0.005) showed significant association with colon cancer suggesting influence may be dependent on the tumor location. Both variant alleles have
previously been shown to modulate chemopreventive activity of aspirin in
colorectal adenoma occurrence, but this is the first dataset to indicate their
effect on carcinoma risk. This data suggests that the presence of these variants modulates CRC risk which in turn may provide insight into the variation
in aspirins chemopreventive efficacy.
P12.040-M
Identification of novel candidate genes for early-onset colorectal
cancer susceptibility
Approximately 25-30% of colorectal cancer (CRC) cases are expected to result from a genetic predisposition, but only in 5-10% of these cases highpenetrant germline mutations are found. Therefore, the majority of CRC
heredity is still unexplained. This missing heritability is thought to result
from the presence of rare variants with a moderate-penetrant risk. Wholeexome sequencing (WES) has made it possible to identify such risk factors.
As hereditary CRC is marked by an early age of onset, we performed WES on
a cohort of CRC patients (n=55) with an onset of disease before 45 years of
age. To identify potentially pathogenic variants, we searched for rare protein-truncating variants or highly conserved missense variants. A total of 272
protein-truncating variants and 1537 missense or in-frame insertions and
deletions were identified. Recurrence filtering and selection of genes potentially involved in CRC development revealed three novel candidate genes:
EMR3, PTPN12 and LRP6. A screen of the coding region of these genes in
a cohort of early-onset or familial CRC patients (n=181) revealed additional rare variants in PTPN12 and LRP6. PTPN12 encodes a protein tyrosine
phosphatase and is described to be a driver of cancer development. LRP6
encodes a component of the Wnt-Fzd-LRP5-LRP6 complex that triggers
beta-catenin signaling. This study contributes to the further understanding
of the heterogeneity of the genetic susceptibility for CRC. Follow-up studies
on the frequency of mutations in these genes in larger cohorts, and their
functional assessment, will provide conclusive evidence for the role of these
genes in CRC development.
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P12.041-S
Whole-exome sequencing identifies rare coding variants in new
predisposition genes for familial colorectal cancer
245
ABSTRACTS POSTERS
of Genetics, Rouen University Hospital, Rouen, France, 6Inserm U1079 and Department
of Genetics, University Hospital, Rouen, France.
Objective: Recognizing colorectal cancer (CRC) patients with Lynch syndrome (LS) can increase life expectancy of these patients and their close relatives. To improve identification of this under-diagnosed disease, experts
suggested raising the age limit for CRC tumour genetic testing from 50 to
70 years. The present study evaluates the efficacy and cost-effectiveness of
this strategy.
Design: Probabilistic efficacy and cost-effectiveness analyses were performed comparing tumour genetic testing of CRC diagnosed at age 70 or below
(experimental strategy) versus CRC diagnosed at age 50 or below (current
practice). The proportions of LS patients identified and cost-effectiveness
including cascade screening of relatives, were calculated by decision analytic models based on real life data.
Results: Using the experimental strategy, 4 times more LS patients can be
identified among CRC patients as compared to current practice. Both the
costs to detect one LS patient ( 9,437/carrier versus 4,855/carrier) and
the number needed to test for detecting one LS patient (42 versus 19) doubled. When including relatives the experimental strategy was found to be
highly cost effective because CRC prevention in relatives neutralised the extra genetic testing costs, resulting in lower costs (- 2180 per extra tested
patient) and an average of 9 life years gained.
Conclusion: Testing all CRC tumours diagnosed at or below age 70 for LS
is cost-effective. Implementation is important as relatives from the large
number of LS patients that are missed by current practice, can benefit from
life-saving surveillance.
P12.045-S
Clinical relevance of 8q23.3, 15q13.3 and 18q21.1 SNP genotyping to
evaluate colorectal cancer risk
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The aim of this study was to determine if the at risk SNP alleles for colorectal
cancer (CRC), previously identified by GWAS studies, could either alone or in
combination contribute to clinical presentations suggestive of an increased
genetic risk for CRC. We performed a prospective national case-control
study based on highly selected patients (CRC in two first degree relatives,
one being diagnosed before 61 years; or CRC before 51 years; or multiple
primary CRCs, the first before 61 years; exclusion of Lynch syndrome, adenomatous and hamartomatous polyposes), and appropriate controls corresponding to healthy volunteers, between 45 and 60 years of age, without
personal or familial history of CRC. We included 1029 patients and 350 controls. We confirmed the association of CRC risk with 4 SNPs, with odds ratio
(OR) higher than previously reported : rs16892766 on 8q23.3 (OR: 1.89;
p=0.0006); rs4779584 on 15q13.3 (OR: 1.45; p=0.0033), and rs4939827
and rs58920878/Novel 1 on 18q21.1 (OR: 1.49; p=0.0061 and OR: 1.48;
p=0,0039). We found a significant cumulative effect of the at risk alleles or
genotypes with OR at 1.62, 2.11, 2.92 and 3.95 for 1, 2, 3 and at least 4 at risk
alleles and OR at 1.71, 2.32 and 6.31 for 1, 2 and 3 at risk genotypes. This
study shows that genotyping of a limited number of SNPs, such as 8q23.3
rs16892766, 15q13.3 rs4779584 and 18q21.1 rs58920878, should allow to
identify subjects at increased risk of CRC who might benefit from early CRC
detection.
P12.046-M
Diagnostic criteria for constitutional mismatch repair deficiency
(CMMR-D) syndrome: suggestions of the European consortium Care
for CMMR-D (C4CMMRD)
ABSTRACTS POSTERS
P12.047-S
Constitutional mismatch repair deficiency due to a homozygous MSH6
mutation in a 14-year-old boy with colonic polyps, colorectal cancer
and retinal lesions
L. S. Penney1,2, A. L. Rideout1, S. Dyack1,2, M. Bernstein1,2, M. Rashid1,2, W. Huang2, G. R.
LaRoche2;
1
The IWK Health Centre, Halifax, NS, Canada, 2Dalhousie University, Halifax, NS, Canada.
Constitutional Mismatch Repair- Deficiency (CMMR-D) is a rare cancer predisposition syndrome in patients with biallelic mutations in one of MLH1,
MSH2, MSH6 or PMS2. Heterozygous mutations cause the adult onset, dominant cancer syndrome Lynch syndrome. However, CMMR-D causes childhood onset cancers, particularly hematological, brain and gastrointestinal
malignancies. Children with CMMR-D also may have skin pigmentation findings suggestive of neurofibromatosis type 1.
We report on a 14-year-old boy with a previous kaposiform hemangioendothelioma who was diagnosed with at least 100 colonic polyps and 6 invasive
adenocarcinomas of the colon and rectum. He had caf au lait macules, axillary and inguinal freckling and bilateral, multifocal congenital hypertrophy
of the retinal pigment epithelium (CHRPE). Therefore he fit diagnostic criteria for both NF1 and Familial Adenomatous Polyposis. He also had a previous clinical diagnosis and family history of Cleidocranial Dysplasia. There
was no family history of colon cancer and he had 2 siblings.
The colon cancers were MSI-H and by immunohistochemistry the tumour
cells and adjacent normal mucosa were negative for MSH6 and positive for
MLH1, MSH2 and PMS2. A homozygous pathogenic MSH6 mutation was
identified (c.3202C>T) in the patient and both parents were found to be heterozygous. Genetic testing of the APC and MUTYH genes was normal.
To our knowledge, this is the first reported case of CMMR-D with CHRPE
and demonstrates difficulties in making this diagnosis in a patient who fits
the clinical criteria for both NF1 and FAP. It also illustrates ethical dilemmas
around genetic testing of at risk siblings.
P12.048-M
Recurrent copy number alterations in prostate cancer: an in silico
meta-analysis of publicly available genomic data
J. A. Squire1, P. Greer2, J. Williams2;
1
University of Sao Paulo, Ribeiro Preto, Brazil, 2Queens University, Kingston, ON,
Canada.
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with 1-3 different kits. Following clinical standards, we evaluated the analytical sensitivity, the analytical specificity, false positive and negative rates,
the assay robustness and the precision for each of the considered capturing techniques. Our results demonstrate that technical and functional differences exist between capturing methods that could compromise clinical
diagnosis if specific method-associated deviations are not considered.
P12.051-S
Mutually exclusive RAS-RTK and JAK2 mutations drive sub-clonal
expansions in the majority of acute lymphoblastic leukaemia cases in
Down syndrome
P12.049-S
In-depth comparison of available targeted resequencing strategies for
BRCA gene panels
S. Zuiga, O. Rodriguez, G. Marco, J. Durban, L. Cabornero, S. Santillan, C. Collado, V.
Fernndez-Pedrosa, J. C. Trivio;
Sistemas Genomicos, Valencia, Spain.
247
ABSTRACTS POSTERS
P12.052-M
Neoantigen in esophageal squamous cell carcinoma for dendritic cellbased cancer vaccine development
Ewing sarcoma is a cancer that often presents in the second decade of life
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Breast and ovarian cancer predisposition has been associated with a number of high-, moderate-, and low-penetrance susceptibility genes. Here we
report on the results of high-resolution custom array-CGH for deletion/duplication analysis and panel-based screening of 94 genes associated with
hereditary cancer predisposition.
Selection criteria for the 100 patients were defined by the German Consortium for Breast and Ovarian Cancer. NGS was performed on an Illumina MiSeq
and target enrichment was done with the Illumina TruSight cancer panel,
whereas custom array CGH was performed using Agilent technology. In 28
% of the patients, BRCA1 or BRCA2 variations have been found. These were
either clearly pathogenic protein truncating mutations (12 %) or very rare,
unclassified missense variations with high probability of effect (16 %). In
39 % of the patients we found rare, unclassified missense variants in low
penetrance susceptibility genes, especially NBN and ATM. In one case with
early onset of breast cancer and no familial history, a putative splice relevant
mutation in TP53 could be identified, which is currently being investigated
on cDNA level. 33 % of the patients did not reveal any convincing sequence
variation. In 10% of the patients we identified deletions in either BRCA1,
CHEK2, or ATM.
The extension of mutation screening beyond BRCA1 and BRCA2 reveals
disease-causing mutations in high-penetrance genes, like TP53, as well as
mutations in low-penetrance susceptibility genes. However, the enormous
number of unclassified sequence variants and the detection of carriers for
other hereditary diseases pose a huge challenge for genetic counselling.
P12.057-S
First evidence for FANCM as a novel breast cancer predisposing gene
ABSTRACTS POSTERS
ponent of the Fanconi anaemia core complex and promotes the recruitment
of the S-phase checkpoint machinery after binding to DNA damage sites. The
nonsense mutation removes the ERCC4-like domain (aa1802-1922) of the
2022aa FANCM protein. A subsequent Sequenom-based case-control study
including 1896 independent BRCA1/2-negative BC index cases and 1726
controls revealed a significant association with BC occurrence (9/1896 cases vs. 2/1726 controls; p<0.05). The carrier frequency of the p.Gln1701Ter
mutation in controls (0.12%) is similar to that identified in the framework
of the NHLBI Exome Sequencing Project (5/4300; european ancestry;
0.12%). Taken together, our data provide first evidence for FANCM as a novel BC predisposing gene (9/1896 cases vs. 7/6026 controls; p=0.00242) at
a moderate penetrance level (OR: 4.101; CI95%:1.401-12.199; RR=2.357;
CI95%:1.27-3.326). Due to the low frequency of the FANCM p.Gln1701Ter
mutation, however, large collaborative studies are required to quantify the
RR of FANCM alterations for BC and potentially other cancer entities.
P12.058-M
First clinical experience with screening formalin fixed and paraffinembedded (FFPE) archival tissue for mutations in BRCA1 and BRCA2
- A new paradigm in genetic investigation and counseling
A. H. Petersen1, M. M. Aagaard1, M. Waldstrm2, A. Bojesen1;
1
Vejle Sygehus, Department of Clinical Genetics, Vejle, Denmark, 2Vejle Sygehus,
Department of Clinical Pathology, Vejle, Denmark.
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Introduction: The main causes for gastric cancer are Helicobacter pylori (H.
pylori) infection, diet and genetic factors. Germline CDH1 mutations underlie a gastric cancer predisposition syndrome with autosomal dominant inheritance, characterized by early-onset diffuse gastric cancer. In the majority of cases with early-onset or familial gastric cancer the underlying genetic
cause remains unknown. We aimed to find new genetic predisposition genes
for gastric cancer. Patient: A 23-year old female patient with diffuse-type
gastric cancer and without a germline CDH1 mutation also suffered from
recurrent fungal infections. Family history revealed a consanguineous relation of her parents. There were no other cases of gastric cancer in the family.
Results: Whole exome sequencing of the patients germline DNA identified
a homozygous missense variant (c.712C>T, p.Arg238Cys) in the gene encoding MYD88, which plays a central role in the immune response against
H.pylori infections. Immunological assays on peripheral blood mononuclear
cells revealed normal immune responses to Staphylococcus aureus, but impaired immune responses upon stimulation with H.pylori and Candida albicans, characterized by a specific defect in production of Th17 cytokines
IL-17 and IL-22. This was due to defective IL1-beta responses, a crucial cytokine for Th17 development. Conclusion: Impaired Th17 mucosal host defense against H.pylori, which is associated with gastric cancer development,
was observed in a patient with early onset gastric cancer and a homozygous
MYD88 variant. These defective Th17 responses were also likely the cause of
fungal infections. Our data suggest that genetic defects leading to defective
anti-Helicobacter innate pattern recognition predispose to gastric cancer.
P12.062-M
Prohibitin 3 untranslated region 1630 C>T polymorphism and copy
number variation contributes to its gene expression deregulation in
gastric cancer
Gastric cancer is the second leading cause of cancer-related deaths worldwide. PHB has been reported as an oncogene and tumor suppressor in several
neoplasias, including in gastric cancer. Here, we evaluated whether the PHB
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ABSTRACTS POSTERS
copy number and the rs6917 polymorphism affect its expression in gastric
cancer. Forty-eight pairs of gastric cancers and corresponding non-neoplastic gastric samples were evaluated. PHB expression was analyzed by real-time quantitative PCR and by immunohistochemistry. Gene copy number was
investigated by quantitative PCR. Allele-specific expression was determined
by sequencing and by TaqMan assay. Down-regulation and up-regulation of
PHB was observed in the tumors (45.5% and 20.5%, respectively). Reduced
PHB expression was associated with dedifferentiation (P=0.029), lower invasion (P=0.002), absence of lymph node metastasis (P=0.040) and early
gastric cancer (P=0.002). In all cases, the PHB immunoreactivity was detected in neoplastic and non-neoplastic cells. PHB was mainly expressed in
the cytoplasm. PHB amplification was observed in 34.2% tumors. PHB gain
was associated with higher gene expression (P=0.003) and late-onset gastric cancer (P=0.022). In our sample, 22% patients were heterozygous and
4.17% were homozygous for the minor allele in the rs6917 polymorphism.
The presence of the T allele in this polymorphism was associated with reduced expression in GC (P<0.001) and in non-neoplastic samples (P<0.001).
Only one patient presented a higher C/T ratio in both tumor and non-neoplastic samples. In most cases, lower C/T ratio was detected. Thus, PHB copy
number variation and differential expression of the rs6917 polymorphism
may have a role in PHB transcriptional regulation.
P12.063-S
Extrachromosomal driver mutations in glioblastoma and low grade
glioma
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have also confirmed LOH at 8q21.3 and 14q13.3 with a frequency of 17.9%
(5/28) and 60.0% (9/15), respectively, which other authors have reported.
P12.065-S
Effects of NDRG2 downregulation on glioblastoma patient survival
G. Steponaitis, D. Skiriute, P. Vaitkiene, M. Mikuciunas, A. Kazlauskas;
Lithuanian University of Health Sciences, Neuroscience institute, Laboratory of
Neurooncology and Genetics, Kaunas, Lithuania.
Molecular changes associated with the relapse of glioblastoma after standard radiochemotherapy remain poorly understood. Here we compared
genomic profiles of 27 pairs of primary and recurrent IDH1/2 wild-type
glioblastomas by genome-wide array-based comparative genomic hybridization. By bioinformatic analysis, primary and recurrent tumor profiles
were normalized and segmented, chromosomal gains and losses called taking the tumor cell content into account, and difference profiles deduced.
Seven of 27 (26%) pairs lacked DNA copy number differences between
primary and recurrent tumors (Equal pairs). The recurrent tumors in 9/27
(33%) pairs contained all chromosomal imbalances of the primary tumors
plus additional ones, suggesting that a major subclone sequentially accumulated aberrations (Sequential pairs). In 11/27 (41%) pairs, the profiles
of primary and recurrent tumors were divergent, i.e. the recurrent tumors
contained additional aberrations but had lost others, suggesting a polyclonal composition of the primary tumors and considerable clonal evolution
(Discrepant pairs). Losses on 9p21.3 harboring the CDKN2A/B locus were
significantly more common in primary tumors from non-Equal pairs. NonEqual pairs showed ten regions of recurrent genomic differences between
primary and recurrent tumors harboring 46 candidate genes associated
with tumor recurrence. In particular, copy numbers of genes encoding apoptosis regulators were frequently changed upon recurrence. In summary,
approximately 25% of IDH1/2 wild-type glioblastoma pairs have stable
genomic imbalances. In contrast, approximately 75% of IDH1/2 wild-type
glioblastomas undergo further genomic aberrations and alter their clonal
composition upon recurrence impacting their genomic profile, a process
possibly facilitated by loss on 9p21.3 in the primary tumor.
ABSTRACTS POSTERS
P12.067-S
Targeted resequencing for analysis of gene mutations in pediatric
Glioblastoma Multiforme
Glioblastoma multiforme (GBM; WHO-grade IV), the most frequent primary malignant brain tumor in adults, accounts for approximately 7-9% of
all central nervous (CNS) tumors in childhood. Adult and paediatric GBMs
(pGBMs) have distinct genetic and molecular pathways of tumorigenesis and
different studies, based on array-CGH analysis, reported that there are significant differences in Copy Number Alterations (CNA). In our previous study
we identified, using array-CGH, recurrent CNA in 8 pGBMs establishing minimum common regions (MCR) of duplication/amplification and deletion.
Based on these results, we developed a next-generation sequencing (NGS)
approach to screen the genetic profile of tumors. NGS has provided a new
paradigm in biomedical research to delineate the genetic basis of human diseases. The panel was designed to cover 420 genes selected within of MCRs
to try to identify new genes involved in tumorigenesis and/or progression
of pGBMs. In 8 patients we found 18 heterozygous mutations in different genes. The same mutations were also found in DNA extracted from blood and
in two cases we demonstrated the parental origin of 12 mutations. Given the
rarity of the disease and the scarcity of data in the literature, our findings
may better elucidate if there is a genomic background in development and
progression of pGBM. The recognition of candidate genes underlying this
disease could then improve treatment strategies for this devastating tumor.
P12.068-M
Distinctive expression profiles of lncRNAs in glioma subtypes
Background: Gliomas are the most malignant and common primary brain
tumors, classified upon malignancy grade and histological characteristics.
Correct diagnosis that relies solely on histopathological characteristics
might be difficult and inadequate, particularly in cases that lack typical features. Glioma subtypes have distinct molecular features and gene expression
analyses could uncover molecular biomarkers for deciphering glioma subtypes. Our purpose was to identify lncRNAs that might play role in gliomagenesis and investigate potential association of differential expression profiles
with different glioma subtypes. Patients and methods: Sixty-four patients
were included in the study. We used quantitative real-time PCR (qPCR) in
order to determine differentially expressed lncRNAs using LncRNA array
profiler on a smaller cohort of pathohistologically evaluated glioma samples normalized to human brain reference RNA. A subset of differential lncRNAs was further validated by qPCR approach on a biger cohort of glioma
samples and statistically evaluated using Mann-Whitney and Kruskal-Wallis
tests. Results: LncRNA profiling revealed a total of 74 among 90 analysed
lncRNAs to be widely expressed and statistical tests identified a subset of
10 significantly differentially expressed lncRNAs. Further analyses of 7 dysregulated lncRNAs (7SL, EGOA, HOTAIR, JPX, MEG3, RNCR3 and ZNFX1-AS1)
have showed statistically significant differences in relative gene expression
ratios implicating distinctive expression profiles in glioma subtypes. Conclusions: Our findings support the concept of lncRNAs as molecular biomarkers by determining distinctive subtype-related lncRNA expression patterns between glioma subtypes, which suggest an important role of lncRNAs
in glioma biogenesis and implicate the potential use of lncRNAs in glioma
histological classification.
P12.069-S
Genomic hallmarks in glioma
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L. Ouyang1, J. Lee2, C. Park2, M. Mao3, Y. Shi1, Z. Gong1, H. Zheng1, Y. Li1, G. Wang1, Y. Zhao1,
H. Fu1, J. Kim2, H. Lim2;
1
BGI, Shenzhen, China, 2Sungkyunkwan University School of Medicine, Seoul, Korea,
Republic of, 3Pfizer Inc., San Diego, CA, United States.
251
ABSTRACTS POSTERS
both primary and metastatic tumors are usually sensitive or resistant to the
same systemic treatments.
P12.073-S
Identification of a novel CDH1 gene mutation in a family with
hereditary diffuse gastric cancer
Malignant gliomas have a highly invasive phenotype, which is a main determinate for the poor prognosis of patients suffering from these tumors
despite multimodal therapy. In previous studies, we used 24-color-FISH
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The Li-Fraumeni syndrome (LFS), due to germline TP53 mutations, represents a remarkable cancer predisposition characterized by the extent of tumour spectrum. For 20 years, analysis of over 1700 families allowed us to
identify 214 French families with TP53 mutation, thanks to the Chomprets
criteria gradually elaborated by the French LFS working group. Updated
data from 322 LFS patients with 552 tumours revealed that the median age
of first tumour onset was 27 years, that the most frequent tumours were
breast cancers (167), soft-tissue sarcomas (103), osteo/chondro-sarcomas
(58), brain tumours (43) and adrenocortical carcinomas (42), and that 43%
of patients developed multiple primary tumours. Germline TP53 mutations
were detected in 50% of children with adrenocortical carcinoma, choroid
plexus carcinoma, or rhabdomyosarcoma. Sixty-four percent of TP53 alterations were missense mutations and 4% genomic rearrangements. We found
that TP53 mutation carriers harbouring the MDM2 285-309GG haplotype
developed tumours 5 years earlier than others. The most striking observation was that patients with dominant-negative missense mutation developed
first-tumour earlier (22 y) than patients with null-mutation (42 y). Twenty
ABSTRACTS POSTERS
years after the identification of LFS molecular basis, this report reveals that
2 forms of LFS probably exist: an aggressive, characterized by early-onset tumours, due to highly penetrant dominant-negative missense mutations, and
a moderate, characterized by later age of onset, due to mutations with lower
penetrance. The remarkable high incidence of multiple primary tumours is
probably explained by genotoxic effects of chemo- and radio-therapy and
the key role of p53 response to DNA damage.
P12.078-M
Identification of a characteristic copy number alteration profile
by high-resolution SNP arrays associated with metastatic sporadic
colorectal cancer
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It is becoming evident that Lynch Syndrome (LS) is one of the most common
cancer syndromes. Diagnosis is not always trivial and may be costly. Knowledge of incidence, spectrum of mutations and genes involved in specific populations, facilitates the diagnostic process and contributes to clinical workup. AIM: To report a cohort of LS families in the Israeli population, gene
distribution, mutations detected and co-occurrence of related syndromes.
METHODS: Patients were studied at high risk clinics. Diagnostics followed a
multi-step process. It included testing for founder mutations, tumor testing,
gene sequencing and MLPA. LS was defined by positive tumor analysis and/
or positive mutation testing. RESULTS: We describe a cohort of 318 carriers from 169 families with LS. We have identified 61 different mutations in
102 families; 3 founder mutations occurred in over 70% of Ashkenazi LS
families, where mutations in MSH6 were more common than in MLH1. We
identified founder mutations also among Jewish families from Georgia, Iran
and Afghanistan. MSH2 was mutated in 62% of the cases, one third of these
mutations were large deletions. Constitutional mismatch repair deficiency
(C-MMRD) was identified in 6 families. CONCLUSIONS: Mutation spectrum
and gene distribution in the Israeli population is unique. Mutations in MSH2
and MSH6 cause the majority of cases. Six founder mutations contribute to
LS in 4 different ethnic sub-groups. C-MMRD occurs either due to founder
mutations or consanguinity. These features affect the phenotype, the diagnostic process, risk estimation, and genetic counseling.
P12.081-S
Genotype and Parent of Origin Effect on Colorectal and Endometrial
Phenotypes in Patients with a PMS2 mutation
Introduction Mutations in the PMS2 gene are responsible for Lynch syndrome, a genetically inherited disorder with an increased risk of foremost
colorectal cancer (CRC) and endometrial cancer (EC). Possibly, the reported
variability in cancer prevalence and age of diagnosis between patients and
families can partly be explained by genotype and/or parent of origin effects
(POE). Methods The genotypes and clinical data of 383 European PMS2 mutation carriers were available for analysis. Mutations with loss of RNA expression (group 1) and retained RNA expression (group 2) were compared.
A one-way Anova test and Cox regression were done to compare mean age
of cancer diagnosis and calculate hazard ratios (HR). Results Mean age of
CRC diagnosis was 50,99 years (CI 48,09-53,89) for group 1 and 60,00 years
(CI 52,50-67,50) for group 2 (p=0,032). For EC no significant differences in
mean age of diagnosis were found. Cox regression showed slightly higher
non-significant HRs for both CRC (HR: 1,274, p=0,440) and EC (HR:1,216,
p=0,722) when comparing group1 to group 2. No significant HR for CRC or
EC depending on whether the mutation was inherited from father or mother
was found. Discussion A higher mean age at CRC diagnosis and a non-significant higher CRC risk in the group with loss of RNA expression was identified. No significant evidence of a POE was found. Larger studies are needed
to confirm findings. Possible information on genotype and other modifying
risk factors might lead to individual risk stratification and surveillance programs in the future.
P12.082-M
Bi-allelic MSH6 mutations in a case of early onset multiple primary
tumors resembling Lynch and Turcot syndrome
253
ABSTRACTS POSTERS
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Multiple primary tumors of the gastrointestinal tract, uterus, ovaries, central nervous system and other organs are hallmarks of Lynch syndrome,
caused by heterozygous germline mutations in the mismatch repair (MMR)
genes MLH1, MSH2, MSH6 and PMS2. Bi-allelic mutations of the same genes
have been associated with a rare childhood cancer syndrome characterized
by hematological malignancies, sarcomas, brain and gastrointestinal tumors
together with caf-au-lait spots resembling Neurofibromatosis 1. Here we
report on a female patient who developed multiple primary tumors between
21 and 27 years of age including two metachronous colorectal cancers, a
fillodes tumour of the breast, a glioblastoma and a clear cell carcinoma of
the ovaries. Genetic testing identified two germline heterozygote MSH6
mutations: a frameshift mutation (c.1610_1613delAGTA) inherited from
the father and a suspected deleterious missense mutation (p.Arg1076His)
inherited from the mother. No TP53, MLH1 or MSH2 mutations were found.
Microsatellite analysis revealed instability of BAT26 and BAT40 in both the
colon and the ovarian carcinomas. The MLH1, MSH2 and PMS2 proteins showed nuclear staining in all specimens while MSH6 was completely absent in
tumor and normal cells, including the fillodes tumor of the breast and the
normal mucosa of the colon. Germline mutations in the MMR genes, either
mono-allelic or bi-allelic, can therefore underlie a wide spectrum of cancer
syndromes characterized by variable age at onset and severity of the cancer
risk. Development of multiple primary tumors in young adults appears to be
suggestive for the presence of bi-allelic mutations of the MMR genes.
c.1187G>A (p.G396D) variant and was diagnosed as MAP syndrome. Predictive test for relatives unveiled an unexpected finding, we found one of
her sons with an apparent wild type sequence. Further analysis evidenced
a large deletion comprising exon 4-16 in both mother and son. Breakpoint
characterization of this deletion allowed us to precisely define this alteration as c.348+33_*64+146del4285insTA with a precise size deletion of 4,285
kb. The same deletion has been published in other two unrelated families
from French and Portuguese origin suggesting a founder effect. Large rearrangements at MUTYH locus are rare although probably under-diagnosed.
Based on these findings we recommend first, to include MUTYH gene in the
testing strategy for LS and second, to test for MUTYH large rearrangements
in heterozygous cases after whole gene sequencing, as well as in apparent
homozygous cases.
Background: In male breast cancer (MBC) the prevalence of BRCA1/2 mutations varies considerably between countries. In the Netherlands data were
collected of all MBC patients diagnosed in the last 20 years. The nationally
agreed criteria for DNA testing are rather broad, implying that many MBC
cases with or without a family history for breast cancer have been tested.
Aim of this study is to get more insight in the percentage of BRCA1/2 mutations among an unselected cohort of MBC patients. Methods: All diagnosed
MBC patients between 1989-2009 (n=1487) were linked to databases of
all clinical genetic centers. Data of BRCA testing, family history and tumor
characteristics were collected. Results: 334 (22%) of MBC patients were
tested for BRCA1/2. Ten (3%) BRCA1, 51 (15%) BRCA2 mutations were
identified and also 7 (2%) variants of uncertain significance (VUS). At least
one first or second degree relative with breast cancer <50 yr was seen in
80% of BRCA1, 20% of BRCA2 and 6% of non-BRCA1/2 MBC patients. Preliminary studies did show that the majority (86%) of BRCA associated MBC
were ductal carcinomas of no special type. Conclusion: Roughly one fifth
of unselected MBC patients had undergone DNA testing, in 18% of these a
BRCA mutation was found. Most striking family histories were seen in BRCA1 families, whereas the majority of BRCA2 MBC patients had no strong
family history. Based on our results and previous studies genetic testing for
BRCA1/2 should be recommended for any MBC case, regardless of family
history for breast cancer.
P12.083-S
Constitutional epimutation of MLH1 gene coexisting with a genomic
deletion in Lynch Syndrome
Significant phenotypic similarities among Lynch syndrome (LS) and MUTYH-associated polyposis (MAP) has been reported.
We describe a family suspicious of having LS. A 69 years old female diagnosed of an endometrial cancer (T1N0M0) and right CRC (T3N0M0) with
multiple polyps at ages 60 and 65, respectively. Endometrial tumor had loss
of MLH1 and PMS2 expression, MSI, no BRAF_V600E mutation, absence of
MLH1 methylation, and KRAS_G12C mutation. No pathogenic variant was
found in the MLH1 mutation screening and MAP suspicion was considered.
MUTYH testing was approached by Sanger sequencing screening of recurrent variants at exons 7 and 13. Patient did show homozygous pattern for
254
P12.085-S
Male breast cancer in the Netherlands: uptake and outcome of BRCA
testing. Results of study the Dutch Breast Cancer Research Group
(BOOG 2009-04) in collaboration with the EORTC 10085 study.
C. J. van Asperen1, N. Dijkstra2, P. J. M. van Hees1, W. A. G. van Zelst-Stams3, J. C.
Oosterwijk4, M. G. E. Ausems5, R. A. Oldenburg6, M. A. Adank7, E. W. Blom8, M. Ruijs9, T.
A. M. van Os10, L. A. M. Janssen1, C. van Deurzen11, S. Roshani11, J. W. M. Martens6, C. P.
Schroder4;
1
Dept. of Clinical Genetics, LUMC, Leiden, Netherlands, 2BOOG Study Center,
Amsterdam, Netherlands, 3RadboudUMC, Nijmegen, Netherlands, 4UMCG, Groningen,
Netherlands, 5UMCU, Utrecht, Netherlands, 6ErasmusMC, Rotterdam, Netherlands,
7
VUMC, Amsterdam, Netherlands, 8MUMC, Maastricht, Netherlands, 9NKI, Amsterdam,
Netherlands, 10AMC, Amsterdam, Netherlands, 11Dept. of Pathology, ErasmusMC,
Rotterdam, Netherlands.
P12.086-M
Role of MAP/Microtubule Affinity Regulating Kinase 4 in the
regulation of cell cycle progression and cytoskeleton dynamics
ABSTRACTS POSTERS
intermediate filaments. Overexpression of kinase-dead mutants indicated
that the effects on both cytoskeleton compartments are due to MARK4 kinase activity. The overall data highlight MARK4 as a key component in the
regulation of MT dynamics, demonstrate its role in cell cycle progression,
particularly at the G1/S transition, and point to vimentin as a new plausible
MARK4 interactor.
P12.087-S
Multiple primary melanoma (MPM) as a valid criterion for genetic
assessment : an Italian IMI multi-center study
Back to index
BACKGROUND: Ipilimumab-based immunotherapy has substantially increased survival for patients with advanced melanoma, however, the benefit
is observed only in a small portion of treated patients. It is highly plausible,
yet completely unexplored, that germline genetic factors modulate immunotherapy outcome. In this study we performed whole-exome sequencing
(WES) to discover novel germline determinants of response to ipilimumab.
METHODS: Blood samples were collected from >60 metastatic melanoma
patients treated with ipilimumab at the New York University Cancer Center. WES was performed on objective responders (OR) and non-responders
(NR), defined by immune-related response criteria, using the Nextera platform (Illumina) at average 30x coverage. A novel method for testing the association between OR and NR by variant, gene and enrichment of molecular
networks was implemented. Gene-Set Enrichment Analysis and Pathway
Studio were used to test the pathway associations. RESULTS: The preliminary data comparing an initial subset of 30 ORs and 30 NRs identified significant associations with ipilimumab response for several loci including
RPS6KB1 (p=0.001) and LNX2 (0.001). In addition, the pathway analysis
showed significant associations for SMAD 3 (p=0.04) and interleukin 1
(p=0.04) related pathways. CONCLUSION: Preliminary findings provide promising evidence supporting the presence of germline genetic factors associated with response to ipilimumab therapy. The data suggest in part a role
for immune-related pathways. As we continue to accrue patients, we anticipate an increase in the analytical power of the discovery phase and also an
expanded validation of the current findings, suggesting for the first time that
germline genetic factors modulate immunotherapy response.
P12.090-M
Functional consequences of cancer associated variants in six human
microRNAs
I. Torruella-Loran, A. Gallego, I. Balcells, E. Garcia-Ramallo, Y. Espinosa-Parrilla;
Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Barcelona, Spain.
Non-small cell lung cancer (NSCLC) is the major group of lung cancers. MicroRNAs (miRNAs) are a class of small non-coding RNAs that play a crucial
role in the regulation of mRNA translation and degradation. miRNA expression is affected in many cancers. The expression levels of miRNAs let-7a,
miR-155, miR-205 in tumor and adjacent normal tissue at 2 and 5 cm from
255
ABSTRACTS POSTERS
tumor were measured by real-time PCR with subsequent quantification
using a 2-ddCT method. Obtained results were then analyzed for association
with clinical-morphological parameters: age, cancer stage, and tumor cells
differentiation. The expression of let-7a was significantly decreased in tumors comparing to adjacent tissue at both 2 and 5 cm. Let-7a and miR-155
levels in tumor were substantially lower than in adjacent tissue in patients
under 63 years. The expression of let-7a and miR-155 in tumor was also
suppressed in patients with III-IV stages of NSCLC. Besides that patients
with poorly differentiated NSCLC had significantly lower let-7a level in tumor comparing to adjacent tissue. The levels of let-7a, miR-155, miR-205
were different even in adjacent tissue, in patients with differentiated tumors
it was higher than in the group with poorly differentiated tumors. The study
showed that let-7a expression is suppressed in tumors comparing to adjacent normal tissue at 2 and 5 cm from tumor. The decrease of let-7a and
miR-155 is distinctive for younger patients, III-IV stages of NSCLC, and poor
tumor cells differentiation. Altogether these findings consider miRNAs let7a and miR-155 as markers of unfavorable prognosis for NSCLC patients.
P12.093-S
A germline mismatch repair mutation possibly leading to a de novo
NF1 germline mutation
We report on a female patient diagnosed with breast, colon, and endometrial cancer at the age of 38, 40, and 41 respectively, presenting with caf au lait
macules, multiple neurofibromas and axillary freckling.
With respect to the family history, her paternal grandmother died at the age
of 63 for a colon cancer diagnosed when she was 35, whereas her father died
at the age of 42 because of an accident.
As the proband met the criteria for Neurofibromatosis 1 (NF1), analysis of the
NF1 gene was performed. A frameshift mutation (c.7096_7101delAACTTT)
was identified confirming the clinical diagnosis.
The parents showed no clinical signs of NF1 nor did the two probands siblings, which tested negative.
Because of the co-occurrence of endometrial and colon cancer in our patient, and the presence of an early-onset colon cancer in the paternal grandmother, expression of mismatch repair protein and Microsatellite Instability
(MSI) were analyzed on endometrial and colon cancers.
Both tissue showed loss of staining for the MSH2-MSH6 heterodimer and
high MSI, therefore analysis of MSH2 was performed, leading to the identification of a nonsense mutation of exon 2 (c.289C>T, p.Gln97*), consistent
with the presence of the Lynch syndrome.
As women with NF1 have a fivefold risk of developing premenopausal breast cancer, no further analysis was performed.
Although it was not possible to analyze any member of the paternal family,
an intriguing possibility is that the inherited germinal mutation affecting
the mismatch repair system led to a de novo NF1 germinal mutation in our
patient.
P12.094-M
Whole genome sequencing of mitochondrial DNA in low and high-risk
neuroblastoma patients
F. M. Calabrese1, P. Pignataro1, F. Totaro1, G. Acierno1, F. Cimmino1, R. Gallesio2, G. Tonini3,
L. Longo2, A. Iolascon1, G. Gasparre4, M. Capasso1;
1
University of Naples Federico II, Napoli, Italy, 2IRCCS AOU San Martino-IST, Genova,
Italy, 3Istituto di Ricerca Pediatrica (IRP), Padova, Italy, 4Universitario S. OrsolaMalpighi, Bologna, Italy.
256
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Multiple primary malignant tumours (MPMT) are frequently taken as an indicator of potential inherited cancer susceptibility and occur at appreciable
frequency both among unselected cancer patients and referrals to cancer
genetics services. Analysis of a referral based series of 212 MPMT cases sho-
ABSTRACTS POSTERS
wed that only around 40% of those who underwent genetic testing and 20%
of referrals overall were identified as having a pathogenic germline mutation conferring predisposition to malignancy. Comparison of individuals who
tested positive and negative revealed considerable overlap between the two
groups with respect to clinical characteristics indicative of an inherited cancer syndrome, suggesting that many of the latter group also have a genetic
basis. Analysis of PTEN and TP53, however, did not reveal any significant
variants. Failure to detect a germline mutation may result from mosaicism
for a mutation in a known inherited cancer gene, an unusual phenotype
that leads to the relevant gene being overlooked or mutation in a novel inherited cancer gene. To address these possible explanations, further cases
are being identified and next generation sequencing techniques applied to
blood samples from the series. Initial analysis is being performed using the
Ilumina TruSight cancer panel (of known inherited cancer genes) with subsequent whole exome or genome sequencing if no mutation is identified. Tumour samples will also be analysed for loss of heterozygosity at the relevant
locus where putative mutations are found. Recruitment is currently open.
P12.098-M
Colorectal cancer susceptibility alleles as a possible explanation for
phenotype variance in MUTYH associated polyposis (MAP) patients
Infantile myofibromatosis (IM) is a rare disorder characterized by the development of benign tumors in the skin, muscle, bone, and viscera. The incidence is 1/150,000 live births and the disease is the most common cause of
fibrous tumors in infancy. Although the identification of mutations in genes
that may cause IM is the first step towards the possibility of targeted treatments, the molecular pathogenesis of IM is still poorly understood. Recently,
mutations in PDGFRB and NOTCH3 have been implicated in families with
the autosomal dominant forms of IM. We have performed whole-exome sequencing of a family with a probably autosomal recessive visceral multicentric infantile myofibromatosis. We studied two brothers and their healthy
consanguineous parents. In the two brothers we identified a c.511G>C
(p.Val171Leu) novel homozygous mutation in NDRG4 (N-myc downregulated gene family member 4). The healthy parents were heterozygous for the
mutation. Consistent with the phenotype of IM, NDRG4 is a tumor-related
gene. Its expression has been shown to be decreased in numerous types of
Back to index
neoplasia and there have been proposed that it might be a tumor suppressor gene. Additionally, studies have demonstrated that NDRG4 may have a
role in cell survival, tumor invasion and angiogenesis in tumors. We believe
that this homozygous mutation in NDRG4 may be a good candidate for the
causative variant of the autosomal recessive form of IM in the family studied
and we propose that it should be investigated in other cases of autosomal
recessive infantile myofibromatosis.
Financial Support: CNPq and Capes.
P12.100-M
Next-generation panel based characterisation of breast/ovarian
cancer genetic predisposition
Oral squamous cell carcinoma (OSCC) is the eighth most common cancer
worldwide. Alcohol, tobacco and smoking are well known risk factors for
OSCC. During oral cancer development; inflammation, angiogenesis and
thrombosis are involved which correlate with immune cells involved in the
production of cytokines, growth factors and adhesion molecules. The study
was to evaluate association of cytokine gene variants viz. IL-1RN Variable
Number of Tandem Repeats (VNTR in intron 2), IL-1-511C/T [rs16944], IL6-597G/A [rs1800797] and TNF--308 G/A [rs1800629] with oral cancer in
a north Indian population. Clinical and addiction details of healthy age/sex
matched controls (n=140) and OSCC patients (n=77) were recorded after
ethical clearance and consent. DNA was extracted and SNPs genotyped by
polymerase chain reaction and restriction fragment length polymorphism
(PCR-RFLP). Minor allele frequencies; genotype and allele frequencies were
calculated by chi-square (2) analysis (SPSS v.15.0). Gene-gene interaction,
pairwise linkage disequilibrium (LD) based on D statistics and correlation
coefficient (r2) of frequencies were analyzed using SHEsis (online version).
Genotypic frequencies of IL-1RN, IL-1 and IL-6 while allelic frequency of
IL-6 showed significant association with OSCC (P<0.001). TNF- increases
risk of OSCC upto 1.68 times in alcoholic subjects. GATI* and GGTII* haplotypes increased the risk up to 2.863 and 38.285 times respectively. This is the
first report from India showing the effect of cytokine gene polymorphisms
in OSCC to predict individuals at risk of oral cancer. The knowledge of risk
alleles will enable individuals to take precautionary measures before hand
and prevent or delay the onset of disease.
P12.103-S
Referral of ovarian cancer patients to genetic counselling by
oncologists: room for improvement
257
ABSTRACTS POSTERS
Aims and methods: BRCA1/2 mutations occur in 10-15% of ovarian cancer
(OC) patients, regardless of family history. In November 2012 a pilot protocol was developed by the Unit of Hereditary Cancer (UHC) and the Oncology
Service (OS) caring most OC patients in our Institute, to assess the feasibility
of offering genetic counselling (GC) to all OC women. Oncologists agreed to
propose GC during their clinics and to refer all interested OC patients by
directly arrange a contact with UHC. After the first year we evaluated oncologists adherence to protocol, patients compliance with GC and testing, and
prevalence of BRCA1/2 mutations.
Results: 104 OC women underwent an oncology visit from November 2012
to December 2013. Ten patients were excluded because they had GC in the
past. Only 29 patients (29/94; 31%) were referred to GC; 22/29 attended
GC (76%) and 21/22 had genetic testing (95%). Three pathogenic BRCA
mutations were detected (3/21; 14%); among healthy female close relatives, seven were tested and three were mutation-positive. Referral was much
higher for patients attending the first visit (14/26; 54%) than for follow up
patients (15/68; 22%). The main differences in these two settings are the
time schedule (one vs half an hour) and the checklist in the clinical record
(including or not family history).
Conclusions: Patients compliance to GC/testing was high, and BRCA mutation prevalence was as expected. However, oncologists compliance was
low, mainly because of practical barriers. Efforts are needed to integrate GCfocused tools and procedures in oncology practice.
P12.104-M
Targeted resequencing approach to investigate the mutational
landscape associated to platinum resistance in EOC
Introduction Despite initial response to first line platinum-based chemotherapy, more than 80% of high grade serous ovarian cancer patients relapse and develop resistance. The molecular and genetic features involved in
drug resistance are still unknown. By gene expression profile in a cohort of
patients from which matched biopsies were taken at primary surgery (PS-O)
when tumor was sensitive to chemotherapy and at time of relapse (SCR)
when the tumor was resistant, we identified EMT pathway as a key player
in tumor relapse (Marchini et al., 2013). Here we investigate the genomic alterations driving drug resistance by performing targeted DNA resequencing
on our cohort of SCR and PS-O samples.
Methods DNA libraries enriched in a selected panel of 30 genes encompassing key players of signal transduction, cell cycle and DNA repair were
generated using TruSeq Custom Amplicon kit and sequenced on MiSeq (Illumina). Data were analyzed using a high performance cluster computing
platform (Cloud4CARE project).
Results Analysis identified a total of 166 mutations (152 SNPs and 14 InDels), of which 51 affecting PS-O and 115 SCR. With a 500X coverage, we
observed BRCA1, BRCA2 and TP53 mutated in the majority of cases. In addition, the PI3K pathway was found mutated in SCR samples only.
Conclusions Our preliminary results suggest two main conclusions:
1- genomic alterations (SNPs or InDels) were more frequent in SCR compared to PS-O;
2- most of the mutations affected genes belonging to DNA and PI3K pathway.
P12.105-S
Transcriptome and pathway analysis identifies IRF1 as a predictor of
progression free and overall survival in ovarian carcinoma
258
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Aim: This study investigated the clinical background of Ashkenazi pancreatic cancer patients, either carriers or non-carriers of any of the three founder Ashkenazi mutation in BRCA1/2 genes namely, 185delAG, 5382insC and
6174delT.
Methods: Clinical characteristics including age at onset, smoking behaviour,
physical activity, BMI, and chronic diseases e.g., hypertension and hyperlipidemia was available for 90 pancreatic cancer patients, consecutively referred to our oncogenetic clinic.
Results: Fifteen (16.7%) carried a founder Ashkenazi mutation in either
BRCA1or BRCA2: of these, nine (60%) carried the 6174delT mutation in BRCA2; five (33.3%) and one (6.7%) harbored the 185delAG and the 5382insC
mutations in BRCA1, respectively. Carriers compared to non-carriers were
diagnosed at 617.5 and 63.011.3 years of age, respectively (p=0.520); and
have higher BMI levels (28.97.3 compared to 25.14.9; p=0.112). Although,
not significant, carriers smoked less than non-carriers (3, 20% compared to
28, 37.3%; p=0.197), were less physically active (3, 20% compared to 34,
45%; p=0.092), suffered less from the chronic diseases, hypertension (3,
20% compared to 31, 41%; p=0.072); and hyperlipidemia (0 compared to
11, 14.7%; p=0.080).
Conclusion: Our observation revealed that although not significant, noncarriers tend to have more known risk factors for the development of pancreatic cancer compared to BRCA1/2 mutation carriers.
P12.108-M
Germline FH mutations presenting with Phaeochromocytoma
ABSTRACTS POSTERS
University of Cambridge, Dept. of Medical Genetics, Cambridge, United Kingdom,
University of Cambridge, MRC Cancer Unit, Cambridge, United Kingdom, 3University of
Birmingham, Centre for Rare Diseases, Birmingham, United Kingdom, 4West Midlands
Regional Genetics Service, Birmingham Womens Hospital, Birmingham, United
Kingdom, 5Division of Genetics and Molecular Medicine, Kings College London, London,
United Kingdom.
1
2
At least a third of patients with phaeochromocytoma (PCC) or paraganglioma (PGL) harbour an underlying germline mutation in a known PCC/
PGL gene. Identification of germline mutations and key somatic mutations
is important for the application of personalised medicine. To investigate
the molecular pathogenesis in PCC, PGL and head and neck paraganglioma
(HNPGL) we analysed PCC/PGL/HNPGL by next generation (NGS) and Sanger sequencing strategies. For the NGS studies, 55 tumours were analysed
with the Ion Torrent AmpliSeq Cancer Hotspot Panel v2 which targets 54kb
of mutational hotspots in 50 oncogenes and tumour suppressor genes and
exome resequencing was performed in 12 tumours. Confirmed somatic missense mutations occurring in a single tumour were detected in CDH1, APC
and JAK3. Activating mutations in HRAS (p.Gln61Arg and p.Gly13Arg) and
HIF2A (p.Pro531Thr) were detected in 9.3% and 2% of tumours analysed.
HRAS and HIF2A oncogenic mutations were detected in 6/30 PCC, 0/21
HNPGL and 0/4 PGL (frequency in PCC vs HNPGL P=0.69). Ten tumours
harboured mutations in inherited PCC/PGL/HNPGL genes and no HRAS or
HIF2A mutations occurred in this group. Combining our data with a previous report of HRAS mutations in PCC/PGL (JCEM 2013;98:E1266-71) we
find that the mean frequency of HRAS mutations (7/9 at codon 61) in sporadic PCC/PGL is 10.8% (9/83; 95% CI = 6.3 to 17.9%) and in PCC/PGL with
an inherited gene mutation 0% (0/29; 95% CI 0 to 13.9%) suggesting that
HRAS and inherited PCC/PGL gene mutations might be mutually exclusive.
P12.110-M
Germline mutations in SDHB, SDHD, VHL, RET and TMEM genes in
patients with nonsyndromic pheochromocytoma/paraganglioma in
Serbian population
J. Bankovic, B. Ilic, B. Beleslin Cokic, J. Antic, G. Rodic, I. Milicevic, M. Petakov, S.
Damjanovic;
Clinic for endocrinology, Clinical Center of Serbia, Belgrade, Serbia.
Back to index
rently sporadic PHEO /PGL were screened.Germline mutations were investigated by using direct sequencing for point mutations in RET, VHL, SDHB,
SDHD and TMEM, and multiplex ligation - dependent probe amplification
for gross deletions in VHL gene.
Results: In 30/200 (15%) probands, germ-line variants were identified: 12
heterozygous germline mutations ( 7 novel nonsense: W218X; frameshift:
c.661delG, p.Asp221ThrfsX27; splicing:c.424-12delTCTT; missense: R116M;
frameshift: c. 636_637 ins A, p. Met 213Asn fsX8 and missense: H132R; splicing: c. 424-1insG -7 delA ) of the SDHB gene, 10 in VHL1 novel, V84M,
in 3 families , 6 variants in SDHD, 1 in RET and 1 in TMEM gene. Familiy
members were also tested. Most of the patients with mutation in SDHB gene
were found to have malignant PHEO/PGL. Patients with mutations in VHL,
RET and TMEM genes developed PHEO.
Conclusion:The most commonly mutated gene was SDHB, which carries the
highest risk of malignancy. Our patients with extra-adrenal disease needs
careful follow-up, since they are in higher risk for the development of metastases or novel adrenal/extra-adrenal PHEO. The patients with VHL mutation (V84M) is apparently classified as 2C. These patients may develop some
other tumors than PHEO.
P12.111-S
Post-transcriptional regulation of PHOX2B gene expression
Neuroblastoma (NB) is one of the most frequent and severe solid tumors in
childhood.
Mutations in the genes coding for the transcription factor PHOX2B and for
its transcriptional target ALK have been detected in sporadic and familial
cases of NB and over-expression of the two genes have been identified in NB
samples and cell lines.
In this work, in order to investigate the mechanisms underlying PHOX2B
overexpression in NB, we report in silico and in vitro characterization of the
PHOX2B 3 untranslated region (3UTR).
First, the in silico search for elements known to regulate the mRNA stability
allowed us to identify three AU-RICH elements (AREs) in the more distal
region of the 3UTR, in addition to several putative miRNAs binding sites.
Regions of the distal PHOX2B portion likely responsible for mRNA regulation were eventually defined by combining the above predictions with results
from the phylogenic conservation of the PHOX2B 3UTR.
In vitro experiments in IMR32 NB cells have shown that PHOX2B mRNA is
not stable, thus suggesting it as target of post-transcriptional regulation mechanisms. The successive cloning of the 3UTR PHOX2B downstream the Luciferase gene in the pmiRGlo vector allowed us to confirm such a hypothesis
and, following the generation of constructs containing progressively shorter
deletions of the 3UTR, to map position and extent of the regions responsible
for the PHOX2B mRNA stability.
As a consequence of all the above described observations, we suggest that
regulation and modulation of PHOX2B post-transcriptional stability may be
considered a pharmaceutical target in NB.
P12.112-M
In vitro drug screening approaches targeting PHOX2B overexpression in neuroblastoma
PHOX2B is a transcription factor involved in the regulation of neurogenesis and in the correct differentiation of autonomic nervous system. Several
evidence report a pathogenetic role of PHOX2B in neuroblastoma (NB): (i)
somatic and germline gain of function mutations in familial, sporadic and
syndromic cases of NB; (ii) the finding of ALK, a transcriptional target of
PHOX2B, as major familial NB predisposition gene; and (iii) the observation of ALK and PHOX2B over-expression in tumor samples and NB cell lines. Starting from these observations, we have performed an in vitro drug
screening targeting PHOX2B over-expression as a potential pharmacological means in NB. First, we have evaluated the effects of a (i) small subset
of molecules and an (ii) epigenetic library in a IMR-32 cell line stably expressing Luciferase gene under the control of PHOX2B promoter to identify
molecules able in down-regulating PHOX2B expression. Curcumin, SAHA
and trichostatin A showed a down-regulation of PHOX2B promoter activity
and a decrease of both protein and mRNA expression. To deepen into curcumin mechanisms of action, we have investigated the role of transcription
factors (TF) predicted by in silico analysis to bind the PHOX2B promoter.
259
ABSTRACTS POSTERS
Among these TF, we have demonstrated that treatments with 8 or 20 M
curcumin led to a decrease of PBX1/MEIS1 expression and modulated the
activity of NFB and AP-1. Moreover, combined drug treatments showed
successful effects of down-regulation of the expression of both PHOX2B and
its target ALK, thus supporting the notion of the effectiveness of molecule
combination in tumor therapy.
P12.113-S
PIK3CA mutations in non-small cell lung cancer
Results: The 1634A>C mutation which has already been identified in many
cancers and NSCLC was determined in 7,5% of the tumor tissue samples.
This mutation rate is higher than reported in the literature. Interestingly
a second mutation (1658_1659delGTinsC) was identified in these patients.
The concurrence of these two mutations has been reported as Cowden
syndrome in the literature which is known to be a cancer predisposition
syndrome. Conclusion: This finding is quite important since it can be an
indication of underlying cancer predisposition syndrome in NSCLC patients.
Besides previously reported PIK3CA mutations some novel mutations have
been defined.
P12.114-M
Development of Acquired Resistance to Anti-EGFR Therapy in
Colorectal Cancer Identified by Whole-Genome Plasma DNA
Sequencing
Introduction: EGFR-targeting monoclonal antibodies, cetuximab and panitumumab, are important therapeutic options in KRAS wild-type metastatic
colorectal cancer (mCRC) patients. However, secondary resistance inevitably ensues in all patients within 3-12 months from the start of therapy.
The mechanism and timing of emergence of resistance which limit the efficacy of these drugs, is highly relevant for designing therapeutic strategies.
Methods: We examined the plasma DNA of 10 KRAS wild-type mCRC patients who received anti-EGFR therapy, using a high-throughput whole genome sequencing (Plasma-Seq) and ultra-deep sequencing of genes associated
with resistance to anti-EGFR therapy such as KRAS, BRAF, PIK3CA and EGFR
with Illuminas MiSeq. Results: Genome-wide characterisation of the plasma DNA and corresponding primary tumor revealed several tumor specific
aberrations such as over-representation of chromosomes 8q, 13 and 20q,
and losses of 8p, 4 and 18. The development of resistance to anti-EGFR therapy was observed to be associated with novel focal amplification of KRAS
(n=3), MET (n=2) and ERBB2 (n=1) or high level polysomy of 12p which
includes KRAS (n=1). Overrepresentation of EGFR gene was associated with
initial good response to therapy. However, ultra-deep sequencing of KRAS,
BRAF, PIK3CA and EGFR did not reveal any novel mutations. Conclusion:
260
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Overall, predictive biomarkers associated with the anti-EGFR therapy efficacy, correlating well with treatment response was identified in 70% of our
patients. The tumor genome is prone to continuous changes and plasmaSeq, a fast and affordable tool enables identification of novel clones and guide real-time modification of treatment regimen to delay or prevent disease
progression.
P12.115-S
PMS2 mutation detection and PMS2 mutation spectrum in the
Netherlands
The genetic basis for Familial Colorectal Cancer Type-X (fCRC-X) is unknown.
Mutations at the proof-reading domains of DNA polymerase (POLE) and
(POLD1) have been recently identified in families with multiple colorectal
adenomas and CRC.
We aimed to assess the prevalence of POLE and POLD1 mutations in
fCRC-X.
A total of 63 index cases fulfilling Amsterdam II criteria with normal expression of mismatch repair proteins and microsatellite stable tumors were
included. Sanger sequencing of POLE-exon13 and POLD1-exon11 was performed.
None of the cases carried mutations in POLE. We found one case with a new
POLD1 variant in heterozygosis: c.1421T>C (p.Leu474Pro). The patient was
a woman who underwent a synchronous CRC and large bowel gastrointestinal stromal tumor at age 36. Her maternal aunt had metachronous CRC
(dx33y) and endometrial cancer (dx56y) and was a carrier of this variant.
Therefore, the index cases mother, who underwent endometrial cancer
(dx52y), was an obligate variant carrier. It has been described that the homologous residue of POLD1 p.Leu479Pro mutation in S. cerevisiae causes a
mutator phenotype. Furthermore, this is the paralogous residue of the hot
spot at POLE (p.Leu424). In silico prediction analysis strongly suggests a
pathogenic nature for this variant. Integration of all these evidences drove
us to classify this new variant as probably damaging.
POLD1 mutations might explain about 1.6% of fCRC-X. POLD1 testing should
be included in the diagnostic strategy for unexplained familial CRC.
ABSTRACTS POSTERS
Back to index
P12.117-S
Polymerase Proofreading - Associated Polyposis (PPAP): an unusual
family with the highly penetrant POLE p.Leu424Val mutation with
colorectal cancer, polyposis, duodenal cancer and diabetes
P12.119-S
DNA methylation profiles of PDGFB and FGF2 are potential
biomarkers of disease progression in primary myelofibrosis
P12.118-M
Association of variant -765G>C in the PTGS2 gene promoter with
melanoma in Italian patients and its relation to gene expression in
dermal fibroblasts
Aim: The production of prostaglandins, especially prostaglandin E synthetase (PGE2) is hypothesized to influence carcinogenesis by promoting cell
proliferation, inhibiting apoptosis, stimulating angiogenesis, and mediating
immune suppression. Cyclooxygenase-2 (Cox-2), the inducible isoform of
cyclooxygenase coded by the PTGS2 gene, is the key enzyme in the production of prostaglandins involved in inflammatory processes including cancer.
In melanoma skin cancer, Cox-2 is overexpressed in primary malignant melanoma and in their corresponding metastases. Aim of this study was to investigate if polymorphisms -765G>C (rs20417), and -1195A>G (rs689466)
in the PTGS2 gene impact on its expression in dermal fibroblasts and are associated with individual susceptibility to malignant cutaneous melanoma.
Methods: Two hundred forty patients presenting melanoma and 342 control individuals were genotyped for polymorphisms -765G>C (rs20417) and
-1195A>G (rs689466) by restriction fragment length polymorphism (PCRRFLP) analysis. PTGS2 gene expression was performed by Real Time PCR
using Sybr Green.
Results: The allele -765C was associated with an increased prevalence of
melanoma. No association of -1195A>G polymorphism was observed. Haplotype analysis of both variations showed that the haplotypes carrying the
minor alleles were associated to a higher risk of melanoma (p=0.02). Expression analysis indicated that allele -765C is associated to a higher gene
expression and thus could represent a risk allele by affecting the functionality of the promoter.
Conclusion: In conclusion, variant -765G>C may be associated to malignant
cutaneous melanoma with a low penetrance effect and this effect could be a
consequence of altered gene expression.
P12.120-M
Synergistic effect and VEGF/HSP70-hom haplotype analysis:
relationship to prostate cancer risk and clinical outcome
Background: BRAF mutation analysis is a useful adjunctive tool in diagnosing thyroid nodules. The BRAF V600E (c.1799T>A) mutation comprises
over 95% of all BRAF gene mutations in papillary thyroid carcinoma (PTC).
The clinicopathological association of rare BRAF variants other than V600E
261
ABSTRACTS POSTERS
mutation is still obscure.
Methods: We evaluated a total of 1067 consecutive patients with malignant
or indeterminate thyroid nodules by ultrasonography. All fine needle aspiration cytology (FNAC) samples were tested for BRAF mutation using mutant enrichment with 3-modified oligonucleotide (MEMO) sequencing with
real-time PCR concurrently. Rare BRAF variants were evaluated with regard
to cytology and/or histology results.
Results: BRAF mutations were detected in 37.9% (404/1067) of all samples.
The V600E mutation was detected in 98.3% (397/404), and six rare variants were detected by MEMO sequencing. Three types of variants were identified: c.1799_1801del (p.Val600_Lys601delinsGlu), c.1794_1795insGTT
(p.Ala598_Thr599insVal), and c.1801A>T (p.Lys601*). The former two are
known mutations to be associated with PTC and three patients with these
mutations were diagnosed as PTC histologically. The third variant was novel.
The case with c.1801A>T was diagnosed as a benign follicular nodule.
Conclusions: Out of 1067 thyroid nodule cytology samples, six (0.56%) had
rare BRAF variants. A novel variant was identified in a cytologically benign
thyroid nodule. Targeted testing for detecting only the V600E mutation may
be good enough to increase the diagnostic value of FNAC, however, identification of other variants in BRAF gene may provide additional valuable
information on the nature of thyroid nodules in the future.
P12.122-M
Association between the cytogenetic profile of tumor cells and
response to preoperative radiochemotherapy in locally advanced
rectal cancer
Neoadjuvant radiochemotherapy to locally advanced rectal carcinoma patients has proven efficient in a high percentage of cases. Despite this, some
patients show non-response or even disease progression. Recent studies
suggest that different genetic alterations may be associated with sensitivity
vs. resistance of rectal cancer tumor cells to neoadjuvant therapy. We investigated the relationship between intratumoral pathways of clonal evolution as assessed by iFISH (51 different probes) and response to neoadjuvant
radiochemotherapy, evaluated by Dworak criteria in 45 rectal cancer tumors before (n=45) and after (n=31) treatment. Losses of chromosomes 1p
(44%), 8p (53%), 17p (47%), 18q (38%) and gains of 1q (49%), 13q (75%)
as well as amplification of 8q (38%) and 20q (47%) chromosomal regions
were those specific alterations found at higher frequencies. Significant association (p<0.05) was found between alteration of 1p, 1q, 11p, 12p and
17p chromosomal regions and degree of response to neoadjuvant therapy. A
clear association was observed between cytogenetic profile of the ancestral
tumor cell clone and response to radiochemotherapy; cases presenting with
del(17p) showed a poor response to neoadjuvant treatment (p=0.03), while
presence of del(1p) was more frequently observed in responder patients
(p=0.0002). Moreover, a significantly higher number of copies of chromosomes 8q (p=0.004), 13q (p=0.003) and 20q (p=0.002) were found after therapy vs. paired pre-treatment rectal cancer samples. Our results point out
the existence of an association between tumor cytogenetics and response
to neoadjuvant therapy in locally advanced rectal cancer. Further studies in
larger series of patients are necessary to confirm our results.
P12.123-S
Epigenetic Profile of Early Relapsed Childhood ALL
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a role in drug resistance and relapse, we performed Infinium HumanMethylation450K BeadChip arrays and to characterize the molecular evolution
of relapsed childhood ALL, we used Illumina HumanCytoSNP-12 arrays to
identify somatic copy number alterations (CNAs) in 16 diagnosis/relapse
pairs. Flow-sorted normal B-cell progenitor subpopulations and CD4+CD8+
T-cell purified from thymus were used as controls.
Analysis variant CpG sites in an unsupervised manner, we identified three
distinct DNA methylation profiles in samples according to their structural
variations. ALL cases did not show significant differences between paired
diagnose/ relapse samples though they had hyper DNA methylation than
control samples. Aberrant DNA methylation had been detected in negative
regulator of cell cycle genes and DNA damage repair genes in ALL. Copy
number analysis of patients revealed varying numbers of genetic lesions
ranging from 0 to 45 CNAs per sample. The vast majority of CNAs observed were shared between diagnosis and relapse in the same patients. One
of the most frequent CNAs involved deletions of CDKN2A/B , occurring in 8
patients, 2 of 8 patients lost the CDKN2A/B at relapse time. Integration of
methylation and genotyping data revealed high concordance.
Early relapse samples were more likely to be similar to their respective diagnostic sample that suggests early-relapse results from the emergence of a
related clone.
P12.124-M
Genome-wide methylation analysis of tubulocystic and papillary renal
cell carcinomas
M. Korabecna1, A. Horinek1, O. Seda1, Z. Halbhuber2, R. Blatny2, O. Hes3, V. Tesar1;
1
First Faculty of Medicine of Charles University and General Faculty Hospital in Prague,
Prague, Czech Republic, 2Central European Biosystems, Prague, Czech Republic, 3Faculty
of Medicine in Pilsen, Charles University, Pilsen, Czech Republic.
This study was undertaken to characterize and compare molecular signatures associated with tubulocystic renal cell carcinoma (TRCC) and papillary
renal cell carcinoma (PRCC).
We performed methylated DNA immunoprecipitation (MeDIP) coupled with
genome wide microarray analysis (Roche NimbleGen) in 6 PRCC and 2 TRCC
together with analysis of control tissues of normal histological appearance
adjacent to each examined tumor sample.
All examined tumors and their control tissues of histological normal appearance share alteration in regulation of specific pathways. In TRCC we
found higher number (48) of methylated tumor suppressor genes (TSG)
than in PRCC (35). Only TSG DPH1 and VHL were found to be distinctively methylated in all PRCC. The methylation of gene sequences SPIB, PLAT,
PCDHB1 and FAF2 was found exclusively in TRCC tumor samples, the methylation in genes KIFC3, MDH2, PAG1, BICD1, PLG and CTRL was limited to
all PRCC tumor samples only.
Using bioinformatics tools (DAVID and GSEA), we found the pathways and
genes which are altered by differential methylation and which may be responsible for developmental divergence of both tumors from precancerous
renal tissue. The genes involved in transmembrane transport of small molecules, lipoprotein metabolism and transmission across chemical synapsis
are significantly overrepresented among genes methylated preferentially in
TRCC, PRCC is characterized by overrepresentation of genes playing roles in
RNA metabolism and processing.
Supported by grants no. PRVOUK P25/LF1/2 of the Ministry of Education,
Youth and Sport of the Czech Republic and no.RVO-VFN 64165 of the Ministry of Health of the Czech Republic.
P12.125-S
Identification of mosaicism in patients with sporadic retinoblastoma
using NGS
Objectives: Retinoblastoma is the paradigm of hereditary cancer. Approximately, 40% of patients carry germline mutations in the retinoblastoma
gene RB1 that predispose to develop retinoblastoma. These mutations can
be detectable in blood DNA using standard techniques. However, these techniques are not sensitive enough to detect mosaicism in sporadic cases of the
disease (without family history). This study was thus designed to determine the frequency of mosaicism in patients with sporadic retinoblastoma by
using next generation sequencing (NGS) technologies.
Methods: We selected for this study 22 patients with sporadic retinoblastoma in which we had previously identified two inactivating mutations in the
RB1 gene in tumor DNA, but in which none of those mutations were detected in blood DNA using Sanger sequencing. Blood DNA of these patients
was analyzed by NGS (Roche/454) in order to identify in low proportion
ABSTRACTS POSTERS
(mosaicism) one of the mutations identified in the tumor.
Results: Roche/454 sequencing was able to detect mutations present at <1%
in blood DNA. In our series, NGS was able to detect, in 5 out of 22 patients
(22.7%), one of the mutation identified in the tumor in the blood DNA. The
frequency of the mutations in blood DNA ranged between 0.8 and 16.8%.
Conclusion: Roche/454 sequencing is very sensitive allowing the identification of mosaicism in patients with sporadic retinoblastoma. The existence
of mosaicism can modify the risk of retinoblastoma and the probability of
transmitting the mutation to offspring. Therefore, the identification of mosaicism in these patients improves genetic diagnosis.
P12.126-M
BRCA1 and TP53 germline mutations are associated with gynecologic
sarcomas
D. E. Nowakowska1, A. Niwiska, A. Kluska, M. Dbrowska, A.D. Czapczak, E.
Kwiatkowska, A. Nasierowska-Guttmejer2;
1
The Maria Sklodowska -Curie Cancer Center & Institute in Warsaw, Warsaw, Poland,
2
Central Clinical Hospital Ministry of Interior in Warsaw, Warsaw, Poland.
Introduction: gynecologic sarcomas comprise less than 1% of all gynecologic malignancies and represent very heterogeneous group. Very little is
known about etiology of these malignancies, the only documented etiologic factor in less then 25% of these tumors is previous pelvic irradiation
and some have been linked to tamoxifen treatment. We report that BRCA1
& TP53 germline mutations are associated with gynecologic sarcomas.<br
/Methods:
During the last ten years 16 patients diagnosed with gynecologic sarcoma
were seen at Genetic Counseling Unit at Cancer Center & Institute of Oncology in Warsaw. All the patients were offered genetic testing, after genetic
counseling and after obtaining the written informed consent. The consent
protocol was accepted by the local ethical committee. BRCA1 analysis was
performed on DNA from the peripheral blood leukocytes. The mutations in
BRCA1 were detected using DHPLC and sequencing of exons 2, 5, 11 and 20,
where the Polish founder mutations are found most frequently. TP53 gene
was sequenced from exon 2 to 10.
Results:
Among 16 patients with gynecologic sarcomas, we found 6 (37%) BRCA1
germline mutation carriers.
We have found only one TP53 mutation carrier, who had been diagnosed
with vulvar angiosarcoma at exceptionally young age.
Conclusions:
In women diagnosed with gynecologic sarcoma screening for the germline mutations in the BRCA1 gene is important strategy. In pediatric patients
gynecologic sarcoma should prompt Li-Fraumeni syndrome diagnosis.
patient ID
diagnosis
number
DN 121
DN 310
DN 1430
DN 1659
AN 245
AN 1263
DN 5698
Table 1
age at
mutation mutation
diagnosis
in BRCA1 in TP53
(years)
67
yes
no
75
yes
no
54
52
60
45
9
yes
yes
yes
yes
no
P12.127-S
Glutathione S-transferase P1 gene in secondary Acute Myeloid
Leukemia
no
no
no
no
yes
Back to index
Hereditary breast cancers account for 5-10 % cases and are predominantly due to BRCA1/2 genes and less commonly due to other high penetrant
(TP53, STK11, PTEN) and less penetrant (CHEK2,ATM,PALB2, BRIP1) genes. Breast cancer is an important component of Li-Fraumeni Syndrome
(LFS) cancer spectrum related to TP53 gene mutation. Genetic testing for
BRC1/2 is generally not offered in young breast cancer women (<30 years)
alone unless they meet the testing criterion ( Manchester score etc). However TP53 testing is suggested for young breast cancer alone cases without
family history of LFs tumours
We analysed our data of past 6 years (2008-2013) of TP53 gene testing in
women with breast cancer. Information was obtained regarding family history of cancers, age of onset, receptor pathology and BRCA1/2 testing. No
TP53 mutation was found in our breast cancer alone cohort. The solitary
TP53 mutation was detected in a woman with family history suggestive of
LFS. The TP53 gene mutation detection rate in our cohort was only 4.5 %.
(1/22).
Although our sample size is small TP53 gene mutation rate is likely to be
better in breast cancer cases with LFS family history and may be influenced
by receptor pathology as published previously. Genetic centres with limited
resources need to consider these factors before requesting expensive send
away genetic tests. Availability of affordable NGS panel testing for breast
cancer genes may address this issue in future and would also help in understanding the contribution of the above mentioned genes to hereditary
breast cancer.
P12.129-S
Expression analysis of TNF related apoptosis inducing ligand and its
receptors in gastrointestinal tumors
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P12.130-M
Discordant molecular breakpoints in an apparent recurrent
translocation (3;12)(q13;p13)
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A fraction of sequence variants found in disease-causing genes induce aberrant splicing. At the moment, reliable splice-prediction tools are only available for variants in the consensus splice site regions, and even these cannot
predict the exact molecular nature of the aberrant transcription. Wet-lab
splicing assays, either RT-PCR analyses of patient RNA or functional splicing
reporter minigene tests, have to be performed to confirm aberrant splicing.
Here, we present results of splicing reporter minigene assays performed for
37 disease-gene variants of unknown significance (VUS), mainly found in
the four DNA mismatch-repair genes MLH1, MSH2, MSH6 and PMS2 that are
associated with Lynch syndrome. Twenty variants are located in the consensus splice site regions, 13 are exonic and 4 are deep-intronic variants.
We used a previously described minigene vector, and transfected HEK293
and/or Hela cells with wildtype and variant constructs. For 32 variants also
results from patient RNA analyses were available, either performed by our
laboratory or presented in literature. For comparison with minigene assay
splicing data, we especially included variants that showed multiple aberrant
transcripts in patient RNA analysis, or another splice effect than the prevalent exon skip. We found 100% concordance between patient RNA analyses and minigene assays in terms of showing an effect on splicing or not.
However, for 6 variants discrepancies in the molecular nature of aberrant
transcription were observed. Possible explanations for these discrepancies, and implications for the assessment of pathogenicity of the variant are
discussed.
P12.134-M
Genetic risk factors in a vulvar cancer cluster among young
Indigenous women in Arnhem Land, Australia
Vulvar cancer is usually rare, and occurs most often in postmenopausal women. Among young (<50 years) Indigenous women living in remote Aboriginal communities in Arnhem Land, however, the incidence of this malignancy
is more than 70 times the national Australian rate for the same age group.
Previously, we found that neither excess human papillomavirus (HPV) incidence nor a particularly virulent strain of HPV could explain the very high
incidence of vulvar cancer in this population. Reports from the Gynaecology
Outreach Service that cases appeared to cluster in family groups suggested
that a genetic susceptibility, either to the effects of HPV or another cause
of vulvar cancer, may be involved in this cluster. To investigate the role of
genetic risk factors, 30 cases and 61 controls, matched on age and community of residence, were recruited to the study. DNA was extracted from saliva samples, and genotyped to provide information on approximately 2.5
million variants. These data were analysed using both genome-wide association and identity-by-descent techniques. We found clear evidence for the
involvement of a genetic risk factor predisposing this population to vulvar
cancer, and identified three genomic regions of interest. Bioinformatic analysis prioritised biologically plausible candidate genes within these regions
and functional studies to further elucidate the role of genetic variants in the
aetiology of vulvar cancer are currently underway. This is the first genetic
ABSTRACTS POSTERS
study of this population, and these findings continue to inform health care
delivery in Arnhem Land, especially vaccination policy and screening strategies.
P12.135-S
Whole exome sequencing identifies novel mutations from DNA repair
pathways in familial esophageal squamous cell carcinoma
Next generation sequencing of the whole transcriptome enables high resolution measurement of gene expression activity in different tissue and cell
types. This methodology provides an in depth study of known transcripts
and depending on the data analysis, allows identification of additional transcript types such as transcript variants, fusion transcripts, and small and
long ncRNAs. In this study we performed RNA-Seq using the Ion Torrent
Proton platform to compare the expression profile of testicular germ cell
tumors (seminoma type, n=3) and normal testis (n=3). Using Partek Flow
and Star or TopHat aligners, we aligned the reads to the human genome and
mapped sequences to the RefSeq database. We identified a large number
of genes that were up and down regulated with high degree of significance
p<0.01, >2X FC). These included genes related to testicular tissue type, stem
cell pleuripotency (NANOG; POU5F1) and proliferation (KRAS, CCND2). In
addition, a number of differentially expressed noncoding RNAs were identified (SNORD12B, XIST). The method was validated on a small set of genes (>20) using qPCR (TaqMan Assays). We used the Open Array platform
to quantitatively screen a larger number of differentially expressed genes
(224) across a number of different testicular germ cell tumor types (nonseminoma).
P12.137-S
Upregulation of key wnt signaling molecules in human astrocytic
brain tumors
N. Pecina-Slaus, A. Kafka, D. Tomas, B. Kruslin;
Medical School University of Zagreb, Zagreb, Croatia.
Back to index
50% of glioblastomas (WHO grade IV) and 56% of astrocytomas (WHO grades II and III) showed upregulation of beta-catenin and nuclear localization
was found in 52.1% of glioblastomas. Transcription factors of the wnt pathway were also upregulated. Strong TCF1 and LEF1 expression was observed in 51.6% and 71% of glioblastomas. Analysis of variances performed on
the total sample indicated significant differences in the values of TCF1 weak
expression (F=2.804; p=0.045), LEF1 weak (F=4.255; p=0.008) and LEF1
strong expression (F=5.498; p=0.002) with regard to malignancy grade. The
F-ratios for two variables (LEF1 strong and LEF1 weak) indicated that differences between astrocytomas (II, III) and glioblastomas were statistically
significant (p<0.02).
Allelic losses of APC gene were frequent with glioblastomas showing 60%
and diffuse astocytomas (grade II) 20%. Allelic losses of AXIN1 were found
in 10% of glioblastomas. In 31% of glioblastomas and 22% of astrocytomas
downregulation of axin proteins was detected. In 31% of glioblastomas axin
was localized in the nucleus.
Our findings contribute to understanding of human astrocytic brain tumor
genetic profile and suggest that molecular changes of wnt signaling play important roles in astrocytic tumor etiology.
P12.138-M
Exome sequencing and deep sequencing reveals ,,mutational storm
in xeroderma pigmentosum (XP) patients
Xeroderma pigmentosum (XP) is a rare genetic disorder with molecular defects in the DNA repair machinery. Clinically, patients suffer from increased
UV-sensitivity and a skin cancer risk that is increased more than 1000-fold.
It has been postulated that due to the repair defect patients show a mutator
phenotype with indicative UV-signature mutations. We have sequenced exomes from sun-exposed and sun-protected tissues from three patients with
XPC. Moreover, two long-range amplicons covering the whole mitochondrial
genome and a comparable nuclear DNA fragment have been sequenced at
>1.000 x coverage. These three XP-C patients have been compared to young
and old healthy controls. The bioinformatics analysis was tailored towards
detection of exposition-specific variants with high sensitivity. A representative subset of identified exposure-specific variants has been validated by
a subsequent deep-sequencing approach. The number of exposure-specific
variants was very high in the XP-C patients (610, 2.088, and 6528, respectively compared to 95%), the third XPC patient had relatively low numbers of exposure-specific variants and no predominance for UV signatures
(~75% in 610 variants). Interestingly, all three samples displayed increase
variation in the nuclear DNA but not the mitochondrial DNA as expressed
by the count of non-consensus bases in amplicon deep sequencing. As expected, XPC hypermutation arises in the nuclear DNA predominantly with
typical signature mutations. Interestingly, not all XPC patients seem to share
the signature phenotype although the hypermutation is a common feature.
In contrast, mitochondrial DNA does not show any differences as compared
with young and old controls.
P12.139-S
YAP1 acts as oncogenic target of 11q22 amplification in multiple
cancer subtypes.
The transcriptional coactivator YAP1 is a critical effector of the human Salvador-Warts-Hippo-pathway. Literature data report apparently discrepant
results on the carcinogenic role of YAP1, which acts as oncogene or tumor
suppressor in different in-vitro and in-vivo models. Furthermore, amplification events of 11q22 locus-encompassing YAP1 gene-have been detected in
multiple tumor types but there is limited direct evidence about the oncogenic role of endogenous YAP1 within in the amplicon.
We screened a panel of human tumor samples and cancer cell lines and
identified that the YAP1 amplification event is actually present in up to 23%
of the cases. We exploited EKVX, CaSki and RO82 cell lines harboring both
genomic YAP1 amplification and YAP1 protein overexpression, in order to
study the effects of downregulation of endogenous YAP1 by RNA-interference strategies. Gene expression profiling data identified 707 statistically
significantly modulated genes (p-value =0.002) that were functionally annotated for cell proliferation and cellular movement ontologies. Mechanistic
studies of the identified perturbed pathways revealed that YAP1 silencing
265
ABSTRACTS POSTERS
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significantly decreased cell proliferation and cell cycle perturbation associated with upregulation of p21 and p27 cell-cycle inhibitors, reduced cell
migration (p<0.048) and anchorage-independent growth (p<0.02). In CaSki
cell line, YAP1 silencing induced significantly increased sensitivity and celldeath response to cisplatin treatment (p=0.011) as well as reduction of invivo tumorigenic potential (p=0.027).
Overall, these results establish that YAP1 is a direct oncogenic target of the
11q22 amplicon in previously unreported cancer types and support the relevance of such genetic aberration in carcinogenesis in a fraction of multiple
tumor types.
P12.140-M
No change in the rate of bilateral mammographies after BRCA1/2
testing among true non-carriers
The majority of women who are true non-carriers of the BRCA1/2 familial
mutation may be reassured that they are no longer considered at high risk
for breast and ovarian cancer. For this reason, most should be encouraged
to adopt the same cancer screening practices as those recommended to women of the same age in the general population. The aim of this study is to
compare the rate of bilateral mammographies after BRCA1/2 testing to that
prior among true non-carriers of BRCA1/2 mutation. Information from the
Quebec Health Insurance Board was used to identify all registered bilateral
mammographies done between May 1, 1998 and March 31, 2012 among a
cohort of 143 French Canadian unaffected true non-carriers. The Cox proportional hazards model for repeated events, with womens age as the time
scale, was used to obtain hazard ratios of bilateral mammographies. The
rate of mammographies did not change after BRCA1/2 testing, neither globally (HR=0.93, p=0.22), nor by age (<50 years HR=0.81, p=0.13; 50 years
HR=1.01, p=0.84). Although women <50 years had a lower rate of mammographies than women 50 years (HR=0.55; 95% CI = 0.43-0.70) after
genetic testing, 74% still continued to be screened, which is not generally
recommended to women of the same age group in the general population.
In conclusion, genetic testing information did not have a significant effect
on mammography screening in our cohort of true non-carriers of BRCA1/2
mutation. Clear-cut recommendations for the follow-up of true non-carriers
of BRCA1/2 mutation are needed.
P12.141-S
EGFR and ALK genes mutation screening in Non-small-cell lung
carcinoma (NSCLC) specimens
266
P12.143-S
SOX2 gene expression and copy number variation in squamous cell
lung cancer
Transcription factor SOX2 might have an important oncogenic role in development and progression of lung cancer. The aim of our study was to estimate copy number variation (CNV) of SOX2 in primary tumours and to analyse
SOX2 expression in primary tumour tissue and in blood samples in relation to CNV status in squamous cell lung cancer patients. 28 patients with
metastatic squamous cell lung cancer were prospectively included between
years 2010 and 2013. Total RNA was isolated from whole blood collected in
PAXgene Blood RNA Tube before any systemic treatment. For 10 patients
primary tumour tissue was available from the same time points; for those
patients RNA and DNA were extracted from FFPE tumours. SOX2 expression
levels and CNV status were determined by quantitative RT-PCR. Copy number analysis revealed high copy number of SOX2 in 3/10 (30%) tumours and
gain of function in another 3/10 (30%) tumours. Median SOX2 expression in
FFPE tumour tissue was 3.9 (0.1-29.1) and in blood samples 5.4 (0.4-17.9).
Elevated copy number of SOX2 correlated with higher SOX2 expression in
tumours (=0.92). Moreover, correlation between SOX2 expression in blood
samples and FFPE tumour tissue was observed ( =0.60); patients with high
SOX2 expression in blood tended to have high SOX2 expression in tumour
tissue. According to our observation genetic alterations in SOX2 seem to be
common event in squamous cell lung cancer. Furthermore, good correlation
between SOX2 expression in blood and tumour samples supports the idea of
liquid biopsy in lung cancer.
P13.01-S
A French series of 731 patients with 22q11.2 microdeletion
The phenotype of the 22q11.2 deletion syndrome (22q11DS) is higly variable with a wide spectrum of abnormalities. About 90% of patients have a 3Mb
deletion spanning LCR22-A to LCR22-D in the 22q11 region containing four
low copy repeats (LCR22).
Methods : On behalf of the ACLF, 27 French Cytogenetics laboratories collaborated to collect data of 731 patients with 22q11DS diagnosed between
1996 to 2013.
Results :
Clinical data: On average over the years 2010-2012, at least 70 patients
were annually diagnosed postnatally. The symptomatology that led to genetic analysis changes with patients age, eg :
Heart defect, facial dysmorphism, hypocalcemia and thymus hypoplasia or
agenesis in newborns
Developmental and speech delay with facial dysmorphism and velopharyngeal insufficiency in children
ABSTRACTS POSTERS
Variable phenotype in adults : some had intellectual disability with typical
gestalt, some had psychiatric disorders, and a minority was almost asymptomatic parents when the diagnosis was first made in their children.
Molecular data: Thirteen cases were diagnosed with array-CGH. The majority of these patients were referred for intellectual disability (n=10/13) and
only 3 had heart defect. The size of the deletion was variable: 745 - 2904 kb
and surprisingly only 46,2% had deletion between LCR22-A and LCR22-D.
The 22q11 deletion was inherited in 22.2% of cases and mainly maternally
(86.7%).
Conclusion : We report the largest series of 22q11DS postnatal diagnosis.
We plan to study more patients using array-CGH to investigate whether the
size of the deletion or the presence of other CNV may explain the phenotype
variability.
P13.02-M
Transgenerational inheritance of 22q11.2 atypical deletion:
instability of LCRs in a family with discordant clinical phenotype
The 22q11.2 deletion syndrome (22q11.2DS) is the most common chromosomal deletion syndrome in humans with an incidence of 1 in 2-4000
live births. The clinical presentation of 22q11.2DS is extremely variable,
but the underlying reason for this variation remains unknown. Individuals
with 22q11.2DS most often have a classically associated 3 MB or 1.5 MB
deletion. However, nested deletions as well as atypical deletions also occur
that can contribute to the broad spectrum of phenotypic abnormalities. It is
also hypothesized that variations in the remaining allele could underly the
phenotypic variability.
To investigate variation within the non-deleted allele we performed targeted resequencing of the 22q11.2 region for 127 patients, identifying multiple deletion sizes, including two atypical deletions. We cataloged over 18
thousand hemizygous variant positions, of which sixty percent were previously annotated. As expected, in this gene dense region more variants
were intronic (52%) than intergenic (36%). Within the coding regions we
identified 213 non-synonymous variants, 6 stop gains, and 5 frameshift insertions. In addition, the observed number of variants per gene was higher
or lower than expected for some genes in both our data and 1000 genomes
data, indicating some genes may tolerate variation more or less than others.
This extensive catalog of hemizygous varaints will serve as a blueprint for
future experiments to correlate 22q11DS variation with phenotype and
serve as an analysis model as we extend to whole genome sequencing of
22q11DS patients.
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P13.04-M
New splicing mutation in SERPINA1 gene causing severe alpha-1
antitrypsin deficiency
Alpha1-antitrypsin (AAT) deficiency is a common hereditary disorder associated with reduced AAT serum level, predisposing to pulmonary emphysema or liver disease. It is caused by inheritance of mutations in the AAT gene
(SERPINA1). Most of the deficiency alleles occur in the coding sequence of
the gene due to aminoacid substitution or deletion resulting in reduced protein level or altered functionality. Rarely mutations affecting RNA splicing in
the AAT gene have been described so far.
We have identified a new null allele, QOmadrid in two siblings with significantly reduced serum levels of AAT. QOmadrid allele results from a duplication of a timine in the position +2 of the donor splice site of exon 1C
(+2dupT). In these patients QOmadrid occurred in combination with another previously described rare null variant, QOporto. These two variants
correspond to splicing mutations in a regulatory region of the gene, both
causing disruption of the normal splicing of intron 1C. Analysis of transcripts in patients samples and in vitro assays using minigenes revealed
abnormal splicing leading to absence of transcription from exon 1C, where
the hepatocytes transcription start site is located. Thus, in these patients
no normally spliced RNA products are expected to be produced in the liver,
causing the AAT deficiency. This mutation constitutes a new null allele, QOmadrid, contributing to explain the disease.
P13.05-S
Upstream open reading frames regulate cannabinoid receptor 1
expression under baseline conditions and during cellular stress
267
ABSTRACTS POSTERS
births. We report 7 patients identified from the cytogenetic registers of the
HIMFG, who attended during the last 8 years. They corresponded to r(4)
(p16q35), r(5)(p15.3q35.3), r(9)(q24.3q34.3), r(14)(p11?q32.33), r(19)
(p13.3q13.4), r(21)(p11q22.3) and idic r(21)(p11.2q22.3) (cases1-7, respectively). Additional chromosomal analyses with GTG banding were performed in all cases, FISH with subtelomeric probes were applied to 4 cases
and molecular karyotyping with SNP array were carried out in 2. As cases
2 and 5 are mosaic with a normal cell line, they should have been originated postzygotically. A dynamic mosaicism was present in all the cases. All
the rings were originated by breaks on both arms with loss of subtelomeric
material and subsequent reunion of free ends with the exception of cases 2
and 5. The r(14) was probably generated by an inv-dup-del mechanism as
there are a deletion and a duplication of 14q. The large dicentric r(21) may
have been formed by breakage and reunion of long arms of an isochromosome. The phenotypes described included short height, microcephaly, variable
facial dysmorphism and psychomotor developmental delay, corresponding
to autosomal ring syndrome. Some patients showed hirsutism, renal abnormalities and hypoacusia, data corresponding to monosomy of the specific
chromosomal region implicated. Cases 1 and 7 presented characteristics of
Wolf-Hirschhorn and Down syndromes, respectively. Patients 2 and 4 had
difficult control seizures. We consider that the variable clinical data reported in these patients are due to the specific chromosomal region involved
and to the ring chromosome instability.
P13.07-S
A new translocation involving 4 and 11 chromosomes in a adult man
Chromosomal translocations occurring between the chromosome 11 (region q12) and other chromosomes are the most recurrent chromosomal aberrations observed in some subtypes of leukemia, lymphoma and sarcomas. In
many cases, identification of these chromosome abnormalities is crucial to
select appropriate treatment protocols.
To our knowledge we are the first group to describe a new translocation,
involving 11q12 and 4q21 regions, in a 35 years old man.
The proband came to our observation to perform cytogenetic analysis because of a suspected infertility and we found 46,XY t (4;11) (q21;q12) karyotype. The man had a cortical dysplasia and seizures from birth.
We extended the cytogenetic analysis to the parents and sister of the proband and the same translocation was found only in the father.
It is known that some types of dysplasia are linked to the presence of genetic
alteration but so far it has not been described any association between dysplasia and chromosomal aberration.
The region 11q12 is involved inmanychromosomaltranslocations such as
t(2;11) (q31; q12), t(11; 17) (q12; p11.2), and t(6; 11) (p21; q12) which are
all related to tumor development . The common breakpoint region (11q12)
has been delineated by FISH and contains the FOSL1 gene which has been
associated with fibroblast growth or cancer development, such as colorectal,
breast, prostate and lung cancer, head and neck squamous cell carcinoma.
Since the region 11q12 seems to be linked to tumor development, would be
appropriate to perform a follow-up to monitor the subject at risk.
P13.09-S
Stable segregation of apparently benign chromothripsis in three
generations
268
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G-CTH involving six breakpoints and a ~108kb deletion within a ~6,4Mb region on 3q22.3-q23. Although six protein-coding genes were affected by the
breakpoints the 10 carriers of the G-CTH do apparently not have common
associated disorders. However, there are several spontaneous miscarriages
in the family records presumably resulting from unbalanced rearrangements
due to G-CTH. Our study is the first report suggesting that G-CTH may not
always be associated with an abnormal phenotype but may also have a neutral effect. It is therefore possible that G-CTH may be found not only among
individuals with apparently balanced rearrangements but also karyotypically normal asymptomatic individuals. We describe CTH on the cytogenetic level based on recommendations of ISCN-2013 (International System
for Human Cytogenetic Nomenclature) as: 46,XX,t(3;5)(q22.3;q23.1).arr
ngs[hg19] 3q23.3(137735949-137845370)x1.ngs 3q22.3q23(135827611142218722)cth fam. We suggest combining this with a molecular description following HGVS (Human Genome Variation Society) nomenclature (see
ESHG abstract by Taschner P. et al.). *Shared first-authors
P13.10-M
A family-based genome-wide scan shows 10q25.2, LRFN2, TGFB2 and
CRISPLD2 loci associated with cleft lip with or without cleft palate in a
high-prevalence cluster in South America
F. M. de Carvalho1, F. A. Poletta2, R. F. Fonseca1, D. Montaner3, J. Mereb4, M. A. M. Moreira5,
H. N. Seuanez6, A. R. Vieira7, E. E. Castilla2, I. M. Orioli1;
1
ECLAMC (Latin American Collaborative Study of Congenital Malformations) and
INAGEMP (National Institute of Population Medical Genetics) at Department of
Genetics, Institute of Biology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil,
2
ECLAMC and INAGEMP at CEMIC (Center for Medical Education and Clinical Research)
(CONICET), Buenos Aires, Argentina, 3Bioinformatics Department Research Center
Principe Felipe, Valencia, Spain, 4ECLAMC and INAGEMP at Hospital Zonal El Bolsn, El
Bolsn, Argentina, 5Genetics Division, National Cancer Institute, Rio de Janeiro, Brazil,
6
Genetics Division, National Cancer Institute; Department of Genetics, Institute of
Biology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, 7Departments of Oral
Biology and Pediatric Dentistry and Center for Craniofacial and Dental Genetics, School
of Dental Medicine, University of Pittsburgh, Pittsburgh, PA, United States.
Background: In South America four cleft lip with or without cleft palate
(CLP) high prevalence regions was detected, one of them being the Patagonia (Argentina). We aim the identification of independent autosomal segments containing polymorphic markers that may contribute to CLP with a
family-based design for genome-wide association scan.
Methods: The study sample included 26 families with isolated CLP (27
affected and 99 total individuals).They were genotyped on the Affymetrix
Genome-Wide 6.0 array. Only independent SNPs were included in the association analysis. We calculated linkage disequilibrium (LD) between each
pair of SNPs into a window of 50 SNPs, shifting the window 5 SNPs forward
and repeating the procedure to scan all autosomes. Then we pruned the data
removing one SNP of each pair that was in strong LD (r >0.8). We perform
the transmission disequilibrium test (TDT). We identified segments of a
maximum length of 250Kb with more than one SNP significantly associated
with CLP.
Results: A total of 88 genomic segments with two or more independent SNPs
significantly associated with CLP were identified. An intergenic region of
33Kb on 10q25.2 showed the most significant association with CLP (p<
0.00007). Furthermore, we found other significant association with 6p21.2
including the marker rs4153154 (p<0.00009). Besides, others genomic segments including the genes TGFB2 (1.4Kb on 1q41) and CRISPDL2 (61.3Kb
on 16q24.1) deserve our attention because they have previously been associated with oral cleft in others populations.
Conclusion: Our results suggest 10q25.2 and some genes spread across autosomes as candidate loci to CLP.
P13.11-S
The FRA14B common fragile site maps to a region of frequent somatic
and germ-line rearrangements within the GPHN gene
D. Zheglo1, L. Brueckner2, L. Savelyeva2;
1
Research Centre for Medical Genetics, Moscow, Russian Federation, 2German Cancer
Research Center (DKFZ), Heidelberg, Germany.
Common fragile sites (cFS) are heritable chromosomal loci that exhibit
non-random breaks on metaphase chromosomes in response to replication
stress. Chromosome breakage at cFSs contributes to cancer genome evolution and de novo pathogenic germ-line alterations. Approximately 90 cFSs
have been identified at cytogenetic band resolution, but just few of them
have been molecularly characterized. Precise mapping of cFSs can reveal
new rearrangement-prone candidate genes critical for the development of
cancer and hereditary conditions.
We performed FRA14B mapping in cultured lymphocytes treated with a
replication inhibitor, aphidicoline, using six-color FISH with contiguous
BAC probes on metaphase chromosomes. FRA14B was restricted to a 765
kb region within 14q23.3, overlapping with a major part of the GPHN gene.
ABSTRACTS POSTERS
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P13.12-M
Signature of germline chromothripsis in a 14-break complex
rearrangement associated with deletions at 7q33-q35/11p13 and
CNTNAP2 gene disruption
Even in the era of exome and full genome sequencing, it will take decades
and tremendous resources to saturate the human genome with mutations
linked to abnormal and normal phenotypes.
As a supplement to exome and full genome sequencing strategies, we will
use already identified balanced chromosomal rearrangements (BCR) to
establish a first, detailed map of mutations covering a significant fraction
of the human genome. In the first reexamination of unselected de novo BCRs
(BCRdn) detected by 40 years of prenatal diagnosis in Denmark, we have
shown that BCRs truncate protein coding genes, ncRNA genes, unannotated
transcripts detected by deep sequencing, as well as developmental regulatory genomic landscapes, mimicing random mutagenesis. In addition, our
P13.14-M
Insight into the mutational mechanism of the recurrent CREBBP
c.3832G>A p.(Glu1278Lys) mutation in patients with RubinsteinTaybi syndrome
269
ABSTRACTS POSTERS
P13.16-M
High frequency of chromosomal anomalies and a novel
chromosomal insertion associated with infertility and recurrent
miscarriages(Reproductive Failure) in west Turkey
270
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ABSTRACTS POSTERS
(IVC) (OR=18.00; CI=2.0-159.09; P=0.009) in DS offspring. We conclude that
the RFC1 A80G polymorphism in DS individuals is associated with the risk
for IAC and BHMT G742A maternal polymorphism is associated with risk for
AVSD and IVC in DS offspring.
P13.21-S
Protective action of NADPH oxidase inhibitors and the role of NADPH
oxidase in the pathogenesis of colon inflammation in mice
V. Salteniene, R. Ramonaite, S. Juzenas, J. Kupcinskas, J. Skieceviciene, L. Kupcinskas;
Lithuanian University of Health Sciences Institute for Digestive Research, Kaunas,
Lithuania.
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Small supernumerary marker chromosomes (sSMC) are structurally abnormal parts of the karyotype with unknown origin that may arise de novo or
be inherited from parents. ~70% of people with sSMC grow and develop
normally, while 30% show different clinical signs and symptoms. Here we
present the clinical and cytogenetic findings in a 1-year-old female referred
for genetic evaluation because of dysmorphic features, including hypotonia,
umbilical hernia, hypertelorism, broad nasal bridge, microretrognathia, low
set ears, and wide spaced nipples. Cytogenetic examination of GTG banded
metaphases showed a female karyotype with mosaicism of an sSMC. Additional molecular cytogenetics analysis (cenM-FISH and subcenM-FISH)
characterized the sSMC to be derived from chromosome 5 including heterochromatic and euchromatic material. The shape of sSMC was not clearly to
define; either it is a ring or centric minute. The karyotype can be reported
as : mos 47,XX,1qh+pat,+der(5)?r(5)(::p1?4q11.1::)[3]/der(5)?min(5)
(:p1?4q11.1:)[1]dn[38]/46,XX,1qh+pat[62]. Probands twin sister and
parents have normal karyotypes with respect to the sSMC. According to
the literature, there are several cytogenetically similar cases described but
with different clinical features. The most cases of proximal partial trisomy
5p arose in connection with familial translocations, while just five cases are
due to a pure sSMC(5). The critical region for trisomy 5p syndrome seems to
be located in the distal part of the short arm as the symptoms are similar to
those seen in cases with pure trisomy 5pter to 5p13. The most common features are mental retardation, facial dysmorphism and hypotonia according
to webpage Small supernumerary marker chromosomes (http://ssmc-tl.
com/sSMC.html).
P13.26-M
Defining the role of CGGBP1 protein in FMR1 gene expression
271
ABSTRACTS POSTERS
ved protein which binds specifically unmethylated CGG tracts. The role of
CGGBP1 on FMR1 transcription is yet to be defined. Sequencing analysis and
expression studies through quantitative PCR of CGGBP1 were performed in
cell lines with different allele expansions (wild-type WT, premutation, methylated full mutations FXS and unmethylated full mutation UFM), demonstrating no differences between them. ChIP assays showed that CGGBP1
binds unmethylated CGG triplets of the FMR1 gene proportionally to the
length of the repeats. We also observed that CGGBP1 binding to the FMR1 locus was restored after pharmacological demethylation with 5-azadC of FXS
alleles, suggesting a possible role for CGGBP1 in FMR1 expression. CGGBP1
silencing with siRNA (reaching ~ 85% of CGGBP1-mRNA depletion) did not
affect FMR1 transcription in WT and UFM fibroblasts. Although the strong
binding to the CGG tract could suggest a relevant role of CGGBP1 on FMR1
gene expression, our results demonstrate that CGGBP1 is not a direct regulator of FMR1 transcription.
Supported by Telethon Onlus, FRAXA Foundation and Italian Association for
fragile X syndrome.
P13.27-S
G6PD-Meyer: a new mutation causing compensated chronic
haemolysis
An updated database reports 186 different G6PD mutations. Many of these are polymorphic in various populations; many are sporadic, having been
discovered because they cause chronic non-spherocytic haemolytic anemia
(CNSHA) associated with severe enzyme deficiency (WHO class I).
A little boy with a history of neonatal jaundice treated with phototherapy,
and of anemia (Hb 80-90 G/L) during his first semester, was seen at age 3.4
years. He is clinically well, with no significant physical findings; Hb 110 G/L,
MCV 95, MCH 31, MCHC 33, reticulocytes 271x109/L, bilirubin 22 mol/L:
indicating a well compensated haemolytic condition. G6PD activity was 0.89
IU/GHb (ref values 7-10 IU/GHb). Sequencing the G6PD gene revealed an
A->G change in exon 7 at nucleotide 655, predicting an Arg->Gly change at
codon 219. The mother was shown to be heterozygous for the same mutation: her G6PD assay was normal. This mutation is not present in the database
and therefore it is a new sporadic class I mutation. We interpret the CNSHA
phenotype as related to the fact that Arg->Gly is a rather drastic amino acid
change, since it entails a charge change as well as a steric change: such changes are likely to cause decreased stability of the G6PD protein and enzyme
deficiency. It is interesting that Arg219 is not a conserved residue: we have
previously reported that in terms of clinical phenotype the consequences in
such cases would not be so drastic, and this may explain why this child does
have haemolysis but only mild anemia.
P13.28-M
Large duplication implicating only exon 1 of F8 gene in mild
hemophilia A phenotype
272
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P13.29-S
Subtelomeric chromosomal breakages characterization in patients
with intellectual Disabilities/Congenital Anomalies and mechanisms
for formation
G. M. Novo-Filho1,2, E. A. Zanardo1,2, R. L. Dutra1,2, A. T. Dias1, M. M. Montenegro1, F. A.
S. L. Souza1, A. M. Nascimento1, N. A. Fernandes2, C. O. Santos1, T. V. M. M. Moura1, F. B.
Piazzon1, C. A. Kim2, L. D. Kulikowski1;
1
Department of Pathology, Cytogenomics Laboratory/ LIM 03, HC-FMUSP, So Paulo,
Brazil, So Paulo, Brazil, 2Genetics Unit, Department of Pediatrics/ LIM 36, HC-FMUSP,
So Paulo, Brazil, So Paulo, Brazil.
Abnormal CNVs are frequently found in subtelomeres of patients with intellectual disabilities (ID) and congenital anomalies (CA). The subtelomeric rearrangements do not usually present recurrent breakpoints (BPs) and involve
several different chromosomes ends. Although these regions encompass approximately 30% of pathogenic CNVs, the causes of subtelomeric breakages
and repair have not yet been investigated comprehensively. We investigated
105 unrelated patients with ID/CA using MLPA, FISH and arrays (BeadChipIllumina/CGH-array-Agilent) in order to characterize the subtelomeric BP.
Within the set of subtelomeric rearrangement studied, the deleted regions
ranged from 137 kb to 29 Mb, and the duplicated regions from 155 kb to 32
Mb. We identified 38 BPs, from 19 different regions. Our analysis showed
repetitive elements in 31 BPs encompassing SINEs (Alu; MIR), LINEs (L1;
L2), and LTRs. We also found six BPs presenting simple tandem repeats and
two BPs with interstitial telomeric sequences (ITs). Rearrangements with
exclusively deletions are suggested to be caused by non-homologous end
joining (NHEJ) or microhomology-mediated end joining (MMEJ) mechanisms. Complex rearrangements are likely caused by fork stalling and template
switching (FoSTeS) or microhomology-mediated break-induced replication
(MMBIR). Misalignments during replication due the repetitive sequences in
these loci can lead to MMBIR and generate the complex dup/del rearrangements observed in 7 of our patients. Furthermore, genomic architectural
features, like sequence motifs, non-B DNA conformations, and repetitive elements may increase the susceptibility for DNA breakage or promote FoSTeS
in these regions. Mapping subtelomeric BPs should help to clarify the mutational mechanisms involved in these genomic rearrangements.
P13.30-M
Two cryptic duplications detected in an apparently balanced
inversion posing a diagnostic challenge
MARK4, a centrosomal serine-threonine kinase that phosphorylates microtubule associated proteins, takes part in cell cycle regulation and proliferation. In glioma we pointed out an imbalance between MARK4 isoforms,
proportional to cellular de-differentiation and tumor grade. This imbalance
ABSTRACTS POSTERS
is triggered by decreased expression of MARK4S, the canonical isoform, associated with overexpression of MARK4L, the alternative isoform with skipping of exon 16.
Having ruled out mutations, CNVs and transcription defects as cause of deregulated MARK4 expression, we searched alterations in alternative splicing
at the origin of the observed isoforms imbalance.
Bioinformatic analysis of MARK4 genomic sequence revealed three binding sites for the splicing factor PTB in introns flanking MARK4 alternative
exon 16. A functional role for these sites is suggested by the conservation
in mouse and the surrounding polypyrimidine rich context. Since in glioma
PTB overexpression drives an oncogenic splicing switch favoring exon
skipped-isoforms, we performed Western blot on glioma and glioblastomaderived cancer stem cell samples and found a significant overexpression of
PTB, correlating with MARK4L expression. Splicing assay of the designed
and differently deleted minigenes revealed that IVS15 contains a functional
intronic splicing silencer (ISS). However, mutagenesis of the PTB binding
site in this region does not affect minigene splicing, suggesting PTB binds to
a non-canonical ISS. Electrophoretic mobility shift coupled to mass spectrometry confirmed the PTB binding to MARK4 IVS15 and its involvement in
MARK4 alternative splicing.
Alternative splicing thus emerges as an oncogenic mechanism that through
the PTB-mediated regulation of MARK4 isoforms expression fosters proliferation and de-differentiation in glioma.
P13.32-M
Normal and oncogenic proliferation under control of microRNAs: a
functional high content screening
Cancer cells share several characteristics with normal stem cells especially
those concerning cell cycle regulation. Therefore it is believed that cancer
cells might arise from stem cells possessing self-renewal capabilities. MicroRNAs (miRs) are small RNA molecules that act regulating gene expression post-transcriptionally by targeting hundreds of mRNAs simultaneously.
With this in mind, we hypothesized that significant changes in the cell cycle
could be a good indicator for miRs capable of regulating normal and oncogenic proliferation. To test this hypothesis, 2.000 BJ foreskin fibroblasts were
transfected with 50nM of 28 microRNAs mimics (pre-miR) and inhibitors
(anti-miR) in 96-well microplates. After 5 days, proliferation was measured
by XTT assay. Cell cycle classification (EdU assay) and viability (Sytox Green
staining) following miR transfection were performed in a High-Content
Screening platform. Nineteen treatments significantly altered the cell proliferation. Interestingly, while transfection of pre-miR of miR-20b, miR-101
and miR-181d decreased the proliferation, the corresponding anti-miRs had
the opposite effect. Pre-miR-181d, pre-miR-20b and pre-miR-101 had the
most cytotoxic effect. Pre-miR-181d and pre-miR-101 induced a significant
reduction in the percentage of cells in S phase. Cyclin D1 expression was
elevated by anti-miR-101 and by pre-miR-24, indicating that this cell cycle
regulator might be the mediator of these miRs effects. These results indicate that these miRs can be good candidates for inducing alterations of interest
in the cell cycle, such as speeding it up or slowing it down as desired. Also
miR-101 acts as tumor suppressor in colorectal cancer and this role is under
evaluation in our lab.
P13.33-S
The evaluation of apoptotic gene expressions, telomerase activity
and the effects of insulin like growth factor-I and erythropoietin on
apoptosis in an experimental necrotizing enterocolitis mice model
Necrotizing enterocolitis (NEC) is a major cause of mortality among premature infants. Apoptosis, which occurs after hypoxia/reoxygenation (H/R),
has an important role in the pathogenesis of NEC. Telomerase activity may
also be important in the recovery process. The aim of this study is to evaluate the apoptotic gene expressions and telomerase activity in the H/Rinduced intestinal mucosa and to investigate whether pre-treatment with
insulin-like growth factor-I (IGF-I) and erythropoietin (EPO) could protect
the intestinal cells from apoptosis or intestinal injury.
The study set was divided into 4 groups, each containing 10 young Balb/c
mice. Group 1 mice were exposed to H/R. Group 2 and group 3 mice were
pre-treated with IGF-1 and EPO for 7 days respectively before H/R. Group
4 served as a control group. Intestinal injury was evaluated by histological
scoring. TUNEL test and caspase-3 activity was perfromed to asses apopto-
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sis. Pro-apoptotic (p53, Casp3, Tnf, Bax, Fas, Bad) and anti-apoptotic (Bcl2,
Bcl-W, Bcl-XL, NF-kB) gene expressions and telomerase activity were studied by Real-Time RT PCR.
IGF-1 and EPO treated animals showed decreased histological damage and
decreased apoptosis which was confirmed by TUNEL test and caspase-3 activity. Telomerase activity increased in these groups in addition to increased
expression of anti-apoptotic genes (p< 0.01). However pro-apoptotic gene
expressions were not statistically different (p>0.05). In conclusion, the protective effects of IGF-1 and EPO in H/R damage may be due to increased
expressions of anti-apoptotic genes.
P13.34-M
Phenotype variability in Slovak NF1 patients related to some
mutation characteristics
One of the most frequent autosomal dominant disorders is neurofibromatosis type 1 (NF1). In NF1 patients are usually found at least two of seven
consensus clinical features: caf au lait macules, freckling, Lish nodules,
bone dysplasia, glioma of optical pathway, different types of neurofibromas
and the first degree relative with confirmed NF1. Our cohort included 108
unrelated Slovak NF1 patients and we identified in 39% (42/108) patients
frameshift, in 13% (14/108) missense, in 18.5% (20/108) splicing, in 17%
(18/108) nonsense mutations, moreover in 4,5% (5/108) deletion of entire
gene type I, in 6% (7/108) large deletions and in 2% (2/108) small in frame
deletions. 23% (25/108) of mutations were located in Ras-GRD (Ras GAP
related) domain and 21% (23/108) in CSRD (cystein serine rich) domain.
Caf au lait macules were present in all patients, freckling in 87%, Lish nodules in 28%, glioma of optical pathway in 31%, neurofibromas in 56%, and
bone dysplasia in 6% of patients. Patients with nonsense mutations show
the lowest occurrence of Lish nodules (11%) and the highest incidence of
neurofibromas (72%). There are no significant differences between clinical
features of patient with mutations in Ras-GRD and CSRD domains. Complete
mutation analysis in the first-degree relatives was performed in 51 families.
Interestingly, we showed that 63% (32) of the patients from these families
carry de novo NF1 mutation. Age of the patients was from 2 to 69 years, 41%
patients were younger than 18 years.
P13.35-S
In vitro analysis of alternative splicing of OLR1, a gene involved in
atherogenesis and tumorigenesis
273
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P13.36-M
Exploring the role of a ceRNA-based regulatory network, involving
GBA and GBAP1, in the pathogenesis of familial Parkinson disease
Parkinson disease (PD) is a complex neurodegenerative disorder characterized by the loss of dopaminergic neurons of the substantia nigra, which
causes motor impairment and resting tremor. To date, mutations in the glucocerebrosidase (GBA) gene represent the most frequent cause of genetic
PD. A widespread deficiency of GBA activity was recently demonstrated in
the brains of PD patients carrying GBA mutations. Most interestingly, also
PD patients without GBA mutations had lower levels of GBA activity in the
brain, suggesting that dysregulated GBA expression could contribute to PD
susceptibility. Possible mechanisms modulating GBA expression include
post-transcriptional networks involving microRNA (miRNAs) and competing-endogenous RNAs (ceRNAs). ceRNAs are a class of regulators that titrate away miRNAs from their targets, thus influencing mRNA expression. The
highly-homologous and expressed GBA pseudogene (GBAP1), located 16 kb
downstream of GBA, is particularly suited to act as a GBA ceRNA. To verify
this hypothesis, we first bioinformatically selected five miRNAs potentially targeting both transcripts, and demonstrated that one of them is significantly overexpressed in whole blood from PD cases vs. controls (2-fold increase, P=0.0028). Then, the miRNA precursor was overexpressed in HeLa/
Hek293/HepG2 cells; in all cases, both GBA and GBAP1 endogenous mRNA
levels were significant decreased (up to 70%). The specific interaction between the miRNA and its targets was demonstrated by luciferase-based reporter assays. Finally, preliminary overexpression experiments showed that
the GBAP1 3-untranslated region is able to act as a molecular sponge of
the identified miRNA, thus suggesting the actual existence of a ceRNA-based
regulatory network modulating GBA expression.
P13.37-S
Non-coding PAH gene alterations act as new transcription regulators
M. Stojiljkovic, K. Klaassen, B. Zukic, M. Ugrin, V. Spasovski, N. Novcic, N. Kotur, S.
Srzentic, G. Nikcevic, S. Pavlovic;
IMGGE, University of Belgrade, Belgrade, Serbia.
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ABSTRACTS POSTERS
3UTR variants may be responsible for variable susceptibility to RET-related
pathologies. Furthermore, to identify AU-rich regions, microRNAs and binding factors involved in the post-transcriptional control, the RET 3UTR was
fragmented in three parts and cloned into a luciferase vector. Preliminary
results show different effects on mRNA stability for these fragments both in
RET-positive and RET-negative cell lines.
P13.41-S
Mosaic ring chomosome 17 : a new case
Ring 17 syndrome is a rare disorder and only 19 cases have been reported
so far. Severity of the associated phenotype is influenced by the presence
or deletion of the Miller-Dieker critical region (MDCR) with presence of the
MDCR being associated with milder phenotypes, including growth delay , intellectual disability, epilepsy, caf au lait skin spots and minor facial dysmorphism. We report a patient born at 41 gestation weeks after an uneventful
pregnancy, with normal birth parameters - weight : 3kg350, length : 49 cm-.
The Initial psychomotor development was normal. When he was 3 years old
the patient developed fronto-parietal epilepsy. Cytogenetic investigations
from peripheral blood cells showed a mosaic ring chromosome 17 karyotype :mos 46,XY,r(17)(p13;q25)[42]/45,XY,-17[5]/46,XY[3]. At 6 years of age,
he had generalised epilepsy, developmental delay, school difficulties, and attention deficit disorder. Clinical examination showed no facial dysmorphism
and no growth delay. Skin changes showed sparse caf au lait spots but no
axillary or inguinal freckling, and 2 small achromic spots. Cerebral MRI,
cardiac ultrasound and ophtalmological fundus were normal. Molecular cytogenetic investigations confirmed that no terminal deletion had occurred,
and an array-CGH ( Agilent 44K) did not detect any submicroscopic deletion
or duplication. Chromosome analysis were also obtained on caf au lait skin
spot and normal skin, and are currently being studied. Data from the patient
will be compared to the literature, including the recent reports which raised
the hypothesis of telomere shortening and telomere position effect in mild
ring 17 syndrome.
P13.42-M
Somatic structural genome variations (chromosome rearrangements)
are common in children with intellectual disability, congenital
malformations and autism
Back to index
common among children with intellectual disability, congenital malformations and autism. Supported by the Russian Federation President Grant (MD4401.2013.7).
P13.43-S
Assessing the impact of exonic variants on splicing regulation by
functional analysis and bioinformatics predictions: BRCA2 exon 18 as
a model system
275
ABSTRACTS POSTERS
mother performed amniocentesis because of advanced maternal age. Karyotyping was confirmed by FISH five years ago. The woman with a known balanced translocation between chromosomal bands 14q13 and 18q12.2 was
referred for prenatal diagnosis by amniocentesis at 17 weeks of gestation.
Cytogenetic analysis of the fathers lymphocytes showed a normal male karyotype 46,XY. Their first baby had normal karyotype 46,XX, also prenatally observed. We present the case of prenatally diagnosed tertiary trisomy
with supernumerary derivative chromosome 14, due to a 3:1 segregation of
maternal reciprocal translocation. The fetal karyotype was 47,XX,+der(14)
t(14;18)(q13;q12.2)mat, resulting in partial trisomy 14(pter-q13) and partial trisomy 18 (q12.2-qter). There was no evidence of fetal malformations
by ultrasound examination. After the amniocentesis procedure, amniotic
fluid has leaked and the mother miscarried. In reciprocal translocation carriers these chromosomes can arise in different segregation modes, such as
alternate, adjacent-1, adjacent-2, 3:1 or 4:0, resulting in the formation of 32
possible zygotes with different chromosome complements. This case supports the thesis that the incidence of 3:1 segregation is higher in female than
in male carriers, more often occurs when one of the derivative chromosomes is relatively small, and also suggests that translocation with acrocentric
chromosomes tends to produced 3:1 segregation.
P13.46-M
Mosaic Uniparental Isodisomy (UPD) of 11p, presenting as a regular
beta-thalassemia carrier.
C. L. Harteveld, M. J. v. Belzen, C. A. L. Ruivenkamp, M. J. V. Hoffer, M. Phylipsen;
Leiden University Medical Center, Leiden, Netherlands.
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P13.48-M
Patterns of X inactivation in abnormal X chromosome
P. S. Fahmy;
National Research Center, Cairo, Egypt.
Background: X inactivation is a dose compensation mechanism which results in silencing majority of genes on one of the two X chromosomes in
every somatic cell of human females. Early in embryonic development, cells
inactivate all their X chromosomes except one. Once an X is chosen, it is
stably inherited through subsequent somatic mitotic divisions. The process
of X inactivation is under the control of X inactivation center. Purpose: Study of X inactivation patterns in cases with abnormal X chromosome and its
correlation with patients phenotype. Methods: 15 selected patients having
abnormalities of the X chromosome were subjected to Clinical examination,
GTG banding, FISH technique to detect origin of some structural X abnormalities and Detection of X chromosome replication pattern (Late Replicating Chromatin) technique. Results: Cases were classified according to their
karyotypes into three groups: Cases with numerical X abnormalities, Cases
with iso X chromosome and Cases with other structural X abnormalities.
In most of X aneuploidies, each cell has only one active early replicating X,
while other extra or abnormal X chromosomes are inactivated late replicating. Regarding the balanced X;autosome translocation, there was a mosaic
pattern; in majority of cells translocated X was late replicating inactive,
while in few cells translocated X was early replicating active X chromosome.
Conclusion: It has been found that at least 30 X-linked genes are expressed
on the inactivated X chromosome. The varying degree of phenotypes within
each syndrome occurs because the genes that escape X inactivation are expressed at varying degrees.
P13.49-S
Uncovering the oligomeric structure of the y+LAT1/4F2hc amino acid
transporter
M. Tringham1, J. Kurko1, O. Simell2, J. Mykknen2;
1
Department of Medical Biochemistry and Genetics, University of Turku, Turku, Finland,
2
Department of Paediatrics, University of Turku and Turku University Hospital, Turku,
Finland.
y+LAT1 and 4F2hc are protein subunits that form a transporter complex for
cationic amino acids in the basolateral membrane of epithelial cells, mainly
in the small intestine and proximal kidney tubules. Mutations of y+LAT1,
56 of which are currently known, cause lysinuric protein intolerance (LPI,
OMIM #222700), a rare metabolic disorder characterised by diminished intestinal absorption of the cationic amino acids lysine, arginine and ornithine
and by severe loss of these amino acids into the urine. The more detailed
structure of this transport complex has so far been unclear - it has remained unelucidated whether the complex is formed as a dimer or a tetramer
of the subunits. What has been known, however, is that the y+LAT1 subunits cannot reach the plasma membrane without forming a complex with
4F2hc. We previously established fluorescence resonance energy transfer
(FRET) microscopy and FRET-FACS as tools in studying the interactions of
y+LAT1 and 4F2hc. We have now applied these techniques together with
immunohistochemistry to the exploration of the heteromerisation status
of the y+LAT1/4F2hc transporter complex. We discovered that when fused
into fluorescent vectors and transfected into the HEK293 cells, the y+LAT1
proteins interact together in the presence as well as absence of 4F2hc. Our
initial results therefore suggest that the holotransporter is a multimer of
y+LAT1 and 4F2hc subunits.
P14.01-S
Technical aspects of ALK FISH test to improve therapy selection of
lung cancer patients
ABSTRACTS POSTERS
243 lung adenocarcinomas collected in three Institutions. A series of standardized ALK-negative lung cancer cell lines (Abbott) were used as negative
controls. A specific scoring system considering not only the splitting of the
signals but also their distance was developed.
Results. We identified 12% ALK_FISH+ patients, with similar rate among the
three Institution (13%, 12%, 9%). ALK_FISH+ cases showed: 53% inversion/translocation, 28% deletion and 18% both patterns. Concordance was
observed between the different operators and between the two probes, both
on patients and on the panel of negative controls. In this last, the cut-off
obtained (by calculating M+3SD) was 14.9%.
Conclusions. Difficulties in interpretation of ALK FISH signals pattern might
be bypassed using our detailed scoring system, that is reproducible among
different operators, probes, and provide experimental evidence that 15% is
a reasonable cut-off for patients selection.
P14.02-M
Allelic drop-out in large Iranian Brugada syndrome family revealed
by new generation sequencing
Background: Sangersequencingisagold standardofDNAdiagnostic currently usedforNGS validation.Nevertheless,ithasits ownlimitations producing false results. The well-known mechanism of allelic dropout is the
presence of SNPs in 3-region of PCR primers. The early recognition of allelic
dropout prevents diagnostic errors.
Materials and Methods: The DNA samples from Brugada syndrome Iranian family were extracted from peripheral blood. Sanger sequencing of SCN5A gene coding areas was performed for proband and relatives. Control
re-sequencing via semiconductor PGM IonTorrent platform using Ampliseq
primer pool encompassing coding area of 10 genes including SCN5A was
performed for family members.
The control group comprised 100 ethnically matched healthy donors.
Results: Sanger sequencing has revealed a novel heterozygous genetic variant p.P1506S in exon 26 of SCN5A gene in proband. Surprisingly, his son
carried p.P1506S variant in homozygous state. Carriage of p. P1506S variant in mother had been excluded by Sanger sequencing and PCR-RFLP
analysis. We performed control re-sequencing via semiconductor PGM
IonTorrent platform for proband and his son. NGS results showed a discrepancy with primary Sanger sequencing results, proving heterozygousity of
p.P1506S variant in probands son. Sanger re-sequencing using alternative
oligonucleotide primers for exon 26 confirmed NGS outcome. Thus, SNP
rs41315501 was identified in 3-region of previously used forward primer.
Prevalence of rs41315501 was assessed by PCR-RFLP in 200 chromosomes
cohort and MAF (minor allele frequency) accounted 9,4%. SNP was detected
in probands wife and son in heterozygous state.
Conclusion: Currently Sanger sequencing is used for validation of NGS techniques. But vice versa, NGS technology can be applied for capillary sequencing quality control.
P14.03-S
Scanning Alpha Haemoglobin-Stabilizing Protein (AHSP) gene by High
Resolution Melting Analysis
S. Pappa1, G. Kellaris1, E. Voskaridou1, P. Kollia2, A. Balassopoulou1, E. Boutou1;
1
Laiko Hospital, Athens, Greece, 2Athens University, Athens, Greece.
Back to index
Hereditary autoinflammatory diseases (AID) are characterized by recurrent bouts of systemic inflammation caused by dysregulation of proteins of
the innate immunity system. Identification of about 25 genes over the last
17 years rendered possible genetic diagnosis, a major tool to discriminate
patients with close phenotype. This genetic heterogeneity and the rarity of
these conditions motivated an International concerted action for the use of
next generation sequencing for diagnosis of AID.
A survey among AID experts was conducted in 2013. Twenty laboratories
declared willing to participate in this network and filled out a questionnaire.
Results were as follows:
Sequencing equipments varied across the laboratories. Illumina MiSeq was
the major device (29%), followed by Lifetech PGM (25%), Illumina HiSeq
(18%), Roche Junior (14%), other (14%).
Sequencing results were analyzed with either a software from the platform, an in house pipeline, a collaboration (21% each), or a combination
(37%).
The number of AID genes included in the panels varied from the five most
frequent to all published genes.
Clinical expectations included ethics: develop new informed consent
(53%) and guidelines (47%), and collaboration: sharing experience for variant interpretation (31%), access to patients and database (28%), harmonization of technology and reports (17%).
Through a close collaboration with clinicians, we could connect this project
with two existing dedicated patient (Eurofever) and mutation (Infevers)
registries. Integration of clinical and genetic data will help 1) elaborate a
consensus for classification criteria and 2) identify new AID genes in orphan
patients.
P14.05-S
Next Generation Sequencing approach to molecular diagnosis of autoinflammatory diseases: from gene panel design to variant call
277
ABSTRACTS POSTERS
mality, BCA) is a common chromosomal structural variation. Next-generation sequencing has been reported to detect BCA-associated breakpoints
with the aid of karyotyping. However, the complications associated with this
approach and the requirement for cytogenetics information has limited its
application. Here, we provide a whole-genome low-coverage sequencing approach to detect BCA events independent of knowing the affected regions
and with low false positives. First, six samples containing BCAs were used to
establish a detection protocol and assess the efficacy of different library construction approaches. By clustering anomalous read pairs and filtering out
the false-positive results with a control cohort and the concomitant mapping
information, we could directly detect BCA events for each sample. Through
optimizing the read depth, BCAs in all samples could be blindly detected
with only 120 million read pairs per sample for data from a small-insert
library and 30 million per sample for data from non-size-selected mate-pair
library. This approach was further validated using another 13 samples that
contained BCAs. Our approach advances the application of high-throughput
whole-genome low-coverage analysis for robust BCA detection, especially
for clinical samples, without the need for karyotyping.
P14.07-S
Using biotin-streptavidin interaction for binding plasmid to a cellpenetrating peptide
D. S. Polyakov;
Institute of Experimental Medicine of the NorthWest Branch of the Russian Academy of
Medical Science, St.Petersburg, Russian Federation.
Gene therapy strategies based on plasmid DNA are more preferable than
viral-based gene delivery. Plasmids are more stable in vivo and known to
be less immunogenic. Retroviral-based vectors integrate in actively transcribed genes that can lead to disruption of tumor suppressor genes. Gene
delivery methods based on plasmid DNA do not cause mutagenesis and they
do not integrate in chromosomes providing increased safety. Despite the
potential advantages of using plasmid DNA, delivery issues with plasmid
vectors exist. One of the ways of plasmid delivery into a cell can be biotinstreptavidin interaction of the plasmid to a cell-penetrating peptide (CPP).
Streptavidin binds the small molecule biotin with femtomolar affinity. We
have created a biotinylated pEGFP-N3 circular plasmid DNA to further its
binding to the streptavidin-CPP fusion protein. Few thymines were replaced
by biotinylated uracils in the plasmid. The presence of biotinylated uracils
in purified plasmid was verified in restriction analysis followed by dot blot
procedure. Streptavidin-conjugated alkaline phosphatase was used to detect the biotin-uracils. The ability to express green fluorescent protein was
tested in Hela cell culture. We have created a genetic construct for synthesis
of monovalent tetrameric streptavidin, which consists of one WT streptavidin and three inactive streptavidins. Each inactive streptavidin is fused to
chain of four HIV TAT-peptides. The resulting chimeric protein binds one
biotinylated circular plasmid DNA due to the biotin-streptavidin interaction.
This structure can be used for gene therapy.
P14.08-M
Methyl-DNA detection: a performance comparison of four commercial
kits
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methylation while our comparison shows which kit is the least harsh and
the most efficient.
P14.09-S
Molecular testing of BRCA1&2 genes with NGS technology: a threestep approach
A. Martayan, A. Antenucci, C. Del Carlo, F. De Bellis, L. Conti;
Istituto NazionaleTumori Regina Elena Roma, Rome, Italy.
The aim of our study was to enhance the throughput of mutation detection analysis in order to supply high sensitivity, fast and accurate data on
BRCA1/2 genes mutational status of selected breast and/or ovary cancer
patients. Starting from June 2012 a panel of 8 selected BRCA1/2 pathogenic
mutationprone cases have been analysed on a GS 454 Junior platform (Roche) and 6 out of 8 pathogenic variants were successfully identified. One of
the two unidentified variants was a single nucleotide insertion just adjacent
to a homopolymer stretch of 8 Adenines in the coding region of BRCA2. The
second unidentified variant was a deletion of the whole exon 14 of BRCA1
gene. The latter two unidentified pathogenic variants were revealed subsequently using the BRCA Homopolymer (BRCA HP) kit from MULTIPLICOM
and MLPA analysis. We then decided to adopt a Three-step mutational screening approach. As a first step BRCA1 &2 amplicon libraries were generated
with BRCA MASTRtm assay from Multiplicon, and sequenced on our GS 454
junior platform. Sequencing data are then analysed with Amplicon Variant
Analyzer (AVA) software (Roche). The samples with no pathogenic variants
identified are then involved in the second step which is the processing with
BRCA HP kit in order to uncover insertions or deletions in homopolymeric
coding regions of both genes. The third step is MLPA analysis to uncover
large genomic rearrangements. So far 181 samples were analysed in our
laboratory and 29 pathogenic variants were identified with our three step
testing approach.
P14.10-M
Efficient sharing of BRCA1 and BRCA2 variant and phenotype data
between diagnostic labs
The Dutch and Belgian working group for Breast Cancer DNA Diagnostics
(LOB) has decided to share over 7500 variants detected in the BRCA1 and
BRCA2 genes in breast cancer families since 1997. For this, the data, almost
evenly split among both genes, have been submitted to the LOVD 3 shared
gene variant database installation (1,2). Previously, variants identified in
Dutch and Flemish (Belgian) DNA diagnostic labs were collected yearly and
stored in an Excel data file. Advantages of the LOVD system include: simple
submission of new data in a standardized way, instant updates after curation, easy maintenance and automatic backups. Although most data are publicly accessible online, some data are shared by members only. Others can
see whether such information is available (password protected file links),
giving them the option to contact the submitter for further details. Members can contribute their opinions about variant classification, increasing
its consistency, but being aware of potential misinterpretation they have reservations sharing this information. Data are stored variant-by-variant and
connected to each individual patient and submitting diagnostic lab. Using
existing LOVD functionality, users can perform queries per gene or individual, use other linked resources of interest, get genome browser views of the
data and use web services to access variants stored in other gene variant
databases. In addition, LOVD3 has a new access level, designated collaborator, allowing submitters to share otherwise non-public data with other
submitters, e.g., to share detailed phenotype information with other diagnostic labs only.
1) http://databases.lovd.nl/shared/genes/BRCA1
2) http://databases.lovd.nl/shared/genes/BRCA2
P14.11-S
Validation of a Next Generation Sequencing assay for BRCA1 and
BRCA2
Next Generation sequencing (NGS) is rapidly finding a place in routine diagnostics although, extensive validation of such platforms and the associated
bioinformatics is urgently required before they reach equivalent confidence
as Sanger sequencing. Routine testing of BRCA1 and BRCA2 for Breast Cancer predisposition has resulted in numerous positive and negative controls
for the rigorous assessment of NGS. A further consideration when implementing a new technology for routine testing is its suitability to the diagno-
ABSTRACTS POSTERS
stic laboratory which will be discussed.
This study examines approximately 400 samples referred for BRCA1 and
BRCA2 testing in the context of familial breast cancer and compares the
performance of NGS to Sanger sequencing. The approach used involved
microfluidic PCR (Fluidigm Access Array), followed by NGS on an Illumina
MiSeq instrument and subsequent bioinformatic analysis with NEXTGENe
(SoftGenetics). A single assay is capable of performing 42 BRCA1 and BRCA2
screens in less than 5 days. The failure rate for the assay was 4% (17/399),
primarily contributed by the initial two assays (N=94) with subsequent assays providing a 1.6% failure rate (5/305). The sensitivity of the assay compared to Sanger sequencing was estimated at 99.7%. Overlapping amplicon
design was the most significant problem encountered with bioinformatic
analysis, requiring several rounds of optimisation. Validation of the workflow consisted of ~400 samples distributed across retrospective (including
a reproducibility component) and prospective arms as well as a blinded/
inter laboratory sample assessment and a concurrent testing period. The
validated assay has been accredited to international standards and is now
in routine use.
P14.12-M
Initial evaluation of the cardiomyopathy-specific content of Illuminas
TruSight ONE next generation sequencing assay
S. Waldmueller, K. Imbrich, S. Junker, M. Sturm, P. Bauer, O. Riess, M. Bonin;
University Hospital Tuebingen, Tuebingen, Germany.
Back to index
Celiac Disease (CeD; gluten intolerance) is diagnosed by symptoms, detecting CeD-specific antibodies, and biopsy results. The only available therapy
is a life-long gluten-free diet. Despite the availability of diagnostic tools, as
many as 7 out of 8 adult CeD patients are either not or incorrectly diagnosed,
highlighting the need for novel biomarkers.
Circulating miRNA profiles have been shown to be disease-specific, even
disease-stage-specific, in patients with cancer or gastro-intestinal disease.
We examined whether circulating miRNAs in plasma samples of CeD patients can be used as CeD biomarkers. By next generation sequencing we profiled miRNAs in 95 plasma samples from 12 CeD patients, 5 patients positive
for CeD-associated antibodies and 5 control subjects from the Prevent CD
cohort. In this cohort, newborns at risk for CeD were challenged with low
levels of gluten between 4-6 months of age to induce gluten tolerance, and
plasma samples were taken every 3 months until diagnosis. Comparing data
from samples taken at diagnosis to samples taken at 3 months of age (first
available sample), we found 62 miRNAs significantly differently expressed
(FDR<0.05). Of those, 4 miRNAs were previously reported to be associated
with CeD, 12 were implicated in other autoimmune diseases and 7 miRNAs
have been implicated in immune signaling. One of these immune related
miRNAs is miR-23b, which appears to be downregulated 2-fold in CeD patients at time of diagnosis. This miRNA is known to limit tissue inflammation.
A downregulation of this miRNA would therefore contribute to the pro-inflammatory state in active CeD.
P14.15-S
Northern Lights Assay of cfDNA damage in body fluids
Structural damage in cfDNA molecules in body fluids has been little studied.
Such damage may reflect normal and abnormal cell turnover, genome instablity or exporsure to genotoxic agents. We analyzed cfDNA damage in
plasma, urine and saliva. Standard methods of isolation of cfDNA in plasma
and urine are based on inducing ssDNA with a chaotropic agent and selective cordination binding of lone pair electrons on guanine to silica. These
methods were not usable. In contrast, we found that selective ion exchange
chromatography allowed gentle isolation of DNA without inducing damage.
Damage in isolated DNA was assessed with the Northern Lights Assay. This
assay is based on Two-Dimensional Strandness-Dependent Electrophoresis
(2D-SDE) in premade microgels. Each sample was run in duplicate i.e. uncut
and cut with Mbo I, an enzyme which cuts both single- and double-stranded
DNA. Single-stranded breaks, either nicks or gaps, were detected as horizontal streaks from uncut DNA molecules. Double-stranded breaks generated
an arc in the gel. DNA molecules with interstrand crosslinks (ICL) migrated
as an arc behind normal dsDNA molecules. DNA with intrastrand crosslinks
and bulky adducts were bent and migrated in front of that arc. Single-stranded DNA molecules, too damaged for complementary strand binding, formed a diagonal line. Patterns of cfDNA in plasma of normal subjects showed
an apoptosis pattern with single- and double-stranded breaks of nuclesomal
fragments. cfDNA in urine showed composite patterns of apoptosis and nonspecifc degradation. The most extensive damage and variable patterns were
seen in saliva including prominent single-stranded breaks.
P14.16-M
A retrospective view on External Quality Assessment of Charcot-Marie
Tooth disease over 16 years
B. W. Rautenstrauss1, U. Schoen1, A. Benet-Pages1, J. M. Hertz2;
1
Medizinisch Genetisches Zentrum, Munich, Germany, 2Odense Universitetshospital,
Odense, Denmark.
279
ABSTRACTS POSTERS
Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous disorder of the peripheral nervous system. CMT1A is the most frequent autosomal dominantly inherited form caused by a 1.4-Mb tandem
duplication in chromosome 17p12. In 1997 the first annual German external quality assessment (EQA) was performed for CMT1A. At this time the
molecular genetic analysis was mainly based on 5 methods: RFLP Southern
blotting, STR markers, PCR for junction fragments within the CMT1A-REP
elements, PFGE and FISH. 10 laboratories in Germany participated. In the
year 2000 the scheme was offered to a wider audience with 22 participating
laboratories. Only one genotyping error occurred. Since 2008 the European
Molecular Genetics Quality Network (EMQN) has organised the EQA scheme. In 2012 the number of participating laboratories increased to 66 from
22 countries (198 reports) representing countries from around the globe.
Methods like FISH, PFGE and Southern blotting have disapeared; PCR, qPCR
and MLPA are now used. Next generation sequencing (NGS) allowing simultaneous sequence analysis as well as gene dosage determination is a future
perspective.
Currently at least 60 genes are associated with disorders of the peripheral
nervous system. Consequently the scheme scope has been extended to sequence analysis of GJB1 (CX32) (CMTX1). In 2012 again only 1 genotyping
error occured. The efforts that have been made for an EQA in molecular
genetic diagnosis for Charcot-Marie-Tooth disease demonstrate very good
laboratory analytical performance. Nevertheless national laws affecting human genetics are different, thus harmonization in political terms (e. g. predictive testing) remains as important task.
P14.17-S
Comparative-High Resolution Melting - a novel method of
simultaneous screening for small mutations and copy number
variations
P. Borun, A. Plawski;
Institute of Human Genetics, Poznan, Poland.
Efficient and cost-effective screening for DNA sequence changes, both small
mutations and copy number variations (CNVs), is a crucial aspect for routine
genetic diagnostics as well as for basic research. In this study we present
a development and evaluation of comparative high resolution melting (CHRM), a new approach for the simultaneous screening of small DNA changes
and gene CNVs. In contrast to other methods, relative quantification in CHRM is based on the results obtained during the melting process and calculations of the melting peak height ratio in the multiplex reaction. Validation
of the method was conducted on DNA samples from 50 individuals from Duchenne muscular dystrophy (DMD) families, 50 probands diagnosed with
familial adenomatous polyposis and a control group of 36 women and 36
men. The results of analyses conducted on fragments of the DMD and APC
genes correspond completely (100%) with the results of previous studies.
C-HRM sensitivity in CNV detection was assessed through the analysis of
mixed DNA samples with different proportions of a deletion carrier and
wild type control. The results are presented as a linear regression with R2 of
0.9974 and imply the capability of the method to detect mosaics. C-HRM is
an attractive and powerful alternative to other methods of point mutations
and CNV detection with 100% accuracy in our studied group.
P14.18-M
New molecular technique to monitor minimal residual diseased in
CML
Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the Philadelphia chromosome (Ph), resulting from the t(9;22)
(q34;q11) balanced reciprocal translocation that generates the BCR-ABL1
fusion protein. The first line therapy of CML is Imatinib Mesylate, which targets BCR-ABL1 protein, inhibiting proliferation pathway. Residual leukemia
is assessed by a sensitive molecular quantitative manner evaluating levels
of BCR-ABL1 transcripts by real-time reverse transcriptase PCR (qRT-PCR).
Undetectable levels of chimeric transcript, however, can reflect either an
effective elimination of leukemia cells, or the presence of a quiescent leukemic stem cells transcriptionally silent. We developed a novel highly sensitive
method to identify quiescent leukemic cells and possible candidates for discontinuation of therapy through quantitative real-time PCR (Q-PCR) based
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on the DNA. Detection of BCR-ABL1 fusion gene is critical for the diagnosis
of chronic myeloid leukemia and to follow the disease progress in patients
under therapy. We applied targeted next-generation sequencing (NGS) to
resolve sequence breakpoints in BCR-ABL1 fusion gene in CML patients and
K562 cell line. In conclusion, DNA genomic Q-PCR is a very sensitive and
direct technique to identify quiescent leukemic cells and patients that could
be possible candidates to stop Imatinib therapy. We identified junction sequences in all samples using Agilent SureSelect enrichment and Illumina
paired-end sequencing.
P14.19-S
Rapid Serial PCR Instrument with High Speed Melting (HSM) Analysis
High throughput sequencing technologies enable efficient large-scale genetic analysis, dramatically accelerating the genetic research and diagnostics.
Our study aims to determine the proportion of familial nonsyndromic congenital heart defects (CHD) that can be explained by pathogenic mutations
in currently known CHD associated genes, by implementing next-generation sequencing technologies in genetic diagnosis for familial nonsyndromic
CHD. Targeted resequencing of known cardiac genes was performed in 36
patients from 13 nonsyndromic CHD families, using either array-based or
solution-based method to capture the coding regions of 57 genes associated
with CHD. Following variant analysis and Sanger validation, we identified
6 functional deleterious mutations in 3 genes, explaining the defects in 6
families. The genetic heterogeneity of CHD and remarkable variability of
expression make it challenging to interpret the large number of identified
variants in patients. Thus, setting up a well-defined analysis pipeline is
necessary to identify causative mutations. In conclusion, by targeted resequencing of known CHD associated genes in well selected nonsyndromic
CHD families and cautious variant interpretation, likely causative mutations
were identified in 6/13 (46%) families. Reduced penetrance and possibility of phenocopies complicate the interpretation. Targeted next-generation
sequencing is a powerful tool in genetic testing of familial nonsyndromic
CHD, however, variant interpretation remains a major challenge for the diagnostic application.
ABSTRACTS POSTERS
P14.21-S
Whole exome sequencing in congenital myopathies: two case reports
A. Armaroli1, C. Scotton1, J. Sigursson2, C. Passarelli3, M. Neri1, F. Di Raimo1, F.
Gualandi1, E. Mercuri4, M. Pane4, A. Ferlini1;
1
Dept. of Medical Science, Medical Genetic Section, Ferrara, Italy, 2deCODE Genetics,
Reykjavik, Iceland, 3Unit of Neuromuscular and Neurodegenerative Diseases, Dept. of
Neurosciences, Bambino Ges Children Hospital, IRCCS, Rome, Italy, 4Dept. of Pediatric
Neurology, Catholic University, Rome, Italy.
Congenital myopathies are a heterogeneous group of genetic diseases characterized by hypotonia and muscle weakness. Many causative genes have
been identified in the last decade, underlining the high genetic heterogeneity in these pathologies. Sanger molecular diagnosis is an expensive and time
consuming step-by-step process. In addition some genes involved are very
large and high demanding in terms of molecular diagnostics. Here we report
on two families-of-four with congenital myopathy in which we performed
whole exome sequencing approach (WES). The analysis was performed by
deCODE Genetics and we analyzed the families applying candidate genes interrogation and autosomal recessive inheritance; identified variations were
prioritized for the pathogenic prediction and the rare allele frequency under
1% of the population.
In the first family, with one affected child, we found a known pathogenic inframe deletion of the isoprenoid synthase domain-containing protein gene
(ISPD). Mutations in this gene are known to be associated to congenital muscular dystrophy phenotypes. In the other family, with two affected sibs, we
identified a mutation in RYR1 gene. In both families a previous partial molecular diagnosis by Sanger, and not exploring all exons, was performed.
In these two cases WES identified mutations in two known genes correcting a non-exhaustive diagnostic approach. This is an example confirming
the usefulness of next generation sequencing as an accurate molecular diagnostic approach for pathologies with high genetic heterogeneity and/or
large genes.
The Neuromics EU project is acknowledged.
P14.22-M
Detection of Copy Number Variation by PCR under Limiting
Conditions
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extended genomic regions. In this frame, we analyzed 24 selected CF patients by full CFTR gene resequencing (8 with known CFTR genotype, 11 with
only one mutation identified, and 5 with no mutations after conventional
screening). A custom capture SeqCapEZ Library (Roche) and an HiSeq 2000
platform (Illumina) were used. Multiplexing 6 samples in the capture phase
and 12 in the sequencing step, we obtained a mean depth >1,000X, with a
98% coverage. An in-house developed pipeline was used for variant detection and annotation, which allowed the identification of all 14 previously
known mutations. Moreover, 4 genetic lesions undetected by genetic screenings were found, including a large heterozygous deletion. Additionally,
we found new intronic variants, whose possible role on RNA splicing was
excluded by a combination of in-vivo and in-vitro analyses. In conclusion,
NGS increased the percentage of genetically diagnosed patients, being able
to disclose mutations escaped to standard mutational screening. Moreover, read alignment also allowed an immediate definition of large deletion
breakpoints at the nucleotide level. Finally, even after this extensive sequencing strategy, 5 probands did not show any mutation in CFTR (nor in the
SCNN1A, SCNN1B, and SCNN1G coding regions), suggesting the intriguing
possibility of an additional locus for CF. These subjects are currently being
processed for whole-exome sequencing analysis.
P14.24-M
Performance of the Luminex xTAG Cystic Fibrosis kit (EU)
V. Ritchie, C. Dey, L. Lan, D. Himsworth, J. Liu;
Luminex Molecular Diagnostics, Toronto, ON, Canada.
The xTAG Cystic Fibrosis Kit (EU) [herein termed xTAG CFE] is a qualitative genotyping device used to simultaneously detect and identify a panel of
mutations and variants in the Cystic Fibrosis transmembrane conductance
regulator (CFTR) gene in human blood specimens and blood spots. The assay identifies the recommended ACMG/ACOG mutations and variations, as
well as some of the worlds most prevalent mutations. Additionally, xTAG
CFE identifies mutations specific to Europe, such as 4016insT, L1065P and
L1077P in the French and Italian populations, 2184insA in Central Europe,
T338I in the Italian (Sardinia) population, 712-1G>T in the Spanish population, and E585X, R1066H, Q552X and G1244E in Southern Europe. DNA
samples can be screened for 5 variants and up to 75 CFTR mutations, out
of a total pool of 85 available mutations, allowing the user to customize CF
testing to a particular region. The assay is comprised of a single multiplex
polymerase chain reaction which is then used in three separate Allele Specific Primer Extension (ASPE) reactions (A, B, and C). The limit of detection
of the assay is 2 ng/L with an input genomic DNA range of 10 ng to 1.5 g.
Diagnostic accuracy was assessed for the xTAG CFE assay using the xTAG Cystic Fibrosis and bidirectional dideoxy-sequencing as comparator methods.
Over four hundred samples were analyzed that included genomic DNA extracted from whole-blood specimens, dried bloodspots and Coriell samples.
Sensitivity and specificity for the xTAG CFE assay across all mutations was
100%.
P14.25-S
Missing the forest for the trees: integrating traditional and molecular
cytogenetics technology in hematological neoplasia
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ABSTRACTS POSTERS
relevant. Therapy was initiated before release of results in 27%, after cytogenetics in 9%, after FISH in 3%, after array in 1% and supportive care in
60%. The preliminary data suggests a tier testing approach does not compromise patient care if tier testing is completed within 3-4 weeks of initial
diagnosis. In presence of unfavorable prognostic abnormalities by cytogenetics, array testing may not be warranted. Based on this data, we would like to
propose the optimal genetic testing algorithm.
P14.26-M
Real-time and Droplet PCR quantification for non-invasive
determination of RHD incompatibility between mother and foetus
I. Svobodova, S. Pospisilova, E. Pazourkova, A. Horinek, M. Korabecna;
General faculty hospital in Prague, Prague 2, Czech Republic.
The aim of this study was to compare two strategies of DNA quantification: Real-Time PCR (qPCR) and Droplet PCR (dPCR). The benefit of dPCR
against qPCR is represented by absolute quantification of target nucleic acid
molecules without the requirement of calibration curves. The methods were
compared by quantification of standard nuclear DNA of known concentration. DNA was eightfold diluted (2-0.015 ng/ul) and twelve replicates were
realized for each dilution. Concentrations were then measured as levels of
amplicons of two genes, housekeeping gene GAPDH and human RHD gene
(exon 10). The same genes were analyzed in plasma cell-free DNA and also
in cell-free fetal DNA isolated from maternal plasma. Evaluation of these two
methods performance was based on several parameters, such as degree
of linearity (R), accuracy, detection limit, etc. In case of standard nuclear
DNA, both methods showed high accuracy and low detection limit, both genes were detected even in most diluted samples. High linearity degree (R
> 0.99) has been reached by both methods. The correct RHD status of tested women was immunologically verified. Regarding analysis of cell-free
fetal DNA in RHD negative maternal plasma, dPCR failed against the qPCR
to convincingly distinguish RHD positive samples from the negative ones.
For the purpose of prenatal screening, the qPCR method would be probably
the preferable alternative due to very low fetal DNA concentrations (about
0.002 ng/ul) in maternal plasma, which is under the detection limit of dPCR.
Supported by the Ministry of Health of the Czech Republic RVO VFN64165
P14.27-S
Development and evaluation of a gene panel for the diagnosis of
monogenic epilepsies
Next Generation Sequencing (NGS) is a great advance in the field of molecular genetics, especially for diseases with high genetic heterogeneity, allowing parallel testing of many disease-causing genes. It will lead to a better
access of a larger number of patients to molecular diagnosis. In order to
improve the diagnostic yield of genetic testing in patients with epileptic diseases, we developed a panel including 41 genes causing monogenic form
of epilepsy or neurodevelopmental disorders frequently associated with
seizures. Until now, only 13 of these genes have been currently screened by
Sanger sequencing, on a sequentially basis. In the present study we compared two methods: the Haloplex technology (Agilent) and the SeqCap (Roche)
technology. Sequencing was performed using an Ion Torrent PGM sequencer
(Life Technologies). We performed NGS sequencing on DNA samples from
44 patients, including 38 patients in whom a (probably) causative mutation
had been previously found by sequential Sanger sequencing in our routine practice, and 6 novel patients who had not been screened. DNA samples
were anonymized and the first steps of the analysis were performed following a blind protocol. DNA from all the patients was analyzed with the Haloplex technology and only 24 of them were also analyzed with the SeqCap
technology (they were sorted among the 44 patients). Data analysis will be
performed with the NextGene Software (Softgenetics) and also with specific
BWA-GATK pipeline based on the recommendations of BroadInsitute. Our
analysis will include coverage, sensitivity and false positive rate as well as
a cost evaluation.
P14.28-M
Scaling up whole-exome sequencing on Ion Proton using AmpliSeq
I. M. Jonasson, C. Lindau, U. Gyllensten, A. Ameur;
Uppsala University / IGP, Uppsala, Sweden.
The Ion Proton and Ion PGM (Life Technologies) technologies are used in
the National Genomics Infrastructure - Sweden (NGI) to handle the different
282
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EQA is essential to verify the quality and accuracy of the diagnostic service.
When new technologies emerge, EQAs need to be developed to verify the
laboratory diagnostic validation process and enable benchmarking of their
performance. When laboratories introduce new technologies diagnostically,
most critical errors in EQAs are analytical. As laboratories gain more experience in the new technologies, the critical errors usually become limited to
the interpretation of results.
ABSTRACTS POSTERS
EQA is intended to be educational so if a laboratory receives a poor performance, they are asked to review their results and perform an audit so
they can identify the root cause of the error. If required, repeat EQA samples are made available to exclude the rare instances of a problem with a
manufacturers kit. In 2013, a thorough root cause analysis by one participating laboratory identified a potential problem with one manufacturers
probe. An independent verification by CEQAS using multiple FISH probes
and arrays confirmed a 967kb deletion of 13q14.2q14.3. There is a discordant result between the microarray and FISH probes for one of the deletion
breakpoints. As a consequence the manufacturer immediately withdrew the
probe and replaced it with a new product to eliminate the likelihood of future false negative results. A second laboratory identified an internal problem
with their fluorescent microscope filters which they replaced and then verified gave the correct results using validated EQA material.
This presentation will give examples of how different approaches to investigating a poor performance can result in different outcomes including changes to practice.
P14.31-S
Identification of point mutations and deletions in Fanconi anemia
using a next generation sequencing approach
E. Nicchia1, C. Greco2, D. De Rocco1, R. Bottega2, A. DEustacchio2, E. Cappelli3, A.
Pallavicini4, L. Torelli5, V. Pecile2, C. Dufour3, A. Savoia2,1;
1
Department of Medical Sciences, University of Trieste, Trieste, Italy, 2Institute
for Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy, 3Clinical
and Experimental Hematology Unit, G. Gaslini Childrens Hospital, Genova, Italy,
4
Department of Life Sciences, University of Trieste, Trieste, Italy, 5Department of
Mathematics and Geosciences, University of Trieste, Trieste, Italy.
Genetic testing of Fragile X disorders is now performed with PCR-based methods which sensitivity and precision allow detection and accurate sizing of
FMR1 alleles in the entire spectrum of the CGG repeat amplifications. These
molecular tools, designed to analyze a region of DNA with intrinsic characteristics of instability, could lead to new observations that were out of reach of
previous methods. We report the detection of a smaller and less represented
fragment in PCR amplifications of the FMR1 repeat region in independent
samples of unrelated individuals, three males with intellectual disability
and one normal female, with otherwise normal Fragile X alleles and karyotype. A smaller fragment present in PCR reactions from different DNA extractions and samples of each identified individual, has been detected by three
different Italian laboratories with different settings (primers, amplification,
electrophoresis). In one of the cases both parents of the proband resulted to
not carry the smaller fragment, thereby excluding a rare X-chromosome or
autosomal polymorphism as a possible cause. The possibility of random somatic instability as a cause of such finding in our cases is challenged by the
similar size (286-293 bp) and quantitative representation of the peak. The
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fact that the same result has been found in four unrelated subjects (three
Italian and one Israeli) out of 2000 tested, suggests a frequency of about
1/500 individuals. Further characterization of the extra peak is underway to
establish its sequence origin and better understand the nature of what could
give new information on the Fragile X region.
P14.33-S
Development of a Next-Generation-Sequencing based framework for
comprehensive genetic analysis of Neurogenetic disorders
With the advance of sequencing technologies, the capacity to generate accurate genotype data greatly outpaces our ability to analyse it. To facilitate
data intrepretation, a framework were developed for estimating the relative
pathogenicity of variants. We used Tuberous Sclerosis, an autosomal dominant Neurogenetic disorder caused by mutations in either TSC1 or TSC2,
to test our famework. As all kinds of mutations, including single nucleotide
variants (SNVs), small insertions/deletions (indels), and large deletion/duplication mutations have been reproted in TSC1 and TSC2 scattering almost
all along the 65 exons of the two genes without any obvious hotspot, the
Neurogenetic disorders panel including 399 genes, in addition to TSC1 and
TSC2, was used for the genetic analysis of 276 referred TSC, or probably-TSC
patients. All identified variants of potential clinical interest were verified
by Sanger sequencing or MLPA in probands and family members whenever
available. 137 patients (49.6%) were found to carry known disease-causing
mutations, 98 (35.5%) patients carried 93 unique highly-likely pathogenic
variants. Accumulatively, this assay were able to identify the causative mutation in 85.1% (237/276) TSC patients. Of note, 23 large structural variants and 2 cases of somatic mosaicism were identified in our patients which
might otherwise be neglected in sanger-sequencing based genetic testing. In
conclusion, our framework can be readily used to analyze the gene variant
profiles of TSC with high sensitivity, fidelity, throughput and speed. This framework lay the basis for the genetic testing of other complex Neurogenetic
disorders.
P14.34-M
EGFR screening using Gold nanoprobes
283
ABSTRACTS POSTERS
P14.35-S
Development of a next generation sequencing panel to assess
hereditary cancer risk that includes clinical diagnostic analysis of the
BRCA1 and BRCA2 genes
Assessment of hereditary breast and ovarian cancer risk should include germline sequencing of BRCA1 and BRCA2 as well as additional genes
with known associations in breast/ovarian cancer patients. Sanger DNA
sequencing has been the gold standard for molecular genetic analysis but
next generation sequencing (NGS) platforms could provide another sequencing alternative. However, optimized assay design and validation are critical
to maximize the analytical sensitivity and specificity of NGS assays and to
ensure high quality interpretation for clinical decision making. We developed a 25-gene NGS hereditary cancer panel that uses RainDance PCR technology for high-throughput sample preparation, Illumina HiSeq and MiSeq
NGS technologies, and commercially available and lab-developed informatic
tools. Initial assessment of analytical sensitivity and specificity was performed by comparing BRCA1 and BRCA2. NGS was performed on 1864 anonymized patient samples, which had previously undergone BRCA1 and BRCA2
Sanger sequencing. Sanger sequencing identified 15,878 variants, of which
681 were unique and 482 were classified as disease-associated mutations.
We identified 15,877 variants at an initial sensitivity of >99.99% for BRCA1
and BRCA2. One polymorphic variant was missed due to a variant under the
primer. Sensitivity was subsequently optimized through process improvements. No additional variants were found by NGS, yielding a specificity of
100%. This preliminary analysis facilitated assay optimization and validation of all 25 genes in the NGS panel. This analysis indicates that a NGS gene
panel designed to meet rigorous quality standards can be used to provide
clinical sequencing results equivalent to those obtained from Sanger DNA
sequencing analysis.
P14.36-M
In silico analysis of the effect on splicing of single nucleotide changes
at exon-intron junctions is predictive of pathogenicity. A study on
MYBPC3 mutations.
J. B. A. van de Meerakker, M. P. Lombardi, K. van der Lip, M. Alders, M. A. M. M.
Mannens;
Dept. Clinical Genetics, Laboratory for DNA-Diagnostics, Amsterdam, Netherlands.
284
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Background: Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome. Ph chromosome results from
the translocation of ABL protoonkogene from chromosome 9 and BCR gene
on chromosome 22, which leads to the formation of BCR-ABL chimeric gene
with constitutive tyrosine kinase activity. Imatinib (Gleevec) is the first BCRABL tyrosine kinase inhibitor used in the treatment of CML. Second generation drugs such as nilotinib and dasatinib, are now considered as alternative
treatments to imatinib. Point mutations the kinase domain of the BCR-ABL
were detected from 40% to 90% of the Gleevec-resistance cases. These mutations disrupt the binding site of imatinib on the tyrosine kinase, resulting
in a loss of sensitivity to the drugs. Method: We performed a pyrosequencing assay panel for the detection of eleven important mutations in BCRABL kinase domain, which are Y253H, Y253F, E255K, E255V, V299L, T315A,
T315I, F317L, F317V, F359C, F359V. Plasmids coding for respective vari-
ABSTRACTS POSTERS
ants were used to study assay performance and precision. Totally 25 RNA
samples of BCR-ABL positive patients were analyzed by pyrosequencing and
samples, which had mutation were also analyzed with sanger sequencing.
Results: Our assay allows flexible, fast and reliable detection of mutations
in cDNA from both blood and bone marrow with high concordance to Sanger
sequencing. Conclusion: Our pyrosequencing assay for imatinib-resistant
mutations provides a reliable method for the detection of the mutant BCRABL transcripts and also provides a significant value for the follow up of
CML patients on imatinib.
P14.40-M
Next generation tissue profiling
O. Sderberg;
Uppsala University, Uppsala, Sweden.
K. Hayashibara1, A. Jimnez-Velasco2;
1
Thermo Fisher Scientific, South San Francisco, CA, United States, 2Carlos Haya Hospital,
Malaga, Spain.
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chromosomal translocations is growing exponentially. The two main methods to identify and monitor translocations, fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH) are challenging,
labor intensive, the information obtained is limited, and sensitivity is rather
low. Common sample types for these analyses are biopsies or small tumors,
which are very limited in material making the downstream measurement
of more than one analyte rather difficult; obtaining another biopsy, using
a different section or splitting the sample can raise issues of tumor heterogeneity. The ability to study mutation status (DNA) as well as measuring fusion transcript expression (RNA) from the same sample is powerful because
youre maximizing the information obtained from a single precious sample
and eliminating any sample to sample variation. We isolated DNA and RNA
from the same non-small lung adenocarcinoma sample without splitting or
dividing the sample, and both mutation analysis, as well as fusion transcript
detection was performed using the Ion Torrent PGM platform on the same
Ion 318 chip. Using 10ng of DNA and 10ng of RNA input, we applied the Ion
AmpliSeq Colon and Lung Cancer panel to analyze over 500 COSMIC mutations in 22 genes and the Ion AmpliSeq RNA Lung Fusion panel to detect 40
different fusion transcripts.
P14.43-S
Development and verification of an Ion Ampliseq RNA gene fusion
panel for lung cancer: an OncoNetwork collaborative research study
Chromosomal translocations and corresponding gene fusions play an important role in carcinogenesis. The recent establishment of ALK, ROS1, RET
and NTRK1 fusion transcripts as predictive biomarkers for lung cancer therapy has increased the need for a technology that could detect these biomarkers starting from very limited amounts of material. Here we describe
the development and verification of an Ion AmpliSeq RNA fusion panel that
can simultaneously detect ALK, RET, ROS1 and NTRK1 fusion transcripts
in a single reaction. For the development of the panel we used previously
characterized samples comprising 6 cancer cell lines and 62 FFPE lung tumor samples. Upon RNA isolation and amplification using the panel, samples were sequenced on the Ion PGM system. Initially, using serial dilutions
of RNA from cell lines in normal RNA a limit of detection of 1% was established. Next, using the FFPE samples a fusion transcript was detected in 21
of the 24 known positive samples. Two of the non-concordant results were
due to lack of representative tumor material. For the third, although no specific fusion transcript was identified, the internal control for ALK expression
revealed the presence of an unknown ALK fusion transcript. Additionally,
two new RET fusions were detected in samples not previously tested for
RET translocations. These preliminary results are highly encouraging regarding the possibility of a reliable experimental protocol for the detection
of fusion transcripts in the biological material routinely obtained for the
diagnosis of lung cancer. An extensive verification is under way within the
OncoNetwork.
P14.46-M
MicroRNA detection by real-time TaqMan assays for translational
research
L. Wong, T. Gulham, P. Tsang, B. Moy, C. Liu, F. Hu, S. Dong, C. Chen, J. Stevens;
Thermo Fisher Scientific, South San Francisco, CA, United States.
285
ABSTRACTS POSTERS
upstream chemistry allows synthesis of miRNA template library that is used
in the downstream real-time TaqMan qPCR for miRNA-specific detection.
The universality of template synthesis simplifies the workflow and provides the flexibility for scalable content (miRNA coverage). Whether applied
to basic or translational research, profiling, screening, or validation, this
new and robust method allows detection and quantification of miRNAs to
address the unmet needs in workflow and sensitivity that exists today with
next generation sequencing and other qPCR technologies, especially with
clinical samples. Results show miRNA-specific amplification with a 7-log linear dynamic range and sensitivity of 60 copies input to meet the needs of
translational research and clinical/diagnostic application.
P14.47-S
A new strategy for genetic testing of neurodegeneration with brain
iron accumulation (NBIA) using amplicon multiplexing and next
generation sequencing
NGS is predicted to become the method of choice for fast screening of known
mutations as well as resequencing of the CFTR gene. Our diagnosis laboratory, involved in cystic fibrosis testing, is currently developing this new
strategy using a PGM (Life Technologies). The aim of this study was to define a NGS workflow for specific resequencing of the coding and flanking
intron regions of the CFTR gene. We studied the feasability and analytical
performance of two kits designed for amplicon library building and sequencing on PGM: the AmpliSeq CFTR kit from Life Technologies and the CFTR
MASTR v2 assay (multiplicom). The latter was used according two different
PGM protocols, 200 and 400 bp sequencing, due to the length of amplicons.
These two kits and two protocols were compared using 20 patients DNA
samples that have been previously sequenced by capillary electrophoresis
using a 3500xL Dx Genetic Analyzer for CFTR genotyping (27 exons and
exon-intron junctions). Gene rearrangements were searched using SALSA
MLPA kit (MRC- Holland) or custom array CGH. Mutations were deletions,
duplications, splicing variants and different combinations of T and TG tracts
in intron 9. All expected mutations were identified by the two kits while it
remained difficult to characterize intron 9 homopolymers. We discuss about
the reliability of these protocols and of dedicated analysis pipeline in the
context of cystic fibrosis diagnosis. Further improvements of software analysis pipelines would help to allow for automated CFTR mutation detection,
particularly in repetitive and homopolymer sequence regions.
286
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P14.49-S
The road to next generation molecular diagnostics, when NGS takes
over Sanger sequencing
K. De Leeneer1, J. Hellemans2, W. Steyaert1, I. Vereecke1, E. Debals1, B. Crombez1, M.
Baetens1, M. Van Heetvelde1, F. Coppieters1, A. Dejaegher1, S. Lefever1, E. De Baere1, P.
Coucke1, K. Claes1;
1
Center for Medical Genetics Ghent, Ghent University, Ghent, Belgium, 2Biogazelle,
Zwijnaarde, Belgium.
Cystic Fibrosis (CF) is one of the most frequent autosomal recessive disease
among Caucasians (prevalence 1:2500); it depends on mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene that encodes a chloride
exchanger. More than 1500 causative CFTR mutations have been identified,
including missense, frameshift, splice site, nonsense and deletion mutations.
Their accurate detection is important to improve the clinical management of
CF patients and to correctly identify all CFTR carriers. Here, we report the
use of a next generation sequencing screening for the identification of CFTR
germline mutations. The study was performed on 60 subjects, including CF
patients, CFTR carriers and controls, previously analyzed by traditional methods (reverse dot blot and Sanger sequencing). CFTR coding regions (including their flanking sites) were amplified using the CFTR MASTR Assay
kit (Multiplicom) to obtain a library/sample. The subsequent sequencing
reactions were performed with both the GS Junior System (Roche) and the
MiSeq System (Illumina) allowing the simultaneous analysis respectively of
10 and 60 patients/run. Finally, the downstream data analysis was carried
out through the SeqNext tool (JSI Medical Systems). All the features and performances of the Next Generation Sequencing technologies are discussed
in the light of results obtained with the two used systems. Comparative sequence analysis highlighted that the proposed method is reliable, since all
mutations previously identified were confirmed and the time of analysis is
also markedly reduced. Our results assess the feasibility of a next generation
sequencing approach for CFTR mutation detection to be included in a routine diagnostic workflow.
P14.51-S
Molecular analysis of mutations in the CFTR gene by Next Generation
Sequencing
Cystic fibrosis (CF) is the most common autosomal recessive disease in Cau-
ABSTRACTS POSTERS
casians, with an Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians, with an incidence of approximately 1 in 2500 and
a carrier frequency of 1 in 25. The gene responsible for CF, named the Cystic
Fibrosis Transmembrane Conductance Regulator (CFTR), encodes the cyclic adenosine monophosphate (cAMP)-dependent chloride channel found
in the apical membrane of secretory epithelial cells. The known mutations
of the CFTR gene are 1965 (Cystic Fibrosis Mutation Database). The massive sequencing for mutation analysis and copy number variations (CNVs) by
Next Generation Sequencing (NGS) allows investigating at the second and
third level with the scanning of all exons, the flanking regions and the search
for deletions and/or insertions in a single diagnostic test.
To check the reliability of massive sequencing by NGS as many as 100 control cases provided by an European network, were analyzed. All of the mutations previously obtained by Sanger Sequencing were confirmed by NGS
that was shown to be highly sensitivity and specific.
The massive sequencing offers the possibility to carry out a reliable and
comprehensive analysis for all genetic variants in reduced time (16h) testing up to 20 cases-samples in a single run. The costs of genetic analysis are
substantially decreased.
P14.52-M
Enabling high-throughput discovery of the RNA transcription
landscape using a directional RNA workflow and a combinatorial
multiplexing approach
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Trio-sequencing can be used in all disorders, and has particularly proven its
value in finding causes of intellectual disability (ID) or multiple congenital
anomalies. In contrast, we investigated whether sequencing only the affected patient without parents is sufficient to find the causative mutation, leading to a considerable reduce in costs. In this study, we enrolled 36 patients
with unexplained ID, and sequenced the exome. The exome sequences were
analysed with a stringent post-sequencing annotation pipeline including an
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ABSTRACTS POSTERS
ID gene panel of ~500 genes for filtering of the data. All remaining variants
with a potential clinical consequence were validated by Sanger sequencing
and tested in the parents for inheritance.
After variant filtering we noticed an average of 13 variants per patient (range 2 to 27) requiring further clinical interpretation. The majority of these
variants were inherited from one of the parents. Hitherto, we identified 5 de
novo mutations in 36 patients (14%).
Without exome sequencing the parents, a relatively high amount of potentially pathogenic variants remain. All these variants require clinical interpretation which is very time-consuming, while most of these variants were
likely benign because they are inherited from one of the parents. With trioanalysis inherited variants can be filtered out suggesting that this strategy,
at this moment, is more efficient in identifying the causative variant. In the
future when databases are filled with more and more exome data and consequently with more rare benign variants, exome sequencing single patients
will become a more realistic diagnostic approach.
P14.57-S
Detection of large rearrangements in the CFTR gene: Comparison
between custom CGH array and NGS
M. Audrezet, P. Gueguen, S. Redon, A. Despres, C. Le Marchal, C. Frec;
CHRU BREST, Brest, France.
24 years after the discovery of the CFTR gene, more than 1900 anomalies
are described, mainly single base-pair substitutions or micro-insertions/
deletions, but many large rearrangements are also described.
Identification of mutations has important implications for genetic counselling, prenatal diagnosis, cascade screening in families, and for understanding the genotype-phenotype relationship.
Classical approaches for gene analysis aim either to look for point mutations
or to identify large deletions or duplications, but no strategy allows, to date,
to identify both types of mutations.
Since a few years, NGS technologies enable us to overcome the classical approaches of whole coding sequence sequencing at single nucleotide resolution.
The aim of this study is to validate NGS data analysis to detect large rearrangements in the CFTR gene.
23 DNA carrying known large deletions (n=20) or duplications (n=3) previously identified by CGH-array or qFM-PCR and two control samples, one
complete deletion, and the other corresponding to the wild-type sequence
of the gene were included in this study.
Analysis was conducted by SeqNext and Nextgene sofwares and by a simple
spreadsheet by comparing the average depths obtained for each amplicon.
This method allowed us to confirm all the large deletions/duplications affecting more than one exon. Only one short deletion of 1 kb affecting exon
17b alone was not detected by both methods.
In conclusion, NGS technology should be the first technique that allows in a
single step both the identification of point mutations and large rearrangement in a gene.
P14.58-M
The impact of NGS on gentic services: prioritization criteria and
accountability
288
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The study confirms the potential of NGS techniques, indicates caution in interpretation and validation of data and suggests a soft transition between
classical methods and NGS approach in clinical application.
P14.59-S
Application of NextGeneDx, a validated NGS based procedure, in the
clinical practice
M. G. Elferink1, E. de Bruijn2, P. van Zon1, O. Akkermans1, K. A. Kusters1, W. van Harenvan t Woudt1, G. C. M. L. Page-Christiaens1, K. D. Lichtenbelt1, M. J. A. van Kempen1, I. J.
Nijman1, G. van Haaften1, B. van der Zwaag1, E. Cuppen1,2, J. K. Ploos van Amstel1, G. H.
Schuring-Blom1;
1
UMC Utrecht, Utrecht, Netherlands, 2Hubrecht Institute, Utrecht, Netherlands.
Epigenetic modifications have proven to play a significant role in cancer development as well as fetal development. Taking advantage of the knowledge
acquired during the last decade, great interest has been shown worldwide
in deciphering the fetal epigenome towards the development of methylation
ABSTRACTS POSTERS
based non-invasive prenatal diagnostic (NIPD) assays. We hereby highlight
the different approaches implemented such as sodium bisulphite conversion, restriction enzyme digestion and methylated DNA immunoprecipitation,
for the identification of differentially methylated regions (DMRs) between
free fetal DNA found in maternal blood and DNA from maternal blood cells.
Furthermore, we evaluate the use of selected DMRs identified towards the
development of NIPD for fetal chromosomal aneuploidies. In addition, we
perform a comparison analysis; we evaluate the performance of each assay
and provide a comprehensive discussion on the potential use of different
methylation-based technologies in retrieving the fetal methylome, with the
aim to further expand the development of NIPD assays.
P14.62-M
Next-generation variant effect predictions and data integration
B. Vroling, T. van den Bergh, T. te Beek, R. Kuipers, H. Joosten;
Bio-Prodict, Nijmegen, Netherlands.
The interpretation of variants flowing out of next-generation sequencing experiments is one of the largest bottlenecks for DNA diagnostics. Frequently
used tools like PolyPhen and SIFT are not up to this task, and consequently
there is an enormous need within the community for novel tools to reliably
interpret the effects of polymorphisms. In response to the growing demand
for high-quality variant effect predictions we present 3DM; a data integration & mutation prediction platform for protein families.
3DM relieves many of the burdens that researchers face in dealing with the
growing amounts and complexity of biomedical data. For each protein family a large amount of information that is extracted from protein structures,
alignments and scientific literature, among others, is available. All this information is integrated and validated, and can be analysed via a number of
different methods and tools.
By intelligently combining all this heterogeneous information 3DM is able to
provide state-of-the-art predictions about the effects of genetic variations.
Collaborative work with a number of the largest hospitals in the Netherlands
has shown that our solutions represent a major step forward in helping researchers and clinicians making accurate assessments of the pathogenicity
of their variants, both by providing predictions, as well as enabling them to
quickly navigate to literature and other relevant data.
P14.63-S
The Israeli experience of the first 300 Panorama tests that use
19,488 single nucleotide polymorphisms (SNPs) followed by highthroughput sequencing for common trisomies risk assessment
H. N. Baris1, Z. Weiner2, I. Solt2, M. Shohat3, D. M. Behar4;
1
The Genetic Institute, Rambam Health Campus, Haifa, Israel, 2The Obstetrics and
Gynecology department, Rambam Health Care Campus, Haifa, Israel, 3The Recanati
Genetic Institute, Rabin Medical Center, Petach Tikva, Israel, 4The Genetic Institute,
Rambam Health Care Campus, Haifa, Israel.
Background Cell free DNA (cfDNA) has emerged over the last year as an
alternative for amniocenthesis for diagnosis of the common aneuploidies
looking at trisomy 21, 13, 18, sex chromosomes and triploidy. Methods We
present our experience of the first 300 Panorama tests sent from Israel.
This method is based on massively multiplexed PCR amplification of cfDNA
isolated from maternal plasma, targeting 19,488 SNPs, followed by highthroughput sequencing. The fetal fraction is determined. The SNP pattern of
maternal DNA (from buffy coat) is compared to the SNP pattern of free DNA
from maternal plasma, which contains maternal and fetal DNA. Paternal genomic samples, when available, were included in the analysis; in the absence
of a paternal sample, the algorithm considers population allele frequencies.
Combining the maximum likelihood ratio with a priori risk generates a risk
score. Results The results of the first 300 sequential tests performed in Israel were analyzed. Fifteen samples necessitated redraw, two samples failed
analysis. Four samples yielded high risk scores: two cases for trisomy 21,
one for Kleifelter syndrome (KS) (47,XXY) and one for trisomy 18. Confirmation of both trisomy 21 and one KS were done by CVS or amniocenthesis.
The suspected trisomy 18 is in the process of confirmation. There are no
known false negative results. Discussion Panorama test is a reliable tool
for identification of pregnancies at high risk for fetuses with the common
aneuploidies with a high success rate. We recommend confirmation of the
diagnosis for high risk scores pregnancies using invasive tests.
P14.64-M
Quality control procedure for assessing good manufacturing of a
molecular diagnostics test in freeze-dried format
A. Moiana, L. Ventura, L. Turner, M. Incandela, M. Gramegna;
Sentinel CH. SpA, Milan, Italy.
Back to index
A novel prenatal chromosome microarray testing strategy has been developed, that moves away from size-based detection thresholds, towards a more
clinically relevant analysis, providing higher resolution than G-banded chromosomes but avoiding the detection of imbalances of unclear prognosis that
cause parental anxiety. All prenatal samples fulfilling our criteria for karyotype analysis (n=353) were tested by chromosome microarray; only copy
number variants of established deletion/duplication syndrome regions and
any other imbalance >3Mb were detected and reported. A retrospective fullresolution analysis of 249 of these samples was carried out to ascertain the
performance of this testing strategy. Using our prenatal analysis, 28/353
(7.9%) samples were found to be abnormal. Of the remaining samples, 249
were anonymized and reanalyzed at full postnatal resolution; a further 46
regions of imbalance were detected in 44 of these traces (17.7%). None of
these additional imbalances was of clear clinical significance. This prenatal
chromosome microarray strategy therefore detected all CNVs of clear prognostic value. This strategy avoids the problems associated with interpre-
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ABSTRACTS POSTERS
ting imbalances of uncertain prognosis and the parental anxiety that are a
result of such findings.
P14.67-S
Determining the sensitivity of diagnostic methods to maternal cell
contamination (MCC) in prenatal diagnosis (PND)
M. Ho, E. Pontikinas, M. Vo, N. Lerda, M. Attwell, A. Simsek, C. Nicholls;
SA Pathology, Adelaide, Australia.
Primary ciliary dyskinesia (PCD) is a rare, autosomal recessive genetic disorder characterized by defect in the action of the cilia lining the respiratory
tract, fallopian tube and also of the flagella of sperm in males. The impaired
ciliary function results in neonatal respiratory distress, chronic oto-sinopulmonary disease, infertility, and organ laterality defects in approximately 50% of cases. Currently, the diagnosis of PCD involves the study of cilia
morphology, motility and ultrastructure, nasal nitric oxide measurement
and, more rarely, genetic analysis. This is mainly due to the high genetic
heterogeneity of the disease which has been associated to mutations in 26
different genes.
This study aims to improve the genetic diagnosis of PCD that it is routinely
limited to a subset of the most frequently mutated genes. This approach is
expensive, time consuming and ineffective leaving many patients without a
molecular diagnosis. Using the AmpliSeq technology we developed a panel
which allows comprehensive, rapid and cheap analysis of the 26 PCD-causative genes. A total of 160Kb genomic sequence including all the protein
coding sequences, splice sites and 5-3UTRs will be amplified in 1151 amplicons using two primer pools and sequenced in the Ion Torrent platform.
Twenty PCD patients diagnosed according to the protocol of the European
Respiratory Society Consensus Statement have been selected for a test study.
Technical details, results and cost-effectiveness analysis will be presented.
P14.69-S
Public Health Genomic and genetic tests. Cost evaluation analysis and
quality standards as relevant factors in health care planning
Genomic era has improved a lot molecular diagnosis, but in the last five years
the new revolution of next generation sequencing has open a new window
not only on research but also on diagnostic testing.
The scientific framework and the laboratory activities are completely
changed: the number of known disease genes has increased exponentially,
automation is now applied, certification and accreditation procedures are in
place in a growing number of laboratories.
Both public National Health systems and private laboratories or companies
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are facing with an increasing demand of genomic tests for the most common
diseases. Correct information and awareness among public and all stakeholders are crucial.
Since it has not been clearly defined how many genomic tests have enough
clinical utility, the investigation of their costs could be a way to establish a
correct public health policy.
Activity-Based Costing is the methodology used to assigns the cost of each
activity.
The systematic analysis of activities needed to perform genetics tests has
identified a number of indicators to assess the workload for every professional who participates in the process of diagnosis and the proportion of
material used for each activity. All these parameters have been incorporated
into a software able to split all the lab costs (personnel, material and general costs) for each test provided in order to compare costs among different
laboratories, to compare the performance of the same laboratory in subsequent years and to make a priority list of genetic/genomic tests to provide,
taking into account costs/benefits data.
P14.70-M
Testing homopolymers in pyrosequencing-based next generation
sequencing chemistry
RASopathies are a group of clinically and genetically related disorders, including Noonan, LEOPARD CFC, Costello, Legius and neurofibromatosisNoonan syndrome as well as NF1. These disorders result from mutations
affecting the RAS-MAPK signaling pathway, which explains the clinical overlap within this group of syndromes. In routine diagnostics, RASopathy-associated genes are usually tested sequentially using Sanger sequencing until a
likely causative mutation is identified, which is laborious, time consuming
and expensive.
We have developed a cost-effective multi-gene sequencing panel for RASopathies using HaloPlex target enrichment, sample barcoding and sequencing with MiSeq. The panel targets approximately 40kb of the coding sequences of NF1, PTPN11, SOS1, CBL, BRAF, RAF1, SHOC2, MAP2K2, MAP2K1
, SPRED1, NRAS, HRAS and KRAS. Eleven RASopathy-individuals with previously Sanger-validated variants were sequenced. Bioinformatic analysis
was performed using the commercial softwares; NextGENe and BENCHlab
NGS, as well as an in-house bioinformatic pipeline for HaloPlex data. The
results obtained from the two bioinformatic anlaysis pipelines were similar. The gene panel was highly specific; 93% of sequencing reads aligned
to the targeted regions. The average read depth in region of interest (ROI)
was >2000 and ~98% of targeted bases in ROI had at least 30x coverage. All
known variants were identified, including a 4 bp deletion and a splice-site
mutation in NF1. Conclusively, the RASopathy-multi-gene panel presented
here enables rapid and cost-effective high-throughput screening of RASopathies. The results furthermore show that NextGENe and BENCHlab NGS are
ABSTRACTS POSTERS
suitable for analyzing HaloPlex data in clinical diagnostics. Data from screening of additional patients will be presented.
P14.72-M
Next Generation Sequencing: New approach for diagnosis of
autosomal dominant Retinitis Pigmentosa patients
P. Fernandez-San Jose1,2, F. Blanco-Kelly1,2, R. Rosa Riveiro-Alvarez1,2, M. A. LopezMartinez1,2, B. Garcia-Sandoval3, I. Snchez-Navarro1,2, O. Zurita1,2, M. Corton1,2, C.
Ayuso1,2;
1
Department of Genetics, Instituto de Investigacion Sanitaria-Fundacion Jimenez Diaz
(IIS-FJD), Madrid, Spain, 2Centro de Investigacion Biomedica en Red de Enfermedades
Raras (CIBERER), ISCIII, Madrid, Spain, 3Department of Ophthalmology, IIS-Fundacion
Jimenez Diaz University Hospital, Madrid, Spain.
An X-inactivation (XIA) analysis in females is commonly performed by metaphase R-banding or methylation-specific PCR (M-PCR) technique. The
late-replicating X-chromosome can be recognized under a microscope at the
single cell level by R-banding using BrdU incorporation, but there are limitations: 1) Not so many metaphases can be observed, and 2) Only an abnormal
X which has deletion or duplication more than 10Mb can be detected. M-PCR
can detect random or non-random XIA pattern in cell population, but the
parental samples are needed to identify which is normal or abnormal chromosome X. A subject was a female with an X-autosome balanced translocation; 46,X,t(X;19)(p21.1;q12), and having clinical manifestations of Duchenne
muscular dystrophy. The breakpoint of the derivative X was onto the DMD
gene. We selected three kinds of BAC clones and used simultaneously for our
RNA-FISH study; a clone consistent with XIST gene for detecting inactivation
chromosome X, a clone consistent with UTX gene which is escaping XIA as
a reference probe for both normal and abnormal Xs, and a clone consistent
with HDHD1A gene which is escaping XIA for distinguishing between normal and abnormal Xs of this subject. We successfully visualized XIST gene
expression at the single cell level in this subject. Normal X was inactivated in
67 cells, abnormal X was inactivated in 5 cells, and unclear XIA pattern was
in 28 cells. This RNA-FISH results were in concordance with the phenotype
of this subject, as an affected female of X-linked disorder.
P14.74-M
Quality control for SureSelect Strand-Specific RNA library preparation
protocol using the Agilent 2200 TapeStation system
E. Schmidt1, A. Padmanaban2, A. Inche3, R. Salowsky1;
1
Agilent Technologies, Waldbronn, Germany, 2Agilent Technologies, Bangalore, India,
3
Agilent Technologies, Edinburgh, United Kingdom.
Transcriptomics reveal global changes in gene expression that may contribute to the pathogenesis of a particular disease or help drive a fundamental
biological process. RNA sequencing (RNA-Seq) has emerged as a promising and rapidly growing method for studying and characterizing cellular
transcripts at single base pair resolution. Among different approaches for
generating libraries for transcriptomics study, strand-specific library pre-
Back to index
paration method has added advantage over other methods. The Agilent SureSelect strand-specific RNA library preparation kit generates libraries with
specific adaptors ligated to each strand enabling the identity of the DNA
template strand of origin to be retained in downstream sequencing and data
analysis. The high throughput deep-sequencing based RNA-seq studies generate more data compared to RT-qPCR and array based methods yet they
are expensive and time consuming study. Proper quality control (QC) steps
within the library preparation process are therefore crucial to ensure successful sequencing. The Agilent 2200 TapeStation system offers an easy to
use automated electrophoresis system with rapid analysis time as well as
flexible sample throughput capabilities. Here, we compare the performance
and capabilities of the RNA and D1000 ScreenTape assays for use on the
Agilent 2200 TapeStation system with the respective assays run on the 2100
Bioanalyzer system. The results from the QC of the starting total RNA and
final sequencing libraries generated from Agilents SureSelect strand-specific RNA library preparation kit reveal that the RNA and D1000 ScreenTape
assays are a match to the visual and quantitative results obtained with the
RNA 6000 Nano and DNA 1000 Kits.
P14.75-S
Expanded carrier screening tests currently on the commercial market
There has been a recent rise in the advertising of preconception genetic carrier screening tests directly to consumers via internet. We analyzed the offers with particular focus on comprehensiveness, clinical validity and utility
of the genetic tests proposed.
We gathered data from the websites of Counsyl, InheriGen, Pathway genomics and Recombine in December 2013. Of about 1300 recessively inherited
diseases listed in OMIM (causative gene known), current offer includes a
range of 73 to 206 for a total of 286 diseases of which 30 are included in
the panels of all providers and 92 in at least three providers. According to
the ORPHANET catalogue, 31% of screened diseases are more frequent than
1/100,000 in the general population. On the other hand, there are relatively
frequent recessively inherited diseases (e.g. Duchenne muscular dystrophy,
Friedreich ataxia) not covered by any of the screening tests. 52% of screened
diseases manifest neonatally, 28% during childhood and 3% in adult life(e.g.
Hemochromatosis), while the onset of the others is variable. 34% can be
treated if diagnosed neonatally. On average, companies test for 4% (2,2% to
7%) of all known mutations on a particular gene (HGMD).
Currently, commercial companies offer screening for around 20% of known
recessively inherited diseases. The great diversity of genetic diseases
screened suggests a lack of clear inclusion criteria. Although advertised as
pan-ethnic, all tests include population specific diseases which are extremely rare in the general population. Because of allelic heterogeneity and
relatively poor mutation coverage the tests sensitivity pan-ethnically is
unknown.
P14.76-M
Detection of rare variations in a targeted genomic region in a
population by NGS analysis using pooled DNAs
It is important to find novel rare variations in a targeted region in some populations to get next resources for elucidation of missing heritability. We
developed a method to survey variations including low-frequent variations
in a targeted region in a specific population by next-generation sequencing
(NGS) analysis using pooled DNAs.
We present examples to trace variations and to estimate each frequency in a
targeted region in Japanese and Okinawan people.
Targeted regions were amplified from each genomic DNA of 100 or 200 individuals by LA-PCR. After measuring the quantity and quality of each amplicon, equal amount of amplicon was mixed and amplicons for 100 or 200
individuals were pooled. Then, pooled targeted region was analysed using a
NGS platform. After mapping of reads to the reference, SNPs and indels were
called and the frequency was estimated by count rate of the reads. Then, we
also performed NGS analysis for LA-PCR products from pre-pooled genomic
DNAs. Next, we confirmed each variation and calculated each frequency in
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ABSTRACTS POSTERS
the population by PCR-RFLP, allele specific PCR or direct sequencing in each
individual.
Allelic frequency estimated by the NGS analysis using both pooled DNAs almost correlated the real frequency calculated by the individual analysis. For
sensitivity to detection of allelic frequency, less than 0.5% of allele in the
population could be detected.
P14.77-S
Increasing the detection rate of genome-wide high resolution
SNP array analysis by using the right follow-up test procedures in
constitutional genome diagnostics
Inheritance of a variation is often used as a guide for diagnostic classification in microarray analysis. If a variation is inherited from a normal parent,
morbidity is less likely, compared to a variation that has appeared de novo.
Unfortunately paternal DNA is frequently not available for this analysis.
Therefore many variations are never properly classified and the morbidity of the variation remains unknown. To overcome this problem we have
incorporated a workflow that determines whether a variation is located on
the maternal or the paternal allele in the patient. The workflow requires
SNP-array analysis to be done on maternal and patient DNA samples. If a
variation is located on the maternal allele in the patient and the variation is
not found in the maternal DNA sample, then the variation can be classified
as de novo. Utilizing this workflow enhances the diagnostic value of variations found by SNP-array, when paternal DNA is not available.
P14.79-S
EGFR and KRAS mutational profiling in fresh non-small cell lung
cancer (NSCLC) cells
M. Zorzetto, R. Scabini, S. Inghilleri, S. Ferrari, P. Morbini, M. Luisetti;
Fondazione IRCCS Policlinico S. Matteo, Pavia, Italy.
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Whole exome sequencing (WES) allows the detection of a wide range of variant types. With one single test it is possible to detect small variants (SNVs),
and structural variation. Whilst the role of copy number variants (CNVs) in
intellectual disability (ID) is well known, the role of CNVs in other diseases
is less well studied, although it has been suggested that CNVs can be found
in up to 10% of deafness patients. We have performed read depth WES CNV
analysis on 600 patients across 5 heterogeneous disorders including ID,
deafness, blindness, metabolic disorders, movement disorders. This analysis revealed several clinically relevant CNVs exerting a dominant effect, as
well as CNVs unmasking a recessive mutation that lead to pathogenic compound heterozygous events. The cohort of 310 ID patients had previously
screened negative for CNV microarray analysis as well as WES SNV analysis.
Nonetheless clinically relevant CNVs were identified in 2% of patients. Systematic screening of the remaining patient groups identified CNVs in ~4%
of individuals, including in COL6A1, EYS and deletions in USH2A. To further
examine pathogenic structural variation, we performed whole genome sequencing on 50 patient-parent trios. In 7 patients we identified pathogenic
large variants, including two single exon deletions, a tandem duplication, an
inter-chromosomal duplication and one complex inversion/duplication/deletion event. Discordant reads provided positional information for duplicated sequences identifying an in-frame gene fusion. These results show that
structural variation is not only an important cause of neurodevelopmental
diseases but also a much broader range of genetic diseases.
P14.81-S
Targeted exome sequencing as a molecular diagnostic tool for skeletal
dysplasias
D. L. Polla, M. C. B. Silva, M. T. O. Cardoso, R. W. Pereira, R. Pogue;
Catholic University of Brasilia, Brasilia, Brazil.
Skeletal dysplasias comprise a large group of more than 450 clinically distinct and genetically heterogeneous diseases associated with mutations in
more than 300 genes. Clinical and radiological findings are used to diagnose
these diseases. However, due to the genetic heterogeneity of these disorders,
the diagnostic process is complex. With the advances in molecular genetics
and the elucidation of the molecular basis of these diseases, molecular tests
have become useful in the diagnosis of these dysplasias. Since many different genes have been associated with these disorders, standard diagnostic
approaches using Sanger sequencing can be expensive and time consuming.
To overcome these limitations, we used targeted exome sequencing and designed an 1.4Mb panel for simultaneous testing of more than 4800 exons
in 309 genes involved in skeletal dysplasias, and 28 genomic regions which
when deleted are associated with these diseases. DNA from 96 individuals
with previous clinical diagnosis of skeletal dysplasia was sequenced in 8
multiplexed runs using an Illumina MiSeq sequencer. Reproducibility was
tested by repeating the entire procedure for three patients. NGS of the captured exons resulted in an average coverage of 140X. Causative mutations
were characterized in 13 patients so far including de-novo mutations in 7
cases. Analysis of the rest of the data is ongoing. Confirmation with Sanger
sequencing was performed for these variants and all were confirmed. Our
NGS panel provides a fast, accurate and cost-effective molecular diagnostic
tool and can assist in the diagnosis of genetically heterogeneous diseases
like skeletal diseases.
ABSTRACTS POSTERS
P14.82-M
Molecular diagnosis of hereditary spastic paraplegia: comparison of
enrichment strategies for targeted next-generation sequencing
Hereditary spastic paraplegia (SPG) is a group of neurodegenerative disorders with considerable phenotypic heterogeneity, and mutations in >70
genes involved to date. This makes conventional molecular testing arduous.
We decided to develop a targeted next generation sequencing kit for diagnosis purpose.
We tested two enrichment approaches of 10 SPG genes: one using amplicons
(Illumina, TruSeq Custom Amplicon - TSCA) on 48 patients, and the other
using capture (Roche NimbleGen, SeqCap EZ) on 60 patients. Sequencing
was performed on the MiSeq (Illumina). Coverage and variant distribution
were evaluated using the Genomics Workbench software (CLC Bio).
After read mapping on whole genome (Hg19), the mean coverage per patient varied from 444X (sd 98X) for SeqCap EZ, to 587X (sd 127X) for TSCA.
However, the percent of regions with minimal coverage <30X was <15%
with SeqCap EZ, and decreased to <5% when the patient mean coverage reached 600X. With TSCA, this value was stable (15-20%).
We then compared the variants. Those reported in dbSNP137 were similar
on both strategies. Concerning variants unknown in dbSNP137, 16-20 variants with TSCA vs 0-3 with SeqCap EZ were obtained per sample. This was
explained by an enrichment of variants in some amplicons in TSCA suggesting false positives due to Taq polymerase errors.
SeqCap EZ was finally chosen to enrich our targets. We designed a new panel with 74 SPG genes, which gave <0.7% of regions with minimal coverage
<30X. The challenge is now to interpret the variants discovered in our growing cohort of patient with unsolved HSP.
P14.83-S
Development of a specific and sensitive High Resolution Melting
Analysis for detection of beta globin gene mutations
Back to index
Krppel-like factor 1 (KLF1) plays a central role in the developmental globin gene switch; carriers of KLF1 mutations have hereditary persistence of
foetal haemoglobin (HPFH) and show variably elevated (10-40%) HbF. This
variation may be due to differential expression of other modifier genes. The
KLF1 interactome can be further defined by identifying potential molecular
targets and observing their expression at the cellular level in comparison
with HbF expression and distribution in F-cells.
The mean corpuscular HbF (MCHbF) is normally estimated by dividing the
amount of HbF, determined by HPLC, by the number of F-cells, obtained by
flow cytometry. Since HbF may be unequally distributed among F-cells, an
imaging cytometry protocol enables quantification of HbF per F-cell by fluorescent emission measurements. Protocols presented in the literature were
found lacking in efficacy, mostly due to inefficient fixation and high degree of
autofluorescence. A new intracellular anti-HbF antibody-labelling technique
was thus optimized for use with the Nikon Eclipse Ti inverted fluorescence
microscope. This technique resulted in images of superior quality from
which the MCHbF could be determined for each F-cell observed. This protocol will be used to study the association of KLF1 and other genes, obtained
through NGS data analyses, with MCHbF in normal and HPFH individuals, to
identify new therapeutic targets for -haemoglobinopathies.
P14.85-S
Development and verification of an Ion AmpliSeq TP53 Panel
A large amount of data is available on the functional impact of missense mutations in TP53 and on mutation patterns in many different cancers. TP53
direct sequencing is a time-consuming method with limitations in detection
level. Here we describe the development and verification of a Next Generation Sequencing method based on Ion AmpliSeq technology. The panel
covers TP53 coding regions comprehensively using optimized primers to
minimize off-target sequencing. It is comprised of 24 primer pairs across
2 pools requiring only 20 ng of DNA. The design of short amplicons of 125
to 175 bp permits the amplification of DNA starting from formalin-fixed
paraffin embedded material. We verified the panel on 30 breast cancer
samples previously characterized with Sanger sequencing. Sequencing reactions were carried out with the Ion PGM Sequencer on Ion 318 chip.
The average % of mapped reads on target was of 96,8% and average depth
of coverage 20,386X. Loading 10 samples on an Ion 318 chip 95% of the
sequenced amplicons showed coverage higher than 500X. Data analysis
was performed on Ion Reporter Software 4.0 using optimized TP53-panel
workflows. We detected all the expected somatic mutations including single
base substitutions, insertions and deletion and only a complex duplication
of 18bp was not detected. The overall analytical sensitivity was 95,23%. The
same results were obtained using 32 samples on an Ion 318 chip. These
preliminary data demonstrate that the TP53 Ion AmpliSeq panel workflow
and Ion Reporter Software analysis solution meets the requirements of clinical research laboratories.
P14.86-M
Toward rapid identification of coding fusions and structural
rearrangements in cancer genomes: Multiple Myeloma First
Karyotype and FISH have been standard diagnostic tools in monitoring response to treatment and disease progression in hematological disorders,
including multiple myeloma, for over 40 years. However, with the availability of array and NGS technologies in most clinical diagnostic laboratory
settings, it is time to consider evaluated the use of more modern methods
in diagnosing plasma cell dyscrasias. Although we routinely run RNA-Seq,
SNP-CN arrays and a deep sequencing cancer panel as a cost effective workflow for clinical trials, we find the validation of translocations and structural
rearrangement by PCR and/or FISH cumbersome. More recently, we have
identified the BioNano Genomics Irys System which utilizes nanochannel
technology and high resolution imaging for whole genome mapping of
translocations and copy number abnormalities. As proof of concept, we have
used the Irys system to analyze the multiple myeloma cell line, KMS11, and
will demonstrate the ultility, speed and accuracy in detecting structural re-
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arrangements in this line compared to whole genome sequencing, RNA-seq
and SNP-CN datasets. We will also demonstrate the use of the system with
unknown CD138+ enriched bone marrow samples from consented patients
currently on clinical trials compared to FISH, G-band karyotype and SNP-CN
arrays. We are confident that these data demonstrate that the Irys system
is a disruptive innovation with broad applicability to genome research and
refinement of normal variation and disease.
P14.87-S
Mutation analysis of triple negative breast cancer patients using next
generation sequencing
L. A. Pop1, R. M. Petric1,2, O. Virtic1, I. Berindan-Neagoe1;
1
Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania, 2Babes
Bolyai University, Cluj-Napoca, Romania.
Despite the intense research in the field of breast cancer, this disease still
remains the second most common cancer in women worldwide, being the
first cause of death in women in Romania. Among the different subtypes of
breast cancer, triple negative breast cancer (TNBC) needs a special attention
due to its limitations in therapy and to its aggressiveness. Several studies
have tried to identify different new mutation both in oncogenes and tumor
suppressor gene in order to explain these limitations of TNBC treatment.
The present study was aimed at the identification of mutations in 46 genes
involved in cancer in 31 patients with TNBC operated at the Institute of Oncology Prof. Dr. I. Chiricuta, Cluj-Napoca between 2006-2007, using Next
Generation Sequencing. We used FFPE tissue samples which were sequenced using the Ion Torrent Personal Genome Machine and the Ion Reporter
1.6 software for data analysis. After data analysis we obtained 103 mutations in 34 genes of the 46 studied. The clinical assessment of the identified
mutations showed that three mutations were benign, one was likely benign,
42 were likely pathogenic, 28 were pathogenic and 29 had no assessment.
This study also identified KDR, TP53, PIK3CA, FGFR3 and FGFR2 genes as
being the most frequently mutated genes. Our results show that TNBC has
specific mutations leading to resistance to therapy and poor outcome of these patients.
P14.88-M
Four Decade Old Mummified Umbilicus Making Retrospective
Molecular Diagnosis of Ornithine Carbamoyltransferase Deficiency
In inborn errors of metabolism, sampling and preservation of viable specimen is critical in reaching the correct diagnosis. Utility of preserved umbilical cords have been demonstrated in several occasions, but it remains
unclear how long the mummified umbilicus can preserve genomic DNA that
tolerates sequence-based genetic analysis. We report a Japanese family of
successive early deaths presumably due to clinically diagnosed ornithine
carbamoyltransferase deficiency. The exact molecular pathology, c. 394T>C
(p. S132P), was retrospectively delineated using DNA extracted from the
mummified umbilicus that had been kept in a keepsake wooden box at home
for four decades after the deaths of affected individuals. This observation
illustrates the striking stability of DNA preserved in mummified umbilicus,
and supports an efficient way of genetic sample preservation. From cultural standpoint, preservation of mummified umbilical cord is a unique but
common tradition in Japan for at least several centuries. The exact molecular diagnosis of this X-linked inborn error of metabolism of the urea cycle
had an significant impact on the affected family. Hemizygous female carriers
are at risk of hyperammonemic crisis, and this disorder is treatable with
supplemental dietary interventions. Since naturally mummified umbilicus
provides a highly effective way of genetic sample preservation, utilization
such material should be considered in the retrospective genetic investigation, particularly in patients with south east Asian cultural backgrounds.
P14.89-S
Direct trans-differentiation of skin fibroblasts for functional testing of
unclassified variants
J. Pals1,2, K. van der Kuij2, D. Micha1, A. Maugeri1, G. Pals1, M. J. Baars3, V. Everts2, B.
Zandieh Doulabi2;
1
VU medical center, Amsterdam, Netherlands, 2ACTA, Amsterdam, Netherlands, 3AMC,
Amsterdam, Netherlands.
Introduction Unclassified variants (UVs) are a common finding in DNA diagnostic testing. The introduction of next generation sequencing, allowing
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Usher Syndrome (USH) is characterized by phenotypic and allelic heterogeneity requiring a powerful and reliable molecular diagnosis. Thus, a targeted re-sequencing (TS) panel of the 10 USH genes has been developed. It
covers 96% of the targeted regions being characterized by 872 amplicons
using Ion TorrentTM technology (Life Technologies). This protocol has been
applied to the analysis of 30 USH cases divided as follows: 16 with already known mutations were used for a validation step which demonstrated
100% accuracy, while the remaining 14 were analyzed at diagnostic level.
The combination of systematic data analysis using an established bioinformatics pipeline followed by Sanger sequencing confirmation and segregation analysis led to the identification of 15 novel variants (1 in USH2A, 4 in
PCDH15, 1 in MYO7A, 3 in CDH23, 4 in GPR98, 2 in USH1G). All these variants
were predicted as pathogenic using several in silico predictor tools such as
Mutation Taster, Polyphen-2, SIFT and others. Five known pathogenetic mutations were also detected. Overall, these 20 alleles explain 12 patients, one
is heterozygous for a mutated allele and the second is lacking, and the last is
completely negative (a new case of genetic heterogeneity or the presence of
mutations in regions not yet analyzed of the USH genes?). In term of molecular epidemiology, USH2A characterizes 12 patients (40%) MYO7A 6 (20%),
CDH23 4 (13%), PCDH15 3 (10%), GPR98 2 cases, USH1C and USH1G one
each. In conclusion, this methodology clearly provided a reliable strategy for
routine gene diagnosis of USH.
P14.91-S
p.Asp59Gly in the RAB40AL gene is a common allelic variant: no
evidence for a causal relationship with Martin-Probst syndrome
ABSTRACTS POSTERS
who was diagnosed for a genetic cause of isolated optic atrophy using whole
exome sequencing (HiSeq 1500 and TruSeq Exome Enrichment Kit, Illumina). Interestingly, the patient did not present any symptoms of MPS. Trying
to further verify the pathogenicity of the RAB40AL variants, we screened a
set of control DNA samples representative of the background population of
Central Poland (n=788, female:male ratio 1:1) using allele-specific PCR and
confirmed suspicious variants by Sanger sequencing (Applied Biosystems).
The RAB40AL variants were found in 18 out of 788 subjects. It corresponds
to an allele frequency of almost 2.3%. Results of our study have demonstrated that the p.Asp59Gly amino acid change in RAB40AL gene is present at a
high prevalence in the general population that is typical for common polymorphisms. Our data questions the role of p.Asp59Gly as a disease causingchange for MPS.
P14.92-M
Comparative transcriptome and genome analysis down to the
sequence level for individual cells
A. Meier, E. Fricke, E. Fisch, H. Wedler, U. Deutsch, N. Neubauer, C. Korfhage;
QIAGEN, Hilden, Germany.
Cell heterogeneity plays a central role in biological phenomena during normal development or disease (e.g., cancer development). As gene regulation
is a fundamental process, the analysis of transcript profiles and genomes of
single cells to dissect phenotypic variability is of key interest to scientists.
Deep genome and transcriptome analysis of small biological samples using
next-generation sequencing (NGS) is often limited by the small amount of
sample available (6 pg gDNA and 0.5 pg mRNA/human cell). We developed
two new methods for whole genome amplification (WGA) and whole transcriptome amplification (WTA), based on Multiple Displacement Amplification (MDA) technology, from small samples down to just a single cell.
Parallel WGA and WTA reactions begin with lysis of 251000 cells from the
same sample. Both reactions include a ligation and multiple displacement
amplification (MDA) reaction. This results in amplification products (WGADNA, WTA-cDNA) of the highest comparability and can be used for comparative studies of the genome and transcriptome from the same sample.
The second method (WTA) amplifies RNA from 11000 cells directly and
includes an efficient lysis, cDNA synthesis, and amplification strategy that
results in minimal bias and errors. gDNA is effectively removed to prevent
false-positive results.
We amplified a variety of human cells and checked the resulting genome
and transcriptome using NGS or qPCR methods. Discussed are experiments
on cell-to-cell variation, GC content in comparison to genomic DNA, percentage and consistency of protein coding transcripts, genome coverage with
respective error rates, and genome-wide real-time PCR analysis.
P14.93-S
3Gb-Test: Introduction of Whole Genome Sequencing as diagnostic
application
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address all afore mentioned topics and prepare an implementation plan for
the transition to the future of genetic testing in which WGS is embedded.
Substantial investments may be required and the logistic restructuring of
genetic services will be addressed. Monitoring and harmonization at the European level of all developments is therefore of the utmost importance.
3Gb-TEST is actively getting in contact with the genetics community and will
present the initial results and opinions at the ESHG conference.
P14.95-S
Design and evaluation of two novel methods for detection of ATP7B
gene mutations
Wilsons disease is a rare autosomal recessive disorder of copper metabolism (OMIM#27790) leading to copper accumulation in the liver, brain, kidneys and cornea. It is caused by mutations in the ATP7B gene with a carrier
frequency of approximately 1.2%.
We report the design and evaluation of two different screening methods for
the detection of ATP7B gene mutations which could be used in conjunction
in order to minimize the cost and time for genotyping.
1) A specific mutation detection assay by dry-reagent dipstick biosensors
applied for the 10 commonest mutations in the Greek population (H1069Q,
R969Q, Q289X, 2530delA, L936X, 2299insC, I1148T, 845delT, 1708-1G>A,
X1466R) covering 80% of disease chromosomes. Fragments flanking the
10 ATP7B mutations are amplified by multiplex-PCR, followed by multiplex
primer extension (PEXT) reaction using allele-specific primers. The primer
extension products are simultaneously detected by visual multi-allele dipstick type DNA biosensor using anti-biotin conjugated gold nanoparticles.
Optimization studies on the efficiency and specificity of the PEXT reaction
were performed.
2) A gene scanning protocol using High Resolution Melting (HRM) analysis,
designed to analyze all coding ATP7B regions.
The first method was evaluated by analyzing 50 samples of known genotypes (confirmed by sequencing) along with 50 blind samples. The results
were fully concordant with reference methods. HRM was evaluated by testing 50 known genotypes and 100 blind samples.
The proposed methods are simple, rapid, do not require purification of the
PCR products and could be particularly useful in diagnostic laboratories.
The benefits and drawbacks of each will be discussed.
P14.96-M
EAA/EMQN best practice guidelines for molecular diagnosis of
Y-chromosomal microdeletions: state-of-the-art 2013
295
ABSTRACTS POSTERS
P14.97-S
Introduction of a targeted next generation sequencing gene panel for
familial cancer in DNA diagnostics and genetic counselling
H. H. Lemmink, A. H. van der Hout, Y. J. Vos, B. Sikkema-Raddatz, K. van Dijk-Bos, A.
Knopperts, B. Leegte, J. ter Beest, H. Westers, R. J. Sinke, R. H. Sijmons;
Department of Genetics, University Medical Centre Groningen, Groningen, Netherlands.
Aim: Our aim was to develop a targeted next generation sequencing (NGS)
gene panel that enables to analyze large numbers of genes in patients with
familial cancer at relatively low cost.
Methods: A panel of 70 known tumour syndrome genes based on Agilent
Sure Select Target Enrichment for simultaneous mutation detection was
developed and validated in two series of twelve patients for variants of the
MMR or the BRCA genes previously identified through Sanger sequencing.
In an additional set of twelve patients all genes were analyzed anonymously.
The samples were sequenced using 151 base pair paired-end reads on an
Illumina MiSeq sequencer and analyzed using Softgenetics NextGENe
and Cartagenias Benchlab NGS software. Within the NGS panel, three virtual non-overlapping gene subpanels were designed, based on the levels of
preventive options and strength of risk information for the panel genes. In
genetic counselling the genes to be tested were discussed as the 3 subpanels rather than individually. For diagnostic purposes NGS with the targeted
panel was performed on 75 patients, in whom a pathogenic mutation in the
MMR and BRCA genes has been excluded. Only results from chosen subpanels were reported to patients.
Results and Conclusion: In the validation all pathogenic mutations, unclassified variants and neutral polymorphisms detected previously were identified by the NGS panel. In addition, 40 novel variants could all be confirmed
by Sanger sequencing. The results of the first 75 patients will be presented.
Genetic counselling was adopted to discuss genes as groups rather than as
individual genes.
P15.01-S
Swedegene: Genome-wide association studies of adverse drug
reactions
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Asthma treatment response is highly variable and pharmacogenetic markers that predict treatment response would be one step closer to personalized treatment. Polymorphisms in GLCCI1 could be associated with asthma
treatment response. We genotyped for rs37973 in GLCCI1 in 208 adult asthma patients treated with inhaled corticosteroids (ICS). Change in %FEV1
was analysed after 3 months and after at least 3 years of treatment. Treatment success was defined as good when FEV1 decreased less than 30 ml/
year. After 3 months of treatment, change of %FEV1 was higher in patients
with GG genotype than in patients with AG+AA genotype (p = 0.049), and
this genotype dependent difference was only evident and even higher in
non-smokers (p = 0.037). Similar results were found after at least 3 years
of treatment when all patients were analysed (p = 0.041) and in non-smokers (p = 0.034). Even though, no differences in treatment success (good vs.
poor response) were observed when analysing the entire group of patients,
treatment success was highly influenced by genotype and smoking status.
ABSTRACTS POSTERS
GG genotype was overrepresented in non-smokers with good response (p
= 0.030) and on contrast, AA genotype in smokers with good response (p
= 0.015). There were no differences between smokers and non-smokers
when they were not stratified according to genotype. Our results showed
that treatment response to ICS is associated with rs37973 in GLCCI1, but
smoking has great influence on genotype dependent treatment response
and further studies are warranted to elucidate the role of GLCCI1 in treatment response.
P15.05-S
Influence of single nucleotide polymorphisms on deferasirox Ctrough
levels and effectiveness.
Deferasirox (DFX) is the only once-daily oral chelator for first-line therapy of
blood transfusion-related chronic iron overload. DFX pharmacokinetic has
been related with response to therapy. This drug is metabolized in liver by
UDP-glucuronyltransferase (UGT) 1A1 and 1A3, by cytochrome-P450 (CYP)
1A1, 1A2 and 2D6 enzymes, and it is eliminated via biliary-enteric circulation through multidrug resistance protein 2 (MRP2).
Our aim was to evaluate DFX plasma concentrations according to single
nucleotide polymorphisms (SNPs) in genes involved in this drug metabolism and elimination, in a cohort of non paediatric -thalassemic patients.
Further aim was to define a plasma concentration cut-off value predicting
an adequate response to therapy.
DFX concentrations were determined from plasma samples obtained at the
end of dosing interval (Ctrough) using an HPLC-UV method. Allelic discrimination for SNPs in UGT1A1, UGT1A3, CYP1A1, CYP1A2, CYP2D6, MRP2 and
BCRP1 genes was performed by real-time PCR.
DFX Ctrough levels were significantly influenced by UGT1A1C>T (rs887829)
[p=0.045], CYP1A1C>A (s2606345) [p=0.017], CYP1A2A>C (rs762551)
[p=0.014], CYP1A2C>T (rs2470890) [p=0.004] and MRP2G>A (rs2273697)
[p=0.032] SNPs. According to Chirnomas and Galanello efficacy definitions,
a DFX plasma cut-off value of 20,000 ng/mL was identified (ROC curve,
p=0.008). A logistic regression analysis was performed to determine factors
able to predict this value: both CYP1A1 C>A rs2606345 AA (p=0.017) and
CYP1A2 C>T rs2470890 TT (p=0.037) genotypes may forecast drug concentrations below 20,000 ng/mL, suggesting a negative predictive role of
therapy efficacy.
Our data, the first obtained in non paediatric patients, suggest the feasibility
of a pharmacogenetic-based DFX dose personalization.
P15.06-M
ADRB1 and ADBR2 gene polymorphisms and the ocular hypotensive
response to topical betaxolol in healthy Mexican subjects
Background The adrenergic receptors (ADRB) are expressed in the ciliary body and trabecular meshwork, structures involved in aqueous humor
production and outflow, respectively. ADRB are members of the adrenergic
family of G-protein-coupled receptors. Topic blockers have a good local
and systemic tolerance; they reduce the aqueous humor production and eye
strain blocking the ADRB of the ciliary body and interfering with adenylate
cyclase. However, the ocular hypotensive response is not the same in all
patients and could be mediated by the polymorphisms of the ADRB genes.
Material and Methods Seventy two healthy subjects were studied after treatment with topical betaxolol in both eyes. We analyzed ADRB1 and ADRB2
gene polymorphisms by PCR and automated DNA sequencing. Results There
was statistically significant difference between baseline IOP and final IOP of
both eyes (Baseline IOP 16.21.2 - Follow-up IOP 13.62.0 (mean difference
-2.51.3, P<0.001). Gly389 had a higher baseline IOP than Arg389 (17.01.2
mmHg vs. 16.01.2 mmHg; P=0.02) and conversely Arg389 had a greater
magnitude of response than Gly389 to betaxolol therapy (-2.91.1 mmHg
vs -0.70.4 mmHg; P< 0.001). Gln27 had a higher response than Glu27
(-2.71.3 mmHg vs -1.91.0 p=0.02) Conclusion Arg389 polymorphism of
the ADRB1 gene and Gln27 polymorphism of the ADRB2 gene were associated with the hypotensive response to topic betaxolol in healthy Mexican
volunteers.
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P15.07-S
The impact of CYP2C19, CYP3A4, CYP2B6, ABCB1, ITGB3 and PON1
gene variants on the antiplatelet effect of clopidogrel in Turkish
patients with acute coronary syndrome syndrome
miR-155 is oncogenic microRNA upregulated in many tumors including breast cancer (BC). As potential circulating biomarker, expression pattern of
miR-155 can be determined from blood and may discriminate BC patients
from healthy individuals, report on treatment efficacy and further prognosis. Development of BC may be a consequence of BRCA1 tumor suppressor
gene mutations involved in DNA damage response and repair. BRCA1 epigenetically controls miR-155 expression and its pro-cancerous potential. In
this study, we evaluated expression levels of miR-155 in circulation of individuals carrying BRCA1 mutations with no disease manifestation as well as
in BRCA1 BC patients after tumor resection and therapy.
We determined miR-155 expression in lymphocytes and plasma from peripheral blood. To amplify mature form, we used qRT-PCR with 1 RT and
2 qPCR specifically designed primers. Similarly, we determined expression
levels of miR-17, miR-18a, miR-21 and miR-27a, also known to be involved
in BC pathogenesis.
Compared to healthy controls, individuals carrying BRCA1 mutations with
no disease manifestation displayed an increased miR-155 expression in
plasma, but not in lymphocytes. In BRCA1 BC patients, we observed significantly elevated miR-155 expression in both, separated lymphocytes and
plasma, a phenomenon that persisted after tumor resection and therapy.
Other studied miRNAs were also deregulated.
Our results point to epigenetic control of miR-155 by BRCA1 in wide time
scale. Accurate identification of miRNAs from peripheral blood represents
the key point of development of non-invasive blood-based detection platforms for BC diagnosis, staging, therapy monitoring and prognosis.
Supported by European fond for regional development ITMS 26240220074/
OPVaV-2011/4.2/07-SORO.
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ABSTRACTS POSTERS
P15.09-S
CYP1A2 gene non-coding region polymorphisms in Roma and
Hungarian population samples
CYP1A2 enzyme contributes to biotransformation of wide range of therapeutically important drugs, including caffeine, clopidogrel, clozapine, warfarin, procarcinogens and endogenous substrates. The purpose of this study
was to determine and describe the pharmacogenetic profile and interethnic
differences of variants of CYP1A2 gene between Roma and Hungarian population. From genomic DNA, 404 Roma and 396 Hungarian healthy subjects
were genotyped for two non-coding variants of CYP1A2, namely -163C>A
(*1F) and
-3860G>A (*1C). Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) technique was applied. The minor allele frequency
for CYP1A2*1C variant was significantly different between Hungarian and
Roma samples (2.02% vs. 0%, p<0.001). The AA homozygous genotype was
not detectable. For CYP1A2*1F polymorphism we found a remarkable differences in presence of AA genotype in Roma population compared to Hungarians (31.9% vs. 49.5%, p<0.001) and in minor allele frequency (56.9%
vs. 68.6%, p=0.025). The following CYP1A2 genotypes were identified in
Roma and Hungarian samples, respectively: *1A/*1A (18.1% vs. 12.4%),
*1A/*1F (50% vs. 36.9%), *1F/*1F (31.9% vs. 46.7%). In Hungarian population we found the *1C/*1F genotype (4.04%), but it was not present
in Roma subjects. In conclusion, analysis of distribution of CYP1A2 gene
variants revealed further pharmacogenetic differences between Roma and
Hungarian population samples. Hungarians have higher chance for rapid
metabolism of CYP1A2 substrates, intensified procarcinogen activation and
thereby elevated risk for cancers.
This research was supported by TMOP-4.2.3-12/1/KONV-2012-0028.
P15.10-M
CYP2C9*8 frequency distribution in Puerto Ricans: Implications for
warfarin dosing
K. Claudio-Campos1, C. Orengo-Mercado1, L. Berrios2, R. Pagan2, J. Renta-Torres1, C.
Cadilla1, J. Duconge-Soler1;
1
University of Puerto Rico- Medical Sciences Campus, San Juan, PR, United States,
2
University of Puerto Rico at Rio Piedras, San Juan, PR, United States.
Warfarin is an oral anticoagulant that requires individual monitoring since serious adverse events are common. CYP2C9 encodes for the enzyme
mainly responsible of S-warfarins metabolism. Polymorphisms in CYP2C9
have been previously found to be associated with observed warfarin dose
variability in different populations, but not in Caribbean Hispanics. Caribbean Hispanics originated as a result of a complex admixture among Caucasians, Africans and Amerindians ancestors-a characteristic that should be
considered for warfarin management. The rare loss-of-function CYP2C9*8
allelic variant is reportedly more prevalent among individuals with African heritage. Since Puerto Ricans has a significant contribution of African
ancestry in their genetic backgrounds, this cross-sectional study was aimed
to determine the frequency of CYP2C9*8 in a cohort of 150 Puerto Rican
patients undergoing warfarin therapy. DNA specimens were extracted and
genotyped for the CYP2C9*8 using a PCR-based Taqman genotyping assay.
We found 3 heterozygous for the CYP2C9*8 variant in our study cohort, corresponding to a minor allele frequency of 1% (95%CI: 0.0026-0.031). The
observed frequency met Hardy-Weinberg equilibrium. Allele frequency in
our cohort was found to be significantly lower than that from a previous
report in African-Americans (0.01 versus 0.047, respectively, p=0.045 by
two-tailed z-test), with a carrier frequency of 1 in 50 (Puerto Ricans) versus 1 in 11 (African-Americans). Due to the CYP2C9*8 prevalence found
among Puerto Ricans, we concluded that this variant should be included in
any pharmacogenetic-guided algorithm for warfarin dose predictions in this
population.
Approved by University of Puerto Rico, Medical Sciences Campus Institutional Review Board protocol A4070109.
P15.11-S
Extrapyramidal Disorders while Schizophrenia Therapy - analysis of
Cyp2D6*4 allele Influence
Introduction
A lot of clinical investigations of the schizophrenia therapy reveal the dependence of medicament concentration on the individual metabolism peculiarities. The latter depend to a greater degree on the CYP2D6*4 allelic
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state of the person. The discrepancy between the metabolic status of the
patient and definite antipsychotic drug choice and dosage results in various
side effects, among which the extrapyramidal disorders (EPD) are the most
invalidating.
Methods
The CYP2D6*4 genotype ( rs3892097 ) was revealed for 211 patients suffering from schizophrenia that were cured with various antipsychotic drugs:
Fluphenazine, Trifluoperazine, Haloperidol, Flupentixol, Zuclopenthixol,
Sulpiride, Risperidone. PCR with specific primers was used for allele estimation. All the patients from Republican Research and Practice Center for
Mental Health where cured with definite (one) medicine for 2 weeks and
then examined once for the presence of (EPD) with the help of Extrapyramidal Symptoms Rating Scale (ESRS). The analysis of the results was carried
out with the WinPepi package of statistical programs for epidemiology.
Results
In the process of pharmacotherapy the patients fell into two groups: those with (99) or without (112) (EPD). The influence of CYP2D6*4 A allele
as a risk factor for the EPD development was proved for Fluphenazine (P
=0,001), Flupentixol (0,0001). Conclusion We proved the importance of
CYP2D6*4 genotyping for the cohort of the patients we studied that were
cured with special antipsychotic medication. The elaboration of informative
clinical approaches to the adequate drug choice as well as their dosage must
take into account the patients CYP2D6 genotype.
P15.12-M
Towards personalized medicine in Type 2 Diabetes (T2DM): a genetic
risk score for the response to the first line treatment in T2DM,
metformin
N. van Leeuwen1, G. Nijpels2, J. M. Dekker2, L. M. t Hart1;
1
LUMC, Leiden, Netherlands, 2VU University Medical Center, Amsterdam, Netherlands.
ABSTRACTS POSTERS
combination in pipelines, and user interfacing in challenging clinical NGS
diagnostics, we have created MOLGENIS Variant Service, an open-source
web application that can be installed as virtual machine, on local servers, as
shared resource, and in a cloud.
We envision an NGS data exploration app as well as a sharing platform for
unified data formats and pipelines, gold standard data sets, well-curated
reference knowledge-bases, and optimal user interfaces, results of which
can disseminate into research institutes, clinical software companies and
individual labs.
P15.14-M
Personal genomics in Greece: An overview of available direct-toconsumer genomic services and the relevant legal framework
S. Kechagia1, G. Patrinos2, E. Vayena3;
1
University of Geneva, Geneva, Switzerland, 2University of Patras, Patra, Greece,
3
University of Zurich, Zurich, Switzerland.
The aim of this study is to provide an overview of the DTC genomic services
available in Greece and the legal framework within which they operate.
Based on literature review, a questionnaire that was distributed in a genetics conference in Greece and in-depth interviews with human geneticists
in Greece, we assess the landscape of the DTC genomic testing market and
highlight possible particularities of Greek consumers.
Furthermore, we identify the existing legal framework regarding DTC genetic testing. Our interest is not limited only to issues such as consumer
protection laws, lab quality accreditation and the provision of genetic counseling. We also explore the role of medical specialties and their respective
legal responsibilities in the Greek context, since for example the specialty of
Clinical Geneticist does not exist. We also explore the legal authority of the
National Organization of Medicines (E.O.F.) regarding the approval of genetic tests and specific issues relating to paternity tests. We identify gaps in the
current regulatory scheme and conclude with recommendations for a more
comprehensive legal framework.
P15.15-S
2020 vision and beyond - educating tomorrows clinicians for the
genomic era
P. Lunt, S. Gay;
NHS National Genetics & Genomics Education Centre, Birmingham, United Kingdom.
Understanding the person-to-person variability in diseases by using the unprecedented amount of available genomic data may be approached as never
before. We propose to investigate the impact of individual genetic variations
in breast cancers. We performed a meta-analysis of all the publicly available
ER ChIP-Seq datasets in four ER positive cell lines: MCF7, T-47D (breast cancer derived) cell lines and ECC1, Ishikawa (endometrium cancer derived)
cell lines. The number of peaks in intergenic regions is roughly 50 to 55%
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while depending on the cell type 30 to 40% of the peaks are localized in
introns. Some of the genes may have several intronic binding sites. Intronic
binding sites are most likely to regulate their own genes, therefore based on
these statistics and the individual assignment of the binding sites to their
genomic locations, we have identified a set of genes that have ER binding sites in their introns. The number of SNP-s available in the dbSNP database for
the identified intronic binding sites of the identified genes is relatively high,
that is more than one thousand. Based on our investigations a significant
number of SNP-s could influence directly the estrogen dependent regulation
of relevant target genes.
P15.17-S
Circulating levels of FSH in men are genetically determined: study on
the combined effect of polymorphisms in the FSHR and FSHB genes
C. Vinanzi, F. Ganz, M. Pengo, M. Menegazzo, C. Foresta, A. Ferlin;
University of Padova, Padova, Italy.
Recent study have shown that polymorphisms in the FSHR and FSHB genes can modulate circulating levels of FSH. FSHR variants Asn680Ser and
c. -29 G>A have been extensively studied in women, while FSHB variant
-211 G>T seems to influence male serum FSH levels. There are no studies
on the combined effect of these three polymorphism. We have studies 365
subjects: 39 azoospermic, 177 oligozoospermic and 149 normozoospermic.
We evaluated seminal parameters; hormone levels; testicular volume; FSHR
and FSHB polymorphisms. FSHB polymorphism -211 G>T was found significantly associated to FSH levels. FSHR polymorphism -29 G>A and Asn680Ser alone, are not associated to different concentration of FSH. Combined
analysis of the three polymorphisms again highlights that the major determinant in FSH levels is -211 G>T polymorphism, but shows also that this
effect is modulated by -29 G>A polymorphism, as subjects with -211 GG/-29
GG genotype have higher FSH level. Total sperm count and testicular volume is also modulated by the genotype: Homozygotes -211 TT are invariably
azoo-oligozoospermic with a reduced testicular volume and FSH <8 UI/L.
Combined effect of Asn680Ser is negligible. This is the first combined study
on the influence of FSHB and FSHR polymorphisms on male reproduction
and shows that -211 G>T FSHB polymorphism plays an important role, only
slightly modulated by FSHR polymorphisms, on FSH levels, sperm count and
testicular volume. FSHB -211 G>T influences transcriptional FSH gene activity, thus causing an isolated FSH deficiency with azoospermia and thus
represents the best pharmacogenetic marker to FSH treatment.
P15.18-M
Evaluation of microarray gene expression profiling as response to
zearalenone exposure on normal intestinal epithelial cell
299
ABSTRACTS POSTERS
P15.19-S
Taking gene-based diet in real life: a prospective study.
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preparing to enroll newborn infants and their physicians from the neonatal
intensive care unit and the newborn well nursery. In both projects patients
are randomized to receive standard of care with or without GS. Physicians
and patients are followed through surveys, interviews and medical record
review to examine the behavioral and clinical impacts of GS results.
In the GS arms of both projects, GS is conducted and interpreted in CLIAapproved molecular laboratories and the results, including all medically
relevant unexpected findings, are being provided in a digestible report to
the physicians. Reports summarize and present pathogenic variants in rare
dominant conditions, variants indicating carrier status for rare recessive
conditions, pharmacogenomics variants for commonly used medications
and blood group antigens. Interviews, standardized scales and ongoing reviews of medical records are being used to track physician actions, patient
(or parent) behaviors and attitudes, health outcomes and health care costs.
The MedSeq and BabySeq Projects will help to illuminate the benefits, risks
and limitations of GS in the medical care of both adults and infants.
P15.22-M
Single Nucleotide Polymorphisms of the Glucocorticoid-Receptor
Gene Influence the Outcome of Cardiac Surgery
Cardiac surgery triggers systemic inflammatory response which is associated with postoperative morbidity and mortality. Glucocorticoid effects
are mediated by glucocorticoid receptors (GRs) for which a number of common single nucleotide polymorphisms (SNPs) exist that influence GR sensitivity to cortisol. We selected three common SNPs of the GR gene that are
known to affect GR sensitivity and analyzed these data in relation to early
outcome variables in patients undergoing cardiac surgery. We tested the
effects of the following GR-SNPs: rs4123247 (BcII, increased cortisol sensitivity), rs33388 (cortisol hypersensitivity) and rs10052957 (FPB5, cortisol resistance). All study personal was blinded with regard to the patients`
genotype. Study endpoints (primary outcome variables) were the required
dosages of hydrocortisone during surgery and in the Intensive Care Unit.
The study population consisted of 95 patients. Homozygous carriers of alleles associated with increased GR sensitivity (Bcll *G, n=10 and rs33388
*G, n=25) required significantly lower dosages of hydrocortisone in the Intensive Care Unit than non-carriers of the respective alleles. Homozygous
individuals (n=6) for the TT-allele of the FPB5-SNP required significantly
higher dosages of hydrocortisone during surgery and in the Intensive Care
Unit than heterozygous carriers. They also needed significantly higher norepinephrinemax and epinephrinemax dosages in the Intensive Care Unit to
achieve hemodynamic stability and showed a significantly longer duration
of Intensive Care Unit therapy than heterozygous or non-carriers of the Tallele. In summary, hydrocortisone dosages administered according to evidence based algorithm to cardiac patients during and after cardiac surgery
are influenced by SNPs in the GR.
P15.23-S
Genome-wide association study of chemotherapeutic agent-induced
severe neutropenia/leucopenia for patients in Biobank Japan
Chemotherapeutic agents are notoriously known to have narrow therapeutic range that often results in life-threatening toxicity. Hence, it is of clinically
important to identify patients who are at high risk for severe toxicity to certain chemotherapy through pharmacogenomics approaches. In this study,
we carried out 17 genome-wide association studies (GWAS) with 13 122
cancer patients recruited from the Biobank Japan, who received different
chemotherapy regimens, including cyclophosphamide- and platinum-based
(cisplatin and carboplatin), anthracycline-based (doxorubicin and epirubicin), and antimetabolite-based (5-fluorouracil and gemcitabine) treatment,
antimicrotubule agents (paclitaxel and docetaxel), and topoisomerase inhibitors (camptothecin and etoposide), as well as combination therapy with
paclitaxel and carboplatin, to identify genetic variants that are associated
with the risk of severe neutropenialeucopenia in the Japanese population.
In addition, weighted genetic risk score analysis was performed to evaluate the cumulative effects of common genetic variants associated with chemotherapeutic agents-induced severe neutropenia/leucopenia. Instead of
illustrating all 17 GWAS, we will utilize the result from GWAS of paclitaxelcarboplatin combined therapy for further explanation. Although we failed to
identify genetic variants that surpassed the genome-wide significance level
ABSTRACTS POSTERS
(P<5.0x10-8) through GWAS, probably due to insufficient statistical power
and complex clinical features, we were able to shortlist some of the suggestive associated loci. The current study is at the relatively preliminary stage,
but does highlight the complexity and problematic issues associated with
retrospective pharmacogenomics studies. However, we hope that verification of these genetic variants through local and international collaborations
could improve the clinical outcome for cancer patients.
Galactosemia
GALT
Hemochromatosis, type 1
HFE
P15.25-S
Preventive genetic testing for large spectrum of monogenic disorders
using microarray technology in Russian population
Gene
Muscular dystrophy,
CAPN3
limb-girdle, type 2A
Ichthyosis vulgaris
FLG
Galactosemia, duarte
GALT
variant
Hemochromatosis,
HFE
type 1
Mutation
Genotype
.550delA (Thr184fs)
c.1501C>T (Arg501ter)
c.2282del4
Compound heterozygous
state for c.1501C>T
(Arg501ter) and c.2282del4
c.-119delGATC
c.187C>G (His63Asp)
Del/Del
(fs/fs)
C/T (Arg/
ter)
N/Del
Arg/ter;
N/Del
Del/Del
G/G (Asp/
Asp)
Compound heterozygous
His/Asp;
state for c.187C>G (His63Asp)
Cys/Tyr
and .845G>A (Cys282Tyr)
Familial MediterraC/C (Gln/
MEFV
c.442G>C (Glu148Gln)
nean fever
Gln)
Leber optic atrophy MTCYB m.15257G>A (Asp171Asn)
A (Asn)
Total
Frequency,
%
0.5%
0.9%
1.5%
0.5%
2.3%
1.4%
1.4%
0.5%
0.5%
9.5%
Table 2.
Frequency of heterozygous state for recessive disorders in 213 persons genotyped by microarray Ethnogene
Disorder
Gene
Stargardt disease 1
ABCR
Wilson disease
ATP7B
CAPN3
Cystic fibrosis
CFTR
CLCN1
Mutation
c.2588G>C
(Gly863Ala)
c.3113C>T
(Ala1038Val)
c.5882G>A
(Gly1961Glu)
c.3402delC
(Ala1135fs)
.550delA
(Thr184fs)
c.1518delCTT
(Phe508 del)
c.3587C>G
(Ser1196ter)
c.621+1G>T
dele2,3 21080bp
c.1437_1450del14
(Ile479fs)
c.2680C>T
(Arg894ter)
Frequency,
%
3.4%
0.5%
0.5%
2.5%
5.3%
Smith-Lemli-Opitz syndrome
DARS2
DHCR7
GAA
GALT
GJB2
LAMB3
MEFV
PAH
POLG
PTS
SERPINA1
Back to index
c.455G>T
(Cys152Phe)
c.452G>A
(Trp151ter)
IVS1AS-13t>g
c.-119delGATC
c.1430A>G
(Gln188Arg)
c.855G>T
(Lys285Asn)
c.35delG
c.187C>G
(His63Asp)
.845G>A
(Cys282Tyr)
.1903C>T
(Lys635ter)
c.2282G>A
(Arg761His)
c.442G>C
(Glu148Gln)
c.1208C>T
(Ala403Val)
c.1222C>T
(Arg408Trp)
c.752C>T
(Thr251Ile)
.216T>A
(Asn72Lys)
c.1096G>A
(Glu342Lys)
c.863A>T
(Glu264Val)
0.5%
0.5%
0.5%
8%
1%
0.5%
32.3%
0.5%
1.5%
1.5%
0.5%
1%
1.5%
P15.26-M
Assessing the role of known MS genetic variants in the familial
aggregation of MS and other autoimmune diseases
Background: Previous pharmacogenomic studies conducted in Interferonbeta (IFN)-treated Multiple Sclerosis (MS) patients explored the influence
of genetic variants on the short-term response to the drug. The aim of this
study is to perform a genome-wide association study (GWAS) of long-term
response to Interferon-beta observed using a follow-up period of 4 years
after drug start.
Methods: The GWAS study was conducted using the Omni-express Illumi-
301
ABSTRACTS POSTERS
na array on a cohort of 329 IFN-beta treated MS patients followed for 4
years of treatment. We used three different outcome measures of response
to treatment: a disability criterion, measured as the increase of 1 point at
Expanded Disability Status Scale (EDSS), and two composite criteria, which
take into account measures of disability progression and relapse occurrence
(2), on which separate GWAS analyses have been performed.
Results: No genome-wide significant association signal was detected. When
using a cut-off p-value of 10-4, 42 SNPs emerged as associated according to
the disability criterion, while 28 and 41 SNPs for the two composite criteria.
A modest overlap (11.8%) was observed among associations across three
phenotypes.
Conclusions: Our study showed new targets potentially involved in the modulation of the response to IFN-beta treatment. An in silico replication analyses on identified targets is planned on an independent cohort of treated
MS patients.
P15.28-M
Genetic polymorphisms influence the response to adalimumab in
Crohn disease patients
302
Back to index
Psoriasis is a chronic, inflammatory skin disorder affecting 2-3% of the population worldwide. While there are a large number of effective modalities
for treating psoriasis, response to therapy varies among patients which
could be due to genetic factors. Cyclosporine is considered to be a cost-effective first-line systemic therapy for psoriasis, however the response rate
is around 60-70%. The aim of the present study, which is based on a Greek
multi-centre collaboration, was to target ABCB1, which encodes for P-glycoprotein, by selecting polymorphisms that could influence the absorption
and disposition of P-glycoprotein substrate drugs like cyclosporine. In detail,
T-129C (rs3213619), G1199A (rs2229109), C1236T (rs1128503), G2677T
(rs2032582) and C3435T (rs1045642) polymorphisms were selected as
candidate markers of response to cyclosporine treatment after 3 months
of therapy and genotyped in 84 psoriasis patients under cyclosporine therapy. Fifty-two patients (62%) were defined as responders (PASI 75%)
and thirty-two (38%) as non-responders (PASI 50%). Single-SNP and haplotype construction showed that the haplotype for marker C3435T could
account for the prediction of ~20% of the non-responders to cyclosporine
therapy. Notwithstanding the importance of this finding, there is a remaining 80% to be identified, which could be illuminated by systems network
analysis, whereby additional pharmacogenetic targets that may be involved
in cyclosporine transport, processing or metabolism are identified. Overall,
systems biology coupled with experimental validation of specific markers
in large independent cohorts could lead in the creation of a molecular algorithm for the prognosis of psoriasis patients response to cyclosporine as
well as for other therapies.
P15.31-S
Identification of a new late radiotherapy toxicity locus through a
three stage genome wide association study
Increasing evidence supports the role of genetic variants in the development of radio-induced toxicity. Therefore, we performed a three-stage genome wide association study that involved a Spanish cohort of 741 prostate
cancer patients treated with radiotherapy to identify new susceptibility loci.
The replication cohorts consisted of 633 prostate cancer patients from the
UK, and 368 prostate cancer patients from a North-American Caucasian cohort.
Standardized total average toxicity (STAT) scores were derived from individual toxicity endpoints to assess overall acute and late toxicities. Association
tests of genotype with STAT-acute or STAT-late scores were performed with
linear regression. Residuals from multivariate linear regression models, including associated non-genetic covariates, were calculated for each patient
to quantify the toxicity not accounted for by the available non-genetic covariates. To obtain per allele ORs by logistic regression, patients were dichotomized as or > than 1 standard deviation of the acute and late residuals.
Seven and 42 loci were associated (P-value10-5) with overall acute and
late toxicity, respectively. Only one locus associated with overall late toxicity, 2q24.1 (STATlate P-value =6.85x10-9; OR=6.67, 95%CI: 2.25-19.80) was
replicated in the UK cohort (STATlate P-value=2.08x10-4, OR=6.17, 95%CI:
2.25-16.95; meta-analysis P-value=4.16x10-10). The inclusion of the third
ABSTRACTS POSTERS
cohort gave a meta-analysis P-value=4.64x10-11.
Although it is biologically possible that this locus is involved in the regeneration of muscle previously damaged by radiotherapy, future efforts are
needed to identify the causal variant underlying the observed association
and determine the molecular mechanisms involved.
P15.32-M
Genetic determinants of methotrexate toxicity in Tunisian patients
with rheumatoid arthritis: a study of polymorphisms involved in the
folate pathway of methotrexate
Polycythemia vera is a rare bone marrow disorder that leads to an abnormal raising in the number of red blood cells, although the numbers of white
blood cells and platelets are also on high levels. In the background of the disease a gene mutation called JAK2V617F can be found, but the cause of this
and other possible disorder-causing mutations is undiscovered. Our goal
was to examine the rs1048943 polymorphism located in CYP1A1 gene in 90
Roma (Gipsy) with polycythemia vera versus 95 Roma individuals without
this disease. Genotypes were determined with real-time-PCR method, SPSS
20.0 statistical program was applied for evaluating the results. The preliminary data showed 6.11% G allele frequency in Romas with polycythemia
vera, which it was found to be 3.70% in non-affected Roma controls. The
CYP1A1 1384 A/G mutation has never been analyzed in Roma population
before, but further extended studies are required to test the clinical relevance of CYP1A1 in different populations.
P15.34-M
Full resequencing of TRAF3IP2 gene in Mozambican patients with
SJS/TEN induced by Nevirapine treatment: a pharmacogenetics study
Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor, widely prescribed for type 1 human immunodeficiency virus infection. A small
proportion of individuals treated with NVP experience very severe cutaneous adverse events, including Stevens-Johnson syndrome (SJS) and toxic
epidermal necrolysis (TEN).
In the last years TRAF3IP2 gene variants were associated with susceptibility to psoriasis, psoriatic arthritis, systemic lupus erythematosus and cuta-
Back to index
Colorectal cancer (CRC) is amongst the top three most commonly diagnosed
cancer in the world. Majority of these patients require treatment for metastatic CRC since a third are at stage IV during diagnosis and another third of
the curatively resected patients (Stages I-III) will relapse. 5-fluorouracil is
a common drug used for the treatment of CRC but the response rate to this
drug either alone or in combination is less than 40%. Developing a reliable
biomarker that can predict response can facilitate the appropriate tailoring
of treatment for these patients.
Here we report a novel potentially functional Single Nucleotide Polymorphism (pfSNP) approach to identify SNPs predictive of response to 5-FU in
Chinese metastatic colorectal cancer (CRC) patients. 1547 pfSNPs and one
variable number tandem repeat (VNTR) in 139 genes in 5-FU drug (both
PK and PD pathway) and colorectal cancer disease pathways were examined in 2 groups of CRC patients. Shrinkage of liver metastasis measured by
RECIST criteria was used as the clinical end point. We identified a total of
9 novel pfSNPs including 4 non-responder-specific pfSNPs with potential
functional significance that may be able to distinguish non-responders from
responders to 5-FU. These may thus serve as good biomarkers for response
to 5-FU.
P15.36-M
Semiconductor Next Generation Sequencing (Ion Torrent) of the
ABCB1, CYP3A5, and CYP3A4 genes in kidney transplanted patients
treated with Tacrolimus
B. Tavira Iglesias1, C. Diaz-Corte2, D. Coronel2, E. Coto Garcia2;
1
Hospital Universitario Central De Asturias , Oviedo, Spain, 2Hospital Universitario
Central De Asturias, Oviedo, Spain.
303
ABSTRACTS POSTERS
P15.37-S
TGF-B1 -509C>T polymorphism and response to montelukast in
childhood asthma
Human genetic variation underlies a majority of phenotypic differences between individuals, including susceptibility to disease.
Genome wide association studies (GWAS) have identified many susceptibility loci/SNPs for complex traits and diseases. The greatest challenge in
the post-GWAS era is to understand the functional consequences of these
loci. When SNPs occur within a gene or in a regulatory region, they may
play a direct role in disease by affecting the gene function. Therefore SNPs
304
Back to index
Having a good quality geneset is essential for the interpretation of functional genomic, functional transcriptomic, and variation data. The GENCODE
geneset represents the reference human gene annotation for the ENCODE
project and is produced by merging manual annotation and automated Ensembl gene predictions with extensive computational and experimental QC
and validation. Using different reference genesets will inevitably give divergent results and the absence, truncation or misannotation of a gene, exon, or
alternatively spliced (AS) transcript may hinder analysis. We will highlight
some significant differences between the GENCODE and NCBI Reference Sequence Database (RefSeq) genesets. Specifically, we will discuss divergence
in the annotation of alternative splicing (where GENCODE protein-coding
loci have a mean of 7.6 AS transcripts while RefSeq only have 2.1), long noncoding RNAs (GENCODE 2.5 fold more genes and 3.7 fold more transcripts),
pseudogenes (GENCODE 10% more loci), genomic coverage of annotated
exons (GENCODE 1.7 fold greater coverage), degree of manual curation
(GENCODE 4.5 fold more manually curated transcripts), experimental validation, and functionally descriptive biotypes. We will detail the continued
extension and refinement of the GENCODE geneset, including the integration of RNAseq, CAGE, polyAseq, ribosome profiling and epigenomic data, to
ABSTRACTS POSTERS
identify novel loci, define 5 and 3 transcript boundaries and identify novel
translation initiation sites. Finally, we will explain our use of RNAseq data to
determine the expression level of all GENCODE transcripts, allowing us to
present a reduced, but biologically meaningful, set of transcripts including
e.g. only those that are highly expressed or expressed in a particular tissue.
P16.03-S
Jannovar: A Java library for Exome Annotation
Transcript-based annotation and pedigree analysis are two basic steps in the
computational analysis of whole-exome sequencing experiments in genetic
diagnostics and disease-gene discovery projects. Here, we present Jannovar,
a stand-alone Java application as well as a Java library designed to be used in
larger software frameworks for exome and genome analysis. Jannovar uses
an interval tree to speed up the identification of all transcripts affected by a
given variant, and provides HGVS-compliant annotations both for variants
affecting coding sequences and splice junctions as well as UTR sequences
and non-coding RNA transcripts. Jannovar can also perform family-based
pedigree analysis with VCF files with data from members of a family segregating a Mendelian disorder. Using a desktop computer, Jannovar requires a
few seconds to annotate a typical VCF file with exome data.
P16.04-M
Comparison of GWAS and EWAS results to identify loci and genes of
interest in asthma
Asthma is a complex trait with a large phenotypic diversity for which more
than 300 genes have already been associated. In order to better define the
heritability of the disease, omics technologies were used in several studies to explore genetic and epigenetic factors. The large amount of results
obtained with these methods comes with complications for identifying
true genes of interest. This study aims to compare genome-wide association studies (GWASs) and epigenome-wide association studies (EWASs) of
asthma and related phenotypes (atopy and allergic asthma) using a welldescribed asthma familial collection in order to identify loci and genes of
interest. GWASs and EWASs were performed on DNA samples extracted
from whole blood and EWASs were also performed on DNA extracted from
isolated eosinophils. GWASs were analyzed using a quasi-likelihood score
test performed using the MQLS program and EWASs data were compared
using a Mann-Whitney analysis implemented in the GenomeStudio software
by Illumina. Analyses allow identifying 2 common loci between EWAS and
GWAS analyses for asthma phenotype, 5 common loci for atopy and 3 for allergic asthma. Among them 1 principal locus (including 5 CpG sites or more)
was identified in asthma (17q21.2) and 2 principal loci in atopy (5q31.1 and
6p22.1). The three loci identified have already been associated with asthma
in previous studies. Analyses are in progress in order to compare common
genes associated in both omics methods. These results highlight the potential of the combination of omics analyses in order to identify potential
loci of interest in complex traits.
P16.05-S
DNA methylation signature of IL1R1 and IL1R2 genes in asthma
2,3
Back to index
Beckwith-Wiedemann syndrome (BWS), a congenital overgrowth disorder with variable expressivity, results from disordered expression and/or
function of imprinted genes at chromosome 11p15.5. There are no generally agreed clinical diagnostic criteria, with molecular studies commonly
performed to confirm diagnosis. In particular, methylation status analysis at
two 11p15.5 imprinting control centres (IC1 and IC2) detects up to 80% of
cases. In order to evaluate the relationship between the clinical presentation
of suspected BWS and IC1/IC2 methylation abnormalities we reviewed the
results of >1000 referrals for diagnostic testing.
507/1091 (46.5%) referrals had a positive diagnostic test. The rate of a positive diagnostic test increased with increasing numbers of clinical features.
Previously reported genotype-phenotype correlations with paternal uniparental disomy, IC1, and IC2 epimutation groups were confirmed and potential novel associations detected. Predictive values of previously described
clinical diagnostic criteria were compared, and although there were differences in sensitivity and specificity, receiver operating characteristic (ROC)
analysis demonstrated that these were not optimal in predicting 11p15.5
methylation abnormalities. Using logistic regression, we identified clinical
features with the best predictive value for a positive methylation abnormality. We developed a weighted scoring system (sensitivity - 75.9% and specificity - 81.8%) to prioritise patients presenting with the most common features of BWS, and ROC analysis demonstrated superior performance (area
under the curve - 0.85; 95% CI: 0.83-0.87) compared to previous criteria.
We suggest that this novel tool will facilitate selection of patients with
suspected BWS for routine diagnostic testing and so improve the diagnosis
of the disorder.
P16.07-S
Genome-wide methylation analysis on epimutated BWS patients for
detection of known and novel imprinted loci multilocus defects
Loss of imprinting (LOI) through methylation loss (LOM) or gain (GOM) may
result in imprinting disorders (IDs). Beckwith Wiedemann syndrome is a
paradigmatic ID, which arises from dysregulated expression of imprinted
growth regulatory genes at 11p15.5. We pursued a genome-wide methylation study to investigate our cohort of IC2 hypomethylated BWS patients
for the occurrence of Multilocus Methylation Defects (MMD) at imprinted
loci. To this purpose we selected from a cohort of 171 patients with clinical
presentation in the spectrum of Beckwith-Wiedemann syndrome carrying
an 11p15.5 epimutation, 18 cases hypomethylated at IC2: 14 with a complete phenotype, 2 with isolated hemihyperplasia (IH) and two couples of
monozygous twins with discordant phenotype, but concordant for the IC2
defect in blood tissue. The Processing of genomic DNAs from peripheral
blood on an Infinium Human Methylation 450K BeadChip Kit Array (Illumina)showed that 7/16 (44%) of IC2 epimutated BWS and 2/2 IH patients
305
ABSTRACTS POSTERS
had multilocus defects. These comprised hypermethylation at GNAS DMRs
and hypomethylation at GNAS, PLAGL1, DIRAS3, FAM50B and ZNF331 loci,
confirming the view of a network of imprinting deregulated genes. MMD
were detected in blood and saliva from both affected twin members, but
complementary pyrosequencing and MS-MLPA analyses showed that methylation defects were absent/attenuated in the DNA from saliva in the unaffected co-twins. This findings will enhance clinical practice and epigenotype
phenotype correlations. In conclusion 450K genome approach appears a reliable technique to profiling the methylation status of all known imprinted
loci and unravel their recurrent simultaneous deregulation Supported by
2009MBHZPR_003(to LL).
P16.08-M
Comprehensive methylation profiling in Beckwith-Wiedemann
syndrome and Silver-Russell syndrome
R. Helaers, M. Vikkula;
Laboratory of Human Molecular Genetics, De Duve Institute (Universit catholique de
Louvain), Brussels, Belgium.
306
Back to index
The ability to detect low-level genetic variants in heterogeneous populations of cells is necessary for identifying postzygotic or somatic mutations
underlying human diseases such as mosaic birth defects involving the skin.
Existing bioinformatics methods for detecting such variations have been
mainly developed for cancer genomics and are usually based on the analysis
of paired samples. Furthermore, they have limited sensitivity in detecting
certain types of variants such as insertions/deletions. SomatiCaller was developed to systematically detect low-level variants in next-generation DNA
sequencing data. It consists in browsing aligned sequence data (BAM files)
of pairs or trios (i.e. index case and parents) to systematically identify positions with candidate variants. Statistical tests are first performed between
different samples to measure the samples independence of a candidate position for a given variation. In a second time, allelic ratios from all candidate sites are compared to a series of negative controls by Students t-test to
discriminate true positive variants from probable sequencing or alignment
errors. Applied to targeted deep sequencing of a gene associated with mosaic overgrowth syndromes (PIK3CA), SomatiCaller led to identification of
variants with allelic fractions as low as 0.01. Experiments from trio-based
exome sequencing data demonstrated its ability to readily detect variants
with allelic fractions as low as 0.05. Compared to standard variant callers,
SomatiCaller showed increased sensitivity for genetic variants present in
less than 10% of reads. Potential applications of this tool are numerous in
the growing field of the genetics of mosaic diseases, both for research and
molecular diagnosis purposes.
P16.11-S
An extensive, interactive variant-centered annotation browser
Genome-wide association studies and, more recently, next-generation sequencing studies have created a huge amount of genetic associations with
hundreds of common human traits. A still unsolved problem in this context
is the correct annotation of the gene(s) linked to the mostly small effects
observed for genetic variants. The current catalog of genomic annotations
is multi-layered and highly complex, the annotation of variants on the other
hand is often conducted using very basic heuristics. We try to even out this
imbalance by integrating many different genome-wide annotation datasets
into a user-friendly web-accessible resource. Our collection of tools combines linkage disequilibrium data, genetic associations, gene annotations,
expression data and several layers of regulatory annotations. Besides evidence-based prediction of variant-to-gene projections, our resource provides data browsing and retrieval, as well as publication-ready plotting and an
interactive variant-centered genome browser.
P16.12-M
Molecular diagnosis of bladder cancer by detecting gene p16INK4a
from body fluids by methylation-specific polymerase chain reaction
(MSP)
R. Dumache, M. Puiu, R. Bardan, M. Licker, V. Dumitrascu, D. David;
University of Medicine and PharmacyVictor Babes, Timisoara, Romania.
ABSTRACTS POSTERS
Extracellular matrix plays a significant role in tumor development. Our study
focuses on the epigenetic regulation of 12 laminin-encoding genes (LAMA1,
LAMA2, LAMA3A, LAMA3B, LAMA4, LAMA5, LAMB1, LAMB2, LAMB3, LAMC1,
LAMC2, LAMC3), 8 genes of integrins (ITGA1, ITGA2, ITGA3, ITGA4, ITGA6,
ITGA7, ITGA9, ITGB1), 2 nidogen genes (NID1,NID2), 2 genes of the cadherin family(CDH2,CDH3) and the dystroglycan gene DAG1.We have surveyed
109 samples of breast cancer, 109 paired adjacent nonmalignant samples, 6
samples of normal mammary gland from autopsy and 6 samples of breast
cancer cell lines for aberrant promoter methylation of all 25 genes.
Promoters of 11 genes (LAMA1, LAMA2, LAMB1, ITGA1, ITGA4, ITGA7, ITGA9,
NID1, NID2, CDH2, CDH3) have demonstrated abnormal methylation in 1,9%
to 42% samples of breast cancer and/or adjacent tissues. Our results may
be important for understanding of the dramatic changes in the extracellular
matrix during tumor growth and development.
Extracellular matrix proteins genes abnormally methylated in breast cancer
Methylation in
Methylation in
Gene breast cancer and/or normal mammary
symbol adjacent nonmalignant gland from autopsy
samples ( % )
(%)
LAMA1
35
0
LAMA2
42
0
LAMB1
29
0
ITGA1
16
0
ITGA4
21
0
ITGA7
1,9
0
ITGA9
37
0
NID1
36,8
0
NID2
31,5
0
CDH2
5,4
0
CDH3
27
0
Presence(+) / absence(-) of
methylation in breast cancer
cell lines
ZR MCF7 T47D BT HBL HS
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
P16.14-M
Comprehensive transcriptome profiling of breast cancers
Breast cancer is one of the most frequent malignancies among women and
is a major cause of cancer-related mortality, despite advances in early detection and treatment. Breast cancer is a heterogeneous disease with a poorly
defined genetic landscape, which poses a major challenge in diagnosis and
treatment. Recent reports have described an intricate interplay among diverse RNA species, including protein-coding mRNAs and ncRNAs (long ncRNAs, pseudogenes, miRNAs and circular RNAs) which are also implicated in
numerous diseases such as cancer.
By massively RNA-sequencing, we obtained 7.8 billion reads from 55 healthy
and tumour breast tissues belonging to Basal, HER2-positive and Luminal
breast cancers, with or without hereditary predisposition. At the beginning,
we aim to define their comprehensive digital transcriptome. In order to analyse the transcriptomes in specific tumour cells, we used Laser Capture Microdissection system. Furthermore, we performed a RiboZero-based rRNA
depletion in order to focus the sequencing effort on the non-ribosomal portion of the total RNA. Comparative transcriptomic analyses highlighted differentially expressed transcripts, between the different breast cancer groups,
identifying transcripts which may be possible modulator RNAs.
This global transcriptomic profiling can illustrate the intricate internal mechanism of the transcriptome at a very high resolution, allowing us to explore the distinct nature of these breast cancer subtypes, and could provide a
new inventory of diagnostic and therapeutic targets.
P16.15-S
Genetics of Cardiomyopathies: In-silico Analysis of Variants in
Cardiomyopathies-Associated Genes
O. O. Akinrinade1, S. Myllykangas1, J. Koskenvuo2, T. Alastalo1;
1
University of Helsinki, Helsinki, Finland, 2University of Turku, Turku, Finland.
Back to index
rate of missense variation in the two datasets using a window bin of 50bp.
Though most variants are rare (78.7%), and 58.4% (2751/4708) being private, 25.7% (655/2544) of missense variants from 1000 Genomes Project
database are predicted to be pathogenic. Of the overlapping variants, 20.4%
(42/206) and 42.2% (87/206) are known and predicted pathogenic variations respectively. We identified 104 regions distributed among 15 genes, including LMNA, MYH7, TNNT2, and MYBPC3, with high density of pathogenic
variants and no or extremely low variant in the population data. Majority of
the identified loci overlap protein domains that play critical roles.
The identified regions with high density of pathogenic missense variants are
likely hotspots that could predispose to cardiomyopathies, hence no or extremely rare variations have been reported in them in the population data.
P16.16-M
Complex Chromosomal Rearrangements In B-cell Lymphoma:
Evidence Of Chromothripsis?
Genomic instability is a well-known hallmark of cancer. Recent genome sequencing studies have identified a novel phenomenon called chromothripsis in which complex genomic rearrangements are thought to be derived
from a single catastrophic event rather than by several incremental steps.
While chromothripsis is well documented in solid tumors and leukemias,
chromothripsis in lymphoma is rarely reported. We report a case of possible
chromothripsis in a patient presenting with a thyroid mass suspicious for
diffuse large B-cell lymphoma. Chromosome analysis showed a complex karyotype with multiple rearrangements, including a translocation of chromosomes 3 and 7 involving the BCL6 gene region, chromosomes 14, 7 and 22
with involvement of the IGH gene region and an unbalanced structural rearrangement involving chromosomes 8 and 18 involving the BCL2 gene region..
The karyotype was interpreted as 51~56,XX,+X,+2,t(3;7)(q29;p11.2),der(7)
t(3;7)t(14;7;22)(q32;p11.2;q12),+der(7)t(14;7;22),der(8)t(8;18)
(p12;q21),+der(9)t(5;9)(q13;q22),+13,der(14)
t(14;7;22),+21,+1~4r[cp20]. FISH analysis confirmed the BCL6 gene rearrangement, IGH gene rearrangement/extra copies of IGH and 3 copies of
BCL2. Array comparative genomic hybridization studies with Cytochip 60K
custom oligo array showed multiple complex copy number variations including a previously unidentified chromosome 12 abnormality. However, array
analysis did not reveal any imbalance involving the BCL6, BCL2 or IGH gene
regions whose rearrangements were observed by FISH, thus suggesting that
these rearrangements are balanced in nature. Our patients genomic abnormalities show characteristics suggestive of chromothripsis and provides
initial evidence that chromothripsis is not confined to solid tumors, but can
also be seen in B-cell lymphomas with well characterized one or two-step
lymphomagenesis.
P16.17-S
Describing chromothripsis using HGVS sequence variation
nomenclature, suggested extensions
307
ABSTRACTS POSTERS
sequence variant nomenclature checkers (e.g. Mutalyzer, https://Mutalyzer.
nl).
1) ISCN (2013). 2013. An International System for Human Cytogenetics Nomenclature. Shaffer LG, McGowan-Jordan J, Schmid M (eds). Basel: Karger.
2) http://www.hgvs.org/mutnomen/SVtrans_HGVS2013_PT.pdf
3) Taschner PE, den Dunnen JT. Hum Mutat. 32:507-511 (2011).
P16.18-M
Concordance of methods for CNV detection from whole-exome
sequencing as compared with microarray: 100 sample autism cohort
L. J. Culot1, K. Schmitz Abe2, R. Keshavan1, S. Shams1;
1
BioDiscovery, Inc, Hawthorne, MA, United States, 2Department of Pediatrics Harvard
Medical School, Boston, MA, United States.
Copy number variants have been implicated as drivers of many developmental disorders, including autism spectrum disorder. The platform of choice to
detect genome-wide CNVs has traditionally been microarray (including SNP
arrays that can also detect copy neutral LOH regions); however, since samples have often undergone sequencing to discover pathogenic sequence variants, it is desirable to exploit these data to also detect CNVs. Various methods
have been proposed to detect CNVs from NGS data, but little information is
available which compare the success of these approaches to orthogonal methods. Using a large data set of constitutional samples from an autism cohort
that have been subjected to both whole-exome sequencing (WES) and to a
genome-wide SNP microarray, we compare the ability to detect CNVs between these platforms. Several algorithms for CNV detection from the WES
data are examined, and the results are compared with an established HMMbased method for detection of CNVs from the microarray platform.
P16.19-S
De Novo Assembly and Structural Variation Discovery in Complex
Genomes Using Extremely Long Single-Molecule Imaging
De novo genome assemblies using only short read data are generally incomplete and highly fragmented due to the intractable complexity found in most
genomes. This complexity, consisting mainly of large duplications and repetitive regions, hinders sequence assembly and subsequent comparative analyses. As a result of the remaining limitations of DNA sequencing and analysis technologies, it is not feasible to create similarly high quality assemblies
of individuals to detect and interpret the many types of structural variation
that are refractory to high throughput or short-read technologies.
We present a single molecule genome analysis system (Irys) based on NanoChannel Array technology that linearizes extremely long DNA molecules
for direct observation. This high-throughput platform automates the imaging of single molecules of genomic DNA hundreds of kilobases in size to
measure sufficient sequence uniqueness for unambiguous assembly of complex genomes. High-resolution genome maps assembled de novo preserve
long-range structural information necessary for structural variation detection and assembly applications. We have used Irys genome mapping for the
assembly and characterization of several genomes, including human, plant,
fungi, and bacteria.
In addition to describing the technology and analysis approaches useful for
dissecting complex genomes, we demonstrate results from several genomes,
where genome maps span remaining reference gaps, identify known and novel structural variants (including balanced rearrangements) and phase variation within haplotype blocks. We also resolve and measure long tandem
repeat regions that are likely impossible to assemble by other methods.
P16.20-M
Understanding complex gene-environment interactions leading to
impaired DNA methylation in colorectal cancer
We recently applied Methylation Sensitive-High Resolution Melting (MSHRM) technique to evaluate the methylation levels of APC, MGMT, hMLH1,
RASSF1A and CDKN2A genes in over 100 colorectal cancer (CRC) samples
and 80 adjacent healthy mucosa specimens (Copped F et al. Epigenetics
2014, DOI: 10.4161/epi.27956).
Our analysis revealed a correlation between hMLH1 methylation, a marker
of the CIMP high phenotype, with low folate levels, tumor location, increa-
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sing age, female gender, high number of methylated genes, and the TYMS
1494 6bp ins/del polymorphism. Increasing age correlated also with MGMT
methylation levels in CRC, and both tumor stage and the MTR 2756A>G polymorphism with RASSF1A methylation.
The APC gene resulted frequently methylated in both CRC tissues and
healthy adjacent mucosa specimens. APC methylation levels correlated with
the TYMS 1494 6bp ins/del polymorphism in CRC samples and with both
increasing age and the MTR 2756A>G polymorphism in healthy mucosa.
In order to shed some light on complex gene-environment interactions leading to impaired DNA methylation in CRC we are currently elaborating those
data by means of artificial neural networks (ANNs) searching for correlation with lifestyle factors evaluated by means of a detailed questionnaire on
lifestyles and dietary habits filled in by each CRC patient. ANNs are able to
understand non-linear relationships among studied variables and to highlight through a graph the complexity of connections among the studied variables.
The study was supported by Istituto Toscano Tumori (ITT).
P16.21-S
Copy Number Calling for Gene Panels with Next-Generation
Sequencing (NGS)
A. Vadapalli, J. Ghosh;
Agilent Technologies, Santa Clara, CA, United States.
Introduction: Copy number variation (CNV) was more and more investigated in complex diseases. We analysed CNVs of Fc fragment of IgG, low affinity IIIb, receptor (FCGR3B), a candidate gene in Rheumatoid Arthritis (RA).
Droplet Digital PCR was used to quantify CNVs and to identify tandem repeat. Patients and Methods: Thirty-eight RA trio families (one patient with
two parents) and seven control samples (with known copy number) were
genotyped using Droplet Digital PCR (ddPCR, BioRad), which performs an
absolute quantification using up to 20,000 reactions for one sample. Digestion reactions were done in the reaction mix for each sample with several
concentrations of endonuclease FokI enzyme, in order to separate replicated copies in tandem on the same chromosome. Results: CNVs quantification
showed 1 (9.65%), 2 (83.33%) or 3 (7.02%) copies. Comparison of results
from undigested and digested DNAs (2U of enzyme with 33ng of DNA) showed a difference in copy number (CN) estimation for samples with tandem
copies. The CN value obtained without digestion was under estimated in
comparison with digestion. However, when one copy of the gene was located
on each chromosome, the CN estimation was not different between the two
protocols. Conclusion: Use of ddPCR with digested and undigested DNAs allowed CNVs quantification for FCGR3B. We also highlighted recombination
mechanisms (tandem copies) leading to a better genotype characterization.
Further investigation of FCGR3B CNVs and their transmission in 200 trio families will be performed, to better understand the impact of these candidate
gene variations in RA.
ABSTRACTS POSTERS
P16.23-S
Variable R.Msp1 fragmentation in genomic DNA due to DNA
hypomethylation in CRF patients with MTHFR C677Tgene
polymorphism: from genetics to epigenetics
Background and Objective: The role of inflammation, hyperhomocysteinemia, familial genetic markers and epimutations remains incompletely understood in chronic renal failure(CRF). DNA methylation is a post-replicative modification mechanism that is strongly involved in the physiological
control of epimutations and gene expression. In the current study it was aimed to find out the possible role of epigenetic alterations in renal failure due
to functional MTHFR deficiency in CRF patients that requiring long-term
haemodialysis. Method: Current cohort includes 228 CRF patients and 212
healthy individuals from the same population. The MTHFR C677T SNP was
genotyped by real-time PCR analysis and genomic DNA fragmentation sizes
were correlated for wild, heterozygous and homozygous mutated CRF patients after methyl marker cognate enzyme of R.Msp1 digestion. Fragments
were compared by Scion Image histogram plot analysis. Results: Increased T
allele frequency was detected in CRF patients when compared to the health
individuals from the same population. MTHFR 677TT (homozygous) genotype was found 6.1% and the T allele frequency 2.5-fold increased in CRF
when compared with control group. Distinct global DNA methyl patterns
that showed variable R.Msp1 fragmentations were also detected in current
MTHFR gene mutated CRF patients when compare to the wild CRF patient.
Conclusions: The current results indicate that individuals with germ-line
MTHFR C677T mutations have a risk for CRF pathogenesis due to the reduced enzyme activity and global DNA hypomethylation. Results needs to be
confirmed with a larger scale of sample size.
P16.24-M
DNA methylation changes at the 24-dehydrocholesterol reductase
gene are associated with High Density Lipoprotein serum levels
Back to index
Pain is the most unpleasant symptom of illness, which is mediated by a variety agents released from local inflammatory cells. Recent studies points
to the involvement of epigenetic mechanisms both in the development
and maintenance of pain states. One of the most fundamental epigenetic
marks is the methylation of cytosine residues in DNA, catalyzed by DNAmethyltransferase enzymes. The aim of the present study was to analyze the
changes of DNMT1, DNMT3a and DNMT3b in the trigeminal ganglia (TG)
neurons during the maintenance phase of pain states and their modulation
by NSAID.
The Mustard oil ( 10%) was applied to four- month-age rats to induce acute inflammatory pain and responses to mechanical and heat stimuli were
assessed. All animal studies conformed to the Guidelines of International
Association for the study of Pain regarding investigations. The levels of DNMT1, DNMT3a and DNMT3b were measured in nuclear extracts of Trigeminal ganglia (TGs) neurons in study and control groups. DNMTs assay kits
were used to measure the amount of DNA- methyltransferases.
We found that the Mustard oil-evoked pain increased the levels of DNMT3a
and DNMT3b, while it has no significant effect on levels of DNMT1 in the
same regions compare to control sample. Previous administration of the
NSAID revials reduced levels of DNMT3a and DNMT3b.
This study provides the evidence that DNA methylation plays a role in development of pain in animal models. This has important implications for
understanding the mechanisms involved transition from acute to persistent
pain and for pain therapy.
P16.26-M
SNPs identification in duchenne muscular dystrophy female
carriers as biomarkers to discriminate symptomatic/asymptomatic
phenotypes
RNA-seq is a next-generation sequencing method able to characterize thoroughly gene expression profiles and to perform very accurate differential
gene expression analysis. High expression of some genes could both influ-
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ABSTRACTS POSTERS
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The human exome seems to represent a dynamic system in terms of interindividual variability. However, due to the lack of widely accepted criteria for
determining copy number variation (CNV) pathogenic value, the creation of
a map depicting the wide spectrum of benign CNV in the human exome is
hindered. To make a step forward on the road to the map of human exome
variations, we have evaluated 151 unrelated individuals using high-resolution CNV analysis by Affymetrix 2.7M SNP-array and an original bioinformatics technology. CNV affecting exons of intellectual disability genes were
associated with X-linked mental retardation, X-linked dominant conditions
and autosomal dominant intellectual disability. Exome CNV affecting X-linked mental retardation genes in males were as follows: duplication of one
OPHN1 exon (3 individuals) and four DLG3 exons (3 individuals), duplication of twenty six L1CAM exons (7 individuals). Benign intragenic CNV affecting X-linked dominant genes in females were associated with duplication of
twelve SMC1A exons (Cornelia de Lange syndrome 2) in 8 individuals and
duplication of six PORCN exons (Focal dermal hypoplasia) in 3 individuals.
Benign exome variations affecting genes associated with autosomal dominant intellectual disability were associated with deletion of one SMARCA2
exon (3 individuals) and duplication of one TSC1 exon (12 individuals),
KANSL1 duplication (18 individuals) and deletion of two NHEJ1 exons (5 individuals). Our data demonstrate that benign exome CNV affecting intellectual disability genes are relatively common in humans representing a pitfall
for molecular diagnosis of genomic and single-gene disorders. Supported by
the Russian Federation President Grant (MD-4401.2013.7).
P16.28-M
Effects of the culture condition and chromatin remodeling agents on
the epigenetics of organotypic cultures
Alterations of the epigenetic pathways are established as hallmarks of tumorigenesis, together with genetic/genomic variations. Tumor epigenetic
alterations are of increasing relevance to clinical practice, because they are
important druggable targets for cancer therapy using chromatin remodeling agents (CRAs). New evidences highlight the relevance of microenvironment on the epigenetics and the need to use culture models that preserve
the tissue morphology, to better understand the mode of action of CRAs.
We studied the epigenetic response induced by culture condition and CRAs
treatment, in a preclinical model based on organotypic culture from normal
and neoplastic lung specimens, that preserves ex vivo the original tissue
microenvironment and morphology. We assessed the expression pattern of
histone deacetylases (HDACs) and methylation profile of long interspersed
nuclear elements LINE1s and a panel of tumor suppressor genes. We observed a different behavior of the organotypic culture respect to that reported
for other ex vivo models. Interestingly, culture induced an overall increase
of LINE1s methylation, whereas CRAs caused LINE1s demethylation. Differently, culture and CRAs induced opposite effects on the genes: the samples
responded specifically, showing demethylation for some promoters and increased methylation for others. Moreover, we noted that the culture caused
alterations in the HDACs expression pattern.
These overall data reveal the importance of the maintaining the cells in their
original organ architecture to study the mode of action of the CRAs, and suggest that CRAs do not work only in a non-specific way, as previously thought,
in particular on the gene promoters.
P16.29-S
Bioinformatic approaches for next generation sequencing variant
calling in matched sensitive-resistant ovarian tumor pairs
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P16.30-M
Benign copy number variations in the human exome: focusing on
intellectual disability genes
P16.31-S
The Utility of Clinical Diagnostic Exome Sequencing in Adults with
Suspected Genetic Disorders
ABSTRACTS POSTERS
with osteoporosis and aseptic meningitis while the remaining two exomes
were negative. We conclude that CDE is a clinically helpful tool both for the
diagnosis and management of adults with complex phenotypes and that it
should be considered early in the evaluation of such individuals.
P16.32-M
A bioinformatics pipeline to identify candidate disease-causing
variants from exome sequencing data
In a three year pilot study we sequence around 900 exomes of patients suffering from various genetic disorders. In these patients the disease causing
variants are unknown: either known disease-related genes have initially
been found to be unaffected, or the cause of the disease is generally unclear.
Depending on the expected mode of inheritance either patient/parent trios
or only the patients are sequenced.
From the total set of single nucleotide variants (SNVs) and small indels, we
remove common polymorphisms and calling artifacts by comparison to the
1000 Genomes database and an in-house control database. From the remaining variants we identify a candidate set based on the assumed mode of
inheritance, which are then prioritized based on the predicted functional
effect of the variant and the tolerance of the gene to coding mutations. In addition, network analysis of the candidate genes together with a list of genes
known to be associated with the particular disease is performed to identify
the most promising candidate(s) which then undergo functional evaluation.
We found a rare homozygous mutation in the COQ6 gene in a nephrotic syndrome patient. A homozygous mutation in the same gene was reported earlier to be associated with nephrotic syndrome and sensorineural deafness.
Since the patient also suffers from severe hearing impairment, it is highly
likely that the candidate variant is the cause of the symptoms. This case emphasizes the efficiency of our filtering and prioritization strategy.
P16.33-S
Implementation of an IT-platform for the multicenter analysis and
clinical annotation of exomes of 250 children with intellectual
disability
The introduction of exome and whole genome sequencing into clinical diagnostics requires new structures for sequencing and data analysis. In principle, data analysis in this context is as simple as comparing a list of variants
identified in a patient with a comprehensive list of disease causing variants.
In practice, this is currently limited by the substantial number of DNA variants with false annotation or uncertain significance.
We set up an IT environment that supports central sequencing and automated primary data analysis and subsequently provides the results via a web
interface to researchers and geneticists for manual curation, annotation and
experimental validation.
As a pilot project, three cooperating diagnostic teams are investigating 250
trios consisting of patients with severe to mild ID and their healthy parents
for which high coverage exome sequences have been generated.
Variant data including Pindel and CNV calls are generated by an analysis
pipeline and stored in a database. Preliminary analysis revealed 1.7 de novo
non-synonymous coding and canonical splice site mutations per patient.
20% of the investigated cases carried a mutation in genes already known
to cause ID. In addition, evidence for novel ID candidate genes is being generated from the detection of SNVs and indels in specific genes from known
microdeletion regions.
In summary, we show that exome sequencing in a multicenter setting with
an appropriate IT environment can efficiently be used to generate clinical
diagnoses by integrating the advantage of standardized central sequencing
and distributed evaluation of the resulting data in the clinical context.
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P16.34-M
Unbiased functional clustering of gene variants with a phenotypiclinkage network
F. Honti, C. Webber;
MRC Functional Genomics Unit, University of Oxford, Oxford, United Kingdom.
Groupwise functional analysis of gene variants is becoming standard in nextgeneration sequencing studies. As the function of many genes is unknown
and their classification to pathways is scant, functional associations between genes are often inferred from large-scale omics data. Such data types
- including protein-protein interactions and gene co-expression networks
- are used to examine the interrelations of the implicated genes. Statistical
significance is assessed by comparing the interconnectedness of the mutated genes with that of random gene sets. However, interconnectedness can
be affected by confounding bias, potentially resulting in false positive findings. We show that genes implicated through de novo sequence variants are
biased in their coding sequence length and longer genes tend to cluster together, which leads to exaggerated p-values in functional studies; we present
here a method that addresses these bias. To discern molecular pathways
relevant to complex disease, we have inferred functional associations between human genes from diverse data types and assessed them with a novel
phenotype-based method. Examining the functional association between de
novo gene variants, we control for the heretofore unexplored confounding
bias in coding-sequence length. We test different data types and networks
and find that the disease-associated genes cluster more significantly in an
integrated phenotypic-linkage network than in other gene networks. We
present a tool of superior power to identify functional associations among
genes mutated in the same disease even after accounting for significant sequencing study bias and demonstrate the suitability of this method to functionally subcluster gene variants underlying a complex disorder.
P16.35-S
VarElect: phenotype-based variation prioritizer in GeneCards
G. Stelzer, A. Alkelai, T. Olender, S. Zimmerman, M. Safran, D. Lancet;
Weizmann Institute of Science, Rehovot, Israel.
Background WGS/WES translation from research to a clinical setting requires a governance framework that addresses: informed consent for
clinical and research genetic testing management of findings storage of
clinical and genetic data Development of a protocol for governance review
has catalysed local policy formulation in this rapidly developing field which
accommodates clinical and research regulatory regimes, the interests of researchers, clinicians, patients and families, and diverse approaches to the
risk and handling of incidental findings. Methods Extensive discussions
were held between researchers, clinicians, genetic counsellors, clinical scientists and ethicists to gauge expectations and areas of potential divergence
of interests. Reviews were undertaken of current bioethics literature. European and US guidelines and existing WES/WGS patient consent documents
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were used as the basis for discussion and development. Final patient documents were reviewed by patient groups. Results Multidisciplinary discussions have resulted in a protocol which anticipates uncertainties in results
and interpretation. A phased approach to consent will be taken, in which
patients can elect to undergo a process which begins with current clinical
testing, progresses to a targeted analysis of WES/WGS data and ends with
analysis of the full sequence. All findings will be subject to clinical verification and a multidisciplinary committee will determine whether and how incidental findings relating to serious and clinically actionable diseases should
be reported according to consent. Future Work Will involve monitoring and
evaluation of the consent process, MDT decision process, participant engagement and qualitative research into participant understanding, attitudes
and preferences concerning WGS/WGS.
P16.37-S
Genome-wide DNA methylation analysis identifies novel differentially
methylated regions in patients with imprinting disorders
Genomic imprinting is the regulation of gene expression by parent of origin. Imprinting is maintained by epigenetic mechanisms including parent
of origin-specific DNA methylation, and its disruption leads to imprinting
disorders affecting growth, metabolism and predisposition to cancer. Eight
classical imprinting disorders are known, linked to (epi)mutations of specific loci. However, some individuals have methylation anomalies affecting
multiple imprinted loci (HIL) throughout the genome. Here we used epigenomewide analysis to investigate aberrant DNA methylation in HIL patients.
We used the Illumina Infinium Human Methylation450 BeadChip array to
assay genomic DNA methylation at ~475,000 sites in ten HIL patients with
two clinical presentations (Beckwith-Wiedemann syndrome and neonatal
diabetes). We developed a novel informatic pipeline capable of small sample
number analysis, and statistical criteria to quantify DNA methylation.
The pipeline robustly detected hypomethylation at known imprinted loci,
and 25 further candidate imprinted regions (nine shared between patient
groups) including one in the Down syndrome critical region (WRB) and another previously associated with bipolar disorder (PPIEL). Targeted analysis
of three candidate regions (NHP2L1, WRB and PPIEL) confirmed allelic expression, methylation patterns consistent with allelic maternal methylation,
and frequent hypomethylation among HIL patients.
This study has identified new candidate imprinted genes, and shown a remarkable epigenetic similarities between patients with different imprinting
syndromes. Our informatic methods make possible epigenomic profiling of
small groups or even individual patients, and have potential to expand our
understanding of epigenetic regulation in health and disease.
P16.38-M
Genomic imprinting defects: role of cis-acting elements and transacting factors
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role of novel cis-acting elements and trans-acting factors in the origin of the
imprinting defects is being investigated further by high-throughput analysis
of ZFP57 binding in human and mouse cells cells and 11p15.5- and exometargeted resequencing of patient DNAs.
P16.39-S
PECAS: Prokaryotic and Eukaryotic Classical Analysis of Secretome
J. L. Lavin1, A. R. Crtazar1, J. A. Oguiza2, A. M. Aransay1;
1
CIC bioGUNE, Derio, Spain, 2Public University of Navarre, Pamplona, Spain.
A. Zadissa;
EMBL-European Bioinformatics Institute, Hinxton, United Kingdom.
ABSTRACTS POSTERS
del using Akaike Information Content criterion and assigned each mutation
to a specific GMM clone. We calculated mutational signatures of each GMM
clone according to the procedure proposed by Alexandrov (Nature, 2013)
and estimated distance between them. We identified a single clone common
to all relapses but not LLA, we built parent/child relationships with other
clones using distance among signatures. Our analysis reveals a branching
evolution of clones that putatively diverge in response to the clinical treatment. We show GMM is an effective and unbiased technique that can be applied to deconvolve clonal populations in cancer data only using allele frequencies. Clones can be then characterized by their mutational landscape,
this information is sufficient to build clonal relationships when studying the
evolution of the disease.
P16.42-M
H3M2: Detection of Runs of Homozygosity from whole-exome
sequencing data
Runs of homozygosity (ROH) can be defined as sizable chromosomal stretches of homozygous genotypes, ranging in length from tens of kilobases
to megabases. ROHs can be relevant for population and medical genetics,
playing a role in predisposition to both rare and common disorders. ROHs
are commonly detected by SNP microarrays, but attempts have been made
to use Whole Exome Sequencing (WES) data. Currently available methods
developed for the analysis of uniformly spaced SNP-array maps do not fit
easily the sparse and non uniform distribution of the WES target design. To
meet the need of an approach specifically tailored to WES data we developed H3M2, an original algorithm based on Heterogeneous Hidden Markov
Model that incorporates inter-marker distances to detect ROH from Whole
Exome Sequencing (WES) data. We evaluated the performance of H3M2 to
correctly identify ROHs on synthetic chromosomes and examined its accuracy in detecting ROHs of different length (short, medium and long) from
real 1000 genomes project data. H3M2 turned out to be more accurate than
GERMLINE and PLINK, two state-of-the-art algorithms, especially in the
detection of short and medium ROHs. H3M2 is freely available at https://
sourceforge.net/projects/h3m2/.
P16.43-S
Hepatitis C Virus genome variability analysis from high-throughput
pyrosequencing data
High-throughput pyrosequencing enables discovery of rare Hepatitis C Virus (HCV) variants and estimation of viral diversity within a host, which may
play an important role in understanding patients response to personalized
therapy. The aim of our study was to estimate intra-host genomic variation of HCV 1b in nave and Pegylated Interferon-Ribavirin treated patients.
Serum viral RNA from 11 patients was reverstranscribed and amplified to
produce 7.5 kb-long amplicons that were random-fragmented. A sequencing
library was generated by specific adaptor-ligation and analyzed using Roche 454 Titanium chemistry and GS FLX platform. Data were analyzed using
software published by The Broad Institute. Reads from each patient were
assembled into contigs (Vicuna), oriented and frameshift-corrected (V-Fat).
Raw-reads were corrected for pyrosequencing errors (RC454) and sequence
variants were called using V-Phaser 2.0 and analysed with V-Profiler. The
HCV 1b Con1 isolate was used as control sample. The sequencing run resulted in over 1 million passed filter reads with an average length of 397 bases
and 30.81 quality score. Sustained viral response was associated to lower
number of non-synonymous mutations within the initial viral population
compared to non-responders. Also, NS2 mutation frequency seems higher
in non-responding patients. High-throughput pyrosequencing is an effective
tool for assessing intra-host variability of HCV genome, able to reveal differences in genomic variation of viral communities from patients with diverse
response to therapy. Acknowledgement: This work was supported by a grant
of the Romanian National Authority for Scientific Research, CNDI-UEFISCDI,
project number 88/2012, PN-II-PT-PCCA-2011-3.2 (GO).
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P16.44-M
Systems biology approaches to the search for disease genes in
Hirschsprungs disease
The availability of new methodologies of high performance has revolutionized our ability to discovery. However, the high resolution of such technologies can become a double-edged sword. Reduced sample sizes available in
rare diseases are often an obstacle for the detection of candidate genes or
the study of their molecular basis. The limitations of the approaches based
on isolated genes can be overcome using systems biology approaches.
This study shows as a combination of pathway-based analysis (PBA) and
network analysis allowed to discover four new loci (RASGEF1A and IQGAP2,
DLC1 CHRNA7) related to signalling and migration processes associated
with the disease, which were then validated in a cohort of 106 independent
trios.
The further study of an international cohort of 162 HSCR trios allowed us
to confirm the molecular bases of disease using PBA. We found a significant
association of processes related to signalling and its regulation as well as
formation of the enteric nervous system. Although the genes associated
with HSCR varied in different populations, the functions and interaction of
affected networks of proteins were always the same, which reflects the complexity of the disease. This methodology of prioritization of candidate genes
can be extrapolated to any technology of high performance (WES, RNA-seq,
etc.).
P16.45-S
The impact of antisense transcription on epigenetic signals
Amniotic derived cells are ideal seed cells for regenerative medicine proto-
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ABSTRACTS POSTERS
cols since they conjugate a remarkable plasticity to safety properties. This
study investigated the role exerted by human amniotic cells during the process of tendon healing, evaluating by microarray technique, the presence
of transcriptome variations in human amniotic epithelial (hAECs) and mesenchymal (hAMCs) stem cells when xenotransplanted into the preclinical
ovine model of tendon defect, compared to freshly isolated ones. This analysis allowed to understand in which way, after transplantation, hAECs and
hAMCs transcripts have been affected.
Functional analysis of the affected transcripts revealed that the main biological functions involved are: Cell Death and Survival, Cellular Growth and
Proliferation, Inflammatory Response, Cellular Function and Maintenance,
Skeletal and Muscular System Development and Function and Connective
Tissue Development and Function.
These results show that hAECs and hAMCs after xenotranplantation produce
a modulation of the genes involved in their survival, and in the inflammatory response. Intriguingly, it has been observed also the up-expression of
the transcripts related to the connective tissue development and function
as COL11A1 which is involved in the synthesis of collagen type I, the most
expressed protein in the tendon. In conclusion, the present study demonstrates that human amniotic derived cells have a direct involvement in new
tendon matrix remodeling and tissue regeneration. Altogether these results
strongly support the idea that human amniotic cells could be effectively proposed as a prompt stem cell-based therapy that does not require any preliminary in vitro differentiation or any genomic transfection.
P16.47-S
Genomewide characterization of imprinted methylation in humans:
Implication for patients with multilocus methylation defects
Differential methylation between the two alleles of a gene has been observed at imprinted regions, where the methylation of one allele occurs on a
parent-of-origin basis, the inactive X-chromosome in females, and at those
loci whose methylation is driven by genetic variants. We have extensively
characterized imprinted methylation in a substantial range of normal human tissues, reciprocal genome-wide uniparental disomies and hydatidiform moles, using a combination of whole genome bisulphite sequencing
and high-density methylation microarrays. This approach allowed us to define methylation profiles at known imprinted domains at base-pair resolution, as well as identifying 24 novel loci harbouring parent-of-origin methylation, 18 of which are restricted to the placenta. We observe that the extent
of imprinted differentially methylated regions (DMRs) is extremely similar
between tissues, with the exception of the placenta. Further we profiled all
imprinted DMRs in sperm and embryonic stem cells derived from parthenogenetically-activated oocytes, individual blastomeres and blastocysts to
identifying primary DMRs and reveal the extent of reprograming during
pre-implantation development. These results have important implications
for individuals with imprinting disorders with underlying multi-locus methylation defects, including Transient Neonatal Diabetes Mellitus patients
with ZFP57 mutations.
P16.48-M
Computational pipeline to analyze genomic variants with respect to
clinical phenotypes by mining literature. Study of genomic regions
related to intellectual disability
E. Pranckeviien, A. Pranculis, E. Preikaitien, V. Kuinskas;
Department of Human and Medical Genetics, Vilnius University, Lithuania.
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Precise spatiotemporal regulation of splicing is mediated by splicing ciselements on pre-mRNA. The next generation sequencers disclose a large
amount of single nucleotide variations (SNVs) in the human genome. Tools
to analyze the effects of SNVs on the protein functions have been recently
published PolyPhen-2 and SIFT. SNVs affecting intronic cis-elements potentially compromise splicing, but no dedicated tool has been available. We thus
sought for a prediction model. According to the effect size analysis of each
nucleotide at intronic positions -50 to -3 on alternative splicing, we extracted 34 parameters that possibly dictated the strength of splicing signals. We
also partitioned all the alternative 3 splice sites in the human ENSEMBL
annotation into 500 categories and generated a neural network model. The
model predicted the efficiency of alternative splicing events with a correlation coefficient greater than 0.8 for a validation dataset. The model discriminated splicing consequences of intronic single nucleotide variations in
dbSNP134 and the Human Gene Mutation Database. We next compared the
ENSEMBL-based model with models deduced from RNA-seq of normal human brain, cerebral cortex, heart, liver, skeletal muscle, and colon, and found
that a colon-based model yielded the best discrimination. We created a web
service program, IntSplice. IntSplice is the first tool for predicting splicing
consequences of nucleotide variations at intronic positions -50 to -3.
P16.50-M
Genome-wide methylation analysis in Klinefelter syndrome
Background: Epigenetic changes such as DNA methylation have been proposed to play a role in human disorders such as psychiatric, autoimmune
and metabolic diseases. Klinefelter syndrome (KS) is associated with an
increase risk of these disorders, however no study to date have investigated global methylation changes in patients with KS. The aim of this study
is to investigate the global methylation pattern in KS. We hypothesize that
the methylation pattern is changed in KS and that the methylome changes
will explain phenotypic characteristics present in KS. Methods: We performed genome-wide DNA methylation analysis on blood leucocytes from 73
patients with KS and 73 age- and gender-matched controls using the Illumina Infinium Human Methylation 450K BeadChip. Results: 70.525 CpGsites covering over 15.000 genes were found to be differentially methylated
in patients with KS compared to the age-matched controls. Among these
61.567 were on autosomal chromosomes, 8903 were on the X-chromosome
and 55 were on Y-chromosome. One of the genes with promotor associated
differentially methylated CpG sites is the NSD1 gene which is involved in
the androgen receptor (AR) transactivation. Other genes possibly involved
in the phenotype of KS and differentially methylated were ABI3BP, APOB,
C1orf59, CACYBP, DPPA5,GABRG1, HOXA4, LRRC61, NLRP2, PEX10, RPLP1,
RFPL2, SDHAF1, SPEG. Conclusions:For the first time we show that KS is associated with pervasive genome-wide methylation changes, changes which
could play a role in the clinical phenotype seen with KS.
ABSTRACTS POSTERS
P16.51-S
Locus Reference Genomic (LRG) records: reference resource for the
reporting of clinically relevant sequence variants
Back to index
Cutaneous melanoma is the most fatal skin cancer and, although some effective molecular therapies exist, novel targets and drugs are still needed. To
provide new insights for novel targets discovery, we performed an extensive characterization by next-generation sequencing (NGS) of a collection of
melanoma cell lines derived from metastatic cases. Samples were profiled
by whole-exome sequencing (WES) and RNA-sequencing using Illumina
technology. Starting from WES data, we developed a bioinformatics pipeline to catalogue 2,172 novel mutations affecting genes in melanoma key
pathways targeted by current therapies (MAPK and glutamate pathways) as
well as genes never described for melanoma [Cifola, 2013]. Moreover, WES
data were used to perform copy number alteration (CNA) analysis using a
novel software developed by us, called Excavator, which is very sensitive and
precise in DNA copies estimation even in situations of great sample heterogeneity [Magi, 2013]. CNA results were concordant with 250K SNP Array
data and used to explore CN state of mutated genes. To collect and share these results, we created a Melanoma Exome Database (https://155.253.6.64/
MExDB/). On the same samples, we also carried out RNA-sequencing and
performed both a traditional gene expression analysis and more sophisticated structural evaluations. Focusing on fusion transcripts, we identified
72 putative events generated by either inter-chromosomal translocations
or intra-chromosomal rearrangements, recently defined conjoined genes
and representing an additional gene regulation mechanism. Globally, NGS
proved to be extremely powerful to dissect cancer complexity at both genomic and transcriptomic levels, and to identify novel potential targets for
personalized treatment of cutaneous melanoma.
P16.54-M
Integrated sequence analysis pipeline provides one-stop solution for
identifying disease-causing mutations
H. Hu1, T. F. Wienker1, L. Musante1, V. M. Kalscheuer1, P. N. Robinson2, H. Ropers1;
1
Max-Planck Institute for Molecular Genetics, Berlin, Germany, 2Universittsklinikum
Charit, Berlin, Germany.
Next-generation sequencing has greatly accelerated the search for diseasecausing defects, but even for experts the implementation of data analysis can
be a major challenge. To facilitate the data processing in a clinical setting, we
have developed a novel Medical Re-sequencing Analysis Pipeline (MERAP).
MERAP assesses the quality of sequencing, and has optimized capacity for
calling variants, including Single Nucleotide Variant, insertion and deletion, Copy Number Variation, and other structural variants. MERAP identifies
polymorphic and known causal variants by filtering against public-domain
databases, and flags non-synonymous and splice-site changes. MERAP uses
a logistic model to estimate the causal likelihood of a given missense variant. MERAP considers the relevant information such as phenotype and
interaction with known disease-causing genes. MERAP compares favorably
with GATK, one of the widely used tools, because of its higher sensitivity for
detecting indels, its easy installation, and its economical use of computational resources. Upon testing more than 1,500 individuals with mutations in
known and novel disease genes, MERAP proved highly reliable, as illustrated
here for 5 families with disease-causing variants, including a novel ANKS1A
mutation identified in a patient with autosomal recessive non-syndromic
intellectual disability. We believe that the clinical implementation of MERAP
will expedite the diagnostic process of many disease-causing defects.
P16.55-S
Epigenome-wide analysis identified highly significant age-related
DNA methylation changes
Aging is associated with an increased risk for many complex diseases. DNA
methylation represents one of the most promising biomarkers of aging. To
evaluate the effect of age on DNA methylation, we examined the methylation
levels of more than 450K CpG sites in 206 cases and 206 matched controls
belonging to the Italian section of the EPIC cohort. EPIC healthy volunteers
were recruited between 1994-98 and followed up for myocardial infarction
and other diseases. For the CpG methylation level assessment on blood DNA
we used the Illumina HumanMethylation450 BeadChip. Data were analyzed
according to standard procedures (MethyLumi, Bioconductor). All analyses
were corrected for sex, BMI, season, center of recruitment, cellular subtypes
315
ABSTRACTS POSTERS
and batch effect. Linear regression analysis showed that 10,000 CpG sites
were significantly associated with age after Bonferroni correction (p<10-7).
Positive correlation with age was found for 5,687 CpG sites (57%), whereas
4,313 CpG sites (43%) were negatively correlated with age. The top four
significant loci (p<10-28) were located in the CpG islands of ELOVL2 and
FHL2 genes. Methylation levels of the 4 CpG sites were positively correlated
with increasing age, according to previously published results. These results confirm that DNA methylation alterations occur during aging and that
ELOVL2 and FHL2 genes could be used as biomarker of aging. Moreover,
the methylation differences associated with age could help in understanding molecular mechanisms that underlie the development of age-related
diseases.
P16.56-M
Whole genome methylation analysis in idiopathic generalized
epilepsies
Genetic factors play a predominant role in the etiology of common idiopathic generalized epilepsy(IGE) syndromes. An increased rate of maternal
inheritance and excess number of affected females implicate involvement of
epigenetic effects in the etiology of IGE syndromes.
In this study, we have performed a whole genome methylation analysis for
15 parent-offspring trios with vertical inheritance of the IGE trait. DNA
samples were subjected to bisulphite conversion prior to IlluminaHuman
Methylation 450K Beadarray application. Beadchips were scanned through
Illumina iScan platform. We used a comprehensive R-Bioconductor package,
namely RnBeadsV0.99.11, which implements an analysis workflow that
can be used for paired and unpaired differential methylation analysis.
After data normalization, quality control, filtering and methylation profiling
steps, differential methylation analysis revealed 239 genes with p-values
smaller than 0.05. As an alternative approach, we have run a pathway-based
analysis to see if genes with differential methylation values map to common
pathways. This analysis with PANOGA software revealed various pathways
particularly with synaptic and immunological functions.
Analysis of high throughput methylation data has proven to be challenging
where various pipelines with alternative normalization and filtering methods should be applied. Two approaches presented herein have resulted in
identification of new epilepsy related genes and pathways that could as well
be applied to analysis of other trio based methylation studies.
P16.57-S
Molecular characterization of a de novo mutation in a pediatric
patient with isolated ectopia lentis as a diagnostic criterion for
Marfan syndrome
Marfan syndrome is an inherited multisystem disorder that affects the connective tissue. The typical onset is in adulthood and often occurs in children
with isolated clinical signs. The revised Ghent criteria allow to confirm the
diagnosis if ectopia lentis is in concomitance with a mutation in the FBN1
gene definitely associated with Marfan syndrome. Our patient (5 years old)
presents a mutation (p. C154Y) never reported in the literature, hence the
difficulty in establishing a diagnosis. We perform molecular dynamic simulation to investigate how this mutation affects the protein stability. Analysis
was carried out on the mutated form of the N-terminal domain of the human fibrillin-1 (pdb code 2M74) . Structural analyses indicates that mutation perturb the secondary structure of the protein, which reduce the folding
stability of the system. The C154Y mutation disrupts the C154-C166 disulfide bridge and consequently the two antiparallel beta sheets are impaired.
In conclusion, the mutation leads to a destabilization of the native folding
of the protein, and therefore a probably reduction of the physiological activity. The patient is monitored in follow-up because it has all probability of
developing the Marfan syndrome. This study emphasizes the utility to characterize a mutation through molecular modeling in all late-onset diseases
in order to correct follow up of patients.
P16.58-M
Prediction of LHX1-target genes relevant to Mllerian aplasia
316
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LHX1 is a putative transcription factor widely distributed among vertebrates. In mice, Lhx1 acts as a high-hierarchy regulator during urogenital development, as female Lhx1-null mutants lack Mllerian derivatives. Deletions
and point mutations affecting LHX1 have been found in female patients with
Mllerian aplasia (MA). However, the low recurrence of these alterations
indicates that this disorder is multifactorial, and few reports using highresolution genome mapping techniques have been published. Hence, the
identification of candidate genes for MA demands deeper analysis. In order to identify candidate genes for MA, we used a bioinformatic approach
to predict LHX1-target genes, and the results were contrasted against the
set of genes with copy-number alterations compiled from the microarray
reports on MA patients. Starting from 4 documented Lhx1 regulation targets
in Xenopus and their homologs in 5 other species, a highly conserved 50-nt
signature was identified (e-value 1.34e-36). Several known transcription factor-binding sites were contained in such signature, including those of HOXA5 and HNFB1, which have been previously associated to MA. The refined
motif was further searched against the human genome promoter regions,
and 45 putative target-genes of LHX1 were identified. Of these, 38 genes are
expressed in uterine tissues, 19 during embryogenesis, and 15 in both conditions. Two genes, NPHP1 and PIAS3, were found deleted in MA patients. In
conclusion, our approach will be useful to prioritize genes relevant to Mllerian development and may help to establish future validation protocols.
P16.59-S
Age at onset and disease severity in primary progressive multiple
sclerosis: a genome-wide association study, pathway and network
analysis
Thymomas, epithelial neoplasms of the thymus, are present in about 1020% of the patients affected by Myasthenia Gravis (MG), an autoimmune
disease characterized in almost 80% of the cases by auto-antibodies against
the nicotinic acetylcholine receptor (AChRAb). Changes in DNA methylation
might contribute to the risk of thymomas but the role of epigenetics in MG
and MG-associated thymomas is still not clarified.
Polymorphisms in one-carbon metabolism genes, the key pathway for nu-
ABSTRACTS POSTERS
cleotide synthesis and DNA methylation, have been often linked to aberrant
DNA methylation and risk of various types of cancer. We investigated four
polymorphisms in genes of this pathway, namely methylenetetrahydrofolate
reductase (MTHFR) 677C>T, thymidylate synthase (TYMS) 28 bp repeats,
DNA methyltransferase (DNMT3B) -149C>T and DNMT3B -579 G>T, in 110
AChRAb+ MG patients with thymoma (64 females and 46 males, mean age
56.113.1 years) and 429 matched healthy controls (276 females and 153
males, mean age 61.416.8 years). No difference in allele and genotype frequencies was observed between patients and controls in MTHFR and TYMS
gene polymorphisms but an increased frequency of both the DNMT3B
-579TT genotype (P = 0.03) and of the combined DNMT3B -579TT/-149CT
genotype (P = 0.01) was observed in the total cohort of thymoma patients
with respect to controls. After gender stratification both associations resulted significant in males. We are currently evaluating the methylation levels
of the promoters of tumour-related genes, such as CDKN2A, hMLH1, and
MGMT, in thymoma cells of MG patients, searching for correlation between
DNA methylation levels and DNMT3B polymorphisms.
P16.61-S
Behind the scenes: the hidden challenges of exome sequencing in
consanguineous populations
Removing frequently detected variants is one of the most effective approaches to reduce the number of candidate mutations in the data analysis of
next-generation sequencing studies. The incidence of a
rare disorder in a population serves as an upper bound for the allele frequency or genotype frequency that can be used as a filter for dominant or
recessive disorders. However, the frequentist inference
requires that genotypes of a single individual are represented in the database only once. With many and decentralized data submitters the risks increases that samples of the same individual are sequenced multiple times
and are contributed independently under different pseudonyms. We developed a metric that computes the distances to reference samples of the 1000
genomes project. The distance profile of a sample is a unique signature that
may be used to assess whether a
list of sequence variants has already been submitted. We show that this
distance signature is highly specific for a sample but still error tolerant.
This allows the identification of replicates from different enrichment procedures, sequencing platforms and bioinformatics pipelines. Furthermore
the distance signature of a sample provides also a possibility to identify a
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High data quality and accuracy are recognized characteristics of Sanger resequencing projects and are primary reasons that next generation sequencing projects compliment their results by capillary electrophoresis data
validation. We have developed an on-line tool called Primer Designer to
streamline the NGS-to-Sanger sequencing workflow by taking the laborious
task of PCR primer design out of the hands of the researcher by providing
pre-designed assays for the human exome. The primer design tool has been
created to enable scientists using next generation sequencing to quickly
confirm variants discovered in their work by providing the means to quickly search, order and receive suitable pre-designed PCR primers for Sanger
sequencing.
Using the Primer Designer tool to design M13-tailed and non-tailed
PCR primers for Sanger sequencing we will demonstrate validation of 28variants across 24-amplicons and 19-genes using the BDD, BDTv1.1 and
BDTv3.1 sequencing chemistries on the 3500xl Genetic Analyzer capillary
electrophoresis platform.
P16.64-M
Towards integrative family analysis on OMICs data for individual
patient diagnostics
During recent years generating OMICs data became cheaper and faster. Notably DNA sequencing benefited from this change, becoming more popular
and frequently used, specially in the domain of diagnostics. As became apparent, the data from DNA sequencing does often not allow for conclusive
diagnostics, as it only shows one aspect of what contributes to the patients
phenotype. The combination of OMICs data like genomics, transcriptomics
and proteomics presents itself as a solution to this problem, especially in the
context of family analysis which helps to identify interesting features. While
many methods have been developed combining different OMICs sources to
create more complete and comprehensive analyses of a patients genotype,
doing family analysis in the context of OMICs data with an user friendly visualization of the data is still explored very little. We present our vision of a
comprehensive, user friendly application which concentrates on several key
features of OMICs data for comparative analysis. Our vision concentrates on
family analysis of three key features. Individual SNP comparison on genomic data, gene expression comparison with transcriptomic data and protein
variation from proteomics data. While those features are well understood
individually, our vision is to create an easy to use tool to combine those three
data sources based of GensearchNGS, an application that already provides
the required genomics tools and allows for visualization of the analysis and
underlying data. Examples will pertain to genetic disorders in myopathies,
where we recently could show some progress regarding functional understanding and therapy (Walter et al., 2013)
P16.65-S
Transcription as a key determinant of the DNA methylation landscape
in oocytes
L. Veselovska, S. Smallwood, P. Ewels, S. Andrews, G. Kelsey;
The Babraham Institute, Cambridge, United Kingdom.
317
ABSTRACTS POSTERS
in oocytes. Conversely, most hypomethylated domains are transcriptionally
silent. We also conclude that methylated CGIs are predominantly intragenic, while unmethylated CGIs are intergenic or at active TSSs. To test the
association functionally, we investigated the imprinted gene Zac1; deletion
of its oocyte-specific promoter led to absence of DNA methylation across its
entire gene body.
These results suggest that perturbations in transcriptional regulation during oogenesis could have profound effects on the oocyte methylome and
may provide a link between aberrant methylation and ART.
P16.66-M
The influence of genetics on personality development
Personality is known as hereditary to a certain extent. In this work we attempt to classify personality traits as binary traits based on genetic information only. For this we used the 60-item NEO-FFI and over 8 million SNPs
from 6655 Dutch participants. For feature selection we performed a genome-wide association for each personality trait in a five-fold cross validation
setup. All SNPs with a p-value <0.01 were chosen as predictors for a given
fold and a given personality trait, amounting to approximately 2,500 associated SNPs for each trait. An artificial neural network was trained with the
SNPs as input and the personality scores as output. We found it possible to
classify a persons personality to the two sides of the scale significantly better than random. The results of this study prove in a novel way that genetics
have an influence on personality. The next step is to identify, which genes
these SNPs belong to, which hopefully will lead to a greater understanding
of the processes involved in personality development and the onset of personality disorders.
P16.67-S
Analysis of GSTP1 promoter hypermethylation in urine samples of
Bulgarian prostate cancer patients and controls
318
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proportion of common diseases, such as schizophrenia or autism, previously thought to be due to complex multifactorial inheritance, are now thought
to represent a heterogeneous collection of rare monogenic disorders (Mitchell KJ, Porteous DJ, 2011), the large majority of which is still unknown. For
the efficient investigation of genetic mutations next generation sequencing
(NGS) technology has revolutionized molecular diagnostics. Its big advantage is the ability to sequence enormous amounts of nucleic acids in a short
time at an affordable cost. However, the benefits come with a number of
challenges like NGS data management, quality control, mapping, variant calling and their annotation. We have developed a mutation screening and decision support for analysis of data generated by most common sequencing
platforms: Illumina, 454, Ion Torrent, and Sanger Sequencing. The system
furthermore allows the clinician to identify causal mutations through NGS
data analysis and suggests which regions need to be validated with ABI Sanger sequencing. This makes it much easier to the scientist and the clinician
to find causative Mutations. Samples of multiple patients can be processed
and their mutations can be compared with ease. Keeping all gathered information of patients together grants for traceability and enables re-use and
re-analysis of existing data. For questions where a single causative mutation
is to be found in millions of other mutations, comprehensive data management and data analysis is essential.
P16.70-M
From NGS back to Sanger Sequencing: Connecting and Synchronizing
NGS and CE Variant Files with the VR Toolkit
E. Schreiber, S. Berosik, S. Samsani, S. Chang, S. Sharp;
Thermo Fisher Scientific, South San Francisco, CA, United States.
Genomic DNA in single cells can be analyzed using whole genome amplification (WGA) on the Fluidigm C1 Single-Cell Auto Prep System, followed by
next-generation sequencing [1]. This approach is invaluable when screening
for variants, for studying de novo mutations, as well as for looking for defined variants. We present an alternative to WGA for looking at predefined
variants, with a simpler workflow, completed in less than eight hours with
about three hours of hands-on time.
We isolated single GM12752 B-lymphocyte cells, extracted genomic DNA
and preamplified [2, 3] 96 specific loci on the C1 System, using a pool of 96
genotyping assays (all heterozygous) that amplify SNP loci on 22 human
chromosomes. The products were directly analyzed on a 96.96 Dynamic Array IFC (integrated fluidic circuit), and the overall allelic dropout rate was
found to be very low (4% for single alleles).
The approach described here can quickly provide insight into specific genes
or loci from single cells. We believe this technique will find its applications
in single-cell genotyping, variant detection, targeted sequencing, and, potentially, copy number variation.
ABSTRACTS POSTERS
P16.72-M
The influence of structural variants on alternative splicing
E. Ait Yahya Graison1, A. Necsulea2, A. Reymond1;
1
CIG, Lausanne, Switzerland, 2EPFL, Lausanne, Switzerland.
Large structural variations (SVs) are less common than SNPs and indels in
the population but collectively account for a significant fraction of genetic
polymorphism and diseases. Base pair differences arising from SVs are on
a much higher order (>100 fold) than point mutations, however, none of
the existing prevailing methods can comprehensively and effectively detect
them. To address these challenges, we first applied a high-throughput, costeffective genome mapping technology using long single molecule (>150kb)
to discover genome wide SVs and structure differences in the YH genome.
We detected 278 large SVs (>10 kb), of which 251 of 278 (90%) are retrospectively supported by multiple orthogonal methods such as whole genome or fosmid end sequencing (200/278) and historical evidence contained
in the DGV database (51/78). To further investigate the SVs that couldnt
be validated by sequencing based tests, we found that 71 out 78 (91%) intersected with repeat elements, often the blind spot of re-sequencing and
de novo assembly methods.. More than 70% of detected SVs are insertion
events, known to be difficult to detect by sequencing. In this study, genome
mapping also provides valuable information for complex regions (MHC, KIR,
TRB/TRA, IGH/IGL et.al) with haplotypes.
In addition, for the first time, with long single molecule labeling patterns,
inserted exogenous viral sequence and locations can be mapped on a whole
genome scale important for understanding virus induced oncogenesis.
Nanochannel based genome mapping make it now feasible and cost effective to conduct large population-based comprehensive SV studies efficiently
on a single platform.
P16.74-M
Analysis and correction of crosstalk effects in pathway analysis
M. Donato;
Wayne State University, Detroit, MI, United States.
Identifying the pathways that are significantly impacted in a given condition is a crucial step in understanding the underlying biological phenomena. All approaches currently available for this purpose calculate a p-value
that aims to quantify the significance of the involvement of each pathway
in the given phenotype. These p-values were previously thought to be independent. Here we show that this is not the case, and that many pathways
can considerably affect each others p-values through a ``crosstalk phenomenon. Although it is intuitive that various pathways could influence each
other, the presence and extent of this phenomenon have not been rigorously
studied and, most importantly, there is no currently available technique able
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to quantify the amount of such crosstalk. Here, we show that all three major categories of pathway analysis methods (enrichment analysis, functional
class scoring, and topology-based methods) are severely influenced by crosstalk phenomena. Using real pathways and data, we show that in some cases
pathways with significant p-values are not biologically meaningful, and that
some biologically meaningful pathways with non-significant p-values become statistically significant when the crosstalk effects of other pathways are
removed.
We describe a technique able to detect, quantify, and correct crosstalk effects, as well as identify independent functional modules. We assessed this
novel approach on data from four real experiments coming from three phenotypes involving two species. This method is expected to allow a better
understanding of individual experiment results, as well as a more refined
definition of the existing signaling pathways for specific phenotypes.
P16.75-S
EVA: A Completely New Variation Resource at EMBL-EBI
I. Medina;
EMBL-EBI, Cambridge, United Kingdom.
319
ABSTRACTS POSTERS
P16.77-S
Epigenetic mechanisms potentially influencing gene expression in a
rare form of familial Wilms Tumour
Wilms Tumour is the most prevalent type of pediatric kidney cancer. Most
occurrences are sporadic, however approximately 5% of cases are hereditary; one such familial case has been identified in Atlantic Canada. None of the
mutations that are currently known to cause Wilms Tumour are present in
this family. Although our research to date has yielded no obvious causative
mutation, whole-genome sequencing and RNAseq expression data have led
to a long list of genes that could potentially be involved. The need to prioritize this list, coupled with the clinical observation that age of onset differed
depending on whether the disease was inherited maternally or paternally,
have led us to consider epigenetic modifications. We have started by considering DNA methylation. Methylation occurs on cytosine residues of CpG
islands, and can cause gene inactivation by hindering access of transcriptional machinery to the promoter regions of genes. Therefore, gene transcription can be inhibited through hyper-methylation or gene expression may be
promoted by hypo-methylation. We have assessed the methylation status
of three genes (HDAC5, IGF2BP1, and POU6F2) in two patient and three
control kidney samples using a methylation digest assay followed by qPCR.
POU6F2 methylation was significantly decreased in both patient samples,
which correlates with the increased expression we observed through RNAseq. However, the percent methylation varied between digest replicates, so
we are currently attempting to confirm the results using bisulphite conversion. Next, we will assess the methylation status of other candidate genes and
will also investigate another epigenetic mechanism, histone acetylation.
P17.01-S
Genetic relationship of European Roma people and eight ethnic
groups from the Caucasus area which suggest Romas probably
admixed with during their migration
Historical and genetic studies have suggested that Roma people migrated
from India about 1500 years ago and settled in Europe. We investigated the
genetic connection of Central European Romas to populations they could be
connected or admixed with during their migration through the Caucasus.
Population samples were from Abhkasians, Armenians, Chechens, Kumyks,
Kurds, Nogais, North-Ossetians and Tadjiks living in the Caucasus area. We
used publicly available Caucasus datasets and our Roma samples genotyped
on Affymetrix Genome-wide Human SNP Array 6.0 chip. We also used Stanford-HGDP, HapMap Phase 3 and Indian datasets in order to get a better perspective about population relationships. Applying linkage disequilibrium
based pruning method, our datasets contained 88781 SNPs, respectively.
Using advanced algorithms which are capable of processing large autosomal
SNP datasets, we investigated genetic connection of these populations. In
order to infer the structure and relationship of populations, principal component analysis (PCA) and ADMIXTURE analysis were applied. PCA results
show that Romas, compared to Central European and Indian populations,
have more common genetic elements with the 8 investigated populations
of the Caucasus area. Roma samples were most closely to Tadjiks and Nogais, while the others clustered farther from Romas, approximately in the
same distance. ADMIXTURE analysis supports PCA results and shows that
the eight populations from Caucasus contain also more Indian ancestry than
Central Europeans. The data may also suggest that despite the short interval
of contact they still admixed or populations of the Caucasus also have the
same strong Ancestral North Eurasian origin as Romas.
This research was supported by TMOP-4.2.3-12/1/KONV-2012-0028.
P17.02-M
Study of the influence of HLA alleles on the premature biological aging
M. Cenit, A. Blanter, K. Nederveen, A. Serrano, C. Wijmenga, A. Zhernakova;
Department of Genetics, University Medical Centre Groningen, University of Groningen,,
Groningen, Netherlands.
320
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Emerging evidence indicates that population variation in growth and development is influenced by the early life experience of parents and ancestors. For example, paternal smoking in mid childhood is associated with
excess fat mass (5-10kg) in his sons by age 17ys (Northstone et al 2014).
Here we use smoking in pregnancy by either grandmother as the exposure
to test for transgenerational effects on growth. The Avon Longitudinal Study
ABSTRACTS POSTERS
of Parents and Children (ALSPAC) has information on prenatal exposure of
father (n=9677) and mother (n=12,707) with detailed follow up (including
DXA scans) on their children to age 17ys .
With non-smoking mothers, those with prenatal exposure themselves had
larger sons at birth, who went on the have increased lean mass and grip
strength by 17ys; no effects in daughters. By contrast, with non-smoking mothers, paternal prenatal exposure showed no effects at birth but increased
growth (weight, BMI, lean and bone mass) in both sons and daughters by
17ys; the daughters additionally had increased height and fat mass.
With smoking mothers prenatally exposed themselves, there was no effect on
sons at birth or later, whilst daughters had later decreases in height, weight,
and fat, lean and bone mass. The only transgenerational effect with smoking
mothers and paternal prenatal exposure was decreased head circumference
at birth in sons which was associated with reduced IQ at 8ys. Taken together,
these data evoke the transgenerational plasticity theory of evolved human
life history strategy. Interaction analysis with imprinted genes PHLDA2 and
INS VNTR/IGF2 are underway.
P17.06-M
Ancient mtDNA diversity in Bulgaria
The mountain areas of LAquila city, such as Barete and Rocca di Cambio are
characterized by environmental conditions (low temperature and humidity)
that facilitate the preservation of the humans remains and anDNA: this factor
explains the often tempering of a number of paleoanthropological findings.
The migratory events surely resulted in the disappearance of several genes
as well as the introduction of foreign alleles, among which some responsible
for the development of autoimmune diseases HLA-restricted. In this work
we show some preliminary genetic data generated by applying different
bone anDNA extraction protocols, innovative and/or suitably modified from
commercially available kits for forensic analysis. The immunogenetic assays
have showed the positivity for HLA gene HLA-Cw3 (an increased frequency
of HLA-Cw3 and HLA-Dw4 is tipically observed in Rheumatoid Arthritis).
This work has laid the foundation for the design of protocols for extraction
and PCR reaction improved and optimized, to be applied to paleogenetics
and paleopathology samples on which to assess genetic risk factors related
to autoimmune rheumatic diseases HLA-Linked.
References
Fontecchio G., Ventura L., Poma A.M., 2012; Further genomic testing and histological examinations confirm the diagnosis of rheumatoid arthritis in an
Back to index
Italian mummy from the 16th century, 2012; Ann. Rheum. Dis., 71: 630.
Poma A., Carlucci G, Fontecchio G., 2012; Immunogenetics and HPLC analyses contribute to understanding the etiopathology of rheumatoid arthritis
through studies on ancient human remains, Int. J. Immunopathol. Pharmacol., 25: 1075-1082.
This work was partially funded (2013) to A. Poma by ANCE Associazione Nazionale Costruttori Edili LAquila
P17.08-M
Association between Azoospermia Factor c (AZFc) rearrangements
and Y chromosome lineages
Complete deletions of Azoospermia Factor c (AZFc) region are common genetic cause of male infertility, while the contribution of partial deletions and
duplications to spermatogenic impairment is still controversial. Some studies suggested that Y chromosome background affects the formation of AZFc
rearrangements and contributes to spermatogenic failure. The aim of our
study was to investigate the association of AZFc rearrangements and Y chromosomal lineages among 474 men from R. Macedonia (328 Macedonians,
110 Albanians and 36 of other ethnicity). AZFc rearrangements were determined by STS markers and gene dosage analysis. Y lineages were assigned
using multiplex SNaPshot of 26 Y-SNPs and fluorescent PCR of 17 Y-STRs.
Five different AZFc rearrangements (b2/b4, gr/gr and b2/b3 deletions and
b2/b3 and b2/b4 or gr/gr duplications) and 18 different haplogroups were
detected, of which five [E1b1b, I2a1(xI2a1a), R1a, R1b and J2b] were present in 85% of the studied men. The presence of any AZFc rearrangement
was positively associated with R1a (p=3.53x10-19) and negatively with R1b
(p=5.88x10-5) and J2b (p=8x10-3) haplogroups. Most of the b2/b4 duplications were found on R1a, with a higher frequency among Albanians than
Macedonians. The b2/b4 and gr/gr deletions were detected on different haplogroups with gr/gr present almost exclusively among Macedonians. B2/
b3 deletion was present in four men with E1b1b and in the only two men
with N haplogroup. In conclusion, determination of both AZFc rearrangements and Y chromosome lineages in ethnically matched populations may
improve our understanding of their role on male infertility.
P17.09-S
Association BDNF Val66Met genotype and personality traits in
healthy female subjects
Brain-Derived Neurotrophic Factor (BDNF), a member of the nerve-growthfactor family, plays an important role in the regulation of plasticity synaptic
and seems to be involved in the expression of personality traits.
The aim of this study was to understand the effects of the BDNF Val66Met
functional genetic variant on several personality dimensions assessed using
self-reported instruments. We administered the Big-Five-Questionnaire
(BFQ-2) and the Temperament and Character Inventory-Revised (TCI-R)
for the measure of personality; the Emotional Intelligence Scale (EIS) and
the Emotional Quotient Inventory (EQ-I) for the measure of Emotional Intelligence construct; a new instrument for the measure of Fluid Intelligence construct (CAT-FIT). We tested the possible interactions between these
variables on a cohort of 154 healthy female students recruited at the G.
dAnnunzio University, Chieti. A significant and positive correlation has been
observed between the BFQ-2 Agreeableness score and the BDNF 66Met carriers compared with the BDNF Val66Val genotype (r =.17; p<.05). In addition, an association has been evidenced between BDNF 66Met carriers and
the TCI-R Reward Dependence subscale (r = -.20; p < .05); furthermore, the
BDNF 66Met carriers was significantly associated with the TCI-R Self-Trascendence subscale (r = .17; p<.05). No significant association was demonstrated for the BDNF polymorphism and for any of the constructs analyzed.
Our findings support the association between Agreeableness, Dependence
and Self-Transcendence personality traits and the BDNF 66Met variant in
female healthy subjects.
321
ABSTRACTS POSTERS
P17.10-M
Radboud Biobank: a central facility for prospective clinical
biobanking in the Radboud university medical center, Nijmegen
Biobanking is crucial for science-based health care solutions in the 21st century. In order to improve the diagnosis, prevention and treatment of complex,
multifactorial disorders and diseases a better understanding is needed of the
underlying genetic and environmental pathways. Therefore, there is a growing need for large-scale biobanks for biomedical research.
The Radboud university medical center is building a central biobank facility
for disease-based biobanks to optimize the use of biomaterial. The Radboud
Biobank encompasses biomaterial and their descriptions (patient, diseasespecific and phenotypic data) and subsequent data (genotypic data, microarray gene expressions).
For all patient groups included in the Radboud Biobank different samples types are being stored, however, DNA is stored for all groups. The isolation and
storage of DNA-samples takes place at the department of Human Genetics of
the Radboudumc. An automated sample flow for sample handling, storage
and retrieval of all DNA-samples is present . DNA is stored in an automated
-20 storage system. This system will assure sample security over the long
term, sample integrity and efficient and quality based sample management .
At the moment DNA-samples are available of, among others, the following
patient groups: individuals with a confirmed form/an increased risk of colorectal cancer, patients with a cerebrovascular infarct/cerebral hemorrhage/
venous thrombosis, or with rheumatoid arthritis/arthrosis, neurodegenerative disorders, type 2 Diabetes Mellitus, Crohns disease/ulcerative colitis,
leukemia/multiple myeloma/lymphoma and related disorders, chronic (progressive) renal failure and patients who have had a cerebrovascular vascular accident (18-50 years). There are also DNA-samples available of healthy
individuals.
P17.11-S
Establishing of national birth defects registry in Thailand
322
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Background: Previously, relatively high cancer risks were observed in BRCA2 mutation carriers (BRCA2 carriers) over 60 in the Northern Netherlands. We aimed to quantify these regional differences in the breast cancer
risk, and analyzed whether they could be explained by mutation spectrum
or population background risk. Methods: This consecutive cohort study
included all known BRCA1/2 carriers in the Northern Netherlands (N =
1,050). Carrier and general reference populations were: BRCA1/2 carriers
in the rest of the Netherlands (N = 2,013) and the general population in both
regions. Regional differences were assessed with hazard ratios (HR) and
odds ratios (OR). HRs were adjusted for birth year and mutation spectrum.
Results: All BRCA1 carriers and BRCA2 carriers under age 60 had a significantly lower breast cancer risk in the Northern Netherlands, HR were 0.66
and 0.64, respectively. Above age 60, the breast cancer risk in BRCA2 carriers in the Northern Netherlands was higher than in the rest of the Netherlands (HR = 3.99, 95%CI 1.11-14.35). Adjustment for mutational spectrum
changed the HRs for BRCA1, BRCA2 <60 and BRCA2 60 years by -3%, +32%
and +11%, respectively. There was no difference in background breast cancer incidence between the two regions (OR = 1.03, 95%CI 0.97-1.09). Conclusions: Differences in mutation spectrum only partly explain the regional
differences in breast cancer risk in BRCA2 carriers, and for an even smaller
part in BRCA1 carriers. Impact: The increased risk in BRCA2 carriers over
60 may warrant extension of intensive breast screening beyond age 60.
P17.14-M
Immunochip SNP effect on candidate gene expression in Celiac
Disease
ABSTRACTS POSTERS
this study. Merlin 1.1.2 was used for the association test, performed independently in each of the studied groups in order to avoid false associations
due to duplicated genotypes in CD sample pairs. Genotype effect of SNPs
in the expression of many genes was found, but in none of them was the
candidate gene located under the association peak of its putative SNP affected. We analyzed the genomic area around the associated SNPs searching in
online data bases (Haploreg, Ensembl and UCSC) to find possible common
regulatory elements that could be altering the expression of genes far away.
Findings include open chromatin regions, novel protein coding sequences,
novel anti-sense processed transcripts, processed pseudogenes, microRNAs,
novel LincRNAs and some altered motifs in the SNPs showing regulatory power. Further functional studies must be done to determine if those elements
could be real players in the development of CD.
P17.15-S
Gaining access to large European population cohorts through the
transnational biobanking infrastructure BBMRI-LPC
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P17.17-S
Genetic adaptation of the human circadian clock to day-length
latitudinal variations
The temporal coordination of biological processes into daily cycles is a common feature of most living organisms. Day/night cycles represent a major
circadian synchronizing signal and vary widely with latitude. In humans disruption of circadian rhythms is a common feature in psychiatric diseases including schizophrenia, bipolar disorder, depression and autism. We applied
an approach that analyses spatial correlations between genetic variation
and environmental factors. We exploited genotype data from 52 human populations distributed worldwide and determined the annual maximal variation in day-length (photoperiod), this latter used as a measure of selective
pressure. With this approach we analyzed 5 independent sets of genes involved in circadian regulation or in sleep homeostasis. For all sets a significant
enrichment of variants showing signals of photoperiod-driven selection
was detected. Analysis of population genetic differentiation (FST) confirmed strong spatial signatures of natural selection at photoperiod-selected
variants. Also, tests based on haplotype homozygosity indicated that a significantly high proportion of photoperiod-selected SNPs underwent selective sweeps in non-African populations. Finally, using variants identified
in genome-wide association studies we determine that a significantly high
proportion of risk SNPs for schizophrenia and/or bipolar disorder correlates with photoperiod. Notably, many risk variants showing signals of photoperiod-driven selection map to genes with strong evidence of involvement
in circadian rhythm regulation. Thus, human populations adapted to life at
different latitudes by tuning their circadian clock systems. This process also
involved risk variants for neuropsychiatric conditions. Data herein suggest
possible genetic modulators for chronotherapies and candidates for interaction analysis with photoperiod-related environmental variables.
P17.18-M
Using mean inbreeding coefficient to estimate total pathogenic
allele frequency in autosomal recessive disorders results in a biased
estimate of this frequency
Some years ago we suggested to estimate total pathogenic allele frequency (q) of autosomal recessive disorders from the proportion of compound
heterozygotes among affected patients with consanguineous parents. The
method works well when all parental couples in the sample have the same
value of F (provided F>0). The effect of combining data with different Fs,
was never examined, although taking a weighted average was already reported in literature. While testing the use of simulation as a method to examine
the accuracy of estimating q, it was discovered by one of us that taking a
mean value for F would always overestimate q. Here we illustrate the impact
of using an average F in a hypothetical data set consisting of two equally
sized populations, one with an F=0 and the other with a variable F (1/16,
1/64/ 1/256 and 1/1024). It is assumed that 4 different pathogenic alleles
are involved, with relative frequencies 0.4, 0.3, 0.2 and 0.1. The table shows
the resulting biased estimates for two different values of the true q.
True q
0.01
0.02
1/16
0.0419
0.0516
1/64
0.0178
0.0278
1/256
0.0119
0.0219
1/1024
0.0105
0.0205
R. A. Cedeno;
Genetic Medical Institute, Zulia University, Maracaibo, Venezuela, Bolivarian Republic of.
323
ABSTRACTS POSTERS
covery of regularities that are encoded within the data. DM is the search
for new, valuable, and nontrivial information in large volumes of data.The
Registry of Congenital Malformations in Maracaibo, Venezuela is a study for
the clinical and epidemiological This paper presents a future proposal for a
study of Congenital malformation monitoring program in Maracaibo, Venezuela with techniques for Data Mining (DM) and discuss how we carry out
knowledge discovery on the Congenital Malformation database (CMDB) of
the ECLAMC (F01) The aim of this paper is to demonstrate that the techniques of pre-processing dates are the focus of DM processes and we will
present stepby step the CRISP-DM standard approach to help the physician
explore their CMDB
In summary, DM algorithms can be applied using the prepared data. The
adequacy of data preparation often determines whether this data mining is
successful or not speciality in Congenital Malformation database.
Bibliography.
[1] U.M. Fayyad, G. Piatetsky-Shapiro and P. Smyth, The KDD process for extracting useful knowledge
P17.20-M
High frequency of hypoketotic hypoglycemia associated CPT1A
mutation in Northeast Siberia caused by positive selection
Siberian populations represent a unique case for the study of human adaptation to the extreme cold environment. Our previous selection scans based
on genome-wide genotype data highlighted a 3Mbp region on chromosome
11 with 79 genes as the strongest candidate region under positive selection
in Northeast Siberians. However, it was not possible to distinguish which
gene specifically was causing the signal. Here, using whole genome high coverage sequence data and additional neutrality tests, we identify the most likely causative mutation of this selection signal in the CPT1A gene which is a
key regulator of long-chain fatty acid oxidation. We find a non-synonymous
mutation (P479L) at >80% frequency in the coastal Northeast Siberian populations while it is absent in other Siberian populations and public genomic datasets. The only other known ethnic groups with this mutation are Canadian and Greenland Natives among whom the P479L mutation has been
reported in association with hypoketotic hypoglycaemia and high infant
mortality. The mutation is also present in the genome of the Paleo-Eskimo
from Greenland suggesting that the mutation is at least 4KYA. The localised
high frequency and associated haplotype homozygosity of the P479L mutation in the Arctic region supports the hypothesis that its selective advantage
is either due to specific dietary or thermoregulation associated adaptation.
P17.21-S
Genome-wide association study and functional characterization of a
locus associated with CRIPTO serum levels in Cilento isolates
CRIPTO, the founding member of the EGF-CFC genes, plays an essential role
in embryo development and cancer progression. Very little is known about
the variability of serum levels of CRIPTO in humans and the genetic contribution underlying this variability remains still unknown. We performed a
GWAS of CRIPTO serum levels in two isolated villages from Cilento area in
South Italy. The most associated SNPs (p-value<10-8) were all located on the
chromosome 3p22.1-3p14.3, around the CRIPTO gene. Performing a conditional analysis for the best signal (rs3806702, p-value=1.03*10-159) no
loci remain associated at genome-wide significance, while many SNPs were
associated at 5*10-8<p-value<1*10-4, twenty of those were replicated in an
independent sample. Genes closest to the replicated SNPs were included in
a unique network involved in cellular development, death and survival. The
replicated loci explain the 89.8% of the CRIPTO variance, with the 84.9%
explained by the most associated SNP. Among the top associated SNPs, the
rs112481213 variant (p-value=1.53*10-158), located within the 5UTR of
the CRIPTO gene, was predicted to create a binding site for AP-1 transcription factor. To assess a functional role of this SNP, allele effect was tested on
324
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Cystic Fibrosis (CF) is the most frequent severe inherited disease among
Caucasians, having prevalence of 1:3000 and a carrier frequency of 1:27 in
Italy. Three mutations in CFTR gene (F508del, N1303K, G542X) represent
approximately 60% of mutated alleles, while a multitude of additional mutations exhibit a low prevalence. In addition, the distribution of individual
mutations varies throughout the country and several mutations are peculiar
to specific regional areas, i.e, T338I is typical in Sardinia (15.1% of mutated alleles), 2183AA->G and R1162X are frequent in Northeastern Italy (8%
each). We now present data on CF genetic testing in Como, a 0.6 million population regional area of Lombardia (Italy), for which mutation prevalence
information is limited.
From 2002 to 2013, our Unit of Genetics analyzed 3369 individuals for
CFTR mutations, using reverse dot blot-based commercial diagnostic kits
with a detection rate ranging from 75% to 85%. The laboratory successfully accomplished National Health system (ISS)-sponsored external quality
control program since 2003. Most of the individuals were tested for CF in
the context of clinical examinations for infertility or as partners of known
carriers. Additional cases represented symptomatic patients with suspected cystic fibrosis or prenatal diagnosis testing.We detected 147 mutated
alleles (4.3% CF carrier frequency). Frequency of individual mutations were
F508del (40.1%), G542X (11.6%), N1303K (8.8%), 2789+5G->A (6.8%),
2183AA->G (6.1%). Additional 16 mutations comprised 26.6% of mutated
alleles. A survey of mutations in relation with medical indications (including
male infertility) and compared to literature data on different Italian areas
will be presented.
P17.23-S
Polymorphism A2756G at methionine synthase (MTR) and the
maternal risk of Down syndrome: evidence from a meta-analysis of
case-control studies
ABSTRACTS POSTERS
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P17.24-M
FFBSKAT: fast family-based sequence kernel association test
N. M. Belonogova, G. R. Svishcheva, T. I. Axenovich;
Institute of Cytology & Genetics SD RAS, Novosibirsk, Russian Federation.
Nunavik comprises the northern part of Quebec, with 90% of its habitants
being Inuit. The modern Inuit of Nunavik came from the Thule people from
coastal Alaska and traveled eastward along the Arctic tree line and reached
Nunavik around 1500 AD as they became the ancestors of current Inuit
residents. In this study, we performed exome sequencing on 113 Nunavik
Inuit, using Agilent SureSelect V4 capture kit and Illumina HiSeq platform.
Standardized data processing is used to extract all exonic variants. We performed preliminary analysis in order to identify Inuit specific novel or rare
nonsynonymous variants. The analysis yielded 62 protein changing variants
which have allele frequency over 0.5 in Inuit while less than 0.01 in other populations. Among these, a variant p.P479L in the Carnitine Palmitoyltransferase 1A (CPT1A) gene is the most prominent one with allele frequency
0.94. This variant is absent in non-Inuit populations, which frequency also
appears to be the highest in Nunavik when compared to other Inuit populations. Further analysis revealed Nunavik Inuit also have significantly higher
mutation burden in other carnitine acyltransferase family genes compared
to non-Inuit populations such as Asian and Caucasian. The specific genetic
profile of Nunavik Inuit with higher number and frequency of damaging variants in genes regulate lipid metabolism such as carnitine acyltransferase
genes may be associated with the adaptation to their special diet and living
environment in the extreme cold climate. Understanding the role of Inuit
unique variant(s) is very important to the study of Inuit special health care.
P17.26-M
GWAS and candidate gene analysis highlight many novel loci
associated to food preferences
1,2
Food preferences are the first factor driving food choice, nutrition and ultimately diet-related diseases. To understand the genetic component of food
preferences we used two phase approaches: PHASE1: association of bitter
taste receptors and coffee liking and PHASE 2: two-step GWAS of 42 diffe-
Background and aim: Common polymorphisms in the fat mass and obesityassociated gene (FTO) have shown strong association with obesity, metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM) in several populations. In the present study, we explored the association of FTO rs1421085
gene polymorphism with obesity, MetS, T2DM and other biochemical parameters in a larger cross-sectional population-based random sample of
Turkish Adults (TARF Study). Methods: Genotyping was performed using
the Taqman Sistem (ABI PRISM 7900 HT) in 1977 adult individuals (mean
age 50.112.0; 48.3% male). Results: The frequency of the FTO risk-allele
(C) was 0.42, and the genotype distribution was in Hardy-Weinberg equilibrium. Not women but men, carriers of C allele of the rs1421085 polymorphism were at increased risk for the presence of MetS (OR = 1.52; 95% CI:
1.13 to 2.04), elevated insulin level (8.31 1.04, p=0.013), adjusted for age,
smoking status, alcohol consumption, and physical activity. Logistic regression analysis demonstrated a significantly increased likelihood for obesity
in women rs1421085 C allele carriers (OR = 1.56; 95% CI: 1.16 to 2.10), after adjustment for age, cigarette smoking, alcohol usage and physical activity, diabetes mellitus and menopausal status. The rs1421085 polymorphism
was not associated with T2DM in both gender. Conclusion: Our findings indicate that the rs1421085 polymorhism in the FTO gene contribute to obesity
and MetS in the Turkish adults depending on gender.
P17.29-S
Genomic description of the Generation Scotland Cohort: a large family
base genetic study
325
ABSTRACTS POSTERS
of genetic introgression and were able to differentiate between individuals
with a few small exogenous regions in their genome, and those with long
exogenous haplotypes covering a large part of the genome. We found that
the pattern of homozygosity was very similar to that of other European populations and identified an individual carrying a chromosome 1 uniparental
disomy. Overall, there is very limited evidence for geographic differentiation
or stratification of the GS:SFHS sample within Scotland. These findings provide a genomic perspective on the history of the Scottish population, and
have implications for further analyses, such as studying the contributions of
common and rare variants to trait heritabilities and evaluation of genomic
and phenotypic prediction of disease.
P17.30-M
Identification of mutations in the GLI2 gene in Combined Pituitary
Hormone Deficiency (CPHD) in Italian patients
TheGLI2transcription factor is a major effector protein of the sonic hedgehog pathway and it is suggested to play a key role in pituitary development.
GLI2 mutations cause holoprosencephaly or holoprosencephaly-like features. In some cases heterozygous GLI2 mutations have been associated
with hypopituitarism without other anomalies. However GLI2 is not routinely screened in pituitary hormone disorders. The aim of this study was to
determine the frequency ofGLI2mutationsin patients with combined pituitary hormone deficiency (CPHD) in Italian patients that resulted negative
for mutations in other causative genes (PIT1, PROP1, HEXS1, LHX3,LHX4).
In this analysis, we screened the entire gene in 75 CPHD patients. Primers
were specifically designed for the 13 coding exons that were analysed by
direct sequencing in 18 separate fragments. We identified two novel missense mutations: c.731A>T in exon5 leading to the amino acid substitution
p.Asp244Val and c.1157C>T in exon7 resulting in the change p.Pro386Leu.
These two variants were absent in about 13.000 individual present in the
Exome Variant Server. Both these mutations were predicted as probably
damaging using the online tool Polyphen v2, with a high score (1.00 for
Asp244Val and 0.923 for p.Pro386Leu). They fall within the NH2 terminal
repressor domain and an in vitro functional analysis will help to clarify their
significance.
These preliminary results are encouraging and we are planning to increase
the number CPHD patients and extend the analysis also to Isolated Growth
Hormone Deficiency (IGHD).
P17.31-S
A Genome-wide Association Analysis of Lipid traits in a sample of
indigenous population from Mexico
Given the high prevalence of diferent forms of dyslipidemia in Mexican population, in contrast to European populations, it has been suggested that
genetic susceptibility in this population is probably related to its indigenous
component as a result of adaptive processes related to energy savings. It is
therefore of great importance to identify the genetic variation of the native
Mexican population. In this study, 319 individuals from four indigenous populations (174, 70, 25 and 50 from Nahua, Maya, Totonac and Zapotec, respectively) were included. Genotyping was performed using Affymetrix SNP
6.0 microarray. The analysis included a local ancestry estimation to remove
segments infered to be of European origin and mixed linear models using
EMMAX, adjusted for age, gender and two principal components. Two steps
of quality control were performed, before and after the European segments
were removed. Log-transformed values of tryglecides (TG), total cholesterol (TC) and HDL cholesterol (HDL) were analyzed. By removing segments
of European origin in the analysis we identify three suggestive signals, one
for each trait. For HDL, TC and TG, an association was found in intergenic
regions on chromosome 11 (p9.5x10-6), chromosome 21 (p8.8x10-5)
and chromosome 2 (p6.1x10-5), respectively. In the three cases, the risk
allele frequency is 20%, 16% and 10% higher in Indigenuos population than
in European population. This regions have not been identified in previous
GWAS studies thus it is important to replicate these results to confirm the
involvement of these new regions.
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P17.32-M
Selecting significant SNPs in Genome-wide Association Studies using a
global False Discovery Rate
A. Mukherjee, S. Bhattacharjee;
National Institute of BioMedical Genomics, Kalyani, India.
Non-syndromic cleft lip with or without cleft palate (NSCL/P) is one of the
most common congenital anomalies, with a complex and not yet fully elucidated etiology. Therefore, we conducted a genome wide association study
(GWAS) for NSCL/P in a Polish population-based cohort consisting of 288
oral cleft cases and 576 controls. GWAS was carried out with the Illumina
Human OmniExpressExome BeadChip technology. We confirmed the association between the previously identified 8q24.21 locus and the risk
of NSCL/P. The most significant nucleotide variant in this 330-kb region
(rs17242358) had a p-value of 3.21E-09. Under assumption of a dominant
and recessive models, the calculated Odds Ratios for rs17242358 were 2.28
(95%CI: 1.70 - 3.06, p = 2.53E-08) and 2.99 (95%CI: 1.60 - 5.59, p = 3.32E04), respectively. In addition, we found a novel cleft-associated region at
chromosome 22q12.3, with a p value of 1.22E-08 for the most significant
polymorphism (rs210804). The dominant model OR for the rs210804 variant was 8.20 (95%CI: 3.51 - 19.13, p = 1.19E-08). Four other chromosomal
regions 6p12.3 (DEFB112), 22q12.3 (MYH9), 6p24.3 and 11q13.4 (POLD3)
were associated with the NSCL/P risk at close to genome wide significance
level (p values of 5.12E-08, 3.26E-07, 3.40E-07 and 8.15E-07, respectively).
To confirm our GWAS findings further, larger sample size studies in different
populations are needed.
Supported by grant no. 2012/07/B/NZ2/00115 from the Polish Ministry of
Science and Higher Education.
P17.34-M
Family-Control analysis based on Hamming distance for prioritizing
candidate sequence variants
A. Imai1, J. Ott2, Y. Asano1, Y. Sakata1, S. Takashima1;
1
Osaka University, Suita, Japan, 2Rockefeller University, New York, NY, United States.
Linkage analysis has been used to narrow down disease loci for autosomal
dominant (AD) traits. However, small pedigrees are underpowered for significant linkage results. We developed a novel method to detect disease loci in
pedigrees.
ABSTRACTS POSTERS
For a set of basepairs flanking a candidate locus, we calculate Hamming Distance Ratio (HDR, proportion of basepairs differing between two individuals) for all pairs of individuals (an affected family member and 41 control
individuals) and distinguish pairs containing the affected (group 1) from
those that do not (group 2). We assess the difference in distribution of HDR
values between the two groups by the Kolmogorov-Smirnov statistic at sets
of variants 10KB to 100KB around each of ~600 candidate variants. In two
hypertrophic cardiomyopathy families, known pathogenic mutations were
previously detected: c.173G>A (p.R58Q) in MYL2 gene (rs104894369, family A) and c.746G>A (p.R249Q) in MYH7 gene (rs3218713, family B). We
combine results over the 10 regions with suitable test statistics evaluated in
permutation analyses and are able to narrow down known disease variants
as small as 5% (rank as small as 29 out of 606 candidates, p=0.0001).
In a negative control, a restrictive cardiomyopathy pedigree with de novo
mutation in the TNNI3 gene in one affected sibling, TNNI3 mutation was
ranked 221th out of 631 variants.
Our new statistical method for prioritizing disease region by Hamming distance between affected family member and unrelated controls will be useful
for small AD pedigrees and for differentiating AD and de novo mutations.
P17.35-S
Mitochondrial DNA variation analysis in historical provinces of
Romania
Since the Middle Ages, Romanian population lived in three distinct provinces Wallachia, Moldavia and Transylvania until the 19th century. Over the
centuries, their territories were repeatedly invaded by different peoples and
subjected to external political influences resulting in demographic changes
that could affected the genetic structure of populations. We performed mitochondrial DNA analysis in order to visualize the relationships between
Romanian and other populations Europe based on HVSI and HVS II mtDNA
sequence data using Sanger sequencing. We presented here a large scale
mtDNA analysis of 612 Romanians from these historical provinces of Romania. Multidimensional scaling (MDS) plot was constructed from the pairwise Fst values. The results showed that present day Romanians in all provinces share their maternal ancestry with both eastern/central European
and Balkan populations. The three populations of Romania analyzed here
exhibit slightly different mtDNA lineage compositions, mainly consisting of
the haplogroups H, U, J, T, K, N and W, with significant frequency differences
corresponding for H, U and W haplogroups. H haplogroup accounts for 47%
in Moldavia, 35 % in Wallachia and 33 % in Transylvania. Overall, this study
provides a first comprehensive analysis of mtDNA genome variation in Romania revealing the existence of different degrees of provincial differences
of haplogroup frequencies.
P17.36-M
Heritability of age related cognition
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and fine motor skills can be explained by common SNP. Our results support
shared genetic factors for executive function and memory as well as fine
motor skills.
P17.37-S
Involvement of HMGA2 (High-Mobility Group A2) in Idiopathic Short
Stature (ISS).
I. Fusco1, D. Babu1, S. Mellone1, A. Fanelli1, R. Muniswamy1, F. Prodam2, S. Bellone2, A.
Petri2, G. Bona2, M. Giordano1;
1
Laboratory of Human Genetics, Department of Health Sciences, University of Eastern
Piedmont, Novara, Novara, Italy, 2Paediatric Unit, Department of Health Sciences,
University of Eastern Piedmont, Novara, Novara, Italy.
Several lines of evidences point to HMGA2 as a candidate gene in ISS: i) independent GWAS identified some SNPs on chromosome 12q14 in the vicinity of HMGA2 (rs1042725, rs7968682 and rs7968902) as one of the major
determinants of height, ii) microdeletions on 12q14 have been identified in
syndromic patients with short stature as common feature. Among these, one
patient with only severe growth retardation carried the smallest deletion
encompassing HMGA2. The aim of this study was to investigate the involvement of HMGA2 in ISS through the search for mutations/deletions and perform an association study between the HMGA2 SNPs and ISS. One hundredfour patients (48 males and 56 females) with height ranging from -3,8 and
-2 SDS were analyzed by direct sequencing and MLPA. None of the patients
carried mutation/deletion in the coding sequences and intron/exon boundaries. The same 104 patients and 330 normal stature matched controls
were analyzed in an association study with rs7968682. The allele frequency
was significantly different between patients and controls (p=4x10-2). When
the genotypes were considered the TT genotype showed an even stronger
association (p=6x10-3). In conclusion, our cohort of ISS patients did not
show any pathological mutation in HMGA2, suggesting that high penetrance
mutations are not a frequent cause of ISS. However our preliminary results
suggest that HMGA2 might be involved in the susceptibility to ISS. We are
thus planning to replicate the data in an independent cohort and analyze the
tag SNPs surrounding HMGA2 to identify the variations directly responsible
for the association.
P17.38-M
Thioredoxin reductase 1 (TXNRD1) gene variability influences quality
of aging and longevity
G. Rose, S. Dato, P. Crocco, G. Passarino;
Department of Biology, Ecology and Hearth Sciences, Ponte Pietro Bucci cubo 4C,
University of Calabria, 87036 Rende (Cs), Italy.
Oxidative stress is a major determinant of human aging and a common hallmark of age-related diseases. A protective role against free radicals accumulation has been shown for Thioredoxin (Trx) system and in particular
thioredoxin-reductase TrxR, a key selenoprotein antioxidant enzyme, able
to reduce Trx and other substrates, detoxifying cells from oxidative injuries. An effect of this antioxidant system in human aging can be hypothesized from the association reported between TrxR gene (TXNRD1) with
late-life survival in a Northern-European nonagenarian cohort (Soerensen
et al, 2013). Using a tagging approach, we investigated the association of
14 SNPs with longevity and quality of aging, by analyzing their variability
in relation to markers of functional (Activities of Daily Living, ADL; Hand
Grip, HG; Walking speed, WS) and cognitive (Mini Mental State Examination,
MMSE) status, in a Southern-Italian elderly cohort (626 subjects, age range
65-104 years). The work confirms the association of TXNRD1 gene with human survival, with two intronic SNPs, rs7310505 and rs4964728, showing
association with longevity (p<0.04). Furthermore, three other SNPs, two
intronic (rs7962423, rs10861203) and one located in the promoter region
(rs1128446), were significantly associated with ADL, HG and WS (p<0.05).
Haplotypic analyses confirmed the single-SNP results. Moreover, bioinformatic analyses indicated the associated SNPs as putative regulatory sites,
whose function should be further experimentally investigated.
On the whole, this study confirms a role of Trx antioxidant system on human
age-related functional decline and longevity, possibly mediated by modulation of oxidative stress.
P17.39-S
Genetic analysis of thyroid peroxidase (TPO) gene in patients whose
hypothyroidism was found in adulthood in West Bengal, India
Recent research has revealed that genetic defects due to mutation in the
Thyroid Peroxidase (TPO) gene can lead to thyroid dysfunction in the population. We aimed to study the association between genetic defects in TPO
327
ABSTRACTS POSTERS
gene and patients with hypothyroidism found in adult age. Two hundred
consecutive treatment naive hypothyroid patients (age 18 years) (cases)
who were negative for anti TPO antibody and their corresponding sex and
age matched two hundred normal individuals (controls) were enrolled. The
17 exonic regions of the TPO gene were amplified and sequenced directly.
We identified 6 different previously known single nucleotide polymorphisms (SNPs) and 2 novel deletions in TPO gene. Two of the six SNPs revealed
a significant association with hypothyroidism; Thr725Pro (rs732609) and
Asp666Asp (rs1126797). The c.2173C allele of the Thr725Pro in TPO showed a significant association among hypothyroid patients compared to
controls (p = 0.01; Odds ratio=1.45; 95% CI: 1.091.92) suggesting it to be
a potential risk allele toward disease predisposition. Analysis of genotype
frequencies of the polymorphism between the two groups demonstrated CC
as a potential risk genotype (p = 0.006; Odds ratio=1.95; 95% CI: 1.23.15)
for the disease while another SNP Asp666Asp (c.1998T allele) showed protectiveness towards the disease (p = 0.006; Odds ratio = 0.67; 95%CI: 0.500.89). To our knowledge, this is first study reporting the role of TPO gene
with hypothyroidism in a population of Asian Indian origin. The study threw
up the possibility of TPO gene polymorphisms as a possible pathogenetic
mechanism of hypothyroidism.
P17.40-M
Genes involved in interleukin-1 receptor type II (IL1R2) activities are
associated with asthma related phenotypes
328
Back to index
Aim: This study aims to characterize the genetic variability within Italian
population and the level of inter-population variation between the Mediterranean basin and the Italian peninsula.
Methods: A total of 314 samples, collected from the Italian peninsula and
Sardinia island were genotyped, with more than 2,370,000 single-nucleotide polymorphisms investigated. Newly acquired data were analyzed together with the already available genome-wide data sets of individuals of the
Mediterranean basin. Several analyses have been performed to investigate
intra e inter-population variability.
Results: The analysis highlighted that Italy is one of the genetically most
diverse region within Southern Europe. These results may be due to the
countrys complex demographic history, to the large longitudinal extension
of the territory and because of its position as a sort of natural bridge in the
center of the Mediterranean basin.
Conclusion: We confirmed the high genetic diversity of some areas within
Italy, such as Sardinia, and of other populations located along boundaries of
the Italian territory.
We showed that the current genetic landscape of the Italian Peninsula may
be explained by isolation-by-distance rather than a barrier to gene flow and
may also reflect a genetic exchange for the Southern Italian populations between the 2 coasts of the Mediterranean basin.
P17.43-S
Familial Hypercholesterolemia screening in Patients with Coronary
Artery Disease
ABSTRACTS POSTERS
P17.44-M
Study homozygosity disequilibrium in human genome using the
whole-genome sequencing data
H. C. Yang1,2, Y. T. Lin1;
1
Institutue of Statistical Science, Academia Sinica, Taipei, Taiwan, 2School of Public
Health, National Defense Medical Center, Taipei, Taiwan.
Homozygosity disequilibrium (HD), a non-random sizable run of homozygosity in the human genome, has been found related to the evolution of
populations and able to confer susceptibility to diseases. In this study, we
characterized HD in global populations based on the whole-genome sequencing data of 1,092 individuals from 14 populations of the 1000 Genomes
Project. The whole-genome homozygosity intensity was estimated by using
LOHAS (Yang et al, Genetic Epidemiology, 2011). Using the homozygosity
intensity we identified common genomic regions undergoing HD. We found
a high proportion of regions of HD to be population-specific but also identified functionally important regions of HD shared by multiple populations.
Genetic differentiation of global populations can be characterized using
the patterns of HD. In summary, this large-scale whole-genome sequencing
study derives the distribution of HD in the human genome and proves that
HD carries genetic information of human population important for studying
genetic background. The information also provides important clues for the
differential disease prevalence and drug responses in populations.
P17.45-S
A signal near FRMD4A is associated with lower extremity arterial
disease in patients with type 2 diabetes in GoDARTS
Lower extremity arterial disease (LEAD) is a common macrovascular complication of type 2 diabetes (T2D). Twin studies of ankle brachial index
(ABI), the main diagnostic criterion of LEAD, estimate the heritability of
ABI at ~30%. Two genome wide signals have been identified for LEAD near
CHRNA3 and 9p21.3 irrespective of diabetes status. The aim of this study
was to identify genetic determinants of LEAD in patients with T2D. LEAD
cases were patients with T2D and an ABI < 0.9 or ABI>1.3 and or mid-thigh
to mid-foot amputations and or corrective procedures related to LEAD and
or prescriptions for medication used to treat claudication. Controls were patients with T2D free of LEAD, coronary artery disease and ischaemic stroke.
Allelic effects of 5,882,833 SNPs estimated from 1223 LEAD cases and 5638
LEAD free controls were combined in fixed-effects 1000G meta-analysis. A
signal represented by rs72780858 near FRMD4A reached genome wide significance (p=3.7E-8). SNPs in FRMD4A have been associated with nicotine
dependence which may influence smoking status. Smoking is known to increases the risk of LEAD 10 fold. Other suggestive signals are located near
genes that contain genome wide significant hits for risk factors related to
LEAD: rs271946 (p=8.1E-6) near PCSK1 (T2D); rs34562 (p=8.2E-6) near
EFNA5 (Coronary artery disease) and rs75417257 (p=1.2E-6) near CCSER1
(Glomerular filtration rate and albumin excretion rate). This is the first study of the genetic determinants of LEAD in patients with T2D and several
signals including one at genome wide significance were identified.
P17.47-S
Molecular screening of major thalassemia mutations observed in
2012-2013 in eastern Sicily
In this study we evaluated the incidence of alpha, beta and delta thalassemia
in the province of Messina, in the past two years.
236 people were analyzed (Molecular Genetics Laboratory of Genetics and
Immunology Pediatric UOC), after healthy carrier screening of I and II level of Thalassemia by the diagnostic procedure that provides for the CBC,
the determination of the hemoglobin fractions and the assessment of iron
status. The phenotypic framework (classical and non classical), who had hematological parameters indicative of mutations at the level of alpha clusters
and/or non-alpha-globin, were studied by molecular analysis of related genes. This investigation has shown that in the alpha globin gene, the -3.7/
(genotype frequency 13,1%; allele frequency 7,83%) is the most common
genotype, followed by the 2IVS1:-5nt /WT (genotype frequency 4,66 %;
allele frequency 2,33%), and the -20.5/WT (genotype frequency 2,11%; allele frequency 1,05%) genotypes; instead, in the beta globin gene, the cd39/
WT(genotype frequency 5,08%; allele frequency 2,54) is the most typical
genotype, followed by the IVS1:6/WT (genotype frequency 3,81%; allele
frequency 2,75%) and the IVS1:110/WT (genotype frequency 3,81%; allele frequency 1,90%) genotypes; and finally, in the delta globin gene, the
cd 27/WT (genotype frequency 6,35%; allele frequency 3,17%) is the most
Back to index
LOC284889 is an uncharacterized ncRNA gene on reverse strand to MIF mapped to 22q11.23. MIF, a lymphokine, regulates innate immune response by
up-regulating the expression of TLR4, suppressing the p53 activity and has
been shown to be involved in malaria pathogenesis. In this study, the possible effect of MIF variations on malaria susceptibility was investigated by resequencing the complete MIF gene along with 1kb each of 5 and 3 region in
425 individuals from malaria endemic regions of the Orissa and Chhattisgarh
states of India. The subjects comprised of 160 cases of severe malaria, 101 of
mild malaria and 164 ethnically matched asymptomatic controls. Data were
statistically compared between cases and controls for their possible association with Plasmodium falciparum malarial outcome. It is the first study,
which shows that the allele A (rs34383331T>A) in ncRNA is significantly
associated with increased risk to P. falciparum malaria [severe: OR=2.08,
p=0.002 and mild: OR=2.09, P=0.005]. In addition, it has been observed
that the higher MIF-794CATT repeats (>5) increases malaria risk (OR=1.61,
p=0.01). Further, diplotype (MIF-794CATT and rs34383331T>A) 5T confers protection to severe malaria (OR=0.55, p=0.002) while 6A (OR=3.07,
p=0.001) increases malaria risk. These findings support the involvement of
ncRNA in malarial pathogenesis and further emphasize the complex genetic
regulation of malaria outcome. In addition, the study shows that the higher MIF-794CATT repeats (>5) is a risk factor for severe malaria. The study
would help in identifying people who are at higher risk to malaria and adapt
strategies for prevention and treatment.
P17.49-S
Epidemiology of Meckel-Gruber syndrome in Europe: a registry-based
study
329
ABSTRACTS POSTERS
for Health and Welfare (THL), Helsinki, Finland, 3Hjelt Institute, University of Helsinki,
Helsinki, Finland, 4Heart and Lung Center HUCH, Helsinki University Central Hospital,
Helsinki, Finland, 5Haartman Institute, University of Helsinki, Helsinki, Finland,
6
National Institute for Health and Welfare (THL), Turku, Finland, 7University of Tartu,
Tartu, Estonia.
Later age at menopause has been associated with decreased risk of cardiovascular diseases and increased life expectancy, but associations with diabetes are controversial and no genetic links have been reported. Additionally,
smoking lowers and obesity raises menopausal age, which might disturb the
seen associations. Stolk et al. reported of 17 genetic polymorphisms which
associate with menopausal age (Nat Genet. 2012). We constructed a genetic
score of menopausal age by summing the reported effect sizes (years/allele)
of these polymorphisms in four Finnish cohorts (FINRISK1997, PredictCVD,
Health2000 and Corogene) and studied its associations to common diseases
and their risk factors. Linear, logistic and survival analysis corrected by age
and geographical variables were performed in individual cohorts and combined in meta-analysis.
One-year increase in the genetic score (range -2.3 - 3.1 years) associated
significantly with future diabetes in women (N=2831, hazard ratio=1.46,
P=0.0083) but not in men (P=0.3). Interestingly, the genetic score associated nominally (P<0.05) with prevalent diabetes in men (N=5547, odds
ratio=0.87, P=0.042) but no association was seen in women. In women nominal association of higher menopausal age score was seen also with higher
triglyceride levels and lower HDL cholesterol. Controlling for BMI and smoking did not affect these associations.
Our results suggest that the genetic variants associated with the timing of
menopause in women have different effects on metabolism in different genders. In women polymorphisms raising menopausal age raise also the risk
of future diabetes 1.5-fold whereas in men the same variants have a slight
protective effect on prevalent diabetes.
P17.51-S
Exome-wide association analysis of 4,522 individuals from the Oxford
Biobank study reveals novel low frequency variants influencing
human serum metabolite levels
330
Franco, A. Dehghan;
Erasmus medical Center, Rotterdam, Netherlands.
Back to index
BACKGROUND: While the role of common genetic variants is clearly established from recent studies on sporadic cases of multiple sclerosis (MS),
the contribution of rare variants is unclear. AIMS: To identify rare genetic
ABSTRACTS POSTERS
variants contributing to MS susceptibility in an Italian multiplex family. DESIGN: SNP microarray genotyping and whole-genome sequencing (WGS) in
4 MS patients and 4 unaffected individuals belonging to an Italian multiplex
family descending from a first cousin marriage were performed. RESULTS:
We identified 437 variants with high functional impact, two of which under
one of the two LOD peaks on chromosome 8p21.2 and 11q23.3. The first one
is in the OR8G5 gene, an olfactory receptor gene under positive selection,
while the second one falls within the GRAMD1B gene, causing an aminoacid
substitution (S601P). This second variant is not present in dbSNP and in the
1,000 Genome project, and segregates within the family, being homozygote
in 3 affected and heterozygote in the left MS patient. Sanger sequencing confirmed the segregation with the disease. GRAMD1B is a very conserved gene
from yeast to human. It encodes a membrane protein, which is part of the
GRAM containing domain family protein. In the mouse it is highly expressed
in the CNS and in specific immune cell subtypes, like dendritic cells and neutrophils. CONCLUSIONS: The use uf WGS in an Italian MS multiplex family
has been successful in identifying novel rare genetic variants. Further investigations are ongoing to explore the role of the variant on protein function
and its involvement in MS.
P17.56-M
Statistical methods for the analysis of gene expression in single-family
studies for genetic and complex disease
S. Merella1, F. Esposito2, P. Brambilla2, M. Sorosina2, F. Martinelli Boneschi2, E. Stupka1,
P. Provero1,3;
1
Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute,
Milan, Italy, 2The Laboratory of Genetic of Neurological Complex Disorders, INSPE &
Division of neuroscience, San Raffaele Scientific Institute, Milan, Italy, 3Dept of Molecular
Biotechnology and Health Sciences, University of Turin., Turin, Italy.
The genetic basis of complex diseases often involves multiple linked causative loci. Under such a disease etiology, assuming one disease locus in linkage
disequilibrium mapping is likely to induce bias and lead to efficiency loss in
disease locus estimation. An approach is needed for simultaneously localizing the positions of multiple functional loci. However, due to the increasing
number of parameters accompanying disease loci, these estimates can be
computationally infeasible. To circumvent this problem, we propose to estimate the main and gene-gene interaction effects and a nuisance parameter at
the disease loci separately through a linear approximation. Estimates of the
genetic effects are entered into a generalized estimating equation to estimate disease loci, and the procedure is conducted iteratively until convergence.
The proposed method provides estimates and confidence intervals (CIs) for
the disease loci, the genetic main effects, and the interaction effects between
loci, with the CIs for the disease loci providing useful regions for further
fine-mapping. We apply the proposed approach to a data example of casecontrol studies. Results of the simulations and data example suggest that the
developed method performs well in terms of bias, variance, and coverage
probability, regardless of the underlying number of disease loci.
Back to index
P17.58-M
Evolutionary analysis identifies an MX2 haplotype associated with
natural resistance to HIV-1 infection
Nonsyndromic cleft lip with or without cleft palate (nsCL/P) is one of the
most common congenital malformations and has a complex multifactorial
etiology. In the context of a large collaborative study, whole exome sequen-
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ABSTRACTS POSTERS
cing (WES) was performed in two distantly related cousins of an extended
nsCL/P family of German origin. After stringent filtering of variants shared
between both individuals, one variant in the grainy-head like 3 (GRHL3)
gene was identified as potentially causal. This variant, rs138381915, is located in exon 13 and the potential risk allele mediates an amino acid change
(p.Arg490His) of the GRHL3 protein. Prediction programs (Polyphen, Mutation Taster) predict this alteration to be disease-causing. Sequencing datasets (ESP6500, dbSNP, 1000genomes) report the frequency of the minor
allele at rs138381915 to be below 0.5 %. Literature search revealed GRHL3
as an interesting candidate gene for nsCL/P. Based on the hypothesis that
rs138381915 might be the causal variant in the family, we first confirmed
the variant in the two index probands by Sanger Sequencing. We then tested
all other (29) family members for whom DNA was available for the presence
of the putative risk allele. Of these (10 affected, 19 unaffected), the risk allele was observed in a heterozygous state in seven individuals, only two of
them were affected. Five individuals carried the putative risk allele but were
not affected. Eight affected individuals did not carry the putative risk allele.
These data suggest that rs138381915 is unlikely to be a causal variant regarding the nsCL/P phenotype in this particular family.
P17.61-S
Novel mutations described in Saudi Arabia
J. Y. Alaama1, A. Y. Edrees2;
1
King abdulaziz University, Jeddah, Saudi Arabia, 2Princess Al Jawhara Center of
Excellence in Research of Hereditary Disorders, Jeddah, Saudi Arabia.
Jeddah is the second largest city in the Kingdom of Saudi Arabia with a population of over 5 million. The Genetic Medicine Department at King AbdulAziz University is the major referral center for genetic disorders in Jeddah
and was established in 2005. Over this period 1842 patients and families
with genetic disease were seen. Patients were assessed clinically and where
indicated by genetic testing.They were categorized into the following types
based on etiology: chromosomal, single gene disorders, multifactorial, mitochondrial and others. They were then further subdivided into categories.
Most mutations identified were novel and different from the western literature. We will present the types of genetic diseases present and the novel
mutations identified both locally and nationally and put forward recommendations for future studies including the current set up of a data bank will
discussed.
P17.62-M
Contribution of SNPs (rs9939609 and rs8057044) and haplotypes
of FTO gene to the genetic risk for obesity in children from Yucatan,
Mexico
The fat mass and obesity-associated (FTO) gene has been identified as a
strong candidate for obesity-related phenotypes in several populations.
Significant associations of the SNPs rs9939609 and rs8057044 with body
mass index (BMI) and with the risk for obesity have been suggested in homozygotes AA. The central role of FTO might be through an effect on cerebrocortical insulin sensitivity. Each FTO risk allele increases BMI by 0.26
to 0.66 units (kg/m2), and the odds of being obese by ~1.3. In this study,
we evaluated the association of the SNPs (rs9939609 and rs8057044) and
haplotypes of FTO gene with the risk for obesity in children from Yucatan,
Mexico; where child obesity is the first cause of morbidity. We included 155
obese children, and 189 non-obese healthy children under a case-control
association study. Genotype and allele frequencies between cases and controls were compared using SNPstats software. Genotype and allele frequencies were distributed according to Hardy-Weinberg expectations (p>0.05)
in cases and controls, except for rs9939609-FTO in controls (p<0.05). Significant differences were found for the heterozygous AT genotype of the SNP
rs9939609-FTO (p= 0.03) between cases and controls, suggesting that the
heterozygous AT genotype of rs9939609-FTO might be a genetic risk factor
associated with child obesity in the population of Yucatan. No significant associations were found for the SNP rs8057044 nor for the haplotypes of FTO
gene (p>0.05). However homozygotes for the A allele showed a higher mean
BMI and higher waist circumference than TT or GG allele carriers.
P17.63-S
Molecular Analysis of TYR, OCA2, TYRP1 , SLC45A2 and GPR143 Genes in
158 Patients with Oculocutaneous and Ocular Albinism
332
Back to index
Smell is a versatile mechanism for recognizing different odours and is mediated by olfactory receptors. While collecting phenotypes related to smell
in six countries along the Silk Road, we found an increased rate of failure
to discriminate odorants in individuals from Tajikistan respect to the other
countries. Using haplotype-based association we linked this to a 15 kb region within olfactory receptor gene cluster on chromosome 6 (p-value 3.86e05). This region is embedded in the largest intron of OR5V1 and is downstream OR11A1 and upstream OR12D3. We also analysed genetic variability
in 1,114 unrelated samples either from the Silk Road and ten other worldwide populations at over 300,000 polymorphic sites and characterized population genetic structure of the Silk Road within a worldwide context with
a resolution never obtained before. We identified genetic components peculiar to Central Asia and observed that Tajikistan behaves as an outlier population. Indeed Tajiks share a consistent number of unusually large stretches
of homozygosity and have the lowest effective population size (Ne) among
the studied populations, most likely as the result of past isolation and/or
consanguinity. Altogether these novel findings clarify the complex genetic
patterns of the Silk Road populations and suggest that the smell misperception phenotype observed in Tajikistan might be the result of a combination
of genetic drift and relaxed selection at the olfactory receptors genes.
P17.65-S
Targeting a gene network of ADAMTS genes contributing to pediatric
stroke
ABSTRACTS POSTERS
two sample groups. 32 significant (p<0.05) coding non-synonymous or UTR
SNPs in 14 genes were identified and selected for validation using capillary
sequencing and subsequent genotyping within the full cohort of 270 nuclear
families. The resulting data may help to understand the genetic architecture
of ADAMTS genes and their impact on PS.
P17.66-M
A genome-wide association study of Agreeableness suggests a novel
association in the NAV2 gene in Korean women
H. Kim1, S. Roh1, B. Kim1, H. Kim1, H. Cho1, N. Cho2, C. Shin3, J. Sung4, H. Kim1;
1
Ewha Womans University, Seoul, Korea, Republic of, 2Ajou University, Seoul, Korea,
Republic of, 3Korea University, Seoul, Korea, Republic of, 4Seoul National University,
Seoul, Korea, Republic of.
Data from genome-wide association (GWA) studies have been used to find
the common variants of personality. In a previous study, we reported that
neurotransmitters and the olfactory receptor 1A2 gene are associated with
neuroticism in a cohort of young Korean women. However, many genetic
variants that are highly associated with certain personality traits are still
unknown. Here, we report on a meta-analysis of GWA data for personality
in three cohorts samples (2045 individuals). All participants were of Korean
ancestry. Personality traits were measured with the Revised NeuroticismExtraversion-Openness Personality Inventory to assess five factors: Neuroticism, Extraversion, Agreeableness, Openness, and Conscientiousness. In
either discovery stage, classical association analyses were performed under
an additive model followed by meta-analysis using the weighted inverse variance method. We observed consistent direction of effect and significant
association of the NAV2 gene and Agreeableness in either the discovery and
combined stage (p=7.8510-7, for meta-analysis). NAV2 gene involves in
optic nerve development and sensory perception of smell and sound. We
previously reported that the sensory system may play an important role in
personality, and the present study leads to the same conclusion. The sensory
system affects personality as a filter of the acceptance system, which may
have an advantage to reconstruction.
This study was supported by a grant of the National Project for Personalized Genomic Medicine, Ministry for Health & Welfare, Republic of Korea
(A111218).
P17.67-S
New genetic matching methods for handling population stratification
in genome-wide association studies
A. Lacour1, T. Becker1,2;
1
German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany, Bonn,
Germany, 2Institute of Medical Biometry, Informatics and Epidemiology (IMBIE), Bonn,
Germany.
Back to index
IGF-I, IGF-II, IGFBP-2 and IGFBP-3 in prostate cancer using multiple genetic
variants to construct allelic scores as instruments for measured circulating
IGFs and IGFBPs, in a Mendelian randomization approach.
We investigated 1131 SNPs, previously reported to be associated with IGFs
and IGFBPs, in ~700 population controls from the ProtecT study. We generated allelic scores that consisted of the most strongly -and exclusively- associated SNPs with each biomarker. Finally, we used the allelic scores and instrumental variable analysis to estimate the causal effect of IGFs and IGFBPs
on prostate cancer in ~40,000 cases and controls from 21 studies included
in the international PRACTICAL consortium.
IGF-I and IGF-II were positively associated with prostate cancer risk. The
estimated causal odds ratios for the effect of a standard deviation (SD) increase in circulating IGF-I and IGF-II was 1.15 (95% CI 1.06, 1.26) and 1.14
(95% CI 1.02, 1.26), respectively. Notably, IGF-II increased risk for advanced and high grade disease. Conversely, IGFBP-2 and IGFBP-3 were not associated with prostate cancer susceptibility. In conclusion, this Mendelian
randomization study provides additional support for a causal relationship
between IGF-I and IGF-II and prostate cancer risk.
P17.69-S
Combining different sources of information to optimise genomic
prediction of complex traits
In the last decade there has been substantial progress in identifying genetic
loci associated with complex phenotypes but limited progress in using genomic information to predict phenotypic performance. In this study, we assess
the advantage of using previously published results to inform genomic predictors of complex traits. Our goal is to compare model performance with
respect to trait architecture and latent population structure. We consider
linear, additive models with different sparsity levels, either learned from the
data using lasso or elastic nets, or determined a priori by considering markers and their corresponding effects from large meta-analysis association
studies. We characterise the predictive signal captured by each model and
explore whether prediction accuracy can be increased by combining these
simpler predictors into a meta-model.
We evaluate predictive performance using height, body mass index and high
density lipoproteins in two population cohorts, originating in Croatia and
Scotland. We examine how to maximise prediction accuracy when the target individuals come from the same or a different population to the training
samples. Our results demonstrate that between population prediction is
possible using samples from the target population to perform model selection, subject to sample size and trait architecture. Furthermore, we show
that a model combining the predictions from penalised regression with meta-analysis-based polygenic scores performs better than either model on its
own. Our findings suggest that incorporating previous results into statistical
models as well as exploiting the predictive signal from latent data structure
can lead to improved predictions of complex traits.
P17.70-M
Technical issues of using Next Generation sequencing for rare-variant
association
C. G. F. de Kovel, R. van t Slot, B. P. C. Koeleman;
UMC Utrecht, Utrecht, Netherlands.
333
ABSTRACTS POSTERS
removed batch effects beyond all doubts.
Based on our experiences, the use of control sequences from earlier experiments or even other labs cannot be recommended. It is likely to lead to
spurious associations or lack of associations.
P17.71-S
A Script for Linkage Analysis of Rare variants
I. Zorkoltseva, T. Axenovich;
Institute of Cytology and Genetics, Novosibirsk, Russian Federation.
Sophisticated techniques have been developed for genome wide association studies (GWAS) to correct for population substructure that may cause
substantial inflation of test statistics and spurious associations. For rare
variants in spatially structured populations the existing methods do not
effectively control for stratification. We developed an approach to build
up a control group that is most similar to the individuals in the case group
considering all exomic variants especially also the rare ones. We show with
simulations of real exome data that the power of association studies for rare
variants can be optimized if case-control groups are similarity-matched. We
also found that the power could be further improved when we increased the
control:case group ratio by adding additional exome data.
P17.73-S
Functional linear model for regional association analysis of rare
genetic variants in family-based samples
G. R. Svishcheva, N. M. Belonogova, T. I. Axenovich;
Institute of Cytology and Genetics, Novosibirsk, Russian Federation.
Methods based on the linear regression models are widely used for genomewide association analysis of family-based samples. To use these models for
region-based analysis, a collapsing approach is commonly used. However its
power decreases if effects of causal variants have opposite directions.
We developed a new method for the region-based association analysis of
family-based samples, using techniques of functional data analysis. The individual discrete genotypes and effects of multiple variants in the analyzed
region are considered as the continuous data, which can be described by
stochastic functions constructed on the physical positions of the variants,
using a finite set of basis functions. Thus, under the functional linear model,
the genetic effects of the multiple variants are described by coefficients of
the basis functions. The null hypothesis of zero values for the coefficients is
tested by standard statistical tests. We introduced a covariance matrix defined through a relationship matrix in the trait inheritance model to take into
account a genetic relationship between individuals. High power of our method is provided by the simultaneous consideration of genetic information
on not only genotypes of multiple variants, but also their physical positions,
and by taking into account related structure of the samples. Our method is
implemented in the software package FFB-FLM available for free download
(http://mga.bionet.nsc.ru/soft/FFB-FLM/).
This work is supported by RFBR grants 13-04-00272a, 14-04-00126a.
334
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P17.74-M
Genome wide inbreeding estimation within Lebanese communities
using SNP arrays
Rhesus macaques (Macaca mulatta) are the most widely studied nonhuman
primate model species in biomedicine, critical to various aspects of disease
research. We applied next generation whole genome sequencing for 152 unrelated individuals (144 Indian-origin, 8 Chinese-origin) using either deep
(30X) or low coverage (6X) strategies. Analysis using SNPTools identified
51.6 million SNPs. On average, Indian-origin individuals have >9.5 million
variants, while Chinese animals have >12 million, representing much higher
diversity than humans. Current effective population size (Ne) is estimated
at 65,000 for Indian-origin animals, 79,000 for Chinese-origin. Both estimates are substantially higher than values for humans. Analyses also reveal
dramatic demographic changes over time in details to more than 10 million
years ago. Functional annotation in coding sequences found >250,000 missense variants and >4600 stop-codon-gained mutations. In addition, about
110,000 SNPs mapped to conserved ENCODE transcription factor binding
motifs. We used position weight matrices from the JASPAR database to assess these SNPs and found >25,000 candidate variants that may significantly
affect TF binding, and thus gene expression. We mapped rhesus SNPs to the
4% of the genome identified as conserved across 29 mammals, and found
reduced SNP density and MAF, consistent with negative selection in those
regions. We also applied a number of site frequency spectrum tests and
found significant new evidence for both positive and negative selection in
both coding and noncoding regions in the macaques. Analyses of LD and
local recombination rates are in progress.
P17.76-M
Association of five confirmed risk gene polymorphisms with
Rheumatoid Arthritis in the Algerian population
Objectives: The aim of this study was to investigate the role of five con-
ABSTRACTS POSTERS
firmed gene polymorphisms (PTPN22rs2476601, STAT4rs7574865, IRF5rs2004640, TRAF1/C5rs10818488 and TNFAIP3rs6927172) in Rheumatoid Arthritis risk among the Algerian population.
Methods: The study sample comprised 110 patients with RA and 197 ethnically matched healthy control subjects. Each polymorphism was genotyped
using a predesigned TaqMan assay. Allele and genotype frequencies in
patients and control subjects were compared by chi-square test and odds
ratios with 95% confidence intervals CI.
Results: Statistically significant association of all studied polymorphisms with RA was detected. The strongest signal was obtained for PTPN22
(rs2476601) with an allelic P value = 10-11 (OR = 9.83, 95% CI [4.28 22.56]).
Conclusion: This case/controls study lead, for the first time in the Algerian
population, to highlight the association of five risk genetic factors with RA.
This contributes to the characterization of RA genetic component in a population still under genetic investigation.
P17.78-M
How does this Arab Genome differ from other genome?
I. B. N. M. Alabdulkareem, M. A. Aljumah, M. A. Albalwi;
King Abdullah International Research Center, Riyadh, Saudi Arabia.
Introduction:
Soluble CD40 ligand (sCD40L) is a platelet-derived proinflammatory mediator that accumulates during platelet storage. Unfortunately, in some cases,
high levels of sCD40L cause acute transfusion reactions (ATRs). We investigated 2 polymorphisms previously associated with high levels of plasma
sCD40L in some diseases, in the aim to identify dangerous platelet concentrates (PCs).
Material and methods:
Levels of sCD40L were measured by Luminex in 142 PCs (EFS Auvergne-Loire). We performed a Tetra-primer ARMS-PCR to genotype CD40rs1883832 [C>T] and CD40L-rs3092952 [A>G]. Differences of plasma
sCD40L concentrations among groups of genotypes were compared by
ANOVA followed by Bonferroni correction (significance when P<0.017).
Results:
Genotype frequencies didnt deviate from Hardy-Weinberg expectations.
The distribution of genotypes for CD40L-rs3092952 was 68, 43 and 30 for
AA+A, AG and GG+G respectively. No difference was observed between the 3
genotypes in sCD40L levels.
The distribution of genotypes for CD40-rs1883832 was 68, 52 and 8 for CC,
CT and TT respectively. sCD40L was higher in PCs derived from donors with
TT genotype (TT vs CC: P<0.001; TT vs CT: P<0.001; CT vs CC: P=0.7).
Discussion:
The CD40-rs1883832 polymorphism has been reported to regulate CD40
expression. sCD40L may activate cells expressing CD40 receptor as in platelets. We hypothesize that those with TT genotype may express a high
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amount of CD40 on their surface and thus may capture sCD40L, becoming
more activated, thus secreting more sCD40L.
Further studies are required in larger samples to confirm this result allowing the discard of PCs with high sCD40L amounts and consequently preventing ATRs.
P17.80-M
Inferring rare disease risk variants based on exact probabilities of
sharing by multiple affected relatives
I. Ruczinski1, A. Bureau2;
1
Johns Hopkins University, Baltimore, MD, United States, 2Universite Laval, Quebec, QC,
Canada.
Family based study designs are regaining popularity because large-scale sequencing can help to interrogate the relationship between disease and variants too rare in the population to be detected through any test of association
in a conventional case-control study, but may nonetheless co-segregate with
disease within families. When only a few affected subjects per family are sequenced, evidence that a rare variant may be causal can be quantified from
the probability of sharing alleles by all affected relatives given it was seen
in any one family member under the null hypothesis of complete absence
of linkage and association. We present a general framework for calculating
such sharing probabilities when two or more affected subjects per family
are sequenced, and show how information from multiple families can be
combined by calculating a p-value as the sum of the probabilities of sharing
events as (or more) extreme. We also examine the impact of unknown relationships and propose methods to approximate sharing probabilities based
on empirical estimates of kinship between family members obtained from
genome-wide marker data. We apply this method to a study of 55 multiplex
families with apparent non-syndromic forms of oral clefts from four distinct
populations. Whole exome sequencing was performed by the Center for Inherited Disease Research (CIDR) on two or three affected members from
each family. The rare single nucleotide variant rs149253049 in the gene
ADAMTS9 was shared by affected relatives in three Indian families (p=2e-6),
illustrating the power of this sharing approach.
P17.81-S
A Founder Effect for PPIB-associated recessive osteogenesis
imperfecta in Acadian and Cajun Populations
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ABSTRACTS POSTERS
with arbitrary relatedness structures. The possibility to use population cohorts rather than family structures opens up a multitude of new avenues
also for genetic epidemiological research on time-to-event outcomes. However, connecting time-to-event outcomes to whole-genome sequencing data
has so far not been computationally feasible with hitherto existing software. We introduce an R package (GSM) for heritability estimation, multilocus association testing and genomic prediction of time-to-event outcomes
that is scalable to sequencing data. The package implements a very flexible
piece-wise constant hazard model that contains an individual-specific Gaussian random effect with an arbitrary covariance structure. Computationally,
we transform the problem to a Poisson model, which we analyze by fitting
interconnected Generalized Linear Models. The underlying computational
algorithm is written in C++ to enable analyses with millions of genetic markers and events in thousands of individuals. We demonstrate the runtime
efficiency of our implementation and give an example of heritability estimation and multi-locus association testing for cardiovascular disease related events. Our work extends the computational tractability of linear mixed
models from quantitative traits to time-to-event outcomes and will prove
useful, e.g., for combining information across individuals genomes and their
hospital records.
P17.83-S
Genome- wide association analysis of swallowing symptoms related to
dysphagia in a healthy older adult cohort
A. Raginis-Zborowska, N. Pendleton, S. Hamdy, W. Ollier;
The University of Manchester, Manchester, United Kingdom.
Background: Patients with swallowing difficulties (oro-pharyngeal dysphagia) caused by neurological damage (stroke, Parkinsons disease, ageing) show different recovery patterns which may be caused by genetic
determinants.
Aim: To find an association between human genetic variations and swallowing impairments within the ageing cohort.
Materials and methods: We performed case-control genome wide association study (GWAS) of self-reported swallowing symptoms related to dysphagia. The analysis included 555 community dwelling, unrelated, older adults
(mean years of age = 81.4; SD = 5.349) with known phenotype and genetic
information consisting of 512 806 single nucleotide polymorphisms (SNP).
Gene-based association analyses of these traits was also conducted. The
genetic data underwent quality control procedures prior to the study. This
included analysis of population architecture using Multidimensional Scaling
of the genome wide genotype data.
Results: Analysed cohort showed European ancestry with no major population stratification. The results shown one genome wide significant SNP
rs17601696 (P=4.83x10-8) from non-coding region of chromosome 10.
Analyses of individual genes did not result in any genome-wide significant
association.
Conclusion and future work: SNP rs17601696 may have an impact in
swallowing impairment among elderly individuals. The results require replication in an independent cohort with appropriate phenotype/genotype
data. Presented GWAS results will be replicated in the human model study
using Transcranial Magnetic Stimulation (TMS). Identified genetic loci may
play a role of potential markers to predict individuals outcome from swallowing impairments.
P17.85-S
Investigation of hellenic families with microscopic hematuria reveals
the frequency of collagen IV mutations and evidence for activation of
the unfolded protein response
L. Papazachariou;
University of Cyprus, Nicosia, Cyprus.
Familial hematuria(s) comprise a genetically heterogeneous group of conditions which include heritable glomerulopathies (Alport Syndrome (AS)
and thin basement membrane nephropathy (TBMN)). AS is rare, caused
by X-linked COL4A5 or autosomal recessive COL4A3/A4 mutations (ARAS),
while TBMN is frequent. We sought to evaluate the prevalence of COL4A3/
A4 mutations among patients presenting with microscopic hematuria (MH),
belonging to 91 Hellenic families. Also we studied 28 sporadic patients with
MH and four patients with ARAS. We performed functional studies in cultured podocytes, focusing on the induction of the unfolded protein response
(UPR) after overexpression of wild type or mutant COL4 chains.
Among 91 families, heterozygous mutations were found in 11 (12,1%). Restricting the calculation to 68 families with 3 patients with MH, the positive finding is 11/68 (16,2%). Three heterozygous mutations were found
in three of the 28 sporadic patients (10,7%). Altogether, among 52 heterozygous patients 17,3% reached end-stage kidney disease (ESKD). Of those
aged >50 years, 26% reached ESKD, in keeping with previous findings that
336
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TBMN is not always benign. Functional studies showed that mutant COL4A3/A4 chains expressed in podocytes are preferentially retained in the
cells, compared to wild type chains. Mutant chains differentially triggered
activation of the UPR pathway, as evidenced by activation of BiP, a sensitive
ER stress marker.
TBMN may emerge as a more frequent cause of ESKD than AS. The ability of
mutant chains to elicit the UPR pathway when overexpressed in podocytes
may prove of functional significance and prognostic value.
P17.86-M
Variation in BTBD9 gene is associated with Tourette syndrome in the
Polish population
M. Berdynski1, P. Janik2, M. Kobrys1, K. Safranow3, C. Zekanowski1;
1
Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland,
2
Medical University of Warsaw, Warsaw, Poland, 3Pomeranian Medical University,
Szczecin, Poland.
Tourette syndrome (TS) is a childhood-onset neuropsychiatric disorder manifested by tics. The anatomical location, number, frequency, complexity, and
severity of tics change over time. The etiology of the disorder is unknown,
though the predominant role of genetic factors has been established. Variants of the BTBD9 gene (rs4714156, rs9296249 and rs9357271) were
reported to be associated with GTS in French Canadian and Chinese Han
populations.
The study group comprised 162 TS patients, and the control group consisted
of 180 healthy persons. The rs4714156, rs9296249 and rs9357271 variants
of the BTBD9 gene were genotyped.
Analyzed SNPs indicated a strong linkage disequilibrium (D= 1, r2=0.938-1).
). MAFs, genotype frequency, allelic, genotypic and haplotype association
analysis within examined variants of the BTBD9 gene revealed no significant differences between controls and TS patients. However, there were
significant associations between the BTBD9 gene variants and a clinical
phenotype of TS. Minor alleles of all three SNPs were found significantly less
frequently in patients with ADHD and were more frequent in patients with
no comorbidities. There was a borderline statistical significance for minor
alleles to be less frequent in patients with severe tics. All three SNPs were
not found to be associated with the family history and the age of tic onset.
Our results indicate that the examined variants of the BTBD9 gene are not
associated with the risk of developing GTS, but may be associated with comorbidity and tic severity in the Polish population.
P17.87-S
The investigations of susceptibility genes/variants related to type 2
diabetes in Turkish
ABSTRACTS POSTERS
research center, Research Institute for Endocrine Sciences, Shahid Beheshti University of
Medical Sciences, Tehran, Islamic Republic of Iran, 3Prevention of Metabolic Disorders
Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of
Medical Sciences, Tehran, Islamic Republic of Iran, 4Endocrine Research Center, Research
Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran,
Islamic Republic of Iran.
Being located along the proposed southern migration route and the presence of earliest skeletal evidences of anatomically modern human (37,000
B.P.) with aboriginal Vedda population, the island of Sri Lanka could provide the knowledge of genetic variation of modern humans in South Asia. In
order to reveal the genetic relationship of the Vedda people with the tribal
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groups along the southern coastal migration route, the present study compared the mitochondrial DNA (mtDNA) hypervariable segment 1 variations
from 75 Vedda people from Sri Lanka with 33 world tribal groups from
published data bases. The Vedda people signal out as an exceptional tribal
group of South Asia having less than 30% of individuals sharing haplogroup
M with 64% of haplogroups R30, U1 and U7. The latter two haplogroups
were recognized as West Eurasian ancestry. In principal component analysis (PCA) some Vedda groups occupied separate positions while some were
closely related to South Indian tribal groups. Most interestingly, by viewing
PCA from the point of view of the Southeast Asian foragers, it is evident that
their closely related groups are the Vedda people. This fascinating genetic
footprint is suggestive of yet another piece of evidence of the dispersal of
anatomically modern human out of Africa via the southern migration route.
And also this mtDNA study highlights Sri Lankas strategic location along the
southern migration route, thereby providing a genetic gold mine, which will
offer insight into the initial settlements and peopling of South Asia.
P17.91-S
RNA-Sequencing reveals differential gene expression between visceral
and subcutaneous adipose tissue in Greek women undergoing
abdominal surgery
E. Katsareli1, C. Amerikanou1, K. Rouskas2, M. Reczko2, A. C. Dimopoulos2, S. Glentis2, D.
Bielser3, I. Padioleau3, I. Griniatsos4, T. Diamantis4, E. T. Dermitzakis3, I. Ragoussis5,2, A. S.
Dimas2, G. V. Dedoussis1;
1
Harokopio University of Athens, Department of Nutrition and Dietetics, Kallithea,
Athens, 17671, Greece, 2Biomedical Sciences Research Center Al. Fleming, Vari,
16672, Greece, 3Department of Genetic Medicine and Development, University of
Geneva Medical School, Geneva 1211, Switzerland, 4First Department of Propaedeutic
Medicine, Laiko General Hospital, Athens University Medical School, Athens, 11527,
Greece, 5Genome Quebec Innovation Centre and Department of Human Genetics, McGill
University, Montral, QC, Canada.
Bulgaria is situated on the presumed trajectory of the pioneer colonization of Europe. Since then it has been subjected to a series of demographic
events with disputable impact on the contemporary Bulgarian gene pool.
One of the most controversial issues of the Bulgarian past is the origin of
the proto-Bulgarians, which were previously considered as a sparse Turkic
population.
In order to delve into Bulgarian patrilineal origins we have performed a
survey of Y-chromosome haplogroups followed by meta-analysis of haplogroups C, N and Q distinctive for Altaic populations.
The analysis was performed on a sample comprising 808 Bulgarian males
using RFLP and DHPLC analysis. We have found that only 1.49 % of the contemporary gene pool belongs to haplogroups C, N and Q. Our results were
used to upgrade and extend the distribution maps of these haplogroups and
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ABSTRACTS POSTERS
to compare their frequency in 240 Eurasian (sub-) populations with more
than 20 000 samples.
The comparison reveals a statistically significant difference in the distribution of the studied haplogroups between Bulgarians and Altaic populations
as well as between Bulgarians and Eastern Slavic populations. Based on
the novel historical studies which point to a substantial contribution of the
proto-Bulgarians to the modern Bulgarian gene pool the obtained results
suggest that there is no common genetic ancestry between proto-Bulgarians
and present day Altaic populations as they reject the hypothesis of the Turkic origin of proto-Bulgarians.
P17.93-S
Forensic parameters and allele frequency distribution of 15
autosomic STR loci in a Mestizo population from the State of Yucatan,
Mexico
J. Sosa-Escalante1, A. Acosta-Tun1, P. Chable-Castillo1, C. Herrera-Najera1, M. Rivera-.
Guzman2, L. J. Gonzalez-Herrera3;
1
DIMYGEN Laboratorio, Merida, Mexico, 2Genomelab, Tijuana, Mexico, 3Universidad
Autonoma de Yucatan, Merida, Mexico.
The State of Yucatan is a region with a high density of population with Mayan ascendancy at Southeastern, Mexico. Short tandem repeat (STR) polymorphisms are mainly used in forensic fields for paternity tests and personal identification. Since, there are no STR databases from Yucatan, Mexico,
a sample of 200 mestizos was PCR-typed for fifteen STR loci with the Power
Plex 16 Promega kit (D3S1358, THO1, D21S11, D18S51, Penta E, D13S317,
D7S820, D16S539, CSF1PO, Penta D, VWA, D8S1179, TPOX and FGA). Genotype distribution by locus was in agreement with Hardy-Weinberg expectations for all fifteen STRs. The most highly polymorphic loci were Penta E
and D18S51, showing 19 and 17 alleles, respectively. Heterozygosity index
ranged from 61.5 for D3S1358 to 94.5 for Penta E. For each locus, allele frequencies were obtained, ranging from 0.003 to: 0.455 for D3S1358; 0.380
for THO1, 0.283 for D21S11, 0.203 for D18S51, 0.188 for penta E, 0.480 for
D5S818, 0.243 for D13S317, 0.323 for D7S820, 0.328 for D16S539, 0.363
for CSF1PO, 0.278 for penta D, 0.390 for VWA, 0.345 for D8S1179, 0.468
for TPOX, 0.210 for FGA. The most discriminating loci were Penta E (PD =
0.981) and D18S51 (PD = 0.965). The combined power of exclusion was
0.9999999 in the studied population. Our results suggest that this system
provide powerful discrimination and remarck the importance of the generation of local databases for STRs when these markers are being currently
used in forensic casework.
P17.94-M
Retrospective analysis of live birth prevalence of children with Down
syndrome in Denizli, Turkey
Down syndrome (OMIM: #190685) is the most frequent chromosome abnormality among live births. Its prevalence increases with maternal age,
and can be diagnosed by antenatal screening. The average incidence in the
USA is 1/700-800 live births and 1-3/1000 births in the EU. We examined
prevalence variations of DS in Denizli, Turkey, through a retrospective study.
Sixteen years of survey data were retrieved from the two main state hospital
registry records between 1994 and 2010, subject had diagnosis as ICD-90.
We also obtained some demographical, marital and child bearing trends in
Turkish population from Turkey Demographic and Health Survey and TurkStat. Additionally we search some data from EUROCAT for EU and CASP for
USA. We identified 113 DS live births in Denizli for 16 years. The prevalence
of DS was 9.07 per 10,000 live births before the year 2000 and 9.90 after
2000. The prevalence did not change significantly. The population in Turkey is still young; the fertility rate is high in women under 35 years old, in
contrast to EU, and prenatal screening programs are extensively applied; for
these reasons, the prevalence of DS has remained stable during these 16
years.
P17.95-S
Six novel loci associated with VEGF circulating levels identified by a
meta-analysis of genome-wide association studies
338
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ABSTRACTS POSTERS
P18.02-M
Evaluating a digital information resource for adolescents in genetic
research - adolescent and parent perspectives on information
requirements
There is insufficient evidence regarding adolescent participants information needs in genetic research. Previous information interventions have
yielded little improvement in understanding and confidence in participation decisions, and have not specifically addressed the needs of adolescents.
UK adolescents (aged 12-17) and parents with experience of participation
in genetic research discussed their knowledge, attitudes, and information
needs surrounding genetic research, in focus groups or interviews. A digital information resource (website) was developed following participants
suggestions, and subsequently evaluated for its feasibility and desirability
by adolescents (n=21) and parents (n=5) with and without experience of
genetic research. Thematic analysis was carried out on transcripts of focus
groups and interviews, and on written feedback. Predominant attitudes to
genetic research were that such research is prestigious and complex, and
that participation in genetic research is a relatively simple proposition, until
potential outcomes are explored. The evaluation of the resource suggests
participants favour comprehensive information, presented using modern
technology, which is accessible and manageable. Further, participants recommend that information is customisable to accommodate individual
differences in requirements, concerning specific content and information
quantity. Finally, the analysis suggests participant autonomy is an integral
aspect of information provision for adolescents, and should be explicitly
incorporated into information design. Digital technology can satisfy participants preferences, though information resources should be designed with
the specific needs of the adolescent population in mind. Questions remain
regarding the minimum essential information in genetic research, but participants should be afforded as much choice as possible.
P18.03-S
Adolescent participation in genetic research - motivations and
influences on adolescent and parent participation decisions
Increasing numbers of young people participate in genetic research following changes in international regulations. There is a recognised need to support children and adolescents in making informed participation decisions,
yet there is little evidence concerning how to do so. Moreover, adolescents
are a distinct population from children and adults because of their cognitive development and social context. A thematic analysis of focus groups and
interviews with UK adolescent patients (aged 12-17) who have previously
participated in genetic research (n=7), and their parents (n=7), explored
participants experiences of decisions regarding research participation. The
analysis suggests that participants prioritise subjective and interpersonal
factors when making participation decisions, and that genetic aspects of research are subordinate influences on decisions. Adolescents cited primarily
altruistic motivations, while parents referred to their childrens illness and
healthcare experiences as key to participation decisions. Further, the analysis demonstrates how motivations such as altruism and personal benefit are
mediated by other aspects such as trust and personal histories, and highlights differences between adolescent and parental considerations regarding participation in genetic research. The potential influences of subjective
or interpersonal influences on adolescent and parent decisions should be
taken into account when inviting participation, in order to support autonomous participation decisions. Further, participant information should be
tailored to each groups needs to reflect adolescent and parent priorities,
and to ensure that participants are properly informed concerning genetic
aspects of research which may not be viewed as a priority.
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P18.04-M
Telethon Network of Genetic Biobanks: a key service for diagnosis
and research on rare diseases
339
ABSTRACTS POSTERS
bank, involving various stakeholders concerned;
- the definition of criteria and procedures to disclose individual findings to
biobank participants, in preparing for future research on data collected.
P18.06-M
Do we still need to follow the traditional model of face to face results
disclosure for BRCA predictive testing? An examination of current
practice in the Republic of Ireland (ROI)
R. OShea, M. Meany, N. Coady, D. Healy, A. Green, S. Lynch, C. Debaroid;
National Centre of Medical Genetics (NCMG), Dublin, Ireland.
There is evidence that risk-stratified population screening based on multiple factors including a polygenic risk profile has the potential to be more efficient than age-stratified screening. Therefore, genetic information is likely
to be used for cancer prevention strategies in the general population in the
future. To understand issues of acceptability of such genome based screening in the public, we reviewed issues of genetic risk-communication, how
disease risk is perceived and the behavioural response of people to genetic
risk feedback. A systematic review was conducted based on inspection of
1948 abstracts and use of 25 studies. We found that though the general population has limited genetic literacy, they are interested in being informed
of their genetic risk status. They are generally positive about using genetic
information in disease prevention but not about provision of this information to employers and insurers. There is evidence of positive association
between perceived risk and cancer screening behaviour such as uptake and
mammography, particularly when risk is communicated by categorising
into high, medium and low strata. Yet genetic risk feedback particularly that
conveying small increases of risk has little or no effect on lifestyle changing
behaviour. Also, personalised risk-communication is effective in improving
knowledge and risk perception of the population. Personalised risk information is associated with only short term psychological distress including
anxiety and fear but not with fatalism. Strategies of preventing cancer using
personalised genetic information may potentially be acceptable to the general public. However, before implementation of risk-stratified screening,
further empirical evidence is needed.
P18.08-M
Carrier screening for recessive disorders through exome sequencing
P. Makrythanasis1, A. Massouras1, S. E. Antonarakis1,2,3;
1
University of Geneva, Geneva, Switzerland, 2University Hospitals of Geneva, Geneva,
Switzerland, 3iGE3 Institute of Genetics and Genomics of Geneva, Geneva, Switzerland.
340
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ABSTRACTS POSTERS
P18.11-S
Dynamic Consent - A Patient Interface for 21st Century Research
Networks
Introduction Many findings from NGS diagnostic techniques cannot be interpreted yet, but may provide medically relevant information in the future. However, guidelines on recontacting former patients if new actionable
information arises are lacking. Methods As a first step in developing such
guidelines, we conducted a systematic literature search on recontacting in
clinical genetics in PubMed, Embase, Web of Science, and Google Scholar.
Our search strategy identified 974 articles in all four databases. Fifty fulltext articles in English language met our inclusion criteria, and were included in the review. Results Most literature is from the US (48%), followed by
Canada (22%) and Europe (22%). In the literature recontacting is usually
not regarded a legal obligation in clinical genetics. Most authors do consider
recontacting to be desirable. Many articles argue that the responsibility for
recontacting should be shared with the patient. We found no national or international guidelines, except for the 1999 ACMG policy statement on duty
to recontact. Only four of the fifty articles described practical experiences
with recontacting. These showed that patients were usually positive about
the renewed contact. Conclusion Most authors consider recontacting to be
desirable. The limited empirical evidence indicates that patients appreciate
recontacting. Practical problems of implementing recontacting in clinical
genetics are brought forward most often as argument contra imposing a
duty to recontact. One of the challenges for the future will be to create ways
to overcome these. Legal issues remain important. We therefore consider it
important to develop guidelines on this topic for the NGS-era.
P18.13-S
The ethical dimensions and the tools for data sharing in genetics
within evolving frameworks
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Personal genomic tests (PGT) for disease risk assessment, based on genomewide association study variants, have been offered directly-to-consumers
(DTC) by several companies since 2007. Concerns regarding their potential adverse impact include, among others, lack of counselling, dubious test
quality, unnecessary anxiety and medical interventions based on erroneous/
misinterpreted results. To mitigate worries professional education on PGTDTC has been advocated and the central gatekeeper role of family physicians
has been highlighted.
Relatively few studies have been published on awareness, involvement and
attitudes of healthcare providers on DTC marketing of PGT and, to the best of
our knowledge, none in Italy.
A 2008 CDC survey showed that 42% of healthcare providers were aware of
DTC-PGT, that 42% of them had at least one patient asking questions about
having such a test and 15% had at least one patient who brought test results
in the past year.
The preliminary results of a 2014 survey in the Emilia-Romagna Region (Italy), involving solely general practitioners, show that slightly more than 20%
of the respondents are aware of DTC-PGT and that respectively 85% and
15 % of them feel unprepared or only partly prepared to answer questions
about such tests. About 45 % of them have had at least one patient asking
questions on purchasing or performed DTC-PGT during 2013. These data are
coherent with the limited number of Italian companies directly marketing
PGT and underscore the critical need to enhance primary physicians information on genomics tests provided outside of the clinical setting.
P18.15-S
The research policy regarding disclosure of genetic research results:
A historical perspective in Japan
J. Minari1, K. Kato1,2;
1
Department of Biomedical Ethics and Public Policy, Graduate School of Medicine, Osaka
University, Osaka, Japan, 2Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto
University, Kyoto, Japan.
341
ABSTRACTS POSTERS
P18.16-M
Recent situation about rules of sharing, reuse and circulation of
personal genome data in Japan
N. Yamamoto;
Osaka University, Osaka, Japan.
Centre for patients with Epidermolysis bullosa congenita (EB) works at the
University Hospital Brno since 2001, from 2012 as the highly specialized
medical care centre. EB Centrum CZ is a member of the international network of EB centres and clinical experts. The Centre cooperates with DEBRA
Czech Republic (member of the Czech Alliance for Rare Diseases) witch
supports people with EB and their families and try for 10 years to engage
people with EB to a full life. In EB Center CZ works a multidisciplinary team
of health specialists which provides comprehensive care to all EB patients
in CZ. The DNA analysis is performed for EB simplex (EBS) and dystrophica
(EBD) (analysis of the genes for keratin 5 and 14, and collagen VII) . The
mutation was found in 60% patients with EBS, in 67% patients with the
dominant EBD and in all patients with the recessive EBD. In one patient was
confirmed a rare form of EB caused by a mutation in the gene for plectin.
Genetic counseling in our Centre was performed in more than 90 % families
with the recessive form of EBD, in about 66 % families with the dominant
form of EBD and in about 60 % families with EBS. EB patients from Slovakia,
Russia and Ukraine are interested in consultation, genetic conselling and
DNA analyse in EB Centre CZ. Activity of EB Centre CZ and DEBRA CZ are
also focused on education and awereness for healthcare professionals, patients, their families and the public.
P18.18-M
Enhancing genetic counseling for Familial Alzheimers Disease
through improved phenotyping.
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Available guidelines do not clearly address this issue and thus may not provide sufficient guidance for clinicians and counselors. Based also on experience in other contexts, we propose criteria to improve FAD cases phenotyping aiming at enhancing the quality of genetic counseling activities.
P18.19-S
The efficacy of the teaching of the patients during genetic counseling
session
Studies evaluating the efficacy of the genetic counseling are few in number
in Russia. In our study, we evaluated the efficacy of the genetic counseling for
families with affected children or relatives by a number of parameters, such
as change in the conversance of patients and the impact of genetic counseling on reproductive plans of the patients. The study was conducted in the
outpatient department of the Research Centre of Medical Genetics (RCMG),
the Russian Academy of Medical Sciences (RAMS), in 2007-2011. We interviewed 226 respondents aged 17 to 67 years. The survey was conducted before and after genetic counseling. Questionnaires have been analyzed using
Statistica 10.0 software. We found that the conversance of the risk of an affected child birth after genetic counseling increased significantly (Wilcoxon
test, p <0.05). So did conversance of patients about the possibility of prenatal diagnostics ( - distribution, p <0.05). However, when assessing the
extent to which the patients increased their understanding of the causes of
hereditary diseases (that was assessed by a series of test questions), we did
not detect any changes in conversance of patients before and after genetic
counseling. We concluded that these issues deserve a special attention when
conducting genetic counseling. Also, we found no significant changes in reproductive plans of the patients after genetic counseling. Currently, there
are many reasons that affect the reproductive plans of the family, in addition
to an adequate understanding of the extent and meaning of genetic risk.
P18.20-M
Changes in the publics perception of clinical genetics in Cyprus:
Reflections from the 20 years of experience of the Clinical Genetics
Clinic (CGC)
V. C. Anastasiadou1,2, G. Tanteles1, E. Aristidou1, A. Kotti2, T. Delikurt1;
1
Cyprus Institute of Neurology and Genetics; Clinical Genetics Clinic, Nicosia, Cyprus,
2
Makarios Medical Center; Clinical Genetics Clinic, Nicosia, Cyprus.
Having been established in 1994, the Clinical Genetics Clinic (CGC) is a pioneer in the field of clinical genetics in Cyprus. Over the course of the last 20
years, we have had first hand experience in how peoples perception and
acceptance of the offer of clinical genetics and genetic counselling has evolved. While in its infancy we observed frequently that patients experienced
fear of being stigmatised, characteristic of small societies, if diagnosed with
a genetic condition or if they pursued genetic counselling. Some patients
also considered being referred to CGC as accepting to proceed with prenatal
diagnosis. There was a lack of awareness that genetic counselling would give
them the opportunity to discuss their options, receive psychosocial and educational support to make autonomous decisions. As a result, the CGC embarked on (and still continuous) organising public awareness activities, as well
as educating patients/families and other health care professionals on common/rare genetic disorders and the process of clinical genetics and genetic
counselling. In recent years, patient referrals increased substantially from
wide range of specialists (paediatricians, OBGYN, neurologist, oncologist
etc).There are more informed individuals/patients compared to before, who
may inquire about or instigate referral to genetic counselling themselves as
well. We grew in our team as well as services including the Cancer Genetics
Clinic, established in 2006, to provide tailored genetic counselling to cancer
patients. In this poster, we will elaborate on these points mentioned as well
as others to reflect on our 20 years experience in offering clinical genetics.
P18.21-S
Employment and professional integration of genetic counsellors in
France : a new collaboration in the healthcare sector
ABSTRACTS POSTERS
tics. How are these new non-medical practitioners integrated into the multidisciplinary services of genetics? How are they recruited and with which
status? What are the responsibilities entrusted to them? How are they perceived by geneticists working with them? We performed a literature survey
to trace the history of the creation of this profession. To answer the underlined questions, we used socio-epidemiological studies through the elaboration of surveys regarding the education, the role and practice of genetic counselor. Studies were addressed to both genetic counselors and geneticists.
Among the 122 graduate genetic counsellors, 94 are employed (77%), with
an average monthly salary of 2,001.21 /month. They are able to manage
consultations only when no medical procedures are required. However, the
responsibilities are dependent on the relationship established between the
genetic counsellor and the medical geneticist. Overall, this profession has
been quickly established in France and is the only one governed by a specific
law at European level. Although this survey emphasizes inequalities in the
practice of this new profession, including discrepancies regarding the administrative aspects, genetic counsellors are increasingly being integrated into
all levels of healthcare service delivery.
P18.22-M
Challenges of web-based personal genomic data sharing
M. Shabani, P. Borry;
KU Leuven, Leuven, Belgium.
In order to study the relationship between genes and diseases, the increasing availability and sharing of phenotypic and genotypic data has been
advanced as an imperative within the scientific community. In parallel with
data sharing practices by clinicians and researchers, recent initiatives have
been observed in which individuals are sharing personal genomic data. The
involvement of individuals in such initiatives is facilitated by the increased
accessibility of personal genomic data, offered by private test providers
along with availability of online networks. Personal webpages and on-line
data sharing platforms such as Free the Data, Consent to Research and Genomes Unzipped are being utilized to host and share genotypes, electronic
health records and/or family history uploaded by individuals. Although personal genomic data sharing initiatives vary in nature, the emphasis on the
individuals control on their data in order to benefit research and ultimately health care has seen as a key theme across these initiatives. In line with
the growing practice of personal genomic data sharing, this paper aims to
shed light on the potential challenges surrounding these initiatives. As in the
course of these initiatives individuals are solicited to individually balance
the risks and benefits of sharing their genomic data, their awareness of implications of personal genomic data sharing for themselves and their family
members is a necessity. Furthermore, given the sensitivity of genomic data
and the controversies around their de-identifiability, potential privacy risks
and harms originating from unintended uses of data have to be taken into
consideration.
P18.23-S
Opinion about reproductive decision-making among MMR mutation
carriers of reproductive age
J. Duffour1, A. Combes-Petit2, K. Baudry3, J. Rey3;
1
ICM Val dAurelle cancer institute, Montpellier, France, 2CHU Arnaud de villeneuve,
Montpellier, France, 3CHU Arnaud de Villeneuve, Montpellier, France.
The reproductive techniques such as prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD), although debated, are often legally forbidden in case of HNPCC syndrome, without considering mutation carriers
opinion about their reproductive options
We conducted a study in 43 individuals (probands and relatives) identified
as Mismatch Repair mutation carriers of reproductive age (until 40 years for
women, 50 years for men) exhaustively screened during the genetic oncology consultations in our institution. All of them received a closed questionnaire focusing on socio-clinical, possible causes of distress and reproductive
preferences (natural conception, PND, PDG or adoption) whose answers
were ranked from 0 to 4.
Complete data were available for 29 individuals (22 relatives, 7 probands:13
males, 395.6 years; 16 females, 316.8 years).
Main reasons of distress given in first were fear of transmitting predisposition for 17 individuals (9 men, 8 women) (58%) and fear of premature death
for eight (4 men and 4 women, 27.5%). For 5 persons the fear of passing on
deleterious gene took second place, so more than 22/29(75%) of this population regarded this problem as very serious.
PND / PGD and natural conception were equally reported (52% and 48%,
respectively).
Among the subjects afraid enough by hereditary concern (22), only 13(59%)
chose in first the new reproductive techniques and 9/22(41%) gave priority
to natural conception
Back to index
The hereditary recurrent fevers (HRF) are rare rheumatologic diseases characterized by a course of self resolving inflammatory episodes, inflicted by
cells of the innate immune system, and mainly affecting connective tissue,
skin and/or central nervous system. If untreated, some HRF patients may
develop life threatening, secondary amyloidosis. HRF genetic diagnosis is
increasingly requested for patients with recurrent inflammatory episodes of
unknown origin. More than 10 HRF genes are listed in the Infevers database.
An external quality assessment (EQA) program for the primary tested HRF
syndromes (FMF, CAPS, TRAPS, MKD) has been provided by the EMQN since
2009. Fifty laboratories, mostly from European countries, participate in the
scheme. Data demonstrating improved genetic diagnosis for HRF since 2009
will be presented; genotype error rates have dropped dramatically compared to a prior 3 year survey (2005-2008). Moreover, with low participation
outside Europe, best practice guidelines for the genetic diagnosis of HRF
have also been written by HRF genetic and clinical experts, addressing the
scope of diagnosis and clinical significance of pathogenic, clinically debated, rare, novel or population specific variants. A simple interpretation chart
concludes on the contribution of each variant type to the diagnosis of recessive or dominant HRF disease. Guidelines also addressed minimal details
and indication for referral, technical quality assurance measures, variant
description nomenclature and recommendation for further genetic testing.
Our future goal is to expand the scheme to monogenic autoinflammatory
diseases, and to implement EQA on new genetic diagnostic methods for HRF
such as Next Generation Sequencing (NGS).
P18.25-S
Attitudes of genomic researchers, health professionals and students
on returning incidental findings to whole genome research
participants
A. Carnevale1, M. A. Contreras-Sieck2, M. L. Granados-Riveros2, S. Romero-Hidalgo1, G.
Saruwatari-Zavala1;
1
Instituto Nacional de Medicina Genmica, Mexico City, Mexico, 2Escuela Nacional de
Antropologa e Historia, Mexico City, Mexico.
Introduction
In the last years the ethical and legal management of incidental findings in
343
ABSTRACTS POSTERS
massive parallel sequencing have been discussed. There is a lack of literature concerning research participants perspective. The aim of this study
was to investigate whether research participants want disclosure of IFs and
what kind of IFs they want to know about.
Methods: 127 research participants in a study of gastrointestinal polyps
were informed about Whole Exome Sequencing and the risk of IFs. They
were asked to decide whether they A) wanted disclosure on IFs no matter
whether the mutations were associated with a non-treatable or non-preventable condition, B) wanted disclosure on mutations associated with treatable or preventable conditions or C) wanted no disclosure at all.
Results: Participants who wanted disclosure of all IFs (A) accounted for the
majority (n=78), 45 of the participants only wanted disclosure of mutations,
which could lead to surveillance or treatment (B) and four participants did
not want IFs to be disclosed at all (C).
Conclusion: The study showed that almost all research participants wanted
disclosure of at least some types of IFs. The answers did not depend on
age or sex. We suggest that well-defined IFs are to be disclosed in research
projects and that the type of IFs (non-treatable and actionable) are categorized, discussed with the participant, and incorporated in the research
consent form.
P18.27-S
Disclosure of genetic information to family members: does the French
legal framework solve the dilemma?
E. Rial-Sebbag1, C. Farnos1, S. de Montgolfier2;
1
UMR 1027, INSERM, Universit Toulouse Paul Sabatier, Toulouse, France, 2IRIS (Institut
de Recherche Interdisciplinaire sur les enjeux Sociaux. Ehess/INSERM/CNRS) / Univ.
Paris Est Crteil, Paris, France.
344
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Clinical genetics services are time and labor intensive, since they include
both counseling meetings as well as extensive patient related activities such
as administration, summary letters, and the interpretation of new genomic
technologies. With increasing pressure for cost effective medical care, information is needed to evaluate the time and efforts required for providing
medical genetics services. An online survey was conducted among 151 professionals who practice medical genetics throughout the world (85.75% medical geneticists, 13.12% genetic counselors). The reported average amount
of time required for counseling sessions for pediatric, oncogenetic, pregnancy with a malformed fetus and preamniocentesis counseling was significantly different: ~60,42,51,27 minutes respectively. The average time required
to write summary letters varied from 30 to 41 minutes. The time required
for literature searches was 31-60 minutes and for patient related activities,
42 minutes. The time for patient related bioinformatics search and for test
interpretation was 57 minutes . CMA requires an average of 48 minutes for
analysis and for genetic counseling each. Professionals with less than 10
years experience needed more time than those with more than 10 years
experience. Time devoted to clinical work received the highest percentage
followed by administration, research and teaching. This study emphasizes
the complexity and time consuming demands of the practice of medical
genetics in the era of advanced genomic testing; further consideration and
assessment is required in order to determine how to adapt genetic services
to the demands of cost effectiveness, without compromising the quality of
patient care.
P18.30-M
Project Superhero: the kids are doing it for themselves
J. M. Grey1, A. Metcalfe2;
1
AMEND, Tunbridge Wells, United Kingdom, 2Kings College, London, United Kingdom.
ABSTRACTS POSTERS
Back to index
Orphanet has established strong quality standards over the years considering the different situations of the countries part of the consortium. There
are defined inclusion criteria for each activity.
Information is collected from official sources specific to each country. Once
published data will be checked annually by post-validators, who are professionals working in the field of rare diseases with expert knowledge in
the relevant activity. Orphanet UK has established several partnerships to
post-validate its information. Rare Disease UK and Genetic Alliance validate
patient organisations. ERNDIM validates EQA accredited metabolic laboratories in the UK. UK centres of expertise are validated by experts that are
part of the EUCERD and research activities are validated by relevant patient
organisations. Orphanet UK is also working to establish new partnerships to
validate data about molecular and cytogenetic laboratories and data about
clinical trials too. Every effort will be made to ensure that information is
accurate, comprehensive and up to date.
P18.32-M
From clinical suspect to molecular confirmation of Noonan syndrome;
contribution of best practice genetic counselling
Noonan syndrome (NS) is AD disorder, characterized by variable expressivity of clinical features such as: postnatal growth reduction, congenital
heart disease, characteristic facial dysmorphisms and development delay.
In ~75% of all NS cases, germline mutations involving RAS-MAPK signaling
pathway genes (PTPN11, SOS1, RAF1, KRAS, NRAS, BRAF, SHOC2, MEK1,
CBL) are causative.
We reported a case of 13-year-old girl [born at 36w by CS (BW 3250 g
(~95), BL 48 cm (~75)] referred for genetic counseling due to growth retardation, facial dysmorphisms, development delay and learning disability.
After birth she presented frequent vomiting, with failure to thrive and at
5 months of age underwent surgery for intestinal malrotation. Because of
short stature, Growth Hormone (GH) therapy have been introduced at age
of 3yrs up to 11yrs. Negative molecular testing for PTPN11 and SOS1 genes,
normal female karyotype and aCGH analysis were observed.
Objective examination: H 138 cm, (<3); W 33 kg, (<3), no menarche, hypertelorism, eyelids ptosis with down slanting palpebral fissures, low-set
and posteriorly rotated ears, high-arched palate, micrognathia, short and
webbed neck, low hairline at the back of the neck, pectus excavatum, prominent scoliosis, joint hyperextensibility, bilateral pes planus and mitral valve
prolapse disclosed by US.
Phenotype of our patient was suggestive to NS, thus further mutational
screening has been requested. Missense mutation in exon 2 of KRAS gene
(c.40G>A; p.Val14Ile) has been identified. Even though KRAS mutations are
usually associated with NS severe phenotype with cardiac involvement (hypertrophic cardiomyopathy), this finding is not present in our patient.
P18.33-S
Orphanet UK: Ensuring quality of information
Orphanet is the largest online resource for rare diseases and orphan drugs
for all audiences. It provides free and direct online access to the most comprehensive classification for rare diseases, an assistance to diagnosis tool,
the EUCERDs newsletter, thematic studies and reports and updated information about rare diseases and specialised services in around 40 countries.
Orphanet UK (www.orphanet.co.uk) has been operational since 2005. To
date it lists >300 expert centres, 125 laboratories, >300 patient organisations, >130 patient/mutation registries and >580 research projects and clinical trials.
P18.34-M
Attitudes of adult patients and parents of children with cystic fibrosis
towards carrier screening for cystic fibrosis
S. Janssens1, D. Chokoshvili2,3, C. Binst4, I. Mahieu4, L. Henneman5, A. De Paepe1, F. De
Baets6, P. Borry3;
1
Center for Medical Genetics, University Hospital Ghent, Ghent, Belgium, 2University
Hospital Ghent, Ghent, Belgium, 3Catholic University of Leuven, Leuven, Belgium,
4
University of Ghent, Ghent, Belgium, 5VU University Medical Center Amsterdam,
Amsterdam, Netherlands, 6Ghent CF Reference Center, Ghent, Belgium.
P18.35-S
Predictive genetic testing in hereditary heart diseases: a single-center
series of 304 subjects
C. Bordet1, E. Le Boette1, M. Babonneau1, S. Fosse1, M. Garguilo1, E. Gandjbakhch1, V.
Fressart1, P. Richard1, D. Hron2, M. Komadja1, P. Charron1;
1
Centre of reference of hereditary heart disease, Groupe Hospitalier Piti Salptrire,
Paris, France, 2Department of Genetics; Groupe Hospitalier Piti Salptrire, Paris, France.
Hereditary heart diseases are typically characterized by autosomal dominant inheritance and delayed cardiac expression. Predictive genetic testing
(PGT) is offered to asymptomatic relatives to allow targeted medical care
with early therapeutics in order to reduce the risk of complications. Psychological issues related to PGT are complex and have been poorly studied.
To evaluate our practices regarding PGT for hereditary heart diseases and
study the behavior of relatives after the first information consultation, offering a waiting period before blood sampling. We retrospectively studied
records from 304 consecutive relatives seen in our department and have
requested PGT. Underlying diseases in the families were HCM (60%), DCM
(17%), ARVC (15%), LQT (5%), Brugada syndrome (2%) and other (1%).
There were 260 adults and 44 minors. At the time of the first consultation,
the average age was 37 years, and 83 % of the relatives previously had a cardiac checkup. After multidisciplinary consultation, 22 relatives (8%) dropped out of procedure and 11 relatives (3%) performed blood sampling but
did not come back to know their results. Blood sample was delayed for 70%
of relatives and immediate for 30%. A total of 21 different genes were analyzed and most frequent ones were MYBPC3 (97), MYH7 (77), LMNA (37). A
mutation was present in 36% of relatives and absent in 64%. We observed a
high level of genetic uptake after initial consultation but a minority of relatives decided to stop or delay the procedure. These results suggest the benefit
of a waiting period before blood sampling.
345
ABSTRACTS POSTERS
P18.36-M
Presymptomatic and predictive genetic testing in minors - a minireview in preparation for new Danish best practice guidelines
Aim: In preparation for the creating of Danish guidelines regarding predictive and presymptomatic genetic testing in minors, we have reviewed existing
guidelines and policy papers as well as international conventions. Furthermore we have reviewed existing literature concerning the psychosocial impact of testing children.
Materials: Guidelines on the topic from four of the largest English-speaking
genetic associations were reviewed. The Convention on the Rights of the
Child and the Convention on Human Rights and Biomedicine were also considered.
Results: In the reviewed guidelines it is recommended to offer genetic testing if the test result will be of medical benefit to the child. In some guidelines emphasis is on immediate benefit. If there is no potential medical benefit
it is recommended to defer genetic testing for adult-onset disorders until
minors are able to decide for themselves. Some guidelines suggest that psychosocial factors in certain cases can justify genetic testing for adult-onset
disorders. Requests for genetic testing for childhood-onset disorders can
usually be met. Thorough genetic counseling and consideration of timing of
the test is of importance in the decision-making process.
Discussion: There is a lack of knowledge about the psychosocial impact
of testing children. The recommendations in the reviewed guidelines are
based on the classic medical moral principles: Nonmaleficence, beneficence
and respect for autonomy. The recommendations are in consistency with
international conventions on the field, but statements within these conventions can be subject to interpretation. We call for increased awareness of the
difficulties in defining the best interest of children.
P18.37-S
Presymptomatic testing in minors: Requests and pratices. Evaluation
of pluridisciplinary consultations for the last 20 years
A. Jacquette, M. Fuger, A. Durr, C. Colas, M. Garguilo, A. Herson, M. Babonneau, S.
Fosse, I. Marey, S. Whalen, C. Boucher, E. Schaerrer, C. Bordet, p. Charles, E. Le Boette, J.
Feingold, P. Charron, D. Heron;
dpartement de gntique, Paris, France.
Twenty years ago, the first presymptomatic tests (PST) began for
Huntingtons disease. Since then, PST have been extended to other diseases,
in particular among minors. The practice of these tests is regulated by law
within each European country and follows a number of principles, including
respect for the right to not know and autonomy. In the case of children,
performing such tests is complicated because all of these conditions cannot always be met. In the Department of Genetics in the Piti-Salptrire
Hospital, several protocols of PST are reported to the French Biomedicine
Agency, in accordance with French law. We wanted to explore our practices
regarding children. All patients under 18 years of age at the first consultation for a PST were included in this retrospective study. 175 children met the
inclusion criteria and they were divided into 4 groups (cardiology, myology,
neurology, oncology). Medical data but also access to the test or not, motivations, were collected. The average age of minors at the first consultation was
13 years. 69% of children have performed the test but this varies significantly (p<0.05) according to the pathology (46% in neurology and over 90 % in
cardiology and oncology). 8.6% of children also said they did not want the
test at the first consultation, underlying the importance of parental demand.
The study by group of pathologies also notes that the reflexion time, the reasons given for doing the test vary according to the pathology and emphasize
the importance of a differentiated care.
P18.38-M
Promotion of genetic services in the Slovenia-Italy cross-border
region
346
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Slovenian and Italian institutions have joined the project. One of the major objective of SIGN is to raise awareness in both the general public and
medical specialists on the activities of the genetic services and on the new
diagnostic options patients with rare diseases may be offered. For this purpose, training courses in medical genetics for paediatricians, gynaecologists
and oncologists have been organized and several workshops for high school
students have been conducted. All the project partners were actively involved in these meetings, allowing the reinforcement of the genetic network
and the establishment of new scientific collaborations. In addition, a specific
website, in both Italian and Slovenian, has been developed in order to help
patients, doctors and students to better understand human and medical genetics (www.signgenetics.eu). The section dedicated to the general public
contains information on human genetics, genetic diseases and their impact
on the society described in a simple, non-technical way. The section for doctors and students offers a variety of educational tools, including fact sheets
on several genetic diseases, examples of clinical cases useful to recognize
and correctly diagnose specific diseases, video seminars of experts and clinical protocols. Another section is devoted to updating and promoting the
results achieved within the project: it includes a description of the project
partners and their clinical and research activities.
P18.39-S
The French Foundation for rare diseases: accelerating rare diseases
research
The French Foundation for rare diseases is an innovative cooperative framework dedicated to rare diseases research. Flagship of the second French
National Rare Diseases Plan, co-founded by University Hospitals, Research
organisations and Patients organisations, we act as a federative and strategic hub to accelerate scientific, clinical and social innovation by stimulating
cross-sector cooperation to the benefit of patients affected by rare diseases.
With our headquarters at the heart of the French Platform for Rare Diseases
and seven regional coordinators, in direct contact with researchers all over
the national territory, our priorities are driven by grounded needs and integrated into a national strategy with an international perspective. Our active
support, spanning from basic to translational and clinical research, includes
enhancing the access to high throughput technologies such as NGS, promoting international collaboration via dedicated partnerships, and accelerating the translation of research into clinical development through targeted
links to orphan drug experts. Since rare diseases research is tightly linked
to societal challenges, we are also actively supporting studies on social, economical, ethical impacts of rare diseases. We regularly initiate national working groups, open to international perspectives, to specifically target timely
issues, including professional training needs, ethical and regulatory issues
around data and bio-specimen collections, as well as the impact of new
technologies in genetics, patients consent to genetic testing and their paths
through diagnosis and treatment. Through this unique range of actions, we
aim to contribute to acquainted national public health and research policies
and to the promotion of international multi-stakeholders cooperation.
P18.40-M
Rare Diseases week in Timisoara - a campaign with a good start
Volunteers for Rare Diseases was created in Save the Children Timis in
2007 when students from the University of Medicine Timisoara, supported
by teachers, wanted to work with people with special needs. The campaign
launched on the International day of rare diseases was the opportunity to
spread informations about this topic in community. An extension to a whole
week dedicated to this was the next step.
Increase awarenes in general population, involving the local authorities,
rasing the interes in this field for medical services employees and involving
our students in volunteer work were main objectives.
In all the 5 years we used internet as a tool and mass-media campaign also.
Volunteers made a street march in the dedicated day with flyiers distribution in downtown and placing posters. We organised round table at the local
TVs with specialised medical staff. Each year we met the children in hospitals and settled lessons for parents.
With parents we discussed the common situations of various diseases, followed by monitorization of their skills yearly. Rare Disease Day has taken
many forms over the years: symposiums, conferences, information campaign, march (more than 200 participants/year). The childrens with rare
pathologies have in this way support for their integration into society. The
ABSTRACTS POSTERS
authorities realise the need for social protection with different ways to support this people. All the student involved can use informations about this
pathology and early diagnose cases, encouraging prevention.
P18.41-S
Respecting autonomy while reacting to change: A review of current
policy and empirical evidence on re-consent in longitudinal
biomedical research
S. E. Wallace, E. G. Gourna, O. Shoush, L. Brewster, J. Wright;
University of Leicester, Leicester, United Kingdom.
Back to index
in Innsbruck (Fitzky et al., 1998), first mutations in SLOS patients have been
detected by the same group and also by Wassif et al. (1998). Hence SLOS is
a metabolic and malformation disorder caused by mutations in the DHCR7
gene. This gene encodes the 7 sterolreductase. Now more than 130 mutations are known and all reported patients are included in the DHCR7 database (http://databases.lovd.nl/shared/genes/DHCR7). Mutation spectra
are different in European populations. Time since establishment of common
founder mutations (c.964-1G>C, p.Trp151* and p.Thr93Met) is long enough
(about 100 and 200 generations) to explain frequencies (1:100 for c.964-1G>C) by genetic drift (Witsch-Baumgartner et al., 2007). There are modifiers
of clinical severity of SLOS. Depending on SLOS patients maternal variants
apoE 2, 3 or 4 the severity vary significantly (Witsch-Baumgartner et al.,
2004). The fundamental problem of the disease is the lack of cholesterol
during embryogenesis. Regarding therapies adding HMG-CoA reductase inhibitor (simvastatin) might ameliorate the severity (Jira et al., 2000; Haas et
al., 2007). After 50 years the pathophysiology of SLOS is still not as clear due
to multiple functions of cholesterol. To help patients it is still necessary to
continue research on SLOS.
P18.44-M
Gonadal mosaicism in split-hand/foot malformation: Implications for
genetic counselling
N. Martin1, J. Jessen1, C. Barr2, J. T. R. Clarke2,3, K. Chong1, D. Chitayat1,3;
1
Department of Obstetrics and Gynecology, Prenatal Diagnosis and Medical Genetics
Program, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada, 2Service de
gntique mdicale, Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, QC,
Canada, 3Department of Pediatrics, Division of Clinical & Metabolic Genetics, Hospital
for Sick Children, Toronto, ON, Canada.
B. Tolo1,2, . Lunde1;
1
Department of Global Public Health and Primary Care, Faculty of Medicine and
Dentistry, University of Bergen, Bergen, Norway, 2Center for Medical Genetics and
Molecular Medicine, Haukeland University Hospital, Bergen, Norway.
347
ABSTRACTS POSTERS
ture perspectives to prevent people from discrimination and stigmatization.
In order to protect human rights, it is important to inform patients about
the regulation.
P18.46-M
Chromosomal mosaicism in chorionic villi: implications for prenatal
diagnosis and genetic counseling
F. Maggi, F. Malvestiti, B. Grimi, E. Gaetani, C. Agrati, F. Dulcetti, A. M. Ruggeri, G.
Gallazzi, E. Niada, V. Zanatta, L. Branca, G. Simoni, F. Grati;
TOMA, Advanced Biomedical Assays S.p.A., Busto Arsizio, Italy.
P18.47-S
Realising Genomics in Clinical Practice- Whole Exome Sequencing and
the Patient Pathway
C. Alberg1, A. Hall1, N. Hallowell2, T. Finnegan1, M. Kroese1, J. Whittaker1, G. Hall3;
1
PHG Foundation, Cambridge, United Kingdom, 2Institute of Public Health, Cambridge,
United Kingdom, 3Manchester Centre for Genomic Medicine, Manchester, United
Kingdom.
Whole genome sequencing (WGS) and whole exome sequencing (WES) have
been described as transformative technologies with potential to revolutionise patient care. As part of a bigger project addressing the ethical, legal, social
(ELSI) and operational challenges likely to be raised by these technologies
Realising Genomics in Clinical Practice, the PHG Foundation convened a
workshop to explore the impact of these technologies on patient pathways.
Key features such as the point and source of referral for sequencing will influence how WES/WGS will be translated into clinical practice. By comparing patient pathways currently in use in targeted approaches and whole
exome sequencing, our aim was to illustrate the key operational differences
and translational aspects that are likely to arise. In particular, we focused
on three areas which seem to raise distinctive ELSI challenges: the nature
and scope of consent processes and the degree to which they might need
adaptation; technical aspects including the scope, construction and standardisation of data filters, and requirement for data sharing in order to validate
and interpret findings; and the extent, timing and nature of disclosure of
test results, including incidental or unsolicited information. We review our
findings and evaluate the prerequisites for effective translation, including
requirements for additional genetic counselling, and the educational and
training needs of health care professionals, clinical scientists, patients and
publics. These findings will be contextualised by presenting preliminary
results from the entire Realising Genomics Project. Further information on
the Realising Genomics project can be found at http://www.phgfoundation.
org/pages/realising_genomics.htm
P18.48-M
Revisiting Hook: rates of fetal aneuploidy and other significant
genetic aberrations in women presenting for prenatal care
348
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EES1.1
Responding to guilt and shame in clinical consultations
EPL1.1
The impact of total gastrectomy upon e-cadherin carriers:
experiences of eating
C. Baguley;
Psychological Professions Network - Health Education North West, Manchester, United
Kingdom.
Genetic medicine is an area of health care that often deals with delivering
profound and difficult information; it brings clinicians directly in contact
with individuals and families who sometimes have to make very difficult and
life-changing decisions. Genetic counselling involves the skill of giving information, advice and support to help guide patients through this process.
Adjusting to information and assimilating new knowledge can have significant implications for the life decisions patients make. It is now recognised as normal for the course of adjustment to involve a range of emotional
responses including denial, sadness, anger and guilt before the stage of
acceptance is reached. This process can take time, individuals can vary in
presentation and sometimes they can get stuck often resulting in unhelpful
behavioural consequences and increasing distress. In order to be effective
clinicians need to understand this process and be equipped to recognise and
respond to emotional responses that may occur within routine consultations.
This workshop looks specifically at the nature of guilt and shame in the
context of genetic medicine. Drawing upon a cognitive behavioural model of
emotion we will consider how the difficult and corrosive emotions of guilt
and shame can be understood as an attempt by the individual to assimilate
information and actions into their pre-existing belief system.
Drawing on a combination of theory, reflection on clinical examples, and illustration by video, participants will be encouraged to consider how they
can develop their clinical interview skills to identify and respond to expressions of guilt and shame in routine clinical settings.
EES2.1
Qualitative and quantitative methods in psychosocial research
K. ODoherty1, B. Meiser2;
1
Department of Psychology, University of Guelph, Guelph, ON, Canada, 2Department
of Medical Oncology, Psychosocial Research Group, Prince of Wales clinical School,
University of New South Wales, Randwick, Australia.
349
Introduction - Prophylactic mastectomy in BRCA1/2 mutation carriers reduces the breast cancer risk significantly, but may have a profound impact
on body image and self-esteem. In addition, some women report feelings of
isolation and stigmatisation. Other issues to face are an increased risk for
ovarian cancer, potential cancer risk for offspring, intimacy with the partner, communication with family and ongoing grief. Methods - As a result of
our long-year studies on impact of prophylactic mastecomy a group intervention was developed with regard to the supportive-expressive needs of
these women. With a maximum of 10 members, a closed 8-sessions group
programme was started focussing consecutively on the following themes: 1)
introduction 2) body image 3) social support and coping 4) social support
and loss 5) partner relationship or dating 6) ovarian cancer risk 7) communication within the family 8) evaluation and future plans. Results - Seven
women participated in the first group aged 26-52. The older women advised
the younger and vice versa, for example with regard to mother-daughter
communication issues. The group members reported high satisfaction, par-
350
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ticularly they felt they were no longer isolated and they could share their
experiences and learn from each other. One woman who already had mastectomy for previous breast cancer, felt she was different from the others.
Conclusion - A next group will focus on a more homogenous population
with regard to the experience of having had cancer yet or not. After refining
content and structure of the programme an intervention study will be established.
EPL2.1
Ok for us, not for them: Patients and genetic counsellors experiences
of NIPT and views on wider use
A. Effa1,2, E. Alexander1, S. E. Kelly3, L. Kerzin-Storrar1;
1
Manchester Centre for Genomic Medicine, Institute of Human Development, University
of Manchester and Manchester Academic Health Sciences Centre (MAHSC), Central
Manchester University Hospitals NHS Foundation Trust, Manchester, United Kingdom,
2
Program of Genetics and Metabolism, Winnipeg Regional Health Authority, Winnipeg,
MB, Canada, 3Department of Sociology, Philosophy and Anthropology, University of
Exeter, Exeter, United Kingdom.
Fetal diagnostic testing for chromosomal abnormalities such as Down syndrome (DS) is frequently used in Sweden. Prior to testing expecting parents
do not routinely receive information about the conditions being screened
for. The aims were to assess why expecting parents choose to undergo CUBtesting (combined ultrasound and biochemistry test), their perception of
information, thoughts about invasive procedures, possible termination of
pregnancy and knowledge about DS.
Method: From November 2010 to March 2011, 105 pregnant women and
104 partners answered a questionnaire after completing the CUB-test, at
the Fetal Medicine Unit, Uppsala University Hospital.
Results: The most common reason for choosing a CUB-test is to get a confirmation on a healthy baby or because you should do it. A majority had
not been given information on what it means to live with a child with DS
and many requested more information. Internet is the most common source
for information about DS. A substantial proportion of pregnant women and
partners have little knowledge of the medical, cognitive and social consequences of DS. Twenty-four percent had not yet decided about invasive testing if increased CUB-risk and almost half had not decided what to do about
the pregnancy if DS was diagnosed.
Conclusions: A majority of expecting parents attending CUB-test had not received information about DS and requested more information. A substantial
proportion of expecting parents have varying and in several aspects low levels of knowledge about DS and its consequences. Many had not yet decided
what to do if DS was diagnosed.
EPL2.4
Diagnosis Down syndrome: a cross-cultural study of family
experiences
M. L. Van Riper;
University of North Carolina Chapel Hill, Chapel Hill, NC, United States.
Purpose: Much has been written about family-provider interactions surrounding the diagnosis of Down syndrome (DS) and substantial resources
have been devoted to helping health care providers feel more prepared to
deliver the diagnosis of DS. However reports of parents receiving inaccurate information continue to appear in the literature.Moreover, anecdotal
reports of parents feeling pushed to make unwanted choices, such as undergoing invasive testing or terminating a pregnancy following the diagnosis
of DS, are becoming more common. In addition, limited attention has been
devoted to understanding how family experiences vary from one country to
another. Therefore, the purpose of this presentation is to compare family experiences in four countries (Ireland, Portugal, United Kingdom, USA) where
differences currently exist in terms of options for prenatal testing and options for families following the diagnosis of DS. Method: 1185 parents of individuals with DS completed a survey which includes a variety of questions
concerning family-provider interactions surrounding the diagnosis. In addition, interviews were conducted with a subset of parents. Results: Findings
suggest that 50% of the parents were dissatisfied with family-provider interactions and most health care providers are not following the recommended guidelines regarding how to inform parents. Additionally, important
cultural differences were noted. Conclusion: Findings from this study will
help in efforts to improve parental satisfaction with family-provider interactions surrounding the diagnosis of DS. In addition, they will help ensure that
cultural context is considered during the informing process.
EPL2.5
Dynamics of prenatal screening: blurring boundaries between
normative frameworks
W. Dondorp, G. De Wert;
Dept Health, Ethics & Society / Research Schools CAPHRI & GROW,Maastricht University,
Maastricht, Netherlands.
Back to index
no other possible interventions but abortion, prevention is a morally problematic category. This is why official documents define such screening in
terms of what may be called the autonomy paradigm, where the aim is to
help individual women/couples make autonomous reproductive choices.
This is also reflected in counseling guidelines. Whereas some degree of professional directivity is morally acceptable (or even required) for the first
type of prenatal screening, non-directivity is regarded as absolutely essential for the second. However, this distinction is increasingly under threat.
It was already blurred with the introduction of dual purpose ultrasound
screening, but it will only be further undermined with the present dynamics
of genetic technologies. Think eg. of NIPT for both serious fetal disorders
and for gene expression profiles that are predictive of pregnancy complications. The expanding scope of prenatal screening will also provide more
options for fetal therapy. This presentation will consist of a systematic exploration of the ethical challenges involved in this blurring of frameworks
and conclude with ethics guidance recommendations for the future field of
prenatal personalized medicine (Bianchi).
EPL2.6
Stigma and reproduction: the place of stigma in reproductive
decisions
A. J. Clarke;
Cardiff University School of Medicine, Cardiff, United Kingdom.
This paper reviews the impact of a genetic disorder on family life, reflecting
its mode of inheritance, and then examines the social impact of a specific
sex-linked disorder, hypohidrotic ectodermal dysplasia. The stigmatisation
of HED affected males is as important in the accounts given by their womenfolk as the physical effects of the condition; this impacts on their feelings
about transmission of the disorder to the next generation. Perspectives may
also change over time, with grandmothers expressing more strongly their
sense of guilt at having transmitted the condition, despite there being no
question of moral culpability.
We then consider the broader impact of stigma on reproductive decisions.
They can be impacted by stigma both (i) within families where the practical effects of a specific genetic disorder are well known, and (ii) in couples
faced by decisions in pregnancy with no prior expectation of a fetus affected by a genetic disorder. In the former case, any decision made has implications for the self-esteem of affected family members. In the latter case,
decisions about continuing or terminating the pregnancy may be affected
by the parents remembered past and/or imagined future responses to encountering an affected individual. In this way, parents fantasies may shape
their decisions.
The scope for parental fantasies to influence decisions is greater when
technology amplifies the uncertainty attained in genetic investigations or
ultrasound imaging. How will parents fantasies of uncertainty play out in
practice? What can genetics professionals do to promote respect for affected
individuals?
EPL3.1
How do research participants perceive uncertainty in genomic
sequencing?
351
These last years, many studies investigate the clinical utility of whole exome
sequencing as a diagnostic method. Clinical utility is most commonly established on the basis of data that show the effectiveness or economic value
of an innovation. Given that whole exome sequencing is a very new method,
however, a multidimensional approach to clinical utility seems more appropriate, which also investigates its acceptability for patients .
This paper presents how patients and their families assess the utility of
whole exome sequencing, and the diagnosis it produces. Our case-study is
based on 20 in-depth interviews with patients and their families, who are
involved as participants in a research project which aims to assess the clinical utility of whole exome sequencing as a diagnostic tool for children with
hitherto unidentified developmental delay.
The case study provides insight into why these patients and their families
want a diagnosis, and how they value the diagnosis that they eventually get.
Their evaluations reveal not just the acceptability of WES as a diagnostic
tool, but provide insight into how diagnoses are evaluated with respect to
their daily caring practice and social lifeworld. We will argue that this provides important input to an assessment of the clinical utility of WES, which
may inform how WES is to become part of clinical routine, and how it should
be accompanied by counseling.
EPL3.3
Variants in Practice Study (VIP): High risk womens responses to
receiving genetic test results for genomic variants associated with
breast cancer risk
M. Young1, P. James1, G. Mitchell1, L. Forrest1, S. Sawyer1, N. Hallowell2;
1
Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia, 2University of
Melbourne, Melbourne, Victoria, Australia.
Results from genome-wide association studies (GWAS) have identified common genomic variants that form an important component of the heritability
of breast cancer risk. Data from Victorian high risk breast cancer families
demonstrate that testing for these genetic factors identifies a significant
aetiological group known as polygenic families and provides clinically important information.
It is important to investigate lay and professional understandings of novel,
complex genetic test information generated by SNP testing to identify the
most effective ways for genetic health professionals to communicate this information to patients and the treating medical team.
Our qualitative study aimed to assess patient and healthcare professionals understandings of genomic variant data. This presentation focusses upon womens expereinces of receiving SNP results following their
participation in a GWAS study: Variants In Practice study (VIP).
Forty women attended an appointment at the familial cancer clinic, Peter
Mac, Australia. Women had 1. previosuly been diagnosed with breast cancer
2. undergone BRCA1/2 mutation testing (no mutation found). Subsequently
they were genotyped for 22 common genomic variants from which breast
cancer risks were calculated.
Analysis of interview transcripts has revealed a number of preliminary themes, including study participation is motivated by feelings of altrusim and
specifically responsibility for family members. Receiving SNP information
was viewed very postively, particularly by those women who had previously
undergone risk-reducing surgery, who felt their decision was validated by
their polygenic result. In conclusion, this study suggests that SNP results
are regarded as useful information for both the research participants and
their families.
EPL3.4
To Disclose, or Not to Disclose? The Context Matters
Progress in understanding childhood disease using next generation sequencing (NGS) portends vast improvements in the nature and quality of patient
care. Ethical questions surrounding the disclosure of incidental findings (IF)
persist, as NGS and other novel genomic technologies become the preferred
tool for clinical research. Thus, the need for multidisciplinary discussions
and disclosure practices on the return of results in paediatric research has
never been more immediate. The aim of this study is to explore the views
of investigators concerning the disclosure of IFs in the paediatric oncology context. Our findings reveal at least four contextual themes underlying
the ethics of when and how young participants and their families could be
made aware of these unexpected results during the course of their research
participation: clinical significance of the result, respect for persons, scope of
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Introduction: Parents decisions about enrolling children in Duchenne/Becker muscular dystrophy (DBMD) clinical trials (CTs) may be influenced by
hopes and expectations, which when unrealistic may challenge informed
consent. We explore influences on decision-making. Methods: The study
team used a community-based participatory research approach. Parents of
children in DBMD CTs were recruited for an online survey through Parent
Project Muscular Dystrophy and clinics. Participants viewed a series of benefit and worry statements and rated each statements influence on decision-making, and how much they expected and hoped/worried about each.
Results: From the first 61 participants, CT decision-making was influenced
by potential altruistic and individual benefits: learning generalizable information (97% slightly to strongly agree), CT resulting in a drug that works
(82%), better future for other children (82%), improved quality-of-life for
their child (82%), and parent doing everything to help their child (82%).
CT worries most affecting decision-making were: child bothered by side effects (39%), eligibility for another trial (36%), and child not liking the trial
(29%). Expectation and hope ratings were strongly correlated with decision-making influence ratings (r=0.5 to 0.8, p<0.01). Associations between
expectations and altruistic decision-making influences yielded the highest
correlation values; conversely, associations between hopes and individualbenefit influences yielded the highest values. Conclusions: Anticipated CT
benefits influenced parents decision-making more than worries. The relationships among hopes, expectations and decision-making influences for altruistic versus individual benefits will be further explored, to inform a new
conceptualization of opportunities and challenges during informed consent
that extends beyond knowledge-oriented concepts such as therapeutic misconception.
EPL4.2
The impact on children and parents of participation in clinical
research trials for Morquio A syndrome and Sanfilippo A syndrome
D. L. Holliday1,2, M. Farag2, C. Breen2, S. Jones2, T. Clancy2;
1
Yorkshire Regional Genetics Service, Leeds, United Kingdom, 2Manchester Centre for
Genomic Medicine, Manchester, United Kingdom.
Clinical research trials of enzyme replacement therapy for Morquio A syndrome and Sanfilippo A syndrome are underway. This qualitative study explored the impact of participation on 7 children with Morquio A syndrome
and parents (5/9 eligible families) and parents of children with Sanfilippo
A syndrome (4/6 eligible families). Face-to-face semi-structured interviews
were carried out, the interviews were transcribed and key themes identified through interpretative phenomenological analysis. The main questions
explored were: motivations for taking part, impact on life, and views on the
information received prior to the trial. Children described the key role their
parents played in providing them with information and supporting them
through medical procedures. They talked about what was good (e.g. making new friends) and bad (e.g. fear of needles) about taking part. Many
parents felt there was no real choice as current management options are limited. Some saw the trial drug as treatment rather than potential treatment.
Most felt that too much complex information was given during the consent
process, but discussions with the clinical trial team facilitated understanding and provided emotional and practical support. Parents described negative impacts on employment and family life. Positive impacts include an
improvement in their childs condition and a more optimistic outlook for
the future. Overall, children and parents felt the advantages outweighed the
disadvantages, and would recommend taking part in a similar trial to other
children/families. These findings provide an insight into the impact trials
have on children and parents lives and identify potential improvements for
future clinical trials.
EPL4.3
Why do parents request carrier testing in their healthy children? A
comparison of genetic health professionals and parents views
D. F. Vears1,2, C. Delany3,2, J. Massie4,5, L. Gillam1,2;
1
Melbourne School of Population and Global Health, Centre for Health and Society,
University of Melbourne, Parkville, Australia, 2Childrens Bioethics Centre, The Royal
Childrens Hospital, Parkville, Australia, 3School of Health Sciences, University of
Melbourne, Parkville, Australia, 4Department of Respiratory Medicine, The Royal
Childrens Hospital, Parkville, Australia, 5Department of Paediatrics, University of
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Many parents want to know the carrier status of their other children following the diagnosis of a child with a genetic condition. However, the reasons
behind their desires for this information have not been fully investigated.
The aim of this study was to explore the reasons parents want carrier testing
performed in their healthy children and health professionals understanding
of these reasons.
Semi-structured interviews were conducted with genetic counselors and
clinical geneticists (n=17), and parents (n=25) of children with one of three
genetic conditions (cystic fibrosis, haemophilia and Duchenne muscular
dystrophy). Inductive content and thematic analyses were used to compare
genetic health professionals and parents accounts of the reasons parents
want to know the carrier status of their healthy children.
Genetic health professionals expressed views about parents reasons for
requesting, including that parents primarily want carrier testing to reduce
their own anxiety and be reassured. Several professionals indicated that
some parents just need to know, and others acknowledged that generally
parents request testing with their childs best interests in mind. In contrast,
parents stated they primarily wanted genetic testing in order to convey the
information to their children. Parents felt that disclosing carrier status to
their children would allow the children to make informed reproductive decisions or prepare themselves for having an affected child.
This mismatch in understanding between the genetic health professionals
and parents of the reasons parents want carrier testing in their healthy
children has implications for genetic counselling practice.
EPL5.1
What is the role of genetic counsellors? A systematic review of
evidence
353
Background: Development in genomics have radically changed our self-understanding, and we have become biomedicalized (Plsson 2007). In US,
consumers feel the need for tools to estimate the promises and claims of
genetic testing services (Green et al, 2011). Ministry of Industry in Japan
is preparing best practice guidelines for consumer-targeted genetic testing,
but there is no legal regulation against genetic discrimination. However, few
studies on public attitudes exist in Japan. Purpose: This paper shows Japanese citizens attitudes toward consumer-targeted genetic testing and its regulation and address broad ethical, legal and social implications in East Asia.
Methods: We employed a web-based questionnaire survey to investigate
general perceptions in 2012. In total, 14,718 Japanese citizens completed
(RR=37.0%). Results: 14% of respondents knew consumer-targeted genetic
testing and just 3.1% had purchased before. 48.6% showed interests to use
susceptibility testing on life-style related diseases, 35.9% for congenital disorders and 30.8% for PGx testing. On genetic testing for children, 27-33.3%
of respondents agreed to share results with schoolteachers for personalized education. They show fewer interests in talent identification testing.
56.2% wished to ban genetic discrimination by law. Discussions: Compared
with past studies in South Korea and Taiwan, Japanese respondents showed
fewer interests. We could explore the reasons why Japanese havent been
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Introduction: Many parents experience difficulties in talking to their children about an IGC that affects their family. Parents often want to talk to their
Background: Disclosure of genetic information within families to at-risk relatives is extremely important, but often problematic. A genetic counselling
intervention, delivered post consultation, may result in increased access to
genetic services by family members. This Australian randomised controlled
trial aimed to assess the effectiveness of intense genetic counselling followup on numbers of at-risk relatives utilising genetics services.
Methods: Participants (probands) (n= 95) were recruited when attending
a genetic service for diagnosis or genetic testing. The intervention group
(n=45) received three telephone counselling interventions while the control
group (n=50) received usual care.
After 18 months, clinical files were audited to look for differences in the percentage of at-risk relatives who contacted genetics services. Analyses were
adjusted for clustering within families.
Results: Overall, 142/554 (25.6%) at risk relatives in the intervention arm
contacted genetic services, compared with 112/536 (20.9%) in the control
arm (adj OR 1.30 95%CI: 0.70-2.42). Subgroup analyses of genetic categories revealed substantial differences in contact percentages: cancers - 28%
(intervention) vs 15%(control); cardiac - 32% vs 44%; CF carriers - 10% vs
13% and others (including FraX, translocation carriers, SMA, Duchenne) 39% vs 10%.
Conclusions: The GIF genetic counselling intervention, specifically designed
to enhance family communication was found to:
remain congruent with principles of genetic counselling practice
be delivered successfully by different counsellors
improve the ability of clients to communicate genetic information effectively
Findings have implications for health professionals who wish to assist clients
in effectively communicating new genetic information to at-risk relatives.
EPL6.3
What would you like to know? Patients attitudes towards
communication of incidental findings emerging from new sequencing
technologies
L. Godino, G. Rodella, G. Severi, M. Mirra, G. Lanzoni, M. Romagnoli, A. Tranchina, G.
Tortora, C. Graziano, A. Wischmeijer, M. Seri, D. Turchetti;
Medical Genetics, Bologna, Italy.
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Background
The ability to analyse the genetic code in ever greater detail means that the
possibility of clinically relevant findings, that are unrelated to the original
reason for a test, is increased. These Incidental Findings (IFs), especially
when identified in children, may not bring health consequences for many
years. This greater sensitivity of genetic testing poses challenges to the clinical encounter including consent to, and disclosure of results.
Study aims
1.Identify current practice, including consent and disclosure practices, surrounding IFs in a range of clinical settings in the UK
2.Investigate family and professional experiences and views about the ethical and practical issues raised by the discovery of IFs
3.Inform policy on the consent and disclosure practices of IFs in clinical
practice
Methods
The findings from 23 clinic observations and 52 in-depth interviews were
analysed thematically.
Findings
4 main findings will be presented:
1.The possibility of IFs is currently not discussed in any systematic way at
the time of genetic testing
2.More results with uncertain clinical significance are being reported. HCPs
communicate these to families in different ways
3.There is no clear consensus from HCPs and families on what and how incidental information should be disclosed
4.HCPs believe that current systems do not facilitate the follow up of IFs
long term
Conclusion
Further debate is required to integrate genomic technologies into medicine
whilst addressing the ethical challenges, particularly as genetics is mainstreamed.
EPL6.5
Very often the answers not black or white: Exploring
communication in paediatric clinical genetic consultations
355
Background: Doctors need to investigate if there is an indication for DNAtesting and provide their patients with information. We aimed to gain insight in discussion of cancer genetic topics by gastroenterologists and surgeons as part of an intervention study, comprising a checklist for doctors,
intending to optimize referral to genetic counselling of patients visiting the
Gastro-Intestinal Oncology Centre Amsterdam. Insight into physicians performance can improve referral and optimize patient understanding.
Methods: Following a pre-post design, both before and after introduction of
the checklist, 40 consecutive, new patients completed a short questionnaire
assessing the discussion of cancer genetic topics during the initial consultation. Additionally, after introduction of the checklist, initial consultations
were audiotaped for a qualitative analysis of the discussion of cancer genetic
topics. Data on family history and referral was collected from medical files.
Findings: Discussion of cancer in the family increased from 78% before, to
90% post-intervention (p=0.11). However, doctors infrequently asked about
second-degree family members (pre: 50%; post: 65%; p=0.19) and age at
which family members got cancer (pre: 57%; post: 70%; p=0.29). Qualitative analysis of the audiotapes indicated the use of multi-interpretable and
vague questions.
Discussion: Contrary to expectations, cancer in patients family members
was discussed in most intake consultations. However, the suboptimal quality of the discussion hampers optimal referral for genetic counselling. Development of an alternative intervention might help better discussion of cancer genetic topics. Education for doctors is needed to improve knowledge
and discussion of cancer genetic topics.
EPL7.1
Consent and confidentiality in clinical genetics: a qualitative study
S. Dheensa, A. Fenwick, A. Lucassen;
University of Southampton, Southampton, United Kingdom.
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EPL7.2
Autonomy and emotions: Professional challenges in seeking consent
to genetic testing
H. E. Shipman, A. J. Clarke;
Cardiff University, Cardiff, United Kingdom.
Informed consent is the ethical and legal bedrock of clinical practice and
research involving humans. This paper investigates consent in practice by
exploring how professionals account for consent to microsatellite instability
and immunohistochemical testing for features of Lynch Syndrome and biobanking for research purposes.
Twenty eight semi-structured interviews were undertaken with professionals responsible for seeking consent. Thematic mapping was carried out on
transcribed data, which led to the identification of themes for more detailed
analysis. Data were examined from a rhetorical discourse analysis perspective, which involved micro-examination of the discursive devices drawn on
by participants in their talk.
Two themes encompass the communicative difficulties that were described:
Autonomy (enabling choice) and handling emotional responses in interaction. Challenges pertaining to enabling choice involve different forms of
opposition to respecting the autonomy of the person giving consent, both
within and beyond the individuals involved in communication. Issues concerning handling emotional responses in interaction involve accounts of
strong emotions both of those asked to consent and also of the professionals
involved. These two themes were unevenly balanced between the settings
with challenges of handling emotional responses almost entirely confined to
bio-bank settings. This is likely to reflect the contexts of current and previous
family experiences. In both settings, enabling autonomy becomes complex
when the professional is not involved in a face-to-face interaction with the
person asked to give consent. This work brings an alternative perspective to
accomplishing consent, as an interactive process involving complex moral
negotiations within relationships.
EPL7.3
Randomized controlled trial of a telephone-based peer support
program for female carriers of a BRCA1 or BRCA2 mutation: Impact
on psychological distress
Introduction: Huntingtons disease (HD) is an autosomal dominant, incurable, late onset neurodegenerative disorder. There is an increasing demand for
prenatal or pre-implantation diagnosis by couples in which one partner is
a HD carrier and who want to avoid transmission of the disorder to their
children. This exploratory qualitative study aims to gain insight into the
decision-making process regarding reproductive options by young HD carriers and their partners, and into the way prenatal testing is being experienced. Materials and methods: Using thematic analysis, ten semi-structured
interviews were conducted in HD carriers aged between 18 and 30 years, to
document their attitude on and experience with the decision-making process that preceded their request of a prenatal or pre-implantation genetic
diagnosis. Following topics were addressed: 1/ Which issues arose in the
decision to have children?; 2/ Which issues arose in the decision to have
prenatal testing (PD or PGD); 3/ How did you experience the actual procedure ?; 4/ How do you see the future with your children? Results: For young
HD carriers not to choose for prenatal testing is not an option, nor is refraining from offspring. With regard to the emotional impact, major differences
in the lived experience of PD and PGD are reported: the prospect of a termination of pregnancy causes great distress in women. Conclusions: With this
exploratory study we gain understanding in the complex psychological process linked to procreative decision-making of young HD carriers and their
partners that opt for a prenatal or pre-implantation diagnosis.
EPL8.3
Difficult decisions in prenatal diagnosis - patients experiences
of decision-making under uncertainty, and the implications for
expanding the offer of prenatal testing.
S. Leonard;
University of Bristol, Bristol, United Kingdom.
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gies, a qualitative study was undertaken. In-depth interviews of 26 participants who had received an uncertain prognosis in their pregnancies were
conducted. These interviews identified a number of key themes including
managing information, values and decisional context, and trust. The findings raised a number of questions, regarding for example the usefulness of
the notion of autonomy as a primary ethical principle in the prenatal context
where parents must make decisions based on unclear and often changing
information. These findings have implications for how new fetal anomaly
detection technologies are evaluated and put into practice to ensure that the
maximum benefit is achieved with the minimum harm.
EPL8.4
Offering a choice between 5 Mb and 0.5 Mb prenatal whole genome
SNP array analysis: are pregnant couples able of making informed
decisions?
Background: We implemented whole genome SNP array instead of conventional karyotyping (CK) for prenatal diagnosis (PND). Array detects more
clinically relevant anomalies and more genetic anomalies of yet unquantified risk for neurodevelopmental disorders, so-called susceptibility loci
(SL). Using SNP array ensues the question whether pregnant couples want
to know more and oversee the possible consequences. We explored whether
pregnant couples at increased risk for aneuploidies were capable of informed decision-making when offered a choice between the clinical outcomes
of a SNP array analyzed at 5 Mb resolution (comparable to CK) and a SNP array analyzed at 0.5 Mb resolution with or without SL. Methods: Consenting
pregnant couples (N=143) received genetic counseling by phone and filled
out the Measure of Informed choice (MIC) designed for this study. Choices
based on sufficient knowledge and congruent with attitude were considered
informed. Results : The MIC had sufficient internal consistency (Knowledge
=.74; Attitude =.80). Median MIC knowledge score was 5, range 0-7. We
considered a score 5 on the MIC knowledge scale as sufficient. Based on
this criterion, 66% of the couples had sufficient knowledge and thus made
an informed decision. However, 82% found it difficult to reproduce knowledge about SL. Discussion: Our results show that the majority of couples
were capable of making an informed choice. However, the most complicated
possible outcome of array, SL, was least understood. More research is necessary to assess what the psychological impact of SL is when couples have not
well anticipated this possible outcome.
EPL8.5
SNP Array in prenatal diagnosis; first impressions on the
psychological impact of receiving a susceptibility locus s a test result
357
Background: Array-comparative-genomic-hybridization (aCGH) in pregnancy allows a higher detection rate and shorter turnaround time compared
to karyotyping. However, the test can also reveal findings that are (a) not
related to the reason for which the test was done (b) relevant only much
later in life, and (c) uncertain. Little empirical data exist on health care professionals (HCPs) views about these issues.
Aims: To explore the views of UK HCPs about: the type of information that
should be sought and disclosed; timing of disclosure (i.e. during-pregnancy/
after-birth/when the information is medically-actionable); and who should
decide about these issues.
Methods: Q-methodology, which combines qualitative and quantitative approaches, was used to explore the views of 45 HCPs (Genetic-health-professionals, lab-scientists, fetal-medicine-experts).
Results: Preliminary data suggest that: HCPs either prioritise parentalchoices, or their own judgements about clinical relevance to determine disclosure.
Most HCPs support aCGH testing in pregnancies for further investigations
of abnormalities found on scans, but would not currently wish to offer it
otherwise.
Where aCGH uncovers adult onset predispositions, many argue that these should be disclosed because (a) they might be relevant to one or other
parent, and (b) they are not confident that systems can be set-up to delay
disclosure to nearer the time of clinical relevance.
Conclusion: HCPs support the introduction of prenatal aCGH into clinical
practice in certain circumstances, but are concerned that consent procedures, education of professionals outside of genetics and national-guidelines
are not yet in-place to address all the ethical issues.
EPL9.1
Predictive testing for Huntington Disease: Lessons learned from 24
years experience
F. H. Richards, M. J. Wilson;
Childrens Hospital at Westmead, Westmead, Australia.
M. E. Jones1,2, R. MacLeod1,2;
1
Manchester Centre for Genomic Medicine, Central Manchester University Hospitals
NHS Foundation Trust, Manchester, United Kingdom, 2Manchester Centre for Genomic
Medicine, Institute of Human Development, Faculty of Medical and Human Sciences,
University of Manchester, Manchester Academic Health Science Centre (MAHSC),
Manchester, United Kingdom.
Huntingtons disease (HD) is a progressive neurodegenerative disorder characterised by involuntary movements, cognitive impairment and psychological symptoms and is inherited in an autosomal dominant manner. Predictive testing by direct mutation analysis for individuals at risk of HD has been
available since 1993. Internationally agreed guidelines for predictive testing
have been published and recently updated but variability exists in how testing is delivered between centres. Whilst a substantive literature exists on
the impact of predictive testing, few studies have looked at how the predictive test counselling process is received by tested individuals. This evaluation
study sought the views of individuals who had a predictive test for HD at
our centre over a 5 year period (2007-2012). 44 of 100 eligible individuals
completed and returned a questionnaire designed for the purpose of the
study. Descriptive statistics were used to present the quantitative data and
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a thematic analysis was conduced on the free text comments. Overall, participants were positive about their experience and valued getting information and support as well as building a good relationship with their genetic
counsellor. However, 14/44 participants found the testing process too long
and for 7/44 participants the journey time to the hospital took > 2 hours.
Proposals for improving the service included a more tailored approach that
took greater account of prior experience. In addition participants welcomed
the inclusion of information resources such as video clips highlighting a range of testing experiences, and also advocated more focus on post test follow
up and support.
EPL9.3
Quality issues in genetic counselling practice for presymptomatic
testing: a European Delphi study
Back to index
This qualitative study explores how women who have been diagnosed with
a mutation in BRCA1 or BRCA2 experience living with risk of cancer development, as well as their need for psychosocial and informative follow-up.
The study also investigates whether learning and coping seminars (LCS)
contribute to a greater sense of coping. Focus group interviews were performed with female BRCA1 and BRCA2 mutation carriers when they attended LCS. The interviews were performed immediately before and after
the LCS. 17 women participated in two focus groups in two different LCS
seminars (10+7). The following themes were discussed: Their personal
reactions after being identified as mutation carriers, experiences with the
risk management program, decision making and experiences regarding risk
reducing surgery. In addition, the participants expectations and experience
with participating at the LCS were discussed. They were also encouraged to
expound upon any other issues important to them. The data were analyzed
using content analysis as described by Knodel.
Preliminary results indicate that the participants had experienced random
and different information regarding risk reducing surgery. They regarded
the LCS as a valuable source for information and psychosocial support, and
suggested that future LCS should be available for female BRCA1/2 mutation
carriers shortly after receiving their unfavourable test results. Other themes
as feelings of loneliness and fear of cancer development were also identified
in this study.
EPL9.6
Genetic test declining and high personal colorectal cancer risk
perception in DNA mismatch repair gene mutation families
359
Aim: In 2006, the Israeli Ministry of Health issued directives to expand PGD
to encompass hereditary breast-cancer syndrome. This study aimed to decipher the factors affecting PGD decisions among Israeli BRCA1/2 mutation
carriers or a spouse of a carrier.
Methods: We used a qualitative-phenomenological approach. Participants
were 19 Jewish Israeli women, who requested PGD for BRCA1/2 mutation
detection for their future embryos, at the Sheba Medical Center. All women
were either carriers (n=14) or spouses of a male carrier (n=5), of one of
the three Ashkenazi founder mutations in BRCA1/2. Of carriers, six were
diagnosed with breast cancer. All women are married, and all but three had
at least one child. We invited the women to narrate what motivated their
choice to opt for PGD. Thematic analysis was used to unveil experiences and
preferences related to the issue.
Results: Three significant factors were found to be associated with PGD: Prior infertility treatment (n=12); The existence of frozen embryos after fertility preservation treatment, done before chemotherapy (n=5); and Family
history of ovarian and/or early-onset breast-cancer (n=10). Although most
women underwent PGD (n=16), the majority (n=12) ended by declining the
selection process, primarily for clinical, bureaucratic and financial difficulties.
Discussion: This study details factors associated with the choice to undergo
PGD, in order to prevent the passing on of the `bad` BRCA1/2 mutation. Although, PGD for BRCA1/2 mutation is allowed in Israel, it is not funded by the
Israeli healthcare insurance.
This study was funded by the Israel Cancer Association grant.
EP03-S
Prenatal genetics service: the service users perspective
Prenatal Genetics is about to become yet more complex with the advent of
array comparative genome hybridisation with some centers already having
implemented this approach. Various research studies looking into the process of the prenatal genetic diagnosis interaction highlight that clinicians
and service users can have different goals, purposes and values regarding
prenatal testing. Informed patient decision making requires that information provision is responsive to patient interests. Clinical audit is one method to elucidate the needs of these service users. An advocate in the UK for
this approach of information provision, support and care that is responsive
to the current needs of service users is the national charity Antenatal Results and Choices (ARC), which provides non-directive support to expectant
and bereaved parents throughout and after the antenatal testing process.
ARC provides guidance on the principles of good practice when supporting
parents through a diagnosis of fetal abnormality. The South West Thames
Regional Genetics service is responsible for a population of 2.2 million peop-
360
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Non invasive prenatal testing (NIPT) for aneuploidy using cell free DNA in
maternal circulation has had a dramatic impact on prenatal screening and
diagnosis. In Melbourne, Australia, NIPT is offered by several different service providers with out-of-pocket costs ranging from $500 - $1,200. Testing
is largely performed in the USA and turn around times vary from 10 days to
3 weeks. Some centres provide clients with genetic counselling. At present,
clients attending our public hospital may access NIPT as a first tier screening
option or after first or second trimester screening. We have observed that
many women who receive an increased risk result for Trisomy 21 will utilize
NIPT before committing to an invasive test such as amniocentesis. Personal
experiences that appear to influence this decision include IVF pregnancies,
history of infertility, advanced maternal age, religious/spiritual beliefs and
a desire to further clarify their risk. Counselling for this group of women
requires significant time to explore the various pathways and possible outcomes, and the limitations and benefits of each.
Case examples will be used to illustrate our experiences with clients taking
up NIPT, including management of false positive results, inappropriate use
of NIPT and failure to achieve results. Decision making processes and counselling issues arising from NIPT will be explored.
EP08-M
Monitoring of congenital anomalies in the population of the Republic
of Moldova
V. V. Egorov, N. I. Barbova, E. A. Halabudenco, M. S. Stratila;
National Center of Human Reproduction and Medical Genetics, Chisinau, Moldova,
Republic of.
In the Republic of Moldova, the monitoring of the CA using since 1991, and
from 2009 we are working to incorporate our registry in the EUROCAT.
Were studied the prevalence and sharing of CA on the basis of genetic monitoring for the period from 2008 to 2012. The overall prevalence of the CA
for the 5-year period amounted to 18.92 per 1000 newborns. The maximum
frequency of the CA was noted in 2008 - 20.3 per 1000 births, the lowest
in 2009 - 18.36 to 1000 newborns. Prenatal screening of pregnant women
using non-invasive and invasive diagnostic techniques has allowed to reduce
the average frequency of the birth of children with CA for 7.2% to 17.56 per
1000 newborns. In the structure of the CA by prevalence leading place is
occupied by CA of the musculoskeletal system (22.5 2.58%), multiple malformations (22.02 2.98%) and the CA of the circulatory system (19.12
4.48%). The prevalence of individual nosological forms of CA (esophageal
atresia, cleft lip/palate, omphalocele, Down syndrome) are consistent with
those of the international register of EUROCAT. Results of questionnaire of
158 women given birth to children with CA showed that 85.6% of women do
not take folic acid in the first trimester of pregnancy. 74.5% of the mothers
drank alcohol during pregnancy. Influence of the teratogenic factors noted
at 22.2% of cases.Monitoring data allow to plan and carry out preventive
measures to reduce the birth rate of the children with CA in Moldova.
Back to index
EP09-S
The impact of the perceived severity of genetic conditions on the
attitudes towards genetic testing and termination of pregnancy
A. M. Ciuc, R. Moldovan;
Babe-Bolyai University, Cluj-Napoca, Romania.
Since its foundation 20 years ago, Myriad genetics has become a leading
molecular diagnostic company dedicated to making a difference in patients
lives. Through the discovery and commercialization of transformative tests
to asses a persons risk of developing disease, guide treatment decisions
and assess risk of disease progression and recurrence. These test services
achieve top-quality assessment enabling top-quality information for the patient based on test results. Myriad laboratory operates under the highest
standards of lean efficiency allowing result reporting much faster than average. In its aim to improve patients quality of live and deliver top-quality
information to the patients, Myriad Spain has developed a plan to create
Individualized Medicine Units (IMUs). These multidisciplinary units located
into the health system offer professional cancer counseling services. Different professionals including nurses, geneticists, genetic counselors, oncologists and psychologists will provide genetic testing education to patients
and medical specialists. Thus, IMUs will become single organization structures which will permit a degree of coherence in providing genetic tests to
counseled individuals. With the creation of IMUs access to an integral service with added value will benefit individuals affected of cancer as well as
individuals at risk to develop it. IMUs are patient centered structures which
intend to implement equal opportunity, help specialists to interpret genetic
test results as well as to get genetic guided medical and preventive options
closer to patients.
EP11-S
The disclosure of direct to consumer genetic testing: how to regulate
them?
S. Oliveri1,2, A. Gorini1,2, C. Lucchiari1,2, M. G. Hansson3, G. Pravettoni1,2;
1
Interdisciplinary Research Center on Decision Making Processes IRIDe, Department of
Health Sciences, University of Milan, Milan, Italy, 2Applied Research Unit for Cognitive
and Psychological Science, IEO Istituto Europeo di Oncologia, Milan, Italy, 3Centre for
Research Ethics & Bioethics CRB, University of Uppsala, Uppsala, Sweden.
Direct to the consumer genetic test (DTC GT) service is an indisputable and
increasing phenomenon which portray internet society. It lets hypothesize
that common people search for more health information and more sense of
responsibility on their health behaviors. Indeed, proponents of DTC GT argue that making consumers able to calculate the relative risk of developing
certain diseases may result in improved compliance with health-screening
practices and more healthful lifestyle choices. The advertisements have a
tendency to highlight these benefits and minimize any possible limitations.
Moreover, consumers purchase these tests without the obligatory involvement of the health care provider, leaving free interpretation and use of
genetic data. Despite these aspects, no concrete evidence about the attitudes towards, knowledge and use of DTC GT tests by members of the general population exist. Findings of previous researches indicate a low level
of awareness of direct-to-consumer genetic testing impact, because of the
hypothetical nature of many studies, use of not representative samples of
the population and too little evidence from users. It is necessary to provide
a theoretical framework of DTC tests use and to collect systematic data on
361
As high-throughput genomic technologies became widespread and genetic healthcare workload expands, there is a call for education and training
professionals to translate this changing landscape into appropriate care as
well as for the health education of general population. In that sense, genetic
counsellors are fully skilled professionals that could play an important role
on the provision of safe and ethically adequate practice.
The first Portuguese genetic counsellors have completed their formal training in 2012. The Portuguese course in genetic counselling is accredited by
the European Board on Medical Genetics (EBMG), in accordance with the
proposed standards for education and core competences for professional
practice, and in line with the harmonizing efforts of professionals education across different countries and healthcare settings. Moreover, a national
association of genetic counselling professionals have been created. With this
report we aim to provide evidence on the development of genetic counselling in Portugal, its educational program and current challenges on the establishment of the profession.
A growing body of research focusing diverse settings of the genetic counselling provision has been undertaken recently in Portugal. This includes the
professionals training needs for effective practice, quality issues of counselling, or the set of constraints affecting service delivery. Emergent challenges to continue the development of genetic counselling in Portugal are
the recognition of the genetic counsellor profession, the harmonization of
practice at national level, and the development of a clinical and counselling
supervision network.
EP14-M
Reaction of maternity generations to human genetics in Japan
362
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which were conducted twice. After having an event on genetics with children, further planning and operation were carried out together with the mothers, then FGI were conducted again. Main theme of this program was to
understand and accept a concept, everybody is different therefore everybody is valuable. The transcript data were qualitatively analyzed. The study
protocol was approved by the ethical committee of the affiliated institution.
Results: After series of genetic education, participants naturally accepted
everybody is different. Accordingly, some of them changed their attitude to
have more confidence to themselves, and some have more tolerance to the
children. Most importantly, participants start thinking that learning genetics
is useful for children to stop bullying and discrimination, and want nearby
friends to learn about it.Discussion: Through genetic education, negative
images of heredity that originally participants possessed were drastically
disappeared. Therefore, it is important to educate human genetics and to
give messages to recognize human diversity simultaneously.
EP15-S
And if I announce a genetic rare disease,are there particular
recommendations? That must I know?
K. MBailara1, E. Toussaint2, D. Lacombe2;
1
University, Bordeaux, France, 2Hospital, Bordeaux, France.
Introduction: International guidelines for offering pre-symptomatic genetic testing for Huntington disease (HD) first developed in 1994 have been
recently reviewed (MacLeod et al, 2012). In light of these recent recommendations, the experience and views of clients who had undergone testing
at an Australian HD genetics service operating since 1991 were explored.
Method: Semi-structured telephone interviews with 14 participants were
transcribed, de-identified and coded for thematic analysis using NVivo data
management software. Results: Six main themes were identified: (1) motivations for testing; (2) access to services; (3) information and client-centred
care; (4) perspectives on the guidelines; (5) support person role and impact; and (6) the afterwards. Motivations for testing were similar to previous studies including reducing uncertainty, family planning and to inform
risk status of existing children. Participants felt they were seen promptly
by the service, that they were provided with sufficient information about
testing, their care was client-centred and their needs were met at the time
of testing. Some participants wanted testing immediately, but understood
the rationale in the guidelines for delaying testing until the second counselling session. Involvement of a support person varied, and those who involved their support person only at the results-giving session described a
greater emotional impact of the result on the support person. Some individuals reported a significant long-term psychological impact after receiving
a negative test result. Conclusion: Recommendations for practice include
the involvement of support person/s from the initial session, and to provide
long-term follow-up to all individuals undergoing pre-symptomatic testing,
regardless of result.
EP17-S
Myotonic dystrophy type 1 families: anticipation as a decision making
behavior for presymptomatic DNA testing of asymptomic children
A. Shopova1, S. Shopova2, S. Bichev3, A. Savov3;
1
Department of Neurology, Universiry Hospital Alexandrovska, Sofia, Bulgaria,
2
Department of Neuropsychology, University Hospital St. Naum, Sofia, Bulgaria,
3
National Genetic Laboratory, Sofia, Bulgaria.
Introduction: Pre-Symptomatic genetic Testing (PST) for late onset monogenic neurodegenerative diseases, such as Familial Amyloid Polyneuropathy (FAP), Machado-Joseph Disease (MJD) and Huntingtons Disease (HD),
is available to at-risk healthy individuals. The aims of this study were: 1) to
analyze the motivations for a PST; 2) to access the present clinical situation
of the subjects; 3) to inquire about the individual treatment and preventive
options; 4) to evaluate the reproductive options; 5) to analyze feelings of
regret after PST.
Methods: This retrospective study consisted of telephone interviews of 168
carriers who underwent PST at our Medical Genetics Unit between 2000
and 2013 (145 FAP, 11 MJD, 12 DH).
Results: Most subjects failed to formulate, retrospectively, a motivation for
PST, either than mentioning a positive family history (46%). From all patients that tested positive for FAP, 45% present peripheral neuropathy, 19%
have undergone liver transplantation and 10% are medicated with Tafamidis. 11/23 patients with MJD and HD have psychiatric illness. From 43 pregnancies were reported - 6 patients underwent prenatal genetic testing and
5 couples pre implantation genetic diagnosis. 24 subjects mentioned having
decided not to have children because of the test result. 18 subjects presented feelings of regret after performing PST.
Conclusion:
The complexity of PST calls for discussions about testing that are tailored
to the testing context and the individuals needs and preferences. Hence, it
might be useful to access the long-term psychosocial consequences of PST, as
well as to ensure that adequate health and reproductive care are accessible.
EP19-S
Educational-support groups for daughters of BRCA mutation carriers:
a valuable addition to patient care
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EP20-M
Living with uncertainty: the experiences of young healthy Italian
women who have undergone genetic testing for hereditary breast and
ovarian cancer
M. Franiuk1, N. Rania2, M. T. Ricci3, C. Bruzzone1, P. Buda4, O. Puricelli5, L. Battistuzzi6, P.
Bruzzi7, L. Migliorini8, L. Varesco1;
1
IRCCS AOU San Martino-IST, Unit of Hereditary Cancer, Genoa, Italy, 2University of
Genoa, School of Social Sciences, Department of Education Sciences, Genoa, Italy,
3
IRCCS AOU San Martino-IST - Unit of Hereditary Cancer, Genoa, Italy, 4IRCCS - AOU San
Martino-IST, Unit of Hereditary Cancer, Genoa, Italy, 5IRCCS AOU San Martino-IST, Unit
of Clinical Psychology and Psychotherapy, Genoa, Italy, 6University of Genoa, Section of
Forensic Medicine and Bioethics, Department of Health Sciences, Genoa, Italy, 7IRCCS
AOU San Martino-IST, Unit of Clinical Epidemiology, Genoa, Italy, 8University of Genoa,
School of Social Sciences, Department of Education Sciences, Genova, Italy.
Introduction. Testing of the BRCA1 and BRCA2 genes can identify inherited
mutations that predispose to breast and ovarian cancer, and thus provides
those women with the opportunity to engage in risk-reducing behaviors and
programs. The information derived from the test, however, also exposes women to the responsibility of making difficult decisions and raises important
new issues that may affect how they manage their lives.
Aims and methods. The aim of this qualitative study was to explore, through
in-depth interviews, the experiences and behaviors of 22 young Italian women (11 mutation-positive, 11 mutation-negative, mean age: 35.21) who
participated in BRCA testing. All the women had a known BRCA mutation in
the family but no personal cancer history. Interview data were collected and
analyzed in accordance with the grounded theory approach.
Results and conclusions. The following psychosocial themes were found
to be affected by the results of BRCA testing: childbearing intentions, future projects, family support, feelings towards children and partners, risk
perception, attitudes towards risk management strategies. Test results appeared to have a definite but generally not severe impact on the participants
lives although, in some cases, negative emotions were denied in words but
expressed through behaviors. The results also showed that the main predictor of negative feelings was a previous experience of cancer in the family.
These findings may help clinicians better understand womens experiences
of BRCA testing and thus develop adequate, culturally and ethically sensitive
interventions in the areas of effective communication, support and care.
EP21-S
The initiator and timing of referral to breast cancer genetic
counselling; an exploration of everyday person-centered practice
Objective: The aims of this study were to assess the impact of objective
versus subjective risk on emotional distress and to investigate the extent
to which cognitions mediate the impact of risk perception on emotional
distress in a sample of breast cancer women. Method: In a retrospective
quasi-experiment, a convenience sample of 53 breast cancer women (mean
363
Ovarian cancer is the fifth most common cancer amongst women in the UK,
and accounts for more deaths than all the other gynaecological cancers combined. As early stage symptoms are few and non-specific, ovarian cancer is
often diagnosed at an advanced stage and survival rates remain low. There
is an opportunity to improve outcomes through advances in risk stratification, early detection and diagnosis. A population-based ovarian cancer risk
prediction and stratification program incorporating genetic testing is being
developed. A previous focus group study with individuals from the general
population identified a need and support for the proposed program. This
qualitative interview study was undertaken to explore attitudes of women
at high risk of ovarian cancer (strong family history or BRCA1/2 mutation
carriers). Eight women participated in one-on-one semi-structured interviews to explore their experiences of learning about ovarian cancer risks
and attitudes towards the proposed program. Overall women had very positive attitudes towards the introduction of a population-based risk prediction and stratification program for ovarian cancer. Women felt that it would
be of interest to other women in the general population and would raise
awareness of the disease. Women drew on their own experiences of genetic
testing, screening, risk-reducing surgery and interactions with health professionals to offer suggestions for the successful implementation of the program. Suggestions included providing counselling before and after receiving
risk estimates, different formats for presenting risk estimates and providing
information regarding ovarian cancer symptoms, effectiveness of screening,
preventive options and lifestyle factors to reduce cancer risks.
364
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EP25-S
Genetic counselling for inherited cardiac conditions in Wales: A
service evaluation
Introduction: To optimally inform counselees about their and their relatives risks, information about lifestyle risk factors, e.g. physical activity and
alcohol consumption, might be discussed in breast cancer genetic counselling. This study explored lifestyle discussion in consultations during breast
cancer genetic counselling. Method: First and follow-up consultations with
192 consecutive counselees for breast cancer genetic counselling were videotaped and coded for discussion of lifestyle topics (e.g. physical activity,
diet, alcohol, smoking). Counselees completed online questionnaires before
the first and after the final consultation. Results: With 52 (27%) counselees lifestyle was discussed, either in the first or final consultation, or both.
Counselees mostly raised the topic (60%). Counsellors provided information about lifestyle risk factors to 19% and lifestyle advice to 6% of the counselees. Discussion of lifestyle was not associated with counselees characteristics or causal attributions. Post-counselling, more affected counselees
considered lifestyle as a factor contributing to their breast cancer (29%)
compared to pre-counselling (15%; p=.003). Conclusions: Information and
advice about lifestyle risk factors is infrequently provided, both with breast cancer unaffected and affected counselees and with those who did and
did not consider their lifestyle as a cause of their breast cancer. A debate is
recommended on how to incorporate lifestyle information in breast cancer
genetic counselling. Modifiable lifestyle factors could be discussed more frequently to optimally inform counselees about possible ways to reduce their
risk. Counsellors should be educated about effects of lifestyle and studies
are needed on how to best integrate lifestyle information in breast cancer
genetic counselling.
EP27-S
What ethical and legal challenges are associated with implementing
risk-stratified screening for common cancers?
A cross-sectional survey using a self-administered questionnaire was performed with the aim of exploring the impact of genetic counselling in women
undergoing invasive prenatal procedures because of advanced maternal
age.Out of 637 questionnaires mailed to women who underwent prenatal
diagnosis 1 -6 weeks before, 221 (34.69%) were returned; of those, 52.9%
belonged to women counselled through an individual session, while 47.1%
women had attended a group session.The majority of women (85.5%) defined the information received during counselling as clear (with 15.4% considering it as extremely clear, regardless of the type of counselling received)
and helpful (overall 99.1%).Twenty-two (10%) stated their expectations
about CVS/LA had changed after counselling.Out of 192, 140 (72.9%) reported satisfaction about the counselling process regardless of the type of
counselling received. Although more women (52.6%) stated they preferred,
or would have preferred, individual counselling, the highest satisfaction rates were reported by women attending group sessions.
EP29-S
Cancer genetic counseling in Iceland
Back to index
EP30-M
Is communication with relatives discussed in the final consultation
for breast cancer genetic counselling?
N. Bukvic1, M. Maurizio2;
1
Azienda Ospedaliero Universitaria Consorziale Policlinico di Bari, Bari, Italy, 2Servizio
di Genetica Medica Dipartimento, di Scienze Biomediche Azienda Ospedaliero,
Universitaria di Foggia, Foggia, Italy.
With the surge of genetic tests and technologies, genetic counsellors are faced
with the challenge of translating emerging scientific knowledge into practical
information for patients, clinicians and public health policy makers. The new
tests and technologies also are associated with new psychosocial and ethical
considerations. New guidelines are needed for each new discovery of the genomic impact on phenotype, pathology and disease while old syndromes
and old pathology, continue to require attention. In the new post-Human
Genome Project era, genetic counsellors will be an integral part of translating
genomic discoveries into beneficial impact on human disease, health care,
and medical benefits. The needs for genetic counselling should be designed
into genomic research at the onset. Genetic counsellors need to handle old
while rapidly assimilating new information and the principal challenge is to
be up to date and updated. [World J Med Genet 2013 May 27; 3(2)]
EP32-M
Attitude toward genetic testing of children for common disease risk
in Japan
[Purpose] The purpose of this study was to assess the attitude of Japanese
general public toward genetic testing of children for common diseases, and
to clarify factors related to the attitude by nationwide opinion surveys conducted in 2009 and 2013. [Methods] In the survey in 2009, 4,000 people
(age, 20-69) were selected from the Japanese general population by a stratified two-phase sampling method. In 2013, 2000 people were selected in the
same way. They were queried about the following topics in a mail survey:
attitudes toward genetic testing for disease susceptibilities of children for
common diseases, interest in medical genomic studies, level of genomic literacy and awareness of the benefits and risks of medical genomic studies.
The factors related to the attitude were examined using logistic regression
analysis. [Results] The response rate was 52.5% (2,009/3827) in 2009 and
60.3% (1161/1925) in 2013. The genetic testing for disease susceptibilities
of children was favored by 58.8% people in 2009 and 58.4% in 2013. The
interest in medical genomic studies did not so change between in 2009 and
2013; belief of scientific development has more advantage than disadvantage was a little increased. Interest in medical genomic studies, belief in science and awareness of risks were significantly related to favorable attitudes
365
This paper will show the results, part of longitudinal study /more than 5
years/, of the factors that influence the perception of the information during
the GC and the reproductive decision making process. Till now we explored
the effect of: type of diagnostic procedures, advanced maternal age, family
reproductive history. This study focuses on the pathologic rates of the SA. SA
is inducted and is a result of the concrete situation, not a typical personal
trait. Purpose: Analyzing the anxiety rate during the GC and the reproductive
decision making process. Task: Examination of the SA rate with State-Trait
Anxiety Inventory (STAI). The object of the study are 100 women, who were
pointed for GC because of biochemical screening results and risk pregnancy.
Hypothesis: The need of making reproductive choice after the GC can increase the rates of SA. Results: In most of the cases of women without higher
rates of trait anxiety pathologic rates of the anxiety during the process of GC
are observed. Conclusions: Through the GC process the SA is increased and
this affects the subjective perception of the information and the reproductive decision making process. This suggests creating an algorithm for good
practices for psychological support in the process of genetic counseling.
366
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EP35-S
Content analysis of informed consent for whole-genome-sequencing
offered by direct-to-consumer genetic testing companies
E. Niemiec1,2, P. Borry1, H. C. Howard2,3;
1
Department of Public Health and Primary Care, KU Leuven, Kapucijnenvoer 35 BOX
7001, B-3000 Leuven, Belgium, 2IQhealthcare, Medical Ethics, Radboud University
Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, Netherlands, 3Centre for Research
Ethics and Bioethics, Uppsala University, PO Box 564, SE-751 22 Uppsala, Sweden.
Background: Whole-genome sequencing (WGS) is expected to have a significant impact on the field of human genetics in the near future. The sheer
volume of information generated by WGS raises the question of whether or
not to return such results. This dilemma is exacerbated in the context of
paediatric research as particular issues are raised: the rights of parents to
access their childs genetic information; the best interests of the child; the
right (not) to know; the utility of information; consent, and counseling.
Objective: The aim of this research was to develop guidance on how to
address the return of WGS results in paediatric research. We worked with
the P3G International Paediatric Plateform to build a common tool.
Methods: To formulate the Statement, we: (a) reviewed the legal and ethical
norms applicable to the European and Canadian research communities and
the relevant literature to identify both existing and emerging guidance; (b)
conducted a qualitative study of stakeholder groups; (c) developed recommendations; and (d) validated the recommendations through consultations
with a large number of stakeholders (e.g. genetic researchers, communitybased organizations).
Results: We propose a Statement to address the issues of when WGS results:
i) should be returned; ii) should not be returned (and identify exceptional
circumstances); and iii) described the procedures for the communication
of results.
Conclusion: It is anticipated that the Statement will not only provide a
template for paediatric research using WGS, but also guide researchers and
ethics committees.
EP37-S
Disclosure of genetic information within the family: evaluation of the
model letter accompanying a new French decree
In France, the recent publication of the decree of June 20th 2013 (n 2013527) modifies professional practices in medical genetics. It suggests a protocol regarding the transmission of information to relatives after a genetic
diagnosis of a serious condition with the possibility of preventative or care
measures. If a mutation carrier refuses to directly inform other members of
About 3 million people are concerned by in France and even if the media
and the French Foundation of Rare Disease are proactive in discussing these
diseases, their increased number and specifications are difficult to keep up
with.
A preexisting child presenting with a rare genetic disease will also be a source of psychological disturbances. An overview of the current literature showed that many dimensions are involved in parents with children diagnosed
with rare genetic disease: social, financial, familial, professional, educational, medical and psychological. However this analysis was done on a little
number of studies and very little is detailed on the specificity of the rare
genetic disease. Also these elements are usually studied to individual levels
and not necessarily at the family level. And, if a child is diagnosed with a
rare genetic disease, the whole family will be concerned, will suffer and will
need to adapt to this new situation. It seems that parents are showing signs
of psychic weakness but no prediction can be made on the impact this will
have on the family system nor on the factors of protection of weakness.
But are there specificities in the rare genetic diseases?
EP40-M
The impact of carrier identification on childrens wellbeing from
parents and childrens perspectives
M. Noke, S. Peters, A. Wearden, F. Ulph;
The University of Manchester, Manchester, United Kingdom.
Back to index
367
BRCA1 and BRCA2 carriers who live in the south east of England are offered
an appointment in a one-stop BRCA clinic at Guys hospital following identification of a gene mutation. Carriers are offered a choice of appointments with
specialists in genetics, breast surgery, gynaecology, oncology and psychology.
In addition regular support groups and patient education days are provided.
Dedicated psychological support is provided by a clinical psychologist. We will
discuss the types of interventions offered and the range of issues addressed.
In addition we will present data from a satisfaction survey carried out with patients who opted to see the Clinical Psychologist in the BRCA follow up clinics
between January and March 2014. This survey found that 92% of patients felt
completely listened to and understood, 85% felt completely able to talk about
the things they needed to and 85% felt that the appointment helped them feel
they could move forward a great deal or to some extent with their issues.
UK guidelines recommend that all women considering risk-reducing mastectomy are offered psychological support. The local protocol is that all women
seen in the BRCA multidisciplinary clinic are offered an appointment with the
Clinical Psychologist. An eighteen month audit of the offer of these appointments (by the psychologist) to the women is currently underway. Preliminary
data show that 46% (12) of the total number of women eligible for an appointment (26) were offered an appointment. Possible reasons for this discrepancy
and tentative recommendations for practice will be discussed.
EP44-M
Using Patient Reported Outcome Measures in clinical genetics
services: Pilot studies in six UK clinical genetics centres.
Background: Patient Reported Outcome Measures (PROMs), short selfcompletion questionnaires capturing aspects of health or health-related
quality of life, have recently gained prominence in healthcare evaluation
worldwide. Historically, the quality of UK clinical genetics services has been
evaluated using process measures e.g. patient waiting times, numbers of
patients seen. Whilst current approaches to quality are important, little attention has been paid to patient outcomes because these have been difficult
both to specify and to measure. This paper reports on pilot studies using a
new clinical genetics-specific PROM, the Genetic Counselling Outcome Scale
(GCOS-24) in six UK clinical genetics centres.
Methods: Patients were asked to complete GCOS-24 before and after clinic
attendance. Satisfaction data were also collected in some centres. Data were
analysed using analysis of variance and bivariate correlation. Centres provided feedback on feasibility of PROMs data collection and lessons learned.
Results: Five centres demonstrated statistically significant improvement in
patients GCOS-24 scores following clinic attendance with usable samples
sizes of 42, 45, 54, 55 and 74 patients respectively (p<0.001). One centre
had insufficient matched pre-clinic and post-clinic questionnaires to enable
a useful analysis. GCOS-24 improvement scores correlated significantly with
patient satisfaction.
Conclusions: Findings demonstrated that participating clinical genetics
centres can deliver significant measurable patient benefits and that GCOS24 has potential to be a useful supplement to existing methods of evaluating
quality of routine clinical genetics services. Useful next steps could include
developing methods for optimising patient response rates and for integrating PROMs information into continuous quality improvement cycles in clinical care.
EP45-S
Fitting the Pieces Together - Adoption Issues in Genetic Counselling
M. J. Boon;
Genetic Health Queensland, Brisbane, Australia.
368
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within a genetic counselling session. Primarily, adoption may limit the communication of information between at-risk relatives, with difficulties in notifying either a child who was adopted out at birth, or the biological family
of an adopted individual, of genetic risk information. However, many other
clinical and psychosocial issues can also be raised in these cases which likewise need to be addressed.
In Queensland, Australia, the adoption process is regulated by a single State
Government organisation. The current Adoption Act 2009 legislation came
into effect in February 2010 and aims to balance the privacy of those individuals who do not want to be contacted with peoples right to access information about their birth parents or a child who was adopted. For new couples
wishing to express interest in adopting a child, multiple assessments are
required to determine their suitability as parents, including close examination of their medical history.
Three cases will be presented which highlight some of the various challenges that adoption can present within a cancer genetic counselling context.
The outcome of each case will be discussed, including the specific clinical
and psychosocial issues that were raised. These include the assessment of
eligibility for genetic testing, provision of accurate risk management advice,
facilitating communication of genetic risk, addressing psychosocial issues
and the potential discrimination an individual with a genetic condition may
face if they are wishing to become an adoptive parent.
EP46-M
BRCA isnt all about boobs...or is it? Lessons from a BRCA support
group
EDS patients suffer from a loss of control (body, time, environment, emotions). Inevitably this results in a loss of identity. Four elements stand out:
the danger of living a restricted life, the risk of social isolation, the feeling
of uselessness and the problem of being discredited. Especially the latter
is very hard to endure. Patients are caught in a typical paradox: successful
coping, often at the expense of superhuman efforts, makes other people
doubt about the gravity of the illness. Yet, they have no other choice but to
do their very best. There is always this pending and lurking question: Are
you really trying enough? The underlying assumption is that effort always
yields positive outcomes. If someone does not get any better he is being held
responsible. It is this social response that makes a disease stigmatizing and
tiring, not the disease in itself. We take a social psychological stance: not a
persons character is determining his behavior, it is the context. As a consequence: we should reconsider our notion of empowerment. The traditional
medical belief that rational communication of information and compliance
in following treatment regimens form the basis for patient empowerment
is in line with a social discourse that propagates mastery and responsibility. It overlooks some important elements of illness experience. By listening
to and talking with our patients we, practitioners, by definition, legitimize
their experiences. This is where authority meets vulnerability. Only then are
patients capable of moving forward.
EP50-M
Psychosocial outcomes and uptake of testing in non-pregnant and
pregnant women offered carrier screening for fragile X syndrome
(FXS)
Back to index
We report a family where post-mortem genetic testing identified a predisposition to melanoma and pancreatic cancer. The index case referred to genetic counseling presented a cancer of unclear origin (pancreatic/ovarian)
at age 58. A sister had died of pancreatic cancer at 59, the mother had died
of melanoma at 48 and an aunt was diagnosed with melanoma at 32 and
breast cancer at 52 and died of a head and neck cancer at 60. Analysis of
BRCA 1 and 2 genes was normal. Analysis of CDKN2A gene was discussed
but declined by the patient for different reasons among which were the lack
of efficacy of surveillance measures and clear guidelines. The patient gave
consent for DNA storage for further analysis and research. After her death,
one sister requested genetic testing of CDKN2A through a Canadian genetic team. With the consent of the patients husband, the medical authority
in Switzerland and the ethical committee in Canada, analysis of CDKN2A
was performed in Canada and a pathogenic mutation was identified. Subsequently, the patients adult children who live in Switzerland requested
genetic counseling and opted for testing. Post-mortem genetic testing is
commonly performed in the setting of forensic autopsy (for example following sudden cardiac death) but it presents some specific challenges when
requested by a family member and performed on a stored DNA sample. This
case raised legal and ethical questions about consent, practical questions regarding reimbursement of the test and awareness of psychological aspects
of genetic counseling for offspring.
EP52-M
Reciprocity among genetics professionals in Europe - A UK:Dutch
experience.
A. S. Matadeen1,2;
1
Oxfordshire Regional Genetics Service, Oxford, United Kingdom, 2Leiden University
Medical Centre (LUMC), Leiden, Netherlands.
Recent literature has highlighted that collaborations among genetics professionals in different countries can help to facilitate genetic counselling practice development. Reciprocity is one manner of achieving this. Reciprocity
has been identified by the Transnational Alliance for Genetic Counselling
(TAVC) as a useful entity in allowing for progression of the international genetic counselling community as it allows for reflection on current methods
of genetic counselling in ones own and other countries. Furthermore, the
December 2013 issue of the Journal of Genetic Counselling was dedicated
as a special issue concerning a global perspective of genetic counselling, in
particular highlighting the value and drawbacks of student exchange experiences as a component of study programs. Currently this seems to be the
most common method of gaining such experience. Here, I present the experience of a UK trained and registered genetic counsellor visiting a Netherlands based genetics service and I reflect on the benefits and drawbacks for
staff from genetics centres engaging in these opportunities. This is the first
report of this kind and it is hoped that it may help to facilitate future reciprocity among these two countries. Furthermore, a Dutch perspective of a UK
based service is also provided.
369
Following confirmation of array cgh as first tier diagnostic test for individuals with intellectual/ developmental delay, multiple congenital anomalies
and autism spectrum disorders there has been a lot of discussions regarding
using this test for prenatal diagnosis. In different countries there has been
different approach to this case. Considering the possibility on finding CNVs
of unknown significance leading to counseling dilemmas the general trend
has been to use this technique in cases of ambiguity identified in routine
chromosomal karyotype or where a normal karyotype is reported in a sonographic report of congenital anomalies. In the period of 24 months starting
December 2011 till December 2013 we performed array CGH for 32 amniotic or chorionic villi samples. From these 32 samples, 3were inconclusive
and of the 29, 5 showed a significant CNV. 3 of the 5 had problems in their
sonography and 2 were being studied for de novo markers. It appears that
array CGH Is an instrumentAl tool in clarifying complicated karyotypes and
a significant test for fetuses with abnormal sonography findings.
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J01.04
The AZFc region: crossroad between male infertility and recurrent
pregnancy loss in women
A prospective cytogenetic and molecular study was conducted on 123 Saudi men with azoospermia to estimate the prevalence of common genetic
abnormalities associated with male infertility. Routine cytogenetic studies
were performed to screen for chromosomal abnormalities whereas multiplex PCR methods were employed to screen for submicroscopic microdeletions of the AZFa, b and c regions located on the long arm of the Y-chromosome. Chromosomal abnormalities were detected in 21 patients (17%).
Among the 21 patients with cytogenetic abnormalities, 15 (71%) had the
Klinefelters syndrome 47,XXY karyotype, 4 patients showed different large
deletions of the long arm of the Y-chromosome with or without 45,X cell
line mosaicism, one patient showed low 47,XXY/46,XY mosaicism and one
patient had a chromosomal translocation between chromosomes 2 and 5.
The molecular studies revealed submicroscopic microdeletions of the Ychromosome in 3 (3%) of the 102 patients with a normal male karyotype.
These Y-chromosome microdeletions involved regions AZFc in all three cases and AZFb in two cases.
Our studies revealed that the prevalence of chromosomal abnormalities in
azoospermic Saudi patients is similar to that of major international studies.
However, the prevalence of submicroscopic Y-chromosome microdeletions
in patients with a normal karyotype was noticeably lower than what have
been reported internationally but consistent with a few studies performed
in Arab countries including Saudi Arabia. Our findings therefore strengthen
the assumption that other genetic and/or non-genetic factors may play a
major role in male infertility in Arab patients.
J01.06
The Relationship Between The Index and the Blood Disease
Thalassemia and a Sample Report
Background and Aims: Thalassemia is a severe hemoglobin disease, recognizing by CBC and hemoglobin electrophoresis primarily. At first, CBC and
hemoglobin electrophoresis was performed for the index case recognized as
-Thalassemia but finally experimental studies clearly demonstrated that it
was different type of thalassemia. In the present study, we report a case of
-thalassemia mutations which is called -101 C>T relative to the transcription start site of -globine gene, and is a +-Thalassemia case, but it has been
observed with different indices.
Methods: The patient was admitted based on hematologic indices as
The uncovering of cell free fetal DNA (cffDNA) in maternal serum of pregnant
women (1997) has provided an approach for applying of cffDNA as a noninvasive method for prenatal diagnosis (PND). The determination of fetal
gender is the first step of the PND. It is especially important in fetuses at
risk of sex-linked disorders and in conditions associated with ambiguous
development of the external genitalia.
Peripheral blood sample (10 ml) was obtained from 100 pregnant women
that referred to the diagnostic laboratory for routine pregnancy tests during
6th to 10th weeks of gestational age. Cell-free fetal DNA was extracted from
the maternal serum. The cffDNA was not enriched. The conserve sequences
of SRY and GAPDH genes as a marker for fetal gender and for internal control respectively were amplified by Multiplex-PCR using specific primers for
their reigns. We considered; amplification of just internal control indicates
female fetal gender and amplification of internal control and SRY conserve
sequence indicates male fetal gender. Latterly, all the collected results were
compared with the real gender of the newborns.
Early determination of fetal gender provides the opportunity of deciding and
employing early treatment designed for fetuses at risk. However, this study
aims to validate a simple, reliable and applicable method for non-invasive
Back to index
prenatal diagnosis of fetal gender. In this presentation we will demonstrate our data regarding fetal gender. Furthermore, we will present sensitivity
and specificity of this study.
J01.09
Filling the gap : the prenatal phenotype of two cases of rare
microdeletion syndromes
We describe two cases of prenatal detection of unexpected interstitial deletion syndromes that were recognized by CGH-Array after evidence of ultrasound fetal anomalies and normal karyotype
Case 1: A 43 years old woman in her fourth pregnancy referred for genetic
counseling because of cystic hygroma with normal fetal biometry measurements at the first trimester. Level II ultrasonography: micrognathia, lowset ears, lower limb abnormalities. CGH-Array was performed revealing a
deletion syndrome of 10,6 Mb on chromosome 6 (locus 6q11q14.1). After
termination the fetal pathology showed frontal hypertricosis, small nose,
long filtrum, macrostomia, thin lips, micrognathia, and low-set ears with
abnormal lobes.
Case 2: A 41 year old woman in her third pregnancy. During the second trimester the pregnancy was complicated by intrauterine growth restriction
and unilateral fetal club foot. A microdeletion of 496 kb at 17q21.21 was
detected by CGH- Array. This mutation has been reported in only a limited
number of families and with a variable phenotype. After termination the
physical examination of the fetus demonstrated high forehead, hyperthelorism, bulbous nose, thin lips, pointed chin, low-set simplified ears, camptodactyly, tales equinovarus, and reduced musculature in the lower limbs.
Both deletions were shown to be de novo. Understanding the pathogenesis
of fetal anomalies and correlations between the pre- and post-natal phenotype is difficult and limited because of a lack of prenatal data from cases reported after birth. The clinical spectrum of microdeletion syndromes
could be expanded greatly if more prenatal cases would be described and
published in the open literature.
J01.10
Chromosomal abnormalities in couples with reproductive failures
and/or those included in ART programs
371
BACKGROUND Infertility is a medical problem affecting a significant proportion of the population, up to 15.0% of couples of reproductive age. Genetic pathology is an important part of the general human pathology due to
the increasing number of genetic diseases. The purpose of this study was to
establishing a correlation between the presence of structural and numerical
chromosomal abnormalities in one of the partners and their reproductive
development.
METHODS During the period from August 2007 to December 2011, 2195 patients with reproduction problems, who had received in our hospital, were
investigated for this retrospective study, and the frequency of chromosomal
abnormalities was calculated. The control group consists of 83 fertile couples who had one or more children in history, was investigated by karyotype.
RESULTS Of the 2195 patients investigated by classical cytogenetic techniques 91.12% had normal karyotype and 8.88% had chromosomal abnormalities, the most common chromosomal changes are polymorphisms. Numerical chromosomal abnormalities were detected in the proportion of 0.65%
in infertile men and 0.62% in infertile women in the study group.
CONCLUSIONS: Recently a possible association between infertility and chromosomal abnormalities have been reported with a significant statistically
association. Our study shows that there is not association between chromosomal abnormalities and infertility problems,but this study needs to be
confirmed with further investigations on a larger control group to establish
the role of chromosomal aberrations in the etiology of infertility.
J01.12
FISH assessment of chromosomal aneuploidies in infertile males
Background. Reproductive failure is one of the most important issues for the
population at the age of procreation and approximately 15% of the couple
encounters reproductive difficulties. In the last years it was taken in consideration the hypothesis that not only somatic chromosomal anomalies but
also germ cells chromosomal aberrations could lead to reproductive failure.
Aim: In this study we used multicolor FISH probes for chromosome 13, 18,
21, X and Y to evaluate the aneuploidy incidence in sperm cells from infertile
males.
Methods: The study lot included 35 males with infertility and oligoasthenoteratozoospermia (OAT) and 20 males with normal fertility and normal
semen characteristics for which the conventional cytogenetic investigation
using peripheral blood revealed a normal karyotype. The fluorescent signals
for these chromosomes, were analysed at least 10000 cells/patient.
Results: The overall chromosome disomy and nulisomy in OAT group was
higher than the one identified in the control group. By comparing the incidence of the disomy in the OAT group, the highest incidence was the sex
chromosome disomy, followed by the disomy of chromosomes 13, 21 (equal
values) and then 18. The nulisomy incidence in the OAT group was higher
for sex chromosomes, followed by the nulisomy of autosomes 13, then 21
and 18.
Conclusion: During these days, for patients with OAT, intra cytoplasmic
sperm injection (ICSI) is frequently used, and it is important to inform the
patients that the use of spermatozoa for fertilization could be associated
with an increased risk of aneuploidies in embryos.
J01.13
Cleidocranial Syndrome with Premature Ovarian Failure
N. Almadani, H. Gourabi;
Department of Genetics at Reproductive Biomedicine Research center,Royan institue for
Reproductive biomedicine,ACECR, Tehran, Islamic Republic of Iran.
Our patient family had twenty members that five of them (three brothers,
One sister and father) suffered from oligodontia , enamel hypoplasia , dental
decay, Mild proportionate short stature , frontal bossing , Hx of recurrent
upper respiratory infections , sinusitis, bilateral clavicle bone hypolplasia
, pectus carinatum , short & tapering fingers , lumbar lordosis and thorasic
scoliosis.
My proband was a 33 years old female with premature ovarian failure and
chromosomal abnormality in peripheral blood ( %40 mosaicism of deletion
of 6p2 and %4 45 XO in fifty metaphase ) , other family members had not
any fertility problem and chromosomal abnormality.
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We gussed Our diagnosis in this case is Cleidocranial Dysostosis and ocurrance chromosomal abnormality and this syndrome probability resulted
frome location of this gene (CBFA1) in short arm of chromosome 6 and varibilty of mosaicism 45 XO phenomenon in ovarian tissue.
J01.14
Mutations in cyp21ohb gene of women with reproductive dysfunction
A. Nagimtaeva, A. Borovikova, D. Zhumazhanova, G. Abildinova;
National Research Center Maternal and Child Health, Astana, Kazakhstan.
Introduction Down syndrome (DS or trisomy 21) is a genetic disease resulting from the presence of an extra copy of chromosome 21. Although
advanced maternal age represents the major risk factor for DS, most of DS
children are born from women less than 30 years of age and is seems that
different mechanisms are responsible for chromosome 21 nondisjunction in
young women compared to older ones, each of them potentially affected by
an impaired folate/Hcy metabolism. The objective of this study was to evaluate the correlation between gene polymorphisms MTHFD G1958A and RFC1
G80A and the risk of Down syndrome. Materials and methods Our study
included 26 women that gave birth to DS babies and 46 control mothers
of healthy children. Genomic DNA was isolated from whole blood, using
peqGOLD blood DNA mini kit (ATP Biotech) following the manufacturers
instructions. The MTHFD G1958A and RFC1 G80A mutations were investigated by polymerase chain reaction (PCR) and restriction fragment length
polymorphism (RFLP) analysis. Results The combined RFC-1 80(GA or
AA)/ MTHFD1958GG genotypes compared with the reference RFC-180GG/
1958GG genotype was associated with increased DS risk (OR 0.18 [0.021.41] P= 0. 08), but not statistically significant. Conclusions This is the first
study in a cohort of Romanian mothers of DS children in comparison with
control mothers, analyzing RFC1 G80A and MTHFD G1958A polymorphisms as a maternal risk factor for meiotic nondisjunction of chromosomes 21,
causing DS.
J01.18
Genetic deletion of Pelota in mice leads to embryonic lethality at an
early post-implantation stage
P. Raju, G. Nyamsuren, I. M. Adham;
Institute of Humangenetik, Gttingen, Germany.
Pelota (Pelo) is ubiquitously expressed, and its genetic deletion in mice leads
to embryonic lethality at an early post-implantation stage. In our study, we
conditionally deleted Pelo after the establishment of embryonic stem cells
(ESCs) and showed that PELO depletion did not markedly affect the selfrenewal of ESCs or their capacity to form teratomas. However, Pelo-null
ESCs differentiation into extraembryonic endoderm (ExEn) was severely
compromised during embryoid body (EB) formation studies. Failure of Pelodeficient ESCs to differentiate into ExEn was accompanied by the retained
expression of pluripotency-related genes and alterations in expression of
components of the bone morphogenetic protein (BMP) signaling pathway.
Further experiments have also revealed that attenuated activity of BMP signaling is responsible for the impaired development of ExEn in Pelo-deficient cells. Collectively, our results convincingly show that PELO plays an
important role in the differentiation of ESCs, into ExEn through activation of
BMP signaling. Hence, the observed early embryonic lethality in conventional Pelo-deficient embryos could be attributed to the defects in ExEn formation during early embryonic development.
J01.19
Gene variation of TLR4 in patients with Endometriosis
Back to index
Aim: The endothelial nitric oxide synthase gene (eNOS) polymorphisms have
been associated with reduced vascular NO production or increased level of
homocysteine and related to pregnancy losses. In the current case control
study it was aimed to find out the possible role of eNOS E298D polymorphism in spontaneous pregnancy loss. Material and Methods: Twenty-three
spontaneously aborted fetal materials, 22 mothers who had these abortions and 86 healthy control cases were enrolled in the current results. The
genomic DNAs were isolated from aborted materials and peripheric blood
samples and genotyping for target polymorphic allele was done by real time
PCR technique. Results: Current results showed that eNOS (Glu298Asp)
polymorphisms were significantly associated with spontaneous abortion
(p=0.011). Conclusion: The present study identified the strong association
between eNOS gene polymorphisms and spontaneous pregnancy loss risk.
J01.21
18p partial trisomy with fetal ascites inherited from a mother with
familial 18p deletion syndrome
The deletion 18p syndrome is one of the most common chromosome abnormalities characterized by dysmorphic features, growth and mental retardation with a poorer verbal performance. Until now, no reliable phenotype map
for the characteristic clinical findings such as mental retardation, post-natal
growth retardation and typical facial features has been established yet.
Molecular karyotyping holds the promise of improving genotype-phenotype
correlations for frequent chromosome conditions such as the 18p-syndrome. Until now, there have been 9 reported families with transmission of del
(18p) from a mother to a child (including our study).
We describe a female baby, 46,XX,der(18)t(18;20)(p11.32;p11.2) with fetal ascites, transmission of deletion 18p from a mother and her affected families with partial monosomy 18p of different sizes owing to unbalanced
translocations.
J01.22
Structure of prenatally-identified polyploidies - a retrospective study
C. GUG1,2, R. Mihaescu1, R. Patrascu1, V. Gorduza3, M. Militaru4, A. Trifa4;
1
University of Medicine and Pharmacy Victor Babes, Timisoara, Romania, 2Private
Medical Practice and Genetics Laboratory Dr. Cristina Gug, Timisoara, Romania,
3
University of Medicine and Pharmacy Grigore T Popa,, Iasi, Romania, 4University of
Medicine and Pharmacy Iuliu Haieganu, Cluj, Romania.
Objective: The objective of this study was to identify the frequencies and
types of prenatally-identified polyploidies through amniocentesis or chorionic villus sampling (CVS), and to compare them with polyploidy identified
in terminated pregnancies. Materials and Methods: Fetal karyotypes were
investigated from cell cultures and banded with GTG. Amniocytes were also
screened with FISH and QF-PCR was used in the CVS cases. Results: We report here prenatal diagnostics through amniocentesis for 3 triploid fetuses.
373
FISH screening in uncultured amniocytes, is a standard procedure for the rapid detection of chromosomal aneuploidy in prenatal diagnosis, in order to
detect the most common aneuploidies, involving chromosomes 13, 18, 21, X
and Y. It is easy to perform, dont need cell culture, the risk for misdiagnosis
is low (~0.4%) and, principally, allow results in less than 24 hours.
We report the case of a young pregnant woman with an abnormal ultrasound scan, revealing a foetus with multiple anomalies, including cardiopathy, short femur and clenched hands. It was the first pregnancy of this
young, health, non-consanguineous couple with a normal family history.
Amniocentesis was performed and FISH aneuploidy technique, in uncultured amniocytes, was applied, with normal results. Cytogenetic analysis of
amniotic fluid revealed an unbalanced translocation involving chromosome
11 and 18, with deletion of 11q24-qter and trisomy of 18q11.2-qter, confirmed by FISH.
The foetus had several anomalies consistent with trisomy 18 phenotype.
The FISH rapid prenatal aneuploidy test is, taken in account its possibilities and limitations, a powerful tool for the clinician in the care of pregnant
women. Its limitations, it cannot detect cytogenetic abnormalities such as
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Pregnancy loss and adverse pregnancy outcomes (APO) result from a complex interaction of environmental and inherited factors. Although the etiology of more than half of such events remains unclear, gene mutations leading
to a hypercoagulable state, such as factor V Leiden, factor II prothrombin,
methylenetetrahydrofolate reductase (MTHFR) mutation, are often found
associated with pregnancy loss.
D. Serapinas1,2, J. Jukeviius2;
1
Department of Pulmonology and Imunology, Lithuanian university of Health sciences,
Kaunas, Lithuania, 2Mykolas Romeris university, Vilnius, Lithuania.
D. Serapinas1,2, D. Burkojus1;
1
Department of Pulmonology and Imunology, Lithuanian university of Health sciences,
Kaunas, Lithuania, 2Mykolas Romeris university, Vilnius, Lithuania.
Back to index
Genomic imprinting is an epigenetic phenomenon, which is involved in regulation of embryonic development and placental function. Previously we
have reported that the multilocus methylation defects (MLMD) at imprinted
genes may be among molecular processes, which are responsible for dysfunction of imprinted loci in pathology of early embryonic development.
We hypothesize that MLMD at imprinted genes may cause karyotypically
normal miscarriage, particularly among women experiencing recurrent
miscarriage.Differential methylation of 51 imprinted genes were examined
in first-trimester spontaneous abortions (SA) from women who have recurrent (group RPL, from 9 (data of GoldenGate Methylation Cancer Panel)
to 105 (Methylation-specific PCR (MSP) SA) and single (group SPL, from 6
(data of GoldenGate Methylation Cancer Panel) to 114 (MSP) SA) pregnancy
loss. Sixty induced abortions were investigated as a control group. All spontaneous and induced abortions had the normal karyotype.
Our results provide evidence that MLMD at imprinted genes in the group
RPL was more frequent than that in SPL (15x10-2 and 5.2x10-2, respectively,
p<0.01) with predominance of somatic epimutations (10x10-2 and 3.9x10-2
respectively, p<0.01) and multiple hypomethylation (9x10-2 and 4.4x10-2 respectively, p<0.01). Frequency of MLMD in both groups at maternal loci was
higher than that at paternal one (12x10-2 and 6.4x10-2, respectively, p<0.05
for RPL; 3.6x10-2 and 1.4x10-2, respectively, p<0.01 for SPL). Therefore, the
RPL is characterized by multilocus somatic hypomethylation of imprinted
genes and MLMD at imprinted genes on maternal loci that associated with
abnormal maintenance of maternal imprinting in somatic cells.
J01.32
Genetic association of phase II detoxification genes with recurrent
pregnancy losses among Moldavian women
Background:
Recurrent pregnancy loss (RPL) is a multifactor and distressing disease. In
this study, we aimed to investigate the relationship between the polymorphism of GSTM1, GSTT1, GSTP1 and the pregnancy loss.
375
D. Serapinas1,2, E. Puisyte3;
1
Department of Pulmonology and Immunology, Lithuanian university of Health
sciences, Kaunas, Lithuania, 2Mykolas Romeris university, Vilnius, Lithuania, 3Lithuanian
university of health sciences, Kaunas, Lithuania.
Holt-Oram syndrome (HOS) is caused by TBX5 gene mutation and it is estimated to affect 1 of 100 000 individuals. This condition is inherited in an
autosomal dominant pattern, but most cases result from new mutations in
the gene. HOS is characterized by skeletal abnormalities of the hands and
arms (upper limbs) and heart problems. The diagnosis of HOS can be established clinically. Clinical signs include upper limbs malformations, anomalous heart structure (eg. atrioseptal and ventriculoseptal defects or Fallot
tetrada) and cardiac conduction disease, which can cause bradycardia or
tachycardia. HOS can be confirmed through TBX5 gene mutation molecular
genetic testing - DNA sequence analysis or array comparative genomic hybridization (array CGH). A case report of Holt-Oram syndrome in 6 month
girl is represented from Kaunas hospital of LUHS, when mother has been
taking a valproic acid 500mg/day during pregnancy for epilepsy treatment.
The antiepileptic drug valproic acid has teratogenic side effect and is a potent inducer of neural tube defects and other abnormalities, such as car-
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diac, skeletal and limb deffects. When valproic acid cannot be avoidened in
pregnancy, the lowest possible effective dose should be prescribed. Also it
is recommended to use folic acid to 5 mg/day. A usage of folic acid should
be started before pregnancy, so family planning and prenatal consultation is
recommended for women with epilepsy.
J01.35
Stage-dependent expression of HomeoboxA10 gene in human
endometrium
The inhibitor of DNA binding family members (ID1,ID2,ID3,ID4) inhibit activity of basic helix-loop-helix transcription factor and have an important role
in cell growth ,differentiation and angiogenesis.
Due to the high proliferation and angiogenesis in the endometrial lining of
the uterus during the menstrual cycle, it seems that this gene family may be
involved in the occurrence characteristics of this tissue.
This study focused on expression of ID gene family by real-time PCR in 21
healthy fertile women undergoing tubal ligation surgery, between 20-45
years old to investigate the possible role of this gene family in endometrial
tissue changes during three phases (menstrual ,proliferative , secretory) of
menstrual cycle. For this respect ethical approval and informed patient consent was gained for the use of tissue sample.
Data revealed high levels of mRNA expression for ID1, ID2, ID3 and ID4 in
proliferative phase. Also, all members of this gene family showed more significant expression levels during the secretory phase than the proliferative
phase of the menstrual cycle.
These results suggest for the first time that ID gene family has a dynamic
role in the proliferation and angiogenesis of endometrial cells. We propose
that changes in expression pattern of this family can be reviewed in gynecologic disorder and infertility that related to embryo implantation.
J01.38
Polymorphisms of Toll Like Receptor 2, 3 and 4 in patients that do and
do not enter labour spontaneously at term
T. zl1, Z. Ocak1, S. A. Simavl2, A. Karata1;
1
Abant zzet Baysal University Medical Faculty, Bolu, Turkey, 2zzet Baysal Maternity
and Children Hospital, Bolu, Turkey.
Leber congenital amaurosis (LCA) shows clinically and genetically heterogeneous disorder which is caused by a large number of mutations in at
least 17 different genes. In the present study the application of two genetic
markers including rs11658369 and rs8066853 markers located in AIPL1
genetic region were genotyped and their application were investigated in
prenatal diagnosis of the disease in the Iranian population. The markers
were genotyped using newly designed specific primers by Tetra-primer
ARMS PCR in 150 unrelated healthy individuals in the Iranian population.
Analysis of the genotyping data using GenPop program, indicated the presence of informative haplotypes (>5%) with strong linkage disequilibrium
for the markers and the AIPL1 gene. The efficiency of these haplotypes as
a suitable tool in prenatal diagnosis of LCA was evaluated in two Iranian
families with one affected child with an already diagnosed W278X mutation
in the AIPL1 gene, through an APEX microarry screening and sequencing
analysis. The transmission of the normal and affected alleles form parents
to their affected child and the fetus was confirmed by using the haplotypes
obtained by genotyping of rs11658369 and rs8066853 markers. Interestingly, in line with the mutation results, the genotyping data also confirmed
the transmission of normal alleles to fetuses. The families now were given
birth to children with normal vision as confirmed by ophthalmic diagnosis.
The data suggested that rs11658369 and rs8066853 could be suggested as
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novel informative markers for linkage and prenatal diagnosis of AIPL1 associated LCA in the Iranian population.
J01.40
Analysis of pedigrees of families with reproductive losses
B. Ginzburg;
Regional Hospital of Kaluga, Kaluga, Russian Federation.
377
Male factors are important causes of infertility. Two major causative factors
of male infertility are oxidative stress (OS) and genetic factors. OS damages
the sperm plasma membrane, the genome integrity and expression profile
of genes involved in spermatogenesis. KDM5D or SMCY is one of these genes
which its alteration is associated with male infertility.
In this study the expression profile of KDM5D gene was evaluated in testis
tissues of infertile after OS induction.
Oxidative stress in adult mice testis was induced by injection of the 1:10
concentration of tertiary-butyl hydroperoxide (TBHP). Adult male Balb/c
mice were randomly selected. Case group included treated mice by TBHP
for 2 weeks and control group treated only by injection of dH2O. Induced
ROS levels in testes tissue samples of all mice measured by flow-cytometry.
Consequently the expression of KDM5D gene was quantitatively measured
in samples of both groups by real-time PCR.
According to Flow-cytometry results, an increase of oxidative stress in ROS
treated mice in comparison to control group was observed. Moreover, the
expression level of KDM5D gene was lower in TBHP treated mice than that
of in control mice.
Oxidative stress can have detrimental effects on testicular tissue and alters
the expression of some genes which are involved in spermatogenesis.
J01.44
The case of prenatal diagnosis Pallister-Killian syndrome in
Sverdlovsk region (Russian Federation)
378
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J01.45
Association FBLN5 gene polymorphism with pelvic organ prolapse.
A couple who was referred to us for prenatal diagnosis (PND) for beta thalassemia had experienced 4 consecutive pregnancies with diagnosis of thalassemia major. After four times of therapeutic abortions, the couple decided
to have healthy child using preimplantation genetic diagnosis (PGD).
Ovulation induction and ovum fertilization were performed in Erfan Hospital, Tehran, Iran. In the 3rd day, a single blastomere from each embryo (in
total 8) was removed and given to us. For molecular PGD, a multiplex nested
PCR, using several STRs markers linked to HBB gene and also amplifying
part of the -globin gene, was done. Out of 8 tested cells, 3 blastomers were
thalassemia minor, 2 homozygous normal and one thalassemia major. Also
two embryos did not give us conclusive result on being affected or normal
due to allele dropout (ADO).
The family decided to transfer 3 embryos. Three weeks after embryo transfer, pregnancy test was positive and a single pregnancy was continued up
to the 11th week gestation. The family agreed on fetal testing. CVS was followed. Genetic testing for beta thalassemia and chromosomal aneuplidies
showed a healthy boy who was carrier of thalassemia minor. A healthy boy
was born on the Feb 4, 2014 by cesarean section. Similar test on placental
sample confirmed our findings.
J01.47
Genomic variability study: CNVs analysis in women with premature
ovarian failure (POF)
E. Mashkina, K. Kovalenko;
Research Institute of Biology, Southern Federal University, Rostov-on-Don, Russian
Federation.
15% of human pregnancies are known to end in spontaneous abortion before 12 weeks of gestation. A disbalance of cytokines and growth factors
can affect negatively early stages of human embryogenesis. The aim of the
study is to determine the expression level of the growth factors genes in
miscarriage patients.
Study was performed on the RNA samples from two groups of women. The
first group included of women with a missed abortion or spontaneous abortion. The control group included women with normal pregnancy. The samples of RNA were extracted from decidua and chorion. The level of the gene
expression was assessed using the two-step reverse transcription probedependent fluorescent real-time polymerase chain reaction.
Hypoxia inducible factor (HIF) is the primary molecular sensor responded
to oxygen tension changes. HIF as transcription factor regulated many cellular processes, for examples angiogenesis, invasion, cell survival. HIF in hypoxia condition provides a potent stimulus for VEGF synthesis and is essential for development of maternal and placental vasculation in early human
pregnancy. In the case of miscarriage the level of HIF-1 gene expression was
reduced in the horion. Analysis of gene expression vascular endothelial
growth factor in physiological pregnancy showed that mRNA levels of this
gene was significantly lower in decidua compared to the chorionic tissue (P
= 0.032). In case of miscarriage the level of VEGF gene expression in chorionic tissue was not different from decidual tissue and significantly lower
compared with the level of gene expression in the control.
J01.49
Paternal and maternal origin of Primary ovarian insufficiency (POF/
POI) caused by FMR1 gene premutation - using Repeat Primed PCR
(RP-PCR) method
A. Beke1, V. Karcagi2, B. Nagy1, H. Piko2, M. J. Molnar1, J. J. Rigo1;
1
Semmelweis University, Budapest, Hungary, 2National Institute of Environmental
Health, Budapest, Hungary.
E. Guinchard1, P. Bricca2;
1
EFS Rhne-Alpes site de Lyon-GHE, 69677 Bron Cedex, France, 2EFS Rhne-Alpes site de
Lyon-GHE, 69677 BRON Cedex, France.
During her second pregnancy, a 35 year old patient known with RH:1 status
surprisingly has an anti-RH1 allo-immunization at 24 weeks of pregnancy.
Prenatal non-invasive RHD genotyping on fetal maternal plasma was performed using real-time PCR ( TAQMAN technology ) studying three regions of
the RHD gene ( exons 4, 5 and 10 ) and shows amplification of the 3 exons.
The maternal DNA sequencing allows to identify the type IVa RHD variant
characterized by lesions of exons 2, 3 and 7 : RH1 corresponding antigen
exposed the patient to the risk of anti-RH1 immunization. The titer of the
anti-RH1 increases dramatically in late pregnancy (maximum title 1024),
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however, without sonographic fetal anemia. At birth, the child is RH:1 and
has a minor anemia 120 g / l but a positive direct antiglobulin test (due
to maternal anti-RH1 antibodies on the surface of his erythrocytes). But at
the 12th day, the child presents a deep hemolytic anemia at 52 g / l; requiring 4 CGR transfusions (12th day, 13th day and 30th day). The sequencing of
the RHD gene of the child at birth can highlight the two copies of the RHD
gene: an allele with the standard RHD gene inherited from the father, and
the other with RHD variant ( type 1 DIV ) identical to mothers. This case
shows the possible clinical consequences of anti-RH1 alloimmunization in a
mother carrying a variant of the RHD gene.
J01.51
Diagnostic algorithm for differential diagnosis in high-risk
pregnancies identified by prenatal ultrasound screening: two case
reports
During November 2012-January 2014, 42 samples were analyzed by conventional cytogenetic methods and 5 cases by aCGH technique (Array Comparative Genomic Hybridization). The analyses were performed for early
miscarriages or for induced pregnancy termination due to severe fetal ultrasound abnormalities. The samples evaluated were from products of conception between 7 and 23 weeks of pregnancy, in patients aged between 24
and 41 years.
Depending on the biological sample received, either, chorionic villi, epithelial tissue, amniotic fluid or fetal cord blood was used for analysis. The karyotyping was successfully achieved in 40 cases and the aCGH analysis for
all 5 cases.
Of the 40 cases analyzed by conventional cytogenetics, 23 (57.5%) had abnormal karyotype and in 17 cases (42.5%) no structural or numerical chromosomal abnormalities were identified.
Among the abnormal cases we identified: 13 homogeneous autosomal trisomies, 3 mosaic abnormalities, 2 unbalanced structural chromosomal abnormalities, 1 trisomy and unbalanced structural chromosomal aberration,
one triploidy, one monosomy X, 2 double autosomal trisomies, one clonal
chromosomal instability.
ArrayCGH analysis identified trisomies of chromosomes 21 and 22. In one
case genomic aberrations were identified, including genes involved in embryonic development. For two cases with high gestational age the abnorma-
379
Quantitative fluorescence PCR (QF-PCR) is widely used for the rapid prenatal diagnosis of common aneuploidies by the analysis of short tandem repeats (STR). The amount of each allele is quantified by calculating the ratio
of the peak height or area. In our laboratory for standard risk pregnacies
(screening by combined test or maternal age) we replace the Short Time
Culture (STC) chromosomal analysis of chorionic villi (CV) by (QF-PCR )
while we hold STC+LTC for different indications (parental chromosomal
abnormalities, uncommon aneuploidy in previous pregnancy, ultrasonographic abnormalities, included NT>3,5). In all QF-PCR positive and when
structural or mosaic anomalies are suspected we perform STC too. We also
support the karyotype analysis of amniotic fluid (AF) by QF-PCR in > 19th
weeks pregnancies. 5% of the 417 CV and 10% of the 95 AF gave an aneuploid result, consistent with the classical karyotype of both STC and LTC. In case
of poor sample QF-PCR allowed to resolve maternal contamination dubts
(14 cases). One case of vanishing twin, 9 mosaic karyotype cases, zygosity
of 6 twins, a dubt Array-cgh on Y chromosome ring duplication also were
supported by the QF-PCR results. We further analyzed 13 parental Periferal
Blood (PB), to investigate Primer Binding Site Polymorphism (PSP) vs Sub
Microscopic Duplications (SMD) or Somatic Microsatellite Mutation (SMM)
for D13S1492, D18S386, D21S1442, DXS1187, DXS981, DXS2390, DXYS267,
DXYS218, TAF9/TAF9L, BRAF/BRAFP1)and others microsatellites.
J01.54
QF-PCR: our experience in prenatal diagnosis and recurrent
miscarriages
Our center is responsible for invasive prenatal diagnosis from the 90s, every
year we process by conventional cytogenetic analysis about 1200 samples
between amniotic fluid (AF) and chorionic villi (CVS), and about 70 samples
from recurrent miscarriages. In 2010 we introduced the QF-PCR analysis
for the most common aneuploidies (chromosome 21, 18, 13, X and Y) as
support to cytogenetics in :
1. AF samples: when there is a need to for a rapid response in cases of advanced maternal age, clinical suspicion of fetal anomaly or in amniocentesis
performed late (more than 20 weeks)
2. CVS samples: in all cases, in conjunction with cytogenetic analysis of cytotrophoblast by direct preparation (STC) or to exclude maternal contamination in chorionic villus DNA used in molecular studies. Over the last three
years, we extended QF-PCR analysis to recurrent miscarriages because traditional cytogenetic testing is labour-intensive and has a signicant failure
rate, especially when the sample quality is poor. In this case we investigate
a greater number of chromosomes: 13, 15, 16, 18, 21, 22, X and Y, whose
aneuploidies are the major cases of miscarriage.
In this work we report a summary of our experience, using CE-IVD QF-PCR
systems CY5- labelled. In prenatal diagnosis QF-PCR system proved to be an
effective and specific support to cytogenetic analysis, and a useful and reliable tool to diagnose aneuploidies in spontaneous miscarriages, reaching
a pathologys diagnosis in 45% of cases and were considering to replace
cytogenetic analysis with this molecular system.
J01.55
The correlation between sperm DNA fragmentation and recurrent
abortion in Iranian population
A. Dehghanifard1, M. Noruzinia2;
1
Sarem Cell Research Center- SCRC, Sarem Womens Hospital, Tehran, Islamic Republic
of Iran, 2Sarem Cell Research Center- SCRC, Sarem Womens Hospital, Thran, Islamic
Republic of Iran.
Background: as previous studies have mentioned that sperm DNA quality may be related to unexplained recurrent abortion, this association study
was designed to evaluate the degree of sperm DNA fragmentation using the
SCSA (Sperm Chromatin Structure Assay) on sperm from couples with unexplained recurrent abortion compared to sperm from men of the general
380
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We present a de novo balanced Robertsonian translocation (13;14) identified in prenatal period. The women, aged 32 years, asked the prenatal diagnostic because she had a previous pregnancy with 21 trisomy. The chorionic villous sampling made at age of 12 weeks of amenorrhea was followed by
prenatal analysis by FISH technique, using Aneuvysion kit and confirmed
a 46,XX chromosomal formula. Cell culture indicated an apparently balanced Robertsonian translocation rob (13;14) and a small chromosome marker. The chromosomal analysis in both parents was normal. We repeated the
prenatal analysis after amniocentesis at age of 17 weeks of amenorrhea. The
G banding confirmed the presence of both anomalies: an apparently balanced Robertsonian translocation rob (13;14) and a small chromosome marker. The C banding indicated a possible derivative chromosome der (13;14)
with two centromeres.To identify precisely the chromosomal anomalies we
asked the help of Prof. Thomas Liehr, and the FISH analysis, made at University of Jena, with probes for centromeres of chromosomes 13/21 and 14/22,
and probes for all acrocentric p-arms confirmed that one break appeared in
the short arm of chromosome 13 and the second one in the centromere of
chromosome 14. The latter leads to alphoid DNA derived from chromosome
Sex chromosome structural abnormalities are frequently found in individuals with reproductive failure or disorders of sexual development. X-chromosome rearrangements are identified in patients with Turner syndrome,
usually in a mosaic form. The most frequent structural abnormality in these
cases is the X isochromosome for long arms, although a ring X-chromosome
or other variants can also be found. Additionally, other X-rearrangements
are detected in patients referred for reproductive failure, infertility or even
in woman with no clinical indication, including X translocations, partial deletions or duplications. In balanced X-autosome translocations, breakpoint
position and replication behavior may influence phenotypic outcome. Rearrangements on the Y chromosome include: X-Y translocations, Y-autosome
translocations, isochromosomes, rings, partial deletions and inversions. Dicentric Y isochromosomes for the short arm or ring Y chromosomes are the
most frequent abnormal Y chromosomes found in infertile patients and in
Turner syndrome, in mosaic with 45,X cells. In all these cases, it is important
to well characterize the derivative chromosomes in order to establish a good
genotype-phenotype correlation, to provide information about the role of
different X/Y chromosome regions or loci in the clinical manifestations of
patients.
For this purpose, we have collected patients with different sex chromosome
rearrangements detected with karyotyping: X-autosome translocations (3);
X isochromosome (3), X deletions (4); ring X (1); X inversion (1); Y-autosome translocations (4); Y isochromosome (1). The rearranged chromosomes
have been analyzed using FISH, MLPA or aCGH when appropriate. After a
detailed clinical assessment, genotype/phenotype correlations have been
performed.
J01.60
Management of sex differentiation disorders at University Hospital
Hassan II Fes
I. El Otmani;
Chu Hassan Ii Fes, Fes, Morocco.
Sex differentiation disorders represent all the abnormalities in development of the gonads, the genital tracts, and the external genitalia. Disorders
of sexual differentiation are due to genetic defects or endocrine imbalance.
Sex-determining genes (SRY gene) dictate the gonadal sex whereas the fetal
testicular hormones determine the somatic sex during sex differenciation.
Abnormal sexual development causes unconformity between gender identity and gender role.
The aim of this study was to evaluate the frequency, the genital anatomy
appearance, the diagnostic and the surgical management of disorders of sex
development (DSD) discovered during the neonatal period and the enfance
Between September 2009 and Marsh 2013, 30 patients with abnormal sexual development were identified in our unit. First-line testing included biology measurement and imaging. A surgical management was offered for some
patients. Sexual dimorphic with genital ambiguity was the first reason of
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consultation. One patient had male breast development. All clinical evaluation suggested genital ambiguity.
This presentation points out the need of an accurate diagnosis of sexual
differentiation disorder during the neonatal period. The intervention of a
multidisciplinary team is essential as well for assignment of sex as for therapeutic guidelines.
.
J01.61
Polymorphic locus 820AG of IGF-2 gene as a possible marker of fetal
development disorders
B. Tretjak, H. Makukh, D. Zastavna;
Institute of Hereditary Pathology NAMN of Ukraine, Lviv, Ukraine.
Background:
Many cases of male infertility associated with a severe impairment of spermatogenesis. During the last stage of spermatogenesis (spermiogenesis),
haploid spermatids endures complex changes to differentiate into spermatozoa, this process includes chromatin modifications mediated by different
histone modifying enzymes. Chromodomain Y (CDY) proteins encoded by
the CDY family of genes, are characterized by two functional motifs, a chromodomain and a histone acetyltransferese catalytic domain. A testis specific CDY protein, named CDY1, binds to methylated histone regions through
its chromodomain, and then causes hyperacetylation of genes involved in
sperm chromatin condensation. This study aimed to investigate the relative mRNA expression and chromatin incorporation of chromodomain Y1
(CDY1) protein in the testis tissues of infertile men.
Material & Method:
Local ethical approval was gained for this study and informed consent was
given by patients. Testicular biopsies were collected from 31 infertile men
referred to Royan Institute and underwent testicular sperm extraction
(TESE). These samples distributed into 4 groups: obstructive azoospermia
(positive control), severe oligoasthenoteratozoospermia, non- obstructive
azoospermia and sertoli cell only syndrome.
Using qRT-PCR and nucleosome-ELISA methods, the mRNA levels and chromatin incorporation of CDY1 was evaluated in the tissue samples.
Result(s):
Our data significantly showed lower expression and chromatin incorporation of CDY1 in all 3 sample groups with spermatogenesis defect in comparison to positive control.
Conclusion(s):
This data demonstrated that the defective epigenetic role of the histone acetyltranferase CDY1 may be associated with male infertility.
J01.63
Conventional CGH vs FISH and karyotyping in detection of
chromosomal abnormalities in first-trimester spontaneous abortion
M. Minzhenkova, N. Shilova, Z. Markova, Y. Kozlova, T. Zolotukhina;
Federal State budgetary Institution Research Centre for Medical Genetics under the
Russian Academy of Medical Sciences, Moscow, Russian Federation.
381
1-9
10-21
22
23
24
25-26
27
28
29
30-35
36-42
43
44
45
46-47
48
49
50
51
52-53
54
55
56-59
60
J01.64
Is apolipoprotien E polymorphisms associated with spontaneous
pregnancy loss
Objective: Apolipoprotein E has three major isoforms (Apo E epsilon2, epsilon3 and epsilon4) and has important functions in nerve development
and repair. To evaluate the association of Apo E polymorphisms and spontaneous abortion we studied the genotypes of spontaneously aborted fetuses, their mothers and control cases. Methods: In the current case control
study, three different groups of aborted materials, mothers and healthy
control were compared. Target gene of Apo E2/E3/E4 alleles were analysed
by real time PCR. Results: The E2 and E4 alleles showed high prevalence
in aborted materials comparing both with their mothers and healthy control group (p=<0.0001). The E4 allele was higher in fetuses comparing with
their mothers (p=<0.0001). Conclusion: Apo E2 and E4 alleles seem to be
contributing to the thrombophilic risk factors as a seconder parameter to
spontaneous abortions.
J01.65
Parental idendification of aborted materials for chimerism and other
molecular etiological parameters by microsatellite STR profilling
Aim:Structural and/or numerical chromosomal abnormalities are responsible for 50-60% of the first trimester miscarriages, 20-25% for seconder
and 5-10% for the third trimester miscarriages.In the current study it was
aimed to use the microsatellite STR markers for the parental identifying of
382
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Introduction: More than 95% of beta-thalassemia mutations are point mutations in the beta-globin gene in global population. This study was performed
to determine beta globin gene mutation in blood transfusion dependent patients with in Kurd ethnicity in Kurdistan province in west of Iran.
Materials and Methods: Transfusion dependent patients were enrolled
into the study. Diagnosis of beta thalassemia was confirmed using patients
parents hematologic index and Hb electrophoresis. DNA was extracted
from 5 ml of patients blood using standard salting out method. Common
mutations in Kurdish population were investigated by ARMS-PCR method.
Unknown cases were investigated by direct sequencing of beta globin gene.
Genotype frequency and allele frequency were calculated.
Results: Sixty eight transfusion dependent beta thalassemia patients (35
male and 34 female) with mean age of 166.89 years old were entered in the
study. IVSII-1 had most common allele frequency in 37 (27.21%) chromosomes following by Fr8-9 in 22 (16.18%), IVSI-1 in 13 (9.56%), C36/37(-T) in
11 (8.09%) and IVSI-110 in 7 (3.67%).
Conclusion: IVSII-1 and Fr8-9 were the most common mutations in beta globin gene in Iranian Kurd thalassemia patients, which is in alignment with
previous studies among Kurdish population.
J01.67
Results of prenatal tests in pregnancies after assisted reproductive
technologies
V. Obrikyte1, D. Serapinas1,2;
1
Department of Pulmonology and Imunology, Lithuanian university of Health sciences,
Kaunas, Lithuania, 2Mykolas Romeris university, Vilnius, Lithuania.
P. Bardzilauskas1, D. Serapinas1,2;
1
Department of Pulmonology and Imunology, Lithuanian university of Health sciences,
Kaunas, Lithuania, 2Mykolas Romeris university, Vilnius, Lithuania.
Background: The choroid plexus cysts are one of the fetus ultrasonography
E. Ginzburg, B. Ginzburg;
Regional Hospital of Kaluga, Kaluga, Russian Federation.
The XX male syndrome (OMIM 400045) now termed Testicular DSD (Disorder of Sexual Differentiation) is a rare genetic condition (1:20,000 to 25,000
male newborns) characterized by a spectrum of clinical presentations, ranging from ambiguous to normal male genitalia. In males without genital
ambiguity, the diagnosis is often made during investigation of infertility or
delayed puberty with a frequency of 0,9% among azoospermic males.
Here, we report four observations of 46,XX men detected in our genetic
counselling during genetic evaluation of infertility with azoospermia, hypergonadotrophic hypogonadism and testicular hypotrophy. Chromosomal
abnormality revealed by two karyotypes in different laboratories was refined by molecular investigation of SRY gene using FISH (n=2) and PCR amplification (n=2) as well as AZF loci by multiplex PCR protocol.
The prevalence of XX male syndrome among our azoospermic men serie is
0,97% (4/412). Molecular analyses demonstrated the presence of Yp SRY
gene in the four patients with absence of Yq AZF loci.
Review of literature shows that SRY positive Testicular DSD is the common
variety (85%) and that PCR amplification of SRY is more appropriate, than
FISH, to detect a small amount of Y chromosome translocated on to the X
chromosome and to detect the Y-chromosome material in mosaic forms: XXSRY positive/XX-SRY negative. Moreover, SRY-negative cases (15%) should
undergo further molecular testing to explore the presence of SOX9, SOX3
or Sox3 promoter microduplication or microdeletion, or cryptic mosaicisme
for Y chromosome. Recently, RSPO1 gene seems to be implicated in Testicular DSD associated with hyperkeratosis.
J01.71
Fluorescence in situ hybridization (FISH) on interphase nuclei in the
uncultured chorionic villi samples from spontaneous abortions
I. Tkach1, N. Huleyuk1, T. Liehr2;
1
Institute of Hereditary Pathology, NAMS of Ukraine, Lviv, Ukraine, 2Institut fr
Humangenetik Universittsklinikum Jena, Jena, Germany.
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383
Background:
Spermatogenesis is a unique process in male reproductive system, which
requires precise epigenetic regulation of gene expression. JMJD1A is a critical epigenetic modifier element that highly expresses in male germ cells and
activates expression of genes by demethylation of H3K9me2/me1 modification. This study aimed to evaluate the expression and chromatin incorporation of JMJD1A protein in testicular biopsies of infertile men.
Material &Method:
Ethical approval and informed patient consent was gained for the use of
tissue samples. Testicular biopsies were collected from 31 infertile men referred to Royan Institute and underwent testicular sperm extraction procedure. These samples were classified into the following four subgroups:
obstructive azoospermia (as positive control, n=8), severe oligoasthenoteratozoospermia (n=7), complete maturation arrest (n=8), and sertoli cell only
syndrome (n=8). The expression pattern and chromatin incorporation of
JMJD1A in testicular biopsies were measured by quantitative real-time PCR
and chromatin-ELISA techniques, respectively.
Result(s):
Our finding revealed that the expression and chromatin incorporation of JMJD1A were significantly decreased in severe oligoasthenoteratozoospermia,
complete maturation arrest, and sertoli cell only syndrome groups in comparison to obstructive azoospermia patients.
Conclusion(s):
This study indicates that JMJD1A deficiency in testis tissues can result in
defective spermatogenesis in human infertile males.
J01.76
Sperm head morphology as a predictive mark of DNA fragmentation
E. Shilnikova1,2, M. Mazilina1, I. Fedorova2, A. Gzgzyan2;
1
Saint-Petersburg State University, Saint-Petersburg, Russian Federation, 2Otts
Institution of Obstetrics and Gynecology, Saint-Petersburg, Russian Federation.
384
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N
81
69
Total 150 69 3
1 1^ 13 1
14 1
4
^included in DS; M male, F female, N normal, DS Down syndrome, ES Edward
syndrome, PS Patau syndrome, KS Klinefelter syndrome, TS Turner syndrome, DGS
De George syndrome
J01.78
Association of H3K9ac and H3K9me2 modifications with impaired
spermatogenesis
During post-meiotic stages of spermatogenesis, histones of sperm chromatin are replaced by transition proteins (TNPs) and protamines (PRMs).
Expression of TNPs and PRMs rise at final stages of spermatogenesis to
compact chromatin for normal function of sperm. Any failure of their expression is associated with impaired spermatogenesis. Epigenetic factors
such as histone actyle transferases and histone demethylases are involved
in regulation of these genes. Therefore evaluation of relative modifications
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commended to design a population based STS panel at least for the study of
AZFa region for better detection of microdeletions.
J01.81
An NGS-based test for the identification of individuals carrying
recessive genetic mutations
High incidence of somatic mutations is a hallmark of abnormal embryogenesis. Recent data indicate the significant impact of copy number variations (CNV) into etiology of early pregnancy loss. However mechanisms of
their origin are still poorly investigated. We aimed to estimate the somatic
CNV incidence in placental tissues - cytotrophoblast (CT) and (EM) from 6
anembryonic pregnancies (AP) with normal karyotype using SurePrint G3
Human CGH+SNP 4180K Microarray Kit (Agilent Technologies). Altogether
84 CNVs were found. Twenty-four rearrangements (21 polymorphic and 3
unique, which are absent from the Database of Genomic Variants) were de-
385
Polycystic ovarian syndrome (PCOS) is a complex genetic endocrine disorder among the women of reproductive age. Hyper-androgenemia is one of
the main clinical features of PCOS. Aromatase, the key enzyme for estrogen
biosynthesis, is encoded by CYP19A1 which is comprised of an unusually
large regulatory region including 10 tissue-specific promoters. In human
cells CYP19A1 is expressed in gonads via promoter PII.
The aim of this study is to evaluate the acetylation and methylation levels
of lysine 9 of histone 3 (H3K9ac and H3K9me2), in PII promoter region of
CYP19A1 in granulosa cells of PCOS patients referred to Royan Institute for
in vitro fertilization (IVF).
Six women under ovulation induction treatments for IVF consisting of three
PCOS patients and three controls (women without ovulation problems; nonPCOS) groups were selected. For this respect, ethical approval form was
filled before any experiment. We sequenced PII of CYP19A1 to confirm no
genetic variations. After ovary puncture, follicular fluid was collected and
granulosa cells were extracted via Sil-Select Plus gradient. Specific histone
modifications were quantified via chromatin Immunoprecipitation (ChIP)
coupled with real-time PCR.
Our results clearly demonstrate that in PCOS patients, incorporation of
H3K9ac in PII of CYP19A1 is significantly higher than non-PCOS patients,
386
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Background: Strong association between hypoadiponectinaemia and the occurrence of poly cystic ovary syndrome (PCOS) suggests a pathogenic role
for adiponectin. We aimed to evaluate the expression of the adiponectin and
its receptors (AdipoR1 and AdipoR2) genes in immature and mature PCOS
rat model that were exposed prenatally to androgen excess.
Methods: Four pregnant Wistar rats in the experimental group were treated
by subcutaneous injection of 5 mg free testosterone on day 20 of pregnancy
while the controls (n=4) received only 500 ml of solvent. Female pups (14
cases and 18 controls) of each mother were randomly divided into 3 groups
and sacrificed at 3 stages of life: day 0 (new born, n=10), day10 (10-day-old,
n=10), and day 75-85(adult, n=12). RNAs were extracted from ovarian tissues and relative expression levels for adiponectin and its receptors genes
were measured using TaqMan Real-Time PCR.
Results: The expression levels of investigated genes were not significantly
elevated in newborns (Adiponectin: 1.119, AdipoR1: 1.594, AdipoR2:1.112
fold), while a significant decrease was detected in 10-day-old rats (Adiponectin 0.292, AdipoR1 0.26, and AdipoR2 0.161 fold (p0.05)). We also
observed a marginally significant increase in adiponectin gene expression
at puberty (2.682 fold, p=0.08).
Conclusion: The results of this study showed that the expression of adiponectin and its receptor genes is changed in prenatally androgenized rats.
These changes may alter the normal expression of steroidogenesis regulatory genes and consequently impair the normal development of ovaries and
follicles.
J01.88
The Role of Early Development in Intra-individual Genetic Variation
of Normal Human Fetuses
Noninvasive prenatal testing (NIPT) for fetal aneuploidy detection via massively parallel sequencing has been successfully implemented in a number of
high throughput clinical laboratories. Automation and parallelization of the
complex workflow has reduced turnaround time and labor while maintaining high sensitivity and specificity. As these developments have improved
workflow issues, the availability of new sequencing platforms on the market
has introduced additional flexibility in implementation. Platform flexibility
should encourage competitive pricing, foster innovation and ultimately, improve patient satisfaction.
We examined the performance of the MaterniT21 assay using the Ion
Torrent Proton Sequencer (Life Technologies, San Diego California).
One hundred and fifty-four patient samples, including sixteen from women carrying a known trisomy 21 fetus, as determined by fetal karyotyping, were analyzed. Libraries were prepared and sequenced according to
manufacturers recommendations. Sequenced reads were aligned, filtered
for quality and normalized for GC bias. Robust statistics were then applied
to identify positive samples with a z-score greater than 3.
Fetal aneuploidy status was correctly determined for 154/154 pregnant females, including 16 carrying a T21 fetus. Though the current Proton workflow requires more labor than is optimal for a production environment,
significant improvements in that respect are anticipated in the launch of
the Ion Chef template preparation system. Sequencing time was brief at <
3 hours and data analysis consistent with standard platforms. In summary, the performance of the MaterniT21 assay on the Ion Torrent Proton
platform in this limited study suggests the possibility of its suitability for
implementation in a clinical environment.
J01.90
DNA methylation and demethylation patterns in human
spermatogenesis
We studied the distribution of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in human spermatogenic cells from testicular biopsy
and ejaculate samples. Among analyzed dividing cells several types were
detected: mitotic diploid and polyploid spermatogonia and meiotic spermatocytes at the pachytene, diplotene and diakinese stages. Chromosomes
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Constitutional chromosome abnormalities are among the major contributors to the genetic causes of reproductive disorders. Despite all of worldwide
efforts have been made so far, prognosis for mosaic X chromosome aberration below 30% of aneuploidy has yet to be established. The purpose of this
study was to assess the quantity and quiddity of chromosomal aberrations
that may negatively affect ovarian health causing premature ovarian failure
(POF) and diminished ovarian reserve (DOR). In this purpose, retrospective observational study of clinical features and biological parameters was
performed. A total of 531 individuals who were referred to our ward from
2007 to 2014 because of amenorrhea and poor responders to gonadotropins were selected. High resolution chromosome analysis by GTG banding
was carried out on peripheral blood lymphocytes cultures. Supplementary
tests also were performed when required. Of the 531 cases who were assessed for chromosomal defects, 52 showed abnormal karyotype. 22 cases
were found to have cell lines with different level of X chromosome variation.
Seven cases who were sex reverse sex determining region Y (SRY) negative,
five cases with abnormal X chromosomes, three cases with structurally abnormal autosomes and four individuals carrying X-autosome translocation
were diagnosed. The overall prevalence of chromosomal abnormalities was
9.8% which 2.1% of it belongs to normal variable chromosome features.
The frequencies of chromosomal alterations were 5% and 1.7% in POF and
DOR females, respectively. The results confirm previous observations and
emphasis on the critical role of chromosome abnormalities as one of the
possible etiologies for ovarian follicular attrition.
J01.92
Unique case of fertility in SRY-positive female
387
Purpose: Central serous chorioretinopathy(CSCR) is a mystery characterized by leakage of fluid under the retina that has a propensity to accumulate
under the central macula. SGK1 which has an important role in many epithelial ion transport system may have a role in retinal pigment epithelial pump
function whose disturbance is one of the main mechanism in development
of CSCR. The aim of this study is to investigate whether SGK1gene variants
are associated with CSCR
Materials and methods: We enrolled patients who were diagnosed with
chronicCSCR (n=32) and unrelated individuals as a control group (n=32).
For DNA extraction and PCR amplification followed standard methodologies. SGK1 gene was sequenced using BigDye Terminator v3.1chemistry.
Results: We identified a novel mutation M32V (2/32) in the patient group (6,
25 % and AF.0,031). rs1057293 (p:0,68) is located in the encoder region of
the SGK 1 gene but not associated with CSCR. An intronic rs1743966 (p.028)
is also, not associated. We have also identified 3 more intronic mutations;
143951C>A(1/32; AF: 0,015), 147117A>T(1/32; AF: 0,015) and 145725 del
TTAC(1/32; AF: 0,015).
Discussion: M32V is located on the region of 1-60 amino acids which is
necessary for localization to the mitochondria. This mutation is probably
important for the energy metabolism and plays an important role in the
cellular response to hyperosmotic stress and other stress stimuli. Both
rs1057293(p.0,68) and rs1743966(p.0,27) are not associated with CSCR.
Probably these 7 snp are promising but its difficult to conclude the association between these intronic mutations; 143951C>A, 147117A>T, 145725del
TTAC- and CSCR.
388
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J02.03
The occurrence of the rs61749246, the c.*2GT of the FZD4 gene, in
Slovak patients with retinopathy of prematurity
Z. Lasabova1, K. Matasova2, A. Stanclova3, S. Smolarova4,2, L. Lucanova2, P. Lukac5, A.
Calkovska4, M. Zibolen2;
1
Comenius University in Bratislava, Jessenius Faculty of Medicine, Dept. of Molecular
Biology, Martin, Slovakia, 2Comenius University in Bratislava, Jessenius Faculty of
Medicine, Clinic of Neonatology, Martin, Slovakia, 3Comenius University in Bratislava,
Jessenius Faculty of Medicine, Dept. of Pathological Anatomy, Martin, Slovakia,
4
Comenius University in Bratislava, Jessenius Faculty of Medicine, Dept. of Physiology,
Martin, Slovakia, 5Comenius University in Bratislava, Jessenius Faculty of Medicine,
Clinic of Gynecology and Obstetrics, Martin, Slovakia.
Retinopathy of prematurity (ROP) is a complex disease affecting the development of retinal vasculature in premature infants. The International classification of ROP divides the development of the disorder into 5 stages. The
environmental factors can influence the development of ROP, so the appropriate care may decrease its incidence. However, a genetic predisposition to
ROP is suggested. Recently, mutations in genes FZD4, LRP5, TSPAN12, and
NDP, were ide identified in 3 to 12% of the cases with ROP. Nowadays, our
cohort consists of 11 premature newborns with different stages of ROP (stage 1 -3) treated at the Clinic of Neonatology in Martin. We performed the sequence analysis of the coding exons of the three genes - FZD4, TSPAN12 and
NPD an identified rs61749246 (c.*2GT of the FZD4 gene) in 3 patients with
ROP. Two were heterozygous in stage 1, and one was homozygous in stage 2
(T allele frequency 0.18). The control group (n=50) of premature newborns
without ROP contains two heterozygotes for rs61749246 (T allele frequency 0.02). The rs61749246 (MAF = 0.008 in dSNP) is the polymorphisms of
the second G after the TAA stop codon of the FZD4 gene. It has been shown,
that there are preferred nucleotides around the stop codon necessary for
efficient translational termination. We suggest that this SNP is involved in
the pathogenesis of ROP, probably based on gene expression changes during
the eye development. We will enlarge our cohort focusing on higher stages
of ROP and develop in-vitro translation assay for this SNP.
J02.04
Case report of a patient with sensorineural hearing loss due to
compound heterozygosity of 35delG and G200R mutations in gene
GJB2
Hearing loss is the most common birth defect and the most prevalent sensorineural disorder and affects about one in 1,000 neonates. More than half
of prelingual deafness cases are due to genetic factors. About 70% of all
hereditary hearing loss cases are classified as nonsyndromic and recessive.
Mutations in the genes GJB2 and GJB6, that are located in the locus DFNB1
and encode gap junction protein connexim 26 and connexin 30, are the main
cause of nonsyndromic sensorineural autosomal recessive hearing loss. DFNB1 has digenic patterns of inheritance. The 35delG mutation in gene GJB2
is the most common mutation in DFNB1 in many European populations. We
describe the case of 18 months-old boy attended genetic counsellor due to
profound sensorineural hearing loss. Bidirectional sequencing analysis of
gene GJB2 showed two mutations: c.35delG and p.Gly200Arg. The condition
of heterozygous carrier of G200R mutation was found also in mother, father
found to be a heterozygous cariier of 35delG mutation. Patient with compound heterozygosity of 35delG and G200R mutations in gene GJB2 that we
found has never been described before. Precise genetic diagnosis is crucial
for genetic counselling.
J02.05
Detection of mutations in selected regions of genes GJB6, MT-RNR1
and MT-TS1 in Slovak population associated with non-syndromic
deafness
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poorly understood. It has been suggested that mutations in VSX1 and SOD1
may contribute to disease presentation in some cases. Linkage analysis in an
affected Irish pedigree ultimately in 2011 led to identification of a mutation
in the seed region of MIR184 (+57C>T) as the putative cause of keratoconus
in the pedigree. In a subsequent screening of 790 patients of European or
Indian descent, two novel causative mutations (+3A>G, +8C>A) in MIR184
were identified. Notably, mutations in MIR184 have recently been reported
in two pedigrees, each affected with ocular diseases that affect the cornea.
MiRNA 184 is the most abundant miRNA of the cornea. Here, MIR184 was
screened in 47 unrelated Iranians affected with keratoconus by direct Sanger sequencing. Only one variant allele (+39G>T; rs41280052) was observed in one patient. The same variation has previously been observed in control and keratoconus affected individuals at similar frequencies, suggesting
that it is not a cause of keratoconus. Although the sample size was small, it
is evident that mutations in MIR184 are not a common cause of keratoconus
among Iranian patients. They were not observed among the 94 chromosomes of the patients screened.
J02.09
Comprehensive analysis of keratoconus genetic factors
Neuhauser syndrome, also known as Megalocornea-mental retardation syndrome (MMR), is a very rare autosomal recessive disorder. It can be diagnosed via clinical features: megalocornea, developmental delay, hypotonia
and some dysmorphological signs. Genetic etiology is still unknown. About
40 cases have been reported in the literature. Here we report a further MMR
case. Proband is a 13 month-old girl. Because of macrocephaly, motor and
mental retardation, hypotonia and facial dysmorphic features, she was referred to pediatric genetics subdivision for genetic counseling. She was the fifth
child of nonconsanguineous parents from small village and born at 35 week
of gestation. In the newborn period she was hospitalized for prematurity. At
9 and 11 month of age, she was rehospitalized because of feeding problems
and bronchopneumonia. On admission, her weight was 6,6kg(<3centile),
height 76cm(25-50centile) and head circumference 46cm(97centile). Macrocephaly, broad forehead, hypertelorism, megalocornea, strabismus, low
set ears, short columella and long philtrum were present. Corneal diameters
were higher than 13,5mm bilaterally and intraocular pressures were normal. Laboratory tests revealed hypogammaglobulinemia. Echocardiography
showed secundum ASD (4mm). On MRI, bilateral cerebral atrophy and thin
corpus callosum were observed. Karyotype and subtelomeric FISH were
normal. This case is one of the few MMR cases having immunedeficiency.
389
The aim of this study was to investigate an association of the VDR (vitamin
D receptor) -3731G gene polymorphism with the occurrence of axial myopia in children. We examined 53 girls and 24 boys aged 4-17 yr from Russian population: 19(38 eyes) with high myopia, 44(88 eyes) with medium
myopia, and 14(28 eyes) with emmetropia. The gene polymorphism was
identified with PCR-RFLP. We have found a higher proportion of carriers
of VDR A(-3731)allele in the medium myopia group compared to the emmetropia group (32% and 7%, respectively, OR=4,45, 95%CI 1,13-17,53,
p=0,016). It is known that the variation of the A(-3731) allele frequency is
significant between various populations, being the highest one in Africans
(about 74%) and the lowest one in Caucasians (about 19%). The A(-3731)
allele is an ancestral while the allele G(-3731) is mutant. Null hypothesis for
explaining the mutant allele frequency raise in Caucasians is that this is a
result of a random selection, for example, genetic drift during the period of
human migration out of Africa. Alternative hypothesis is that the difference
in the polymorphism variation is not random, being the result of natural
selection. The latter may be true if the G(-3731) allele gives some benefits
for survival outside Africa. We hypothesize that the vitamin D receptor is a
key regulator of the eye growth, and G(-3731) allele gives more benefits in
preventing against myopic eye growth. The increase in G(-3731) allele rates
in non-African populations might be explained by changing of ultraviolet
radiation intensity.
J02.12
Ophthalmologic status of patients with Waardenburg syndrome
M. O. Mkheidze1, O. K. Yanvareva2;
1
Academy of Postgraduate Teacher Education, St.Petersburg, Russian Federation, 2State
University, St.Petersburg, Russian Federation.
390
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dences such as goiter, vestibular aqueduct enlargement and positive perchlorate discharge test, we report a novel alteration in the 3UTR of SLC26A4
gene led to PS. In addition, bioinformatics analysis showed that the introduced altered allele destabilized RNA structures; although, the Dot matrix
analysis of wild-type and mutant form of selected sequence has not been
showed any difference based on minimum free energy (G= -21.5).
Conclusion: Our data suggest that identified variation might alter mRNA folding and it is conceivable that this new alteration can cause PS in the family
under study.
J02.14
New polyurethane drug delivery system used for bevacizumab - an
alternative to classic treatment of retinopathy of prematurity
Objective: Usher syndrome (USH) is an autosomal recessive disorder illustrated with retinitis pigmentosa (RP) and sensorineural hearing loss with
or without variable vestibular dysfunction. USH is genetically and clinically
heterogeneous. At least fifteen loci and eleven genes identified in USH. Usher syndrome is divided into three subtype: type I (USH1); type II (USH2);
and type III (USH3). USH2 is the most common form of Usher syndrome,
responsible for moderate to severe hearing deficits, retinitis pigmentosa
started around or after puberty associated with normal vestibular responses. Three loci associated with USH2 have been reported: USH2A (USH2A),
USH2C (GPR98), and USH2D (WHRN). Defects in GPR98 are the cause of
Usher syndrome type 2C (USH2C). Here, we report on a consanguineous
Iranian family with two affected individuals for whom we identified a novel mutation in GPR98 (VLGRI) gene. Methods: After performing homozygosity mapping using microsatellite (STR) markers,we approached whole
exome sequencing (WES) to detect the disease-causing mutation of homozygous regions. To confirm the identified homozygote variant in GPR98,
sanger sequencing has performed in all family members. Consequently we
sequenced 100 normal control to ensure detected variant would not be a
polymorphism. Results: We identified a new mutation in GPR98 segregating
with USH2C in this family. The missense mutation c.10019T>G leading to
p.Val3340Gly. Conclusion:This mutation is the second one, which has been
reported for USH2C in Iranian population by our group. To the best of our
knowledge, this is the first report of a genetically confirmed case of USH2C
using WES in Iran.
J02.17
The analysis of the GJA3 gene in patients with hereditary congenital
cataract from Bashkortostan Republic (Russia)
Introduction: Cataracts are one of the leading causes of blindness in humans, and mutations in the connexin 46 (GJA3) and the connexin 50 (GJA8)
genes cause congenital cataract. Different mutations in these genes lead to
the development of distinct cataract phenotypes. The aim of the study was
to analyze the GJA3 gene in patients from Bashkortostan Republic affected
with congenital cataract.
Objective: DNA samples of 40 unrelated patients with isolated form of hereditary congenital cataract from Bashkortostan Republic were analyzed.
Methodology: The analysis was performed by direct sequencing of coding
regions of the GJA3 gene.
Results: Three different nucleotide alterations were detected. In one patient of Tatar ethnic origin with zonular form of cataract the deletion
.del1126_1139 was detected; one patient of Tatar ethnic origin with zonular cataract and microcornea carried the missense mutation . 398 G>A
(p.Arg133Gln). Both alterations were found in the heterozygous state,
hadnt been described in literature earlier, and presumably are functionally
significant mutations. In ne patient new nucleotide substitution .231C>T
(Phe77Phe) in the heterozygous state was found.
Conclusion: Thus, three previously undescribed structural changes in the
gene GJA3 were detected in hereditary congenital cataract patients from
Bashkortostan Republic. To determine their functional significance further
investigation are required. The study was supported by RFBR grant (14-0497007_r_povolgie_a).
J02.18
Mutation screening in autosomal dominant retinitis pigmentosa
family using targeted next generation sequencing
Background: Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous disorder with an incidence of 1 in 3,500, or a total of 1.8 million
people affected worldwide. So far at least 55 genes (RetNet) are known to
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Hearing loss is the most common sensory disorder worldwide which affects
1 of every 500 newborns. At least 50% of cases can be attributed to genetics, most often resulting in nonsyndromic deafness (70%), which is usually
autosomal recessive (80%). Despite the heterogeinity, mutations in GJB2
at DFNB1 locus are the major cause of autosomal recessive nonsyndromic
hearing loss (ARNSHL) in many populations, including Iran (20%). The fact
that many loci are involved together with the heterogeneity of the status, necessitate studying further loci in various Iranian ethnic groups. In this study,
after mutation screening of GJB2 and GJB6, we used homozygosity mapping
to identify regions of autozygosity-by-descent in 23 large pedigrees all originating from a single province of Iran (South Khorasan), using STR markers
for STR markers for 7 loci.
In our study, four out of the 23 families showed GJB2 mutations. Interestingly
heterogeinity within a family were observed, even in large consanguineous
pedigrees. GJB6 deletions were not detected. One family showed linkage to
DFNB3 and one family showed to be linked to DFNB7/11. The remaining did
not show linkage to the studied loci.
Our results once again emphasize the heterogeneity of HL among different
Iranian ethnic groups .These results could provide further insight into the
etiology of HL and may lead to better genetic diagnostics & counseling.
J02.20
Targeted next-generation sequencing for identification of ABCA4 gene
mutations in Polish patients with retinal dystrophies
A. cieyska, M. Odak, A. M. Ambroziak, M. Korwin, R. Poski, J. P. Szaflik, J. Szaflik;
Medical University of Warsaw, Warsaw, Poland.
The ABCA4 gene is one of the most important genes of the human retina. It
encodes an ATP binding cassette (ABC) transporter that is expressed almost
exclusively in the retina. Mutations in the ABCA4 gene cause a wide range of
retinal degenerations. Due to the advent of therapeutic options, identification of a genetic cause of retinal diseases is becoming increasingly important.
The aim of the project was to implement next-generation sequencing (NGS)
for identification of ABCA4 gene mutations in a group of Polish patients with
Stargardt disease, fundus flavimaculatus or cone-rod dystrophy. Genomic
DNA isolated from peripheral blood of 58 patients served as a template. The
introduced variant of NGS is based on the preparation of an amplicon library
containing all coding sequences of the ABCA4 gene (50 exons). Next, the
amplicons were sequenced using the genomic sequencer GS Junior System
(Roche). Presence of allelic variants identified by NGS was confirmed by
Sanger DNA Sequencing. To predict possible functional consequences of the
identified missense variants, two different computational methods, i.e. SIFT
and PolyPhen-2 were used. In the studied group of patients, 32 different
known mutations and 20 different novel potentially pathogenic variants
were found. Our study enabled identification of a genetic cause of retinal di-
391
Alpha1-antitrypsin (AAT) deficiency is an under-diagnosed condition in patients with chronic obstructive pulmonary disease (COPD). The aim of our
study was to evaluate predictive value of quantitative methods of alpha1antitrypsin for Z mutation detection in patients with chronic obstructive
pulmonary disease. Ninety-one AAT deficiency genotypes (40 MZ, 39 MS,
1 SS, 3 SZ and 8 ZZ) were analysed. Calculated sensitivity of quantitative
alpha-1 antitrypsin measurement by nephelometry for heterozygous PI*Z
allele was 45% and for homozygous ZZ genotype - 88%. Specificity of quantitative alpha-1 antitrypsin analysis for heterozygous deficiency was 98%
and for homozygous deficiency - 100%. Thus sensitivity of quantitative alpha-1 antitrypsin analysis is higher than specificity for both - heterozygous
and homozygous deficiency.
The results of the present study support the general concept of targeted
screening for AAT deficiency with adequate laboratory methods in European countries with PI*Z high frequency and large population of COPD
patients with highest diagnostic value - AAT genotyping. A case detection
programme of alpha-1 antitrypsin deficiency in patients with chronic obstructive pulmonary disease using quantitative methods could be used only
in screening programs and exact diagnosis must be confirmed by determining AAT genotype.
J03.02
Identification of a novel missense COL4A5 mutation in Russian family
with X- linked Alport syndrome by next Generation sequencing
Alport syndrome comprises ~3% of chronic kidney disease cases with estimated frequency of 0.02% in human population. It is a genetically determined glomerulopathy caused by mutations in genes encoding the alpha
chains of type IV collagen. The protein protomers are most prevalent component of glomerular basement membrane (GBM). Among them, collagen
345 protomer is the most frequent in a mature GBM, its components
being encoded by COL4A3, COL4A4 and COL4A5 genes. About 85% of all
examined patients with Alport syndrome bear COL4A5 mutations (this type
of nephritis is X-linked and dominant), whereas the remaining 15% demonstrate COL4A3 or COL4A4 mutations (autosomal form, usually recessive).
Genetic test is known to be the only direct diagnostic approach for Alport
syndrome. Here we discuss the research and diagnostic prospects for the
CDS sequencing of collagen type IV genes based on multiplex PCR followed
by next generation sequencing on Ion Torrent platform. We designed a panel
that covers 98% of the CDS with 5 bases of padding around targeted coding
exons. This approach allowed us to identify both previously described and
novel (including missense and frameshift) mutations that cause Alport syn-
392
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Objective: The gasdermin B (GSDMB) gene is located at 17q21.2 and recent reports suggest that GSDMB is associated with childhood asthma in
several populations. We investigated the association of a SNP in GSDMB
(rs2305480C>T) with recurrent wheezing, severity of bronchial obstruction
and family history of asthma and allergy.
Materials and Methods: Family history of asthma and allergy, recurrent
wheezing and atopy were assessed in 93 infants admitted to the hospital for
bronchiolitis. All children were genotyped for rs2305480C>T by PCR RFLP
analysis. The presence of AvaII restriction site was indicated by C-allele and
the absence - by T-allele.
Results: Data were analyzed as a recessive genetic model. Genotype frequencies did not deviate significantly from expected under the Hardy-Weinberg
Equilibrium - T/T genotype was 18%, C/T - 47% and C/C - 35%. Our data
show that the genotype T/T is a possible risk factor for recurrent wheezing
(OR 3,68, 95%, CI= 0,98-13,76). Children homozygous for T-allele are more
likely to have a family history of allergy and asthma (OR 5,41, 95%, CI= 1,4320,47) and early age of first wheezing - 6,88 mo compared to 10,4 mo for
C/C, p= 0,02.
Conclusions: Our results support the role of GSDMB SNP (rs2305480C>T)
for determining asthma phenotypes in preschool children. The study was
financially supported by research grant (2013), Medical University - Sofia
J03.05
Novel deletion in AVPR2 gene causing complete nephrogenic DI
Kartageners syndrome is an autosomal recessive disorder primarily manifesting as ciliary movement disorder. Kartageners syndrome is part of the
larger group of disorders referred to as primary ciliary dyskinesias (PCD).
Although the condition is usually inherited in an autosomal recessive pattern, and some specific gene defects have been recognized, it is clear that
Background:The association of celiac disease (CD) with several autoimmune conditions is well-known.Objectives:To determine the prevalence of CD
among children with autoimmune thyroid disorders(AITD) and insulin-depended diabetes mellitus(IDDM) and to asses the diagnostic role of HLA typing among this patients.Methods:74 children with AITD(lot 1), 98 children
with IDDM(lot 2) and 80 healthy children were screened for CD.In patients
with at least one positive serologic test for CD, intestinal biopsy was performed.All children underwent HLA typing for DQ2/DQ8.Results:CD prevalence in lot 1 was 7%, in lot 2 was 6% and in control lot was 0%.All children diagnosed with CD presented DQ2/DQ8 haplotype.20% of the control
subjects associated heterozygous DQ2 alleles.From 69 children with AITD/
without CD,3 patients (4%) presented heterozygous DQ2 alleles.From 92
children with IDDM/without CD, 25 patients (27%) associated homo or
heterozygous DQ2/DQ8alleles.There were significantly more cases with
IDDM withoutCD but with predisposing haplotype for CD(27%) compared
to the number of patients with AITD seronegative for CD and with DQ2/
DQ8 alleles(4%) p<0,005.Conclusions: Recommending AITD and IDDM as
selection parameters for CD screening in asymptomatic children is justified.
HLA assessment cannot highlight a significant role of a certain allele in the
pathogenesis of autoimmune comorbidity AITD/CD or IDDM/CD. DQ2 and
DQ8 alleles are mandatory but insufficient for CD development.The intervention of environmental factors is very important.Performing as first line
approaching HLA typing in asymptomatic at risk children is important.A
negative result for DQ2/DQ8 alleles will render CD highly improbable and
there will be no need for subsequent CD antibodies testing in such cases.
J03.08
NPHS2 and WT1 mutations in a romanian children Population with
nephrotic syndrome
C. Duicu, V. Moldovan, F. Tripon, A. Crauciuc, C. Banescu;
University of Medicine and Pharmacy Tg. Mures, Romania, Tg. Mures, Romania.
Mutations in NPHS2 and WT1 genes are a frequent cause of steroid resistant nephritic syndrome (SRNS) and occur in 10-28% of children while
mutations are absent from children with steroid-sensitive nephrotic syndrome (SSNS). The frequency and spectrum of mutations in these genes is
unknown for the romanian population. Material and methods This study
comprised 42 pediatric patients with NS. 50 healthy children were enrolled
as a control group. NPHS2 R229Q polymorphisms were determined by the
PCR and RFLP technique utilizing specific primers. Mutation analysis was
performed in WT1 genes, in case of abnormality the corresponding sample
was sequenced. Results In NS group 85.71% were SSNS and 14.29% were
SRNS. Mutation of NPHS2 R229Q gene, was found just in two patients with
congenital NS.Screening of WT1 gene showed one heterozygous mutation
in the donor splice-site of intron 9:c.1228+5 G>A in one SRNS girl with normal karyotype. Conclusion The incidence of NPHS2 mutations in romanian
children with NS is considerably lower than that among European childern
(10-30%). WT1 mutation was present in girl with focal segmental glomerulosclerosis. Therefore screening of WT1 gene in all females with FSGS is
necessary. Because NPHS2 R229Q gene mutation was found in all cases of
CNS, a screening of this gene in children with CNS in the adjacent counties
is required. Further studies with a larger number of patients are needed.
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Chronic obstructive pulmonary disease (COPD) is a multifactorial inflammatory disease primarily affecting distal respiratory pathways and lung parenchyma.
Genome-wide association studies have identified genetic variants influencing the risk of COPD. The aim of this study was to investigate whether
IREB2, CHRNA5, CHRNA3, FAM13A and HHIP polymorphisms would be associated with COPD susceptibility in Russian populations.
Methods: six single nucleotide polymorphisms: rs13180 (IREB2),
rs16969968 (CHRNA5), rs1051730, rs6495309 (CHRNA3), rs7671167 (FAM13A), rs13118928 (HHIP) were genotyped in a case-control study (511
COPD patients and 508 controls from Russia). To estimate the strength of
association, odds ratios were calculated and potential confounding variables were tested by using logistic regression analysis.
Results: Statistical analysis revealed that SNP rs13180 (IREB2) was associated with COPD (P= 0.0004). Analysis showed an association of rs16969968
(CHRNA5) (P=0.0034) and rs1051730 (CHRNA3) (P=0.0015) in additive model and the A-A-G haplotype of rs16969968 (CHRNA5), rs1051730,
rs6495309 (CHRNA3) genes polymorphisms (P= 0.0078) with COPD. The
relationship between the rs13118928 (HHIP) (P=0.0017 for AG genotype)
and COPD risk was found. Significant association with sever COPD were
observed for rs13180 (P=0.0009), rs16969968 (P=0.0071), rs1051730
(P=0.0013), rs13118928 (P=0.003) polymorphisms. Early onset COPD
(before 40 yrs) were associated with rs13180 (P=0.000001), rs16969968
(P=0.0001), rs1051730 (P=0.0004). The SNP rs13118928 near HHIP locus
was significantly associated with pack-years of smoking in COPD patients
(P= 0.029). We demonstrated that rs13180, rs13118928 and rs16969968
polymorphisms were associated with COPD only in smoking subjects.
We confirmed that SNPs the IREB2, CHRNA5, CHRNA3 and HHIP loci were
associated COPD in Russian Population.
J03.10
MTHFR polymorphysms role in diabetic neuropathy
Diabetes Mellitus (DM), the most important public health problem of the
21th century, leads to significant mortality and morbidity due to underlying complications. C677T and A1298C polymorphysisms of MTHFR gene
elevates plasma homocysteine levels, reduces plasma folic acid levels, and
converts gene expression by altering methylation status in genome. These
effects enable the MTHFR gene to be associated with multiple diseases such
as cardiovascular diseases, cancers, and schizophrenia.
The present study looked for a possible association between C677T and
A1298C polymorphysisms of the MTHFR gene in diabetic neuropathy development in diabetic patients and healthy volunteers. Thus a study group
consisting of 103 diabetic peripheral neuropathy patients diagnosed via
electrophysiological examination (ENMG) and a control group of 100
healthy was formed. C677T and A1298C polymorphysisms of MTHFR in the
DNA samples were analyzed by pyrosequencing. MTHFR C677T genotype
distribution was in study group CC: %43.7, CT: %42.7, TT: %13.6, and in
the control group CC: %45.0, CT: 47.0, TT: %8.0 (p>0.05), MTHFR A1298C
genotype distribution was in study group AA: %33.0, AC: %46.6, CC: %20.4
and in the control group AA: %43.0, AC: %44.0, CC: %13.0 (p>0.05). test
was used for statistical analysis and in the study group, the ratio of 2 or more
mutant allele was found to be significantly higher compared to the control
group (p=0.010). The data obtained from this study supports the view that
the increase in the number of mutant alleles in MTHFR gene can increase
diabetic neuropathy susceptibility in patients with diabetes.
J03.11
A novel (Q97R) MEFV gene mutation in a Turkish FMF patient
393
Backround: It is essential to confirm or exclude the diagnosis of cystic fibrosis in time and with accuracy in order to avoid inappropriate testing.
The aims of this study was to determine features of Cl ions concentration
in patients sweat test that were suspected having cystic fibrosis and to evaluate correlation between this concentration and C reactive protein levels
in serum.
Methods: we investigated retrospectively case files of patients with performed sweat test in period of 2011-2012 in Hospital of Lithuanian University
of Health Sciences Kauno Klinikos Paediatrics department. These patients
were suspected having cystic fibrosis (n=48).
Results: 9 out of 48 patients (18,75 % ) showed higher than normal Cl ions
concentration values. 6 of them (12,5%) had a borderline concentration. Mean value of Cl ions concentration in males sweat was 25,37 mmol/l
(p<0,05; PI=21,05-30,40) and there was no statistical significance compared to mean Cl ions concetration in females sweat which was 24,44 mmol/l
(p<0,05; PI=19,96-28,91). In our study no mutations of CFTR gene were
found. CRP test was performed in 17 cases (35,42%). No statisticly significant correlation between concentration of Cl ions in sweat and serum CRP
was found (r= -0,16; p=0,531 ).
Conclusions: 18,75% of checked patients had a higher than normal Cl ions
concetration in sweat test. No statisticly significant difference between Cl
ions concentrations in sweat test according to gender was found in examined group.
J03.13
The prevalence of the cagA and vacA genotypes of H. pylori from
patients with upper gastrointestinal diseases in Uzbekistan
Helicobacter pylori colonizes the gastric epithelium of more than one half of
the worlds human population. This pathogenic bacterium plays a causative
role in the development of chronic gastritis, peptic ulcers, and gastric adenocarcinoma. There is increasing evidence that the genetic variability of H.
pylori have a clinical importance. Several virulence factors of H. pylori have
been described, such as cytotoxin associated gene (cagA) and vaculating cytotoxin (vacA). This study aimed to investigate the prevalence of the cagA
and vacA genotypes of H. pylori from patients with upper gastrointestinal
diseases and the relationship with clinical outcome in Uzbekistan. A total
of 72 patients who underwent endoscopy were included in this study. DNA
was isolated from gastroduodenal biopsy samples and the presence of cagA
and vacA genotypes were determined by PCR. All patients were found to be
H. pylori-positive. The main virulence strain observed in our cohort was the
cagA+ strain identified in 56 patients (77,8%). From the 52 patients with
gastritis, 30(57,7%) harbored cagA+ strains and 12(42,3%) were infected
with strains that lacked cagA. CagA positivity was observed in 26(86,7%)
394
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of patients with peptic ulcer pathology, whereas CagA negativity was observed in 4(13,3%) of these patients. Four VacA genotypes were found: s1/
m1(25,8%), s1/m2(46,8%), s2/m1(14, 5%), s2/m2(12,9%). Comparative
analysis of resulting genotypes showed a high percentage of s1/m2 with a
distribution of 72,7% in patients with peptic ulcer pathology (P < 0.05). The
results of this study might be used for the identification of high-risk patients
harboring H. pylori in Uzbekistan.
J03.14
Methylation of miR-137 in gastric cancer and preneoplastic lesions
INTRODUCTION: MicroRNA miR-137 is an important regulator of gene expression and functions as a tumor-suppressor gene. Expression of miR-137
is downregulated in glioblastoma and colorectal cancer (CRC) due to CpG
island methylation, however, the role of miR-137 methylation in gastric carcinogenesis remains largely unexplored.
AIM &METHODS: The aim of our study was to characterize the epigenetic
regulation of miR-137 in gastric carcinogenesis. We determined miR-137
CpG island methylation level in 81 pairs of primary gastric cancer tissues
samples (T-GC) and corresponding adjacent normal mucosa (N-GC), 20
samples of normal gastric mucosa (N) and 23 gastric tissues from patients
with chronic/atrophic gastritis intestinal metaplasia (CG) using bisulfite
pyrosequencing and compared to 29 colorectal cancers (T-CRC) and corresponding adjacent normal colonic mucosa (N-CRC).
RESULTS: We confirmed the higher methylation level of miR-137 promoter
in T-CRC tissues compared to adjacent normal colonic tissues (p=0.004).
In similar fashion, but to a lesser extent, methylation of miR-137 promoter
region was observed in T-GC tissues compared to adjacent non-cancerous
tissues (N-GC) (p=0.045). When compared to the normal mucosa from controls and mucosa from patients with gastritis, we found gradual increase
in miR-137 methylation (p<0.0001). In subgroup analyses of gastric cancer
tissues, miR-137 methylation was more frequent in tumors localized in antrum compared to cardia and corpus (p=0.07).
CONCLUSION: MiR-137 methylation is a frequent event in gastric carcinogenesis. The gradual increase in miR-137 methylation, as demonstrated for
normal mucosa, chronic gastritis and tumour tissues, may be an early event
in gastric carcinogenesis.
J03.15
Neonatal permanent diabetes caused by mutation INS/Y50C:
integrated system CGMS and insulin pump
Introduction: Obesity is one of the fast spreading diseases. Insufficient physical activity is one of the main environmental factors in the etiology of the
disease. Twin studies have revealed that genetic factors play an important
role in physical activity levels. Although the underlying genetic factors are
not clearly understood, studies have shown that the LEPR gene is among
the candidate genes affecting physical activity level. In our study, we aimed
to show the effect of LEPR gene mutations related with the physical activity in obese women. In addition, we evaluated the impact of LEPR gene on
body composition parameters. Material and Methods: 66 overweight/
obese women were included in our study. Patients were categorized into 3
groups due to physical activity index levels. After the extraction of genomic
DNA from buccal cells, LEPR gene regions were amplified by PCR and PCR
products were sequenced. Physical activity levels and body composition
parameters were calculated by using actical accelerometer and impedance
meter, respectively. Results and Discussion: Three reported polymorphisms, one in exon 6 and two others in intron 7 of the LEPR gene were detected.
No relationship between physical activity levels and LEPR was observed. A
correlation was found between resting metabolic rate and rs12405556 mutation. Furthermore, we determined a significant relation between different
physical activity levels with fat mass, body mass index and total energy expenditure. Further studies are needed to make an interpretation about the
relation between physical activity levels, body composition parameters and
gene mutations.
J03.18
Association and gene-gene interaction analyses of genetic variants
in IL1B, IL1RN, IL8, IL10 and TNFA genes for peptic ulcer disease in
Volga-Ural region of Russian Federation
A peptic ulcer disease (PUD) is an area of damage to the inner lining (the
mucosa) of the stomach or the upper part of the intestine (duodenum). A
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bacterium, Helicobacter pylori, is the main cause of ulcers in this area. The
genes that encode proinflammatory and anti-inflammatory cytokines are
good candidate markers of host susceptibility to gastroduodenal disease.
The present study was performed to evaluate association and gene-gene
interaction of polymorphisms of cytokines genes IL1B (rs1143634), IL1RN
(rs71941886), IL8 (rs4073), IL10 (rs1800872) and TNFA (rs1800629) for
PUD in Volga-Ural region of Russia.
This study enrolled 264 patients with gastric and duodenal ulcers (112 individual were H.pylori-infected), the control group included 277 unrelated
individuals without gastro-duodenal pathology with different ethnic origins
(Russians, Tatars, Bashkirs). Genotyping was performed by PCR-RFLP analysis. To investigate gene-gene interactions, we employed generalized multifactor dimensionality reduction (GMDR) method.
The analysis has revealed a strong association of C allele and CC genotype
of the rs1143634 of the IL1B gene with PUD in Bashkirs (2=7,61, p=0,006;
OR=2,87 2=9,28, p=0,002; OR=4,49; 95%CI 1,78-11,35), confirmed by
meta-analysis. We have also detected that H.pylori-positive PUD-individuals
has significant lower frequency of AA genotype of rs4073 of the IL8 gene
compared with healthy donors (2=5,29, p=0,02; OR=0,46). The 3-locus
GMDR-model of cytokine gene-gene interaction, including rs1143634 of
IL1B, rs4073 of IL8 and rs1800872 of IL10 genes, was shown for male subgroup (p=0,0547).
Thus, we have determined that cytokine genes may contribute in genetic
susceptibility for peptic ulcer disease.
J03.19
Rare germline MEN1 mutation increases susceptibility to pituitary
adenoma
Introduction. Pituitary adenomas are up to 15% of clinically active primary intracranial neoplasms. They are considered benign, non-metastatic
tumors. Although pituitary adenomas have prevalence of 16.7% in general
population, clinically significant tumors affect one individual out of approximately 1000 to 1300 people in general population. There is evidence that
both genetic and epigenetic factors are important to development of human
neoplasms.
Methods. Our study was carried out using 144 cases and 354 controls. Seven
candidate genes (AIP, MEN1, GNAS, SSTR2, SSTR5 and DRD2 and PRKar1a)
were selected based on literature research about pituitary adenoma genetics and 96 tag and non-synonymous SNPs genotyped using Illumina GoldenGate genotyping assay on Illumina BeadXpress system. Basic association
test was used to test difference between cases and controls.
Results. Our data showed that non-synonymous (AT) SNP rs2959656
in MEN1 is strongly associated (OR=17.8 CI95%=2.2-145, P=0.0002) with
development of pituitary tumor. Most patients were diagnosed while their
adenoma were smaller than 10mm in diameter (microadenoma, stage I) as
opposed to trend of pituitary adenomas being diagnosed in macroadenoma
with extrasellar extension (stage II or III) (39 vs. 105 in our sample) suggesting increased secretory activity than typical adenomas of same stage. There was no association with particular hormone secreting profile. Another
notable finding was SNP rs7131056 in DRD2 gene which was associated
with higher risk for adenoma extrasellar growth (OR=2.3 Ci95%=1.4-3.7,
P=0.001) suggesting involvement of dopamine system in aggressiveness of
pituitary adenomas.
Conclusion. Polymorphisms in MEN1 and DRD2 constitute to development
and aggressiveness of pituitary adenomas.
J03.20
The molecular genetic research of chemical hypersensibility among
women in the case of autoimmune thyroiditis
K. Olga, T. Viktorova;
Institute of Biochemistry and Genetics Ufa scientific centre, Ufa, Russian Federation.
395
D. Serapinas1,2, A. Narbekovas2;
1
Department of Pulmonology and Immunology, Lithuanian university of health sciences,
Kaunas, Lithuania, 2Mykolas Romeris university, Vilnius, Lithuania.
Steroid-resistant nephrotic syndrome (SRNS) is a chronic, progressive disorder which affects approximately 10% of all children with nephrotic syndrome. Approximately 10% of all infantile ESRD cases are a consequence of
SRNS, which makes the disease one of the major causes of child morbidity.
The genetic background of SRNS is diverse. The most common mutations are
found in NPHS2 and WT1 genes. NPHS2 codes the protein podocin, which is
an essential structural element of the podocytes slit diaphragm. WT1 encodes a transcription factor with key role for the urinary tract differentiation.
We present here the results from genetic testing of Bulgarian patients with
SRNS. We used direct DNA sequencing to screen 22 children form 21 families for mutations in the coding regions of WT1 and NPHS2. We found several polymorphisms in both NPHS2 and WT1. In one patient we found a
heterozygous R229Q substitution in the podocin gene, while two children
carried novel missense variants in WT1. One of the novel WT1 mutations is
located in exon 8, the other in exon 9. Both affect the zinc-finger structures
of the DNA binding domain. Further tests were carried out for determining
the origin of the two novel mutations and proving their role in disease.
We can conclude that in Bulgarian SRNS patients there is a prevalence of
WT1 mutations above NPHS2 gene defects. Additional investigations need
to be carried out in order to determine the genetic cause of the disease in
the remaining patients.
J03.23
Tight Junction gene expression in intestinal epithelial cell monolayers
A. Jauregi-Miguel, N. Fernandez-Jimenez, L. Plaza-Izurieta, J. R. Bilbao;
BioCruces Reserch Institution/UPV-EHU, Leioa, Spain.
396
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Turner syndrome (TS) is one of the most common genetic disorder affecting
females, occurring in approximately 1:2500 female births. TS occurs when
an X-chromosome is completely or partially deleted or when X-chromosomal
mosaicism is present. It is characterized by short stature, gonadal dysgenesis, primary hypogonadism, congenital heart disease, renal anomalies, and
a variety of somatic features. Girls with TS benefit from early diagnosis and
treatment with growth hormone. However, many girls with TS are not detected until after 10 yr of age, resulting in delayed diagnosis and treatment.
This study aimed to apply PCR-based approach for detection of Turner syndrome and elucidation the parental origin of the X chromosome in patients
from Uzbekistan. We have amplified by polymerase chain reaction five polymorphic markers along the X chromosome (DXS1283E, DYS II, DMD49,
AR and DXS52) and three markers along the Y chromosome (SRY, DYZ3 and
DYZ1). In addition we analyzed patients DNA samples by SYBR Green and
TaqMan probe based real-time PCR assay. The results of our study show that
monosomy(45,) was present in 78% of cases, 45,/46, mosaicism in
18,8%, and 45,/46, in 3,2%. We also determined the parental origin of
the X chromosome in the 2 patients. They had a paternal single X chromosome.
PCR-based approach can be recommended for the screening programs regarding detection of girls with TS in Uzbekistan.
J03.25
Average telomere length as a biomarker in children and adolescents
with type 1 diabetes at the diagnosis
The search for new genetic variants involved in type 1 diabetes and its complications gives the opportunity for personalized medicine. In our study, 58
SNPs were studied in 48 genes which were associated in GWA studies with
one or more of the following phenotypes: fibrogenesis, endothelial dysfunction, diseases of the cardiovascular continuum, type 1 diabetes (T1DM), and
type 2 diabetes. Specimens were evaluated using the Sequenom MassARRAY (USA). Case-control study included 285 patients with T1DM and 300
population controls. All subjects were ethnically Russians living in Siberian
region of Russia (Tomsk and Kemerovo cities). Association with the disease was obtained for the following genetic markers: MMP3 rs679620 (AA,
OR=2,03 (95% CI: 1,19-3,47), p=0,008), ITGA4 rs1143674 (GG, OR=2,03
(1,27-3,26), p=0,003; allele G, OR=1,68 (1,24-2,27), p=0,001), ITGB5
rs1007856 (TT, OR=1,67 (1,10-2,54), p=0,015; allele T, OR=1,31 (1,02-1,70),
p=0,037), ADAMDEC1 rs3765124 (AA, OR=1,76 (1,18-2,65), p=0,005; allele
A, OR=1,31 (1,01-1,70), p=0,040), LIG1 rs20579 (CC, OR=1,95 1,26-3,02),
p=0,002; allele C, OR=1,91 (1,29-2,81), p=0,001). The MMP3, ITGB5, ITGA4
and ADAMDEC1 genes are involved in the formation of extracellular matrix
and collagen metabolism. These associations can be explained by the participation of these genes in the formation of fibrotic changes in the renal tubules and interstitium, which leads to progression of diabetic nephropathy
and deterioration of the patient condition. The LIG1 gene is involved in DNA
repair and recombination. Perhaps this gene is associated with type 1 diabetes through some basic metabolic pathway. Altogether, our results suggest
that T1DM development is influenced by genes involved into fibrogenesis.
J03.27
Association study of promoter polymorphisms of nucb2 gene in
Iranian patients with type 2 diabetes
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Acute liver failure (ALF) is a rare form of Wilson disease (WD) developing
mostly in young female patients. Use of chelating agents and supportive
therapies including MARS (Molecular Adsorbent Recirculating System) in
time may result in a remission in some cases making liver transplantation
unnecessary. We are presenting here a 47 year old male patient with no alcohol consumption, negative hepatits- and autoantibody markers and with
elevated transaminase and ferritin levels. The patient was admitted to our
institution with severe ALF and with the potential diagnosis of haemochromatosis, but the patients coeruloplasmin level was low with very high level
of ferritin. The D-penicillinamin test supported the diagnosis of WD, but the
result of the genetic analysis of the most frequent disease causing mutation
in Hungarian population (H1069Q) was negative (wild type). Targeted NGSbased analysis of the entire coding region of the ATP7B gene showed two
different disease-causing alteration in this patient and made the diagnosis
clear, showing the clinical potential of semiconductor based next generation
sequencing with 36 hours of turn around time.
J03.29
Mutations in genes HFE, SERPINA1, CFTR in Wilsons disease patients
in Latvia
397
Background: HHT affects approximately 1 in 5000 people and has been associated with mutations in ENG, ACVRL1 and SMAD4. Features of this disease are epistaxis, telengectasia, a family history and visceral arteriovenous
malformations (AVMs). International guidelines on the screening of patients
with HHT were published in 2011.
Method: Patients with HHT were identified using the Molecular Laboratory
database at St Marys Hospital, Manchester. Screening on mutation-positive
patients was audited against the International guidelines.
Results: 54 patients were identified and 35 (65%) were found to have a mutation in either ENG or ACVRL1. Pulmonary and cerebral AVM screening was
undertaken in 92% and 62% of patients respectively. Screening for anaemia
in those over 35 years was undertaken in 21%. 2/19 (11%) eligible patients were tested for SMAD4 mutations (both negative). AVMs occurred in
29% ENG and 21% ACVRL1 patients. Lung AVMs were more common in the
ENG group (24% vs 7%). One cerebral AVM was identified and this occurred
in the ENG group. Liver AVMs only occurred in the ACVRL1 group (21% vs
0%). Overall, AVMs occurred more frequently in women than in men (38%
vs 7%).
Conclusion: A consensus on screening for HHT patients is needed in the
UK. It is globally accepted that screening for pulmonary AVMs and anaemia
should be undertaken but cerebral screening is still controversial. SMAD4
mutations should be sought in those patients who are negative for ENG and
ACVRL1. The breakdown of AVMs in this population adds further evidence
to a genotype-phenotype correlation in HHT.
J03.32
Primary hyperoxaluria type 1: Identification of a double mutation in
AGXT gene in Tunisian families
H. Kanoun1, F. Jarraya2, H. Mahfoudh2, Y. Chaabouni2, F. Makni3, J. Hachicha2, F.
Fakhfakh1;
1
Human Molecular Genetic Laboratory, Faculty of medicine of Sfax, Sfax, Tunisia,
2
Research Unit 12ES14 and department of Nephrology, Hedi Chaker Hospital, Sfax,
Tunisia, 3Laboratory of Biochemistry, Hbib Bourguiba Hospital of Sfax, Sfax, Tunisia.
Primary hyperoxaluria type 1 (PH1) is a severe autosomal recessive inherited disorder of glyoxylate metabolism caused by mutations in the AGXT
gene on chromosome 2q37.3 that encodes the hepatic peroxisomal enzyme
alanine:glyoxylate aminotransferase. These mutations are found throughout
the entire gene and cause a wide spectrum of clinical severity. Rare in Europe, PH1 is responsible for 13% of the end stage renal failure in the Tunisian child. In the present work, we identified the double mutation c.32C>T
(Pro11Leu) and c.731T>C (p.Ile244Thr) in AGXT gene in five unrelated Tunisian families with PH1 disease. Our results provide evidence regarding the
potential involvement of c.32C>T, originally described as common polymorphism, on the resulting phenotype. We also reported an extreme intrafamilial heterogeneity in clinical presentation of PH1. Despite the same genetic
background, the outcome of the affected members differs widely. The significant phenotypic heterogeneity observed within a same family, with a same
genotype, suggests the existence of relevant modifier factors.
J04.01
A five case molecular study of 3M syndrome
398
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Objectives: Anterior cruciate ligament (ACL) rupture is a severe multifactorial injury. A familial predisposition toward tearing ACL was demonstrated.
Matrix metalloproteinases and growth differentiation factor 5 (GDF5) are
important physiological mediators of extracellular matrix degradation, remodeling and chondrogenesis. The aim of this study was to determine impact of MMP3 rs679620 and GDF5 rs143383 variations on the risk of ACL
A
0.671
0.596
C
0.543
0.550
G
0.329
0.404
T
0.457
0.450
GDF5 rs143383
AA
0.386
0.369
MMP3 rs679620
CC
0.357
0.312
AG
0.571
0.453
CT
0.371
0.477
GG
0.043
0.178
TT
0.271
0.211
Significant deviation of genotypic frequencies from HWE was detected only for
GDF5 rs143383 in ACL-group (P=0.017). MAFs of tested SNPs in controls were
similar to other Caucasians. Goodness of fit test reveals significant difference of
GDF5 rs143383 genotypic frequencies between groups. GG genotype carriers
had a significantly decreased risk of ACL ruptures versus AG+AA genotypes
(2 5.995, P=0.014, OR 0.196, 95% CI 0.040 to 0.763). This study suggests a
relationship between GDF5 rs143383 variation and risk of ACL rupture in the
Russian population.
J04.05
Filaggrin mutations and atopic dermatitis in Volga-Ural region of
Russia
Aim:The pathogenesis of atopic dermatitis (AD) includes genetic and environmental factors leading to immunological and nonimmunological dysfunctions. The VDR single nucleotide polymorphisms (SNPs), FokI and TaqI
have previously been associated with atopic diseases such as allergic asthma. Methods:In a total of 88 AD patients and 96 healthy controls were included in the current study. The genomic DNA was isolated from peripheral
blood-EDTA, and target VDR gene was genotyped by PCR-RFLP technique
after VDR-FokI (rs2228570) and VDR-TaqI (rs731236) restriction enzymes
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The aim of our study was to investigate the association of four polymorphisms of the VDR gene (FokI, BsmI, TaqI and ApaI) with their susceptibility to
Behets Disease (BD) and their clinical manifestations in respect to the Iranian Azari population. In this cross sectional study we considered the BsmI,
FokI, ApaI and TaqI polymorphisms in 50 Iranian Azary patients with BD
and 50 healthy controls, with the use of Polymerase Chain Reaction-restriction fragment length polymorphism. A significant difference was found for
the FokI polymorphism between the case and control groups. The f allele
frequency of 26% was present in BD patients, compared to only 13% in the
control group. In addition, the f/f genotype was significantly associated with
BD. We found no significant differences between the BD and control groups
regarding the distribution of ApaI, BsmI, and TaqI genotype frequencies. We
found no association between VDR polymorphisms and the clinical manifestations of BD. The VDR f allele and f/f genotype is associated with BD in the
Iranian Azari population.
J04.08
Brooke-Spiegler syndrome: A rare association of thichoepithelioma,
cylindroma and spiradenoma. Report of a familial mexican case
Introduction:
Brooke and Spiegler, independiently described an ephithelioma adenoides
cysticum and skin endotelioma, as distinct entities. Brooke-Spiegler syndrome (BSS, OMIM#605041) characterized by benign adnexal neoplasia. Predominat tumors trichoepithelioma, cylindroma and spiradenoma appear
childhood and early adolescence. BSS is autosomal dominant entity. The tumors located on head and neck, and increase throughout life. Trichoepithelioma showed flesh-colored papules on face; cylindromatosis presents erythematous nodules on the scalp; and blue-colored-painful lesions on trunk
suggested spiradenomas. Pathogenesis is considered a defect in the diferentiation of folliculo-sebaceous-apocrine unit. Mutations have been identified
in CYLD tummor suppressor gene, mapped to chormosome 16q12-q13.
Case Report:
Case I: 9 years-old female, birth: weight 3,400gr, height 53cm. Between 5-6
years-old displayed flesh-colored papules on face. Showed clinical and hystopathological features indicating trichoepithelioma, with keratinizing cystic spaces.
Case II: 42 years-old female showed, flesh-colored-papules on scalp, face
and trunk. Three biopsies were reviewed, 1) right preauricular region revealed trichoepithelioma, 2) scalp region conclusive cylindroma, 3) third
lumbar regin conclusive ecrine spiradenoma. Given cryotherapy treatment
and cosmetic surgery with good results.
Discussion:
Brooke-Spiegler syndrome, sex ratio of F:3: M:1. include skin appendage
tumors such cylindromas, thichoepitheliomas and spiradenomas, share a
common genetic basis. May be associated with other skin disorders such,
basal cell adenomas/carcinomas. Treatment included dermabrasion, cryotherapy and some cases radiotherapy. We present a Mexican family, mother
and daugther similar affected, with the clinical features of the disease. Is the
first mexican case reported with this entity. Molecular studies are needed to
understand the genetic bases of the disease.
J04.09
First molecular analysis of Ehlers-Danlos kyphoscoliotic type in Slavic
population
A. Junkiert-Czarnecka1, M. Pilarska1, O. Haus1,2;
399
The kyphoscoliotic type of Ehlers-Danlos syndrome (EDS) is a heritable connective tissue disorder characterized by a deficiency of collagen lysyl hydroxylase 1 (LH1) due to mutations in PLOD1 gene. According to Villefranche
criteria this type of EDS is characterized by kyphoscoliosis and hypotonia at
birth, severe joint hypermobility as well as skin extensibility and fragility.
2-year old girl examined by us was born by caesarean section because of
pelvic longitudinal position (situs longitudinalis pelvicus). Directly after the
birth, kyphosis, hypotonia, joint hypermobility and unilateral hip dislocation were observed. Parents of investigated child did not present any clinical
features of EDS. The gDNA of affected patient was obtained by isolation from
peripheral white blood cells. Direct sequencing of all PLOD1 exons was carried out, revealing the presence of two mutations. The first one, substitution
of cytosine by thymine in position 1095 (c.1095C>T), was localized in the
exon 10. It was a splice site mutation, described earlier. The second one,
which turned out to be a missense mutation that had never been described
earlier in patients with kyphoscoliotic type of EDS, was localized in exon
17.
Our investigations of genetic background of kyphoscoliotic type of
Ehlers-Danlos syndrome were the first ones in East-Central Europe.
J04.10
Atypical Fibrodysplasia Ossificans Progressiva (FOP) phenotype in a
girl carrying uncommon missense mutation (p.G356D) in ACVR1
Fibrodysplasia ossificans progressiva (FOP, MIM #135100) is a rare genetic condition characterized by progressive transformation of soft tissue into
bone. There are approximately 3 000 individuals living worldwide with this
severely disabling disease, for which there is still no definitive treatment.
Heterozygous mutations in ACVR1 (MIM# 102576) has been identified as a
cause of this condition. All reported patients of various ethnic backgrounds
with classic clinical presentation of FOP have previously been found with
the identical heterozygous activating mutation 617G>A(R206H). Recently
other types of mutations in ACVR1 gene have been described in patients
with variant FOP.
We present a course of FOP in 3,5 years old girl carrying uncommon missense mutation 1067G>A(G356D) in the protein kinase domain of ACVR1.
Identical mutation has been identified in several patients with atypical FOP
among which at least in two with mild clinical symptoms. On contrary, our
patient experiences a severe course of FOP with already significant restriction of movement. Though it should be emphasized, that retrospectively
analyzing evolution of intense heterotopic ossification in our patient, we
have discovered, that most of the lesions were directly triggered by soft tissue injuries resulting from diagnostic procedures and inadequate rehabilitation that she underwent prior to recognition of her genetic disease.
Our presentation may add to the understanding of the variability of clinical FOP presentation in patients with atypical ACVR1 mutations, as well as
help spreading knowledge of this disease among health care professionals,
in hope for the earliest possible diagnosis established in each newly affected
child.
J04.11
Genetic characterization of a Portuguese patient with fibrodysplasia
ossificans progressiva
S. Sousa1, C. Corzo2, I. Alonso1,3, J. Silva1, J. Sequeiros1,3,4;
1
CGPP, Instituto de Biologia Molecular e Celular (IBMC) Univ. Porto, Porto, Portugal,
2
Hospital do Esprito Santo - vora, Unidade de AVC, vora, Portugal, 3UnIGENe, IBMC,
Univ. Porto, Porto, Portugal, 4ICBAS, Univ. Porto, Porto, Portugal.
400
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Despite advances in the treatment of pyoinflammatory diseases of maxillofacial area, the number of diseases is increasing from year to year. In this
connection, the study of the pathogenesis of pyoinflammatory diseases is
one of the most pressing issues in maxillofacial surgery. The purpose of this
study was to investigate the role of the cytokines genes in the development
of odontogenic inflammatory processes.
To estimate the role of polymorphisms of inflammatory mediator genes in
genetic predisposition to pyoinflammatory diseases, the allele and the genotype frequencies distributions f IL1, IL1RA, TNF, TNF and IL10 genes
were investigated. The studied groups included 189 patients with pyoinflammatory diseases of maxillofacial area divided into two groups: odontogenic phlegmon (141) and osteomyelitis (48) and 105 healthy controls.
Genomic DNA was extracted from peripheral blood leukocytes by standard
phenol/chloroform method. Genotyping was performed by the PCR-RFLP
technique.
Studies have revealed that the C*A* genotype (OR = 1,83; 95% CI 1,08-3,10)
of IL10 polymorphic locus 627C>A is associated with increased risk of odontogenic phlegmon, while the G*A* genotype (OR = 0,29; 95% CI 0,08-1,04) of
TNF polymorphic locus 308 G>A is associated with lower risk of osteomyelitis of maxillofacial area. It is possible to suggest that genotype C*A* of IL10
polymorphism (627C>A) and genotype G*A* of TNF polymorphism (308
G>A) may be genetic predictor of odontogenic inflammatory processes.
No significant differences were found between the groups of patients and
Ichthyosis is a group of inherited keratinizing disorders. There are five different types described. This type of genetic disorder can be autosomal recessive or x-link. This disorder has been related with inbreeding and new
mutations. More than 16000 babies are born each year with some form of
ichthyosis. In Manab province, Ecuador (population 1,369,780) there are
more than 230 cases, compared with rates of other countries this is very
high. But when put into context, Ecuador is a developing country, in more
isolated areas marriage between closely related family members is still
common place. We are presenting a case of twin brothers that suffer from
ichthyosis.
Case report: Mother and father are first cousins. They are parents of three
children, one girl and two tween brothers. The boys present the typical fish
scales, alopecia, constant conjunctivitis and skin infections. We found isolates of different microrganisms in all samples taken from eyes, skin and
wounds. They present constant weeping of the eyes and intense itching.
They do not eat pork due metabolic disorders associated with this dissease.
After living in several places in Ecuador, they moved to Banos a small city
in the Andes (1,815 m) because the moderate climate (moisture and temperature) is more favorable to their condition. Their treatment is: Rocutan
(isotretinoin) for 2 weeks and after that Neotigason (retinoids, 25 mg every
day or two). Their pathology has improved a lot since then. They do not
show signs of hyperactivity or any mental problems.
J04.15
Lacrimo-auriculo-dento-digital syndrome, case report
Leri-Weill dyschondrosteosis (LWD; #MIM 127300) is an autosomal dominant hereditary disease, which is characterized by short stature, mesomelic shortening of the limbs, and characteristic bilateral abnormality of the
wrists known as Madelung deformity. LWD is caused by mutations in the
Short Stature Homeobox gene (SHOX). The other diseases which are associated with SHOX gene mutations are Langer Mesomelic Dysplasia (LMD,
MIM #249700) and Turner syndrome as well as nonsyndromic idiopathic
short stature (ISS). The SHOX gene is localized within the pseudoautosomal
region 1 (PAR1) of the X and Y chromosomes. Both alleles of SHOX gene
must be functional for normal growth. While heterozygous mutations of
SHOX gene or its enhancer regions are responsible for both LWD and ISS
syndromes, LMD is caused by homozygous or compound heterozygous mutations in this gene. Herein, four LMD and one LWD siblings and their consanguineous parents with LWD are presented. Mutation analysis revealed
that the parents and one sibling were heterozygous, and the other 4 siblings
were homozygous for the c.42delG (p.Q14QfsX15) in the SHOX gene.
Back to index
J04.17
A novel mutation in LEMD3 gene in a Turkish family with BuschkeOllendorf syndrome
Buschke-Ollendorff Syndrome (BOS, OMIM#166700) is an autosomal dominant disorder of connective tissue with an incidence of 1/20000. Although It
is characterized by multiple subcutaneous nevi or nodules and osteopoikilosis (OPK), some patients may also have melorheostosis. Expressivity of bone
and skin manifestations differs between the cases. Mutations in LEM Domain-Containig Protein-3 (LEMD3, OMIM *607844, chromosome 12q14.3)
have been implicated in BOS. 14-year-old girl was directed to our department for an incidentally realised osteopoikilosis in the X-Ray exploration.
She also has yelowish plaques on her anterolateral thigh. Complete X-Ray
study revealed multiple osteoclastic foci around the bones as implicated in
te BOS. 35-year-old mother has been X-Ray examined and she also has ospteopoikilosis but she has no skin plaques. LEMD3 gene was sequenced from
the DNA of the patient and the mother. Both of them have a heterozygous
c.2405_2406insAGTG mutation in the 12th exon of LEMD3 gene.This mutation results in a premature stop codon at the 802th position of the MAN1
protein encoded by LEMD3 gene. The remaining parts of the gene were also
sequenced and confirmed that there was not another mutation. We submit
the mutation to MutationTaster and see that it causes loss of Interaction
with SMAD1, SMAD2, SMAD3 and SMAD5. To our knowledge, this is the first
study implicating the c.2405_2406insAGTG mutation in the cases of BOS.
J04.18
HRM scanning of mutations in human structural proteins
Mandibuloacral dysplasia with type A lipodystrophy (MADA) is a rare genetic disorder inherited in an autosomal recessive fashion characterized by
hypoplasia of the mandible and clavicles, acro-osteolysis and lipodystrophy
due to mutations in LMNA or ZMPSTE24 gene. Very few families have been
studied for the above genes alteration so far. We have investigated a consan-
401
Background : The clinical and radiographic phenotypes of multiple epiphyseal dysplasia(MED) are heterogenous according to the genetic mutation.
Mutations COMP, MATN3, COL9A1, COL9A2 and COL9A3 result in autosomal
dominant MED, and mutations in the DTDST gene is associated with an autosomal recessive MED. Here in, we present a family with novel COL9A3 gene
mutation.
Case : The proband was a 12-year-old boy born from non-consanguineous
parents. He was referred to the pediatric orthopedic clinic for the evaluation
of intermittent knee pain that occurred from a few months. The radiographic phenotype of COL9-MED is that the epiphysis of the distal tibia, distal
radius and distal ulnae showed lateral shortening, wedge shape and small,
respectively. Direct sequencing of COL9-MED associated genes was performed and identified a novel mutation c.104G>A in exon 2 of COL9A3, which
resulted in p.Gly35Asp. The same mutation was observed in the probands
father. The molecular dynamics (MD) simulation of wild type and mutant
model structure were performed using AMBER11 program and demonstrated that the mutant type collagen was aggregated by themselves while
wild type structure formed hydrogen bonds with its neighboring strand. In
addition, the distances between the mutation site of the wild type and the
mutant collagen are almost twice. The calculated energy state of the mutant
is much lower, causing a self aggregation.
Conclusion : We report a family of MED with novel COL9A3 mutation and
propose that major structural change caused by the mutation of COL9A3
gene induce the malfunction of the type IX collagen.
J04.22
Neurofibromatosis-Noonan Syndrome - clinical and molecular studies
of 4 patients
A. Kutkowska-Kamierczak1, M. Gos1, S. Fahiminiya2, A. Abramowicz1, J. Klapecki1, J.
Majewski2, J. Bal1, E. Obersztyn1;
1
Department of Medical Genetic, Institute of the Mother and Child, Warsaw, Poland,
2
Department of Human Genetics, McGill University and Genome Quebec Innovation
402
Back to index
Osteoreparation is a complex, dynamic and still incompletely revealed process. There are different approaches in evaluation of osteogenic process.
The purpose of our research is evaluation weather and to what extent relative expression of genes that are indicators of young bone growth are in
correlation with histological findings, as commonly used methods for evaluation of osteogenic process. In our research, subcutaneous implantation
model on Balb/c mice was used. The implants were composed from deproteinized bone mineral matrix and bone marrow and/or different components of blood. The animals were sacrificed after 1, 2, 4, and 8 weeks after
implantation. Analysis of implants included comparison of relative expression profiles of the gene for alkaline phosphatase, osteocalcin, osteonectin,
osteopontin and collagen type I (RealTime PCR) and histological analysis of
implants after staining with hematoxylin-eosin and Massons trichrome method. Our results show: the most informative genetics markers are profiles
of expression of genes for osteocalcin and alkaline phosphatase, as indicators of stimulation of young bone growth; histological picture was lagging
in comparison to the findings of gene expression in all terms of sacrifice;
valid assessment of osteogenic process requires a combination of different
methods, because neither gene expression nor histological analysis are sufficient for complete evaluation of osteogenic process.
Key words: RealTime PCR, histology, ectopic osteogenesis
J04.24
Bone mineral accrual and fracture outcomes in children with
osteogenesis imperfecta treated by pamidronate
The purpose of this study was to evaluate the bone mineral accrual and
fracture outcomes in children with osteogenesis imperfecta (OI) treated by
pamidronate (PAM). Material and Methods: in our retrospective study 21
children with different types of OI were included: 7 boys and 14 girls. According to clinical OI classification proposed by D. Sillence, patients were
divided in 3 types: I type - 11, III type - 7, IV type - 3. We divided patients
in 2 groups: mild to moderate (OI I type) and moderate-to severe (III and
IV types). The standard protocol with cyclic PAM infusions (3 consequent
days 3-4 times in a year) was applied in annual cumulative 9-12 mg/kg. All
children received vitamin D and calcium supplementation in physiological
doses. Observation period was 36 months. Bone mineralization parameters
were detected by dual-energy X-ray absorptiometry of lumbar spine L1-L4.
Results: There were no differences in bone mineral accrual between types
D. Stoicanescu1, M. Cevei2;
1
University of Medicine and Pharmacy Victor Babes, Timisoara, Romania, 2University
of Oradea, Faculty of Medicine and Pharmacy, Oradea, Romania.
Many recent reports confirmed, in the disease onset, both genetic and environmental factors, may be different in osteoporosis for women and men.
Mutations in the low-density lipoprotein receptor-related protein 5 (LRP5)
gene cause rare syndromes characterized by altered bone mineral density (BMD). LRP-5 is a transmembrane protein encoded by gene located in
6q25.1. LRP-5 protein takes part in a proliferation and differentiation of
osteoblasts via Wnt signaling. The Wnt/b-catenin signaling pathway stimulates bone formation through a number of mechanisms such as stimulation
of preosteoblast replication, induction of osteoblastogenesis, and inhibition
of osteoblast and osteocyte apoptosis. LRP-5 is widely expressed in most
human tissues, with greater amounts in the liver and pancreas. In bone, it is
mainly expressed by the bone-forming cells as osteoblasts, in the endosteal
and trabecular bone surfaces. Common LRP5 variants were associated with
the osteoporosis risk in males. The aim of this pilot study was to analyze
occurrence of polymorphic variant c.2074G>A (p.V667M, rs4988321) in a
group of 187 male patients with osteoporosis and 203 individuals from Polish population. Genotyping was performed by pyrosequencing technique.
Hardy-Weinberg equilibrium (HWE) were examined for subjected groups by
chi-square distribution and Fisher exact tests. The odds ratios (ORs), 95%
confidence intervals (CIs), and p-values were calculated. Statistical significance was set at p<0.05. We observed statistically relevant higher frequency of minor allele c.2074A in male osteoporosis patients with p=0.01401
(OR=2.162, CI=[1.154-4.050). In conclusion we state that c.2074A in LRP5
gene variant may be one of the risk factor for osteoporosis in males.
J04.27
Association of gene variants in TLR4, TNF-, IL-3 and IL-6 genes with
Perthes disease
S. Srzentic1, V. Spasovski1, D. Spasovski2,3, Z. Zivkovic4, D. Matanovic2,5, Z. Bascarevic2,3,
Z. Terzic Supic2, M. Stojiljkovic1, T. Karan-Djurasevic1, B. Stankovic1, S. Pavlovic1, G.
Nikcevic1, Z. Vukasinovic2,3;
1
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade,
Back to index
Rothmund-Thomson syndrome (RTS) is an autosomal recessive genodermatosis presenting with a characteristic facial rash (poikiloderma) associated
with short stature, sparse scalp hair, sparse or absent eyelashes and/or eyebrows, juvenile cataracts, skeletal abnormalities, radial ray defects, premature aging and a predisposition to cancer. The prevalence is unknown but
several hundred cases have been reported in the literature so far. Two clinical subforms of RTS have been defined: RTSI characterised by poikiloderma,
ectodermal dysplasia and juvenile cataracts, and RTSII characterised by poikiloderma, congenital bone defects and an increased risk of osteosarcoma
in childhood and skin cancer later in life. RTS is genetically heterogeneous:
RTSII is caused by homozygous or compound heterozygous mutations in the
RECQL4 helicase gene (detected in 60-65% of RTS patients), whereas the
403
M. Smerdel1, M. Joergensen2;
1
Vejle Hospital, Department of Clinical Genetics, Vejle, Denmark, 2Vejle Hospital,
Department of Clinical Genetics and Faculty of Health Sciences, University of Southern
Denmark, Vejle, Denmark.
404
Back to index
Langer mesomelic dysplasia (LMD) is characterized by hypomelia with severe hypoplasia of ulnae and fibulae, and bowed, thickened radii and tibiae,
causing deformities of the hands and feet. LMD is caused by homozygous
mutations in the SHOX/SHOXY (short stature homoebox) gene, of which
heterozygous mutations or deletions cause Leri-Weil Dysplasia (LWD).
Phenotype of LWD can be incomplete between and within families. We present a 13 year old female with LMD, the second child of healthy first cousin
parents. She had micrognathia, disproportionate short stature with various
musculoskeletal findings (absence of the distal flexion creases of the 3rd,
4th, 5th fingers on the right, camptodactyly of the 3rd, 4th, 5th fingers on
the left, tibial bowing). X-rays revealed hypoplasia of ulnae, fibulae and the
mandible. Chromosome analysis and FISH investigation by using SHOX gene
probe revealed normal results. Sequence analysis failed due to unsuccessful
PCR amplifications. Array comparative genomic hybridization (a-CGH) study showed a 174 kb homozygous deletion, encompassing the SHOX gene.
Probands parents were heterozygous for the same deletion by a-CGH. FISH
was uninformative, because there was no difference between the intensity
of the signals on both chromosomes. Since the primers used were located
within the deleted region, molecular studies could not be performed. A-CGH
proved to be the most powerful diagnostic tool in this case.
J04.34
Papillon-Lefevre syndrome and an autosomal dominant form of
palmoplantar keratoderma in the same Indian family: mutation
screening and in-depth bioinformatic analyses of the cathepsin C gene
M. Bhavika1, U. Ratnamala2, T. Y. Mehta3, M. Raveendrababu4, J. Banwait5, D. Bastola5, U.
Radhakrishna2;
1
Sree Dattha Institute of Engineering and Science, Hyderabad, India, 2Green Cross
Pathology & Molecular Laboratory, Ahmedabad, India, 3Samarpan Centre for skin,
Sexually transmitted diseases and Research Center, Modasa, India, 4Department
of Zoology, School of Sciences, Gujarat University, Ahmedabad, India, 5School for
Interdisciplinary Informatics, University of Nebraska at Omaha, Omaha, NE, United
States.
Papillon-Lefevre Syndrome (PALS, MIM 245000) is a rare autosomal recessive disorder characterized by palmoplantar keratoderma and early onset
severe periodontitis affecting both deciduous and permanent dentition. Several recessive families and sporadic cases with variable clinical presentation have been reported. Several loss-of-function mutations have been identified in the lysosomal protease cathepsin C gene (CTSC) in PALS individuals
from various ethnic groups. We describe Papillon-Lefevre syndrome and an
autosomal dominant form of palmoplantar keratoderma in the same Indian
family (UR075) and present the mutation analysis of the cathepsin C gene.
Sequence analysis of known exons and splice junctions revealed a homozygous nucleotide substitution G to A at nucleotide position 901, resulting
in a change from glycine (GGC) to serine (AGC) at amino acid position 301
(G301S) was observed in homozygous condition in an affected individual,
and excluded other affecteds with severe palmoplantar keratoderma from
the same family. We re-evaluated family UR075 and excluded chromosome
11q14-q21 region by linkage analysis. This mutation was found to be at a
Back to index
Pediatric Clinic, Skopje, Macedonia, The Former Yugoslav Republic of, 2Department of
Genetics, Erasmus Medical Center, Rotterdam, Netherlands, 3Remedica Hospital, Skopje,
Macedonia, The Former Yugoslav Republic of.
1
Background: Chronic periodontitis (CP) is an oral disease resulting in inflammation within the supporting tissue of the teeth the periodontium,
progressive attachment loss, and alveolar bone loss, it has a microbial aetiology. Recent findings suggest that the genetic factors, such as vitamin D
receptor gene polymorphisms have been also involved in the inflammatory
disease aetiology of CP. Aim of the research: Investigation of the relationship between vitamin D receptor gene polymorphisms and CP among Libyans. Materials and Methods: In this study, we examined 196 unrelated
Libyans between the ages of 25 and 65 years, including 99 patients and 97
controls. An oral examination based on Ramjford Index was performed at
different dental clinics in Tripoli, and informations were obtained using
a self-reported questionnaire. DNA was extracted from buccal swabs, the
VDR ApaI, BsmI, and FokI polymorphisms were genotyped by using polymerase chain reaction (PCR), and were sequenced to read the nitrogen bases
by using Sanger Method. Results: A significant association between ApaI
SNP C/T rs#731236 and CP was found (P value = 0.022), while no significant associations were found between ApaI SNP G/T rs#7975232 , BsmI
SNP A/G rs#1544410, and FokI SNP rs#2228570 and CP (P value = 0.939,
0.466, 0.239) respectively. Conclusion: Vitamin D receptor ApaI SNP C/T
rs#731236 may be related to the risk of CP in the Libyan population.
J04.39
Frequent mutation Ala597Glu in Alox12B gene at autosomal recessive
congenital ichthyosis in patients from Russian Federation
N. N. Vasserman, V. A. Galkina, E. G. Okuneva, G. E. Rudenskaya, A. V. Polyakov;
Research Center for Medical Genetics, Moscow, Russian Federation.
405
PNPLA1 has recently been identified as one of the eight genes (ALOX12B,
ALOXE3, CERS3, CYP4F22, NIPAL4 or TGM1) in which mutations can cause
autosomal recessive congenital ichthyosis (ARCI), although only two families harbouring such mutations (both of North African origin) have been
identified.
In a study of ALOX12B, ALOXE3, CYP4F22, NIPAL4 and TGM1 performed
in 17 ARCI families in Galicia (NW Spain), about 80% of these families had
mutant TGM1 or ALOXE3, while no mutations of NIPAL4, CYP4F22 or ALOX12B were found.
In 2012 a new locus of ARCI mutations, in PNPLA1, was published and we
accordingly investigated the prevalence of PNPLA1 mutations in our series
of 18 ARCI families, which constitute 95% of the 19 Galician ARCI families
identified to date. Four Galician families known to have no deleterious mu-
406
Back to index
The ABCG1 transporter plays an important role in reverse cholesterol transport by mediating the efflux of cholesterol from macrophage foam cells
to high density lipoproteins. Two major ABCG1 isoforms exist in humans,
which differ by the presence or absence of twelve amino acids between the
ATP cassette and the transmembrane domains (ABCG1(+12) and ABCG1(12)). We have reported earlier that expression of the ABCG1 gene was reduced in macrophages of patients with atherosclerosis. However the role
of the ABCG1 isoforms in human atherosclerosis is still undiscovered. The
aim of this study was to evaluate the expression of the ABCG1 isoforms
in macrophages of patients with atherosclerosis and in the control group.
Human peripheral blood monocytes were obtained from 10 patients with
angiographically proved atherosclerosis and 10 healthy blood donors. Monocytes were cultured with macrophage colony-stimulating factor (M-CSF)
for 5 days to get monocyte-derived macrophages. Real time PCR system designed for this study was used for simultaneous detection of ABCG1(+12)
and ABCG1(-12) mRNA levels. Mann-Whitney U-test was used for statistical
analysis of the data. ABCG1(-12) mRNA levels were significantly reduced
in group of patients when compared with the control group: medians were
0.70 (0.15 - 2.02) and 1.61 (0.51 - 2.98) (p<0.05). There were no differences
in ABCG1(+12)/ABCG1(-12) mRNA ratio between patients and controls.
ABCG1(+12)/ABCG1(-12) mRNA ratio tends to be 0.55 in human macrophages. In conclusion, while expression of the ABCG1 gene is reduced in
macrophages of patients with atherosclerosis, the ratio of two main ABCG1
isoforms doesnt change during atherosclerosis.
Abdominal aortic aneurysm (AAA) is an irreversible and progressive dilatation of the abdominal aorta. Known risk factors for AAA include smoking,
male sex, increasing age, and family history, but no genetic markers have
clearly been shown to be responsible for this pathology.
In order to assess the potential role of genetic rearrangements in the pathogenesis of AAA, we analyzed a cohort of 50 patients who had undergone
surgical repair of AAA. DNA from peripheral blood and AAA wall specimens
of each patient was analyzed by CGH array. A common deletion region on
chromosome Xp22 was found in about half of AAA wall specimens and never
in the corresponding peripheral blood. In order to demarcate the boundaries of the identified deleted region, a real time PCR was performed on some
genes located within the deleted region: SHOX, CRFL2 and DHRSX. Specifically, samples deleted for SHOX, CLRF2 and DHRSX genes represented 41.8,
28 and 32.5% respectively.
These results allowed us to identify for the first time a common deletion
region on chromosome Xp22 link to abdominal aortic aneurysm using CGH
array technology. Future characterization of the involved genes within the
deleted region may contribute to define the molecular mechanisms involved
in the structural alteration of vascular wall tissue and predisposing to abdominal aortic dilatation.
J05.04
Mutations in ACTA2 are not a common cause of Bicuspid Aortic Valve
associated with Thoracic Aortic Aneurysm
Back to index
congenital heart diseases. Molecular analysis of genes involved in the RASopathies genes (PTPN11, KRAS, HRAS, NRAS, BRAF, RAF1, SOS1, MAP2K1,
MAP2K2, SHOC2, and CBL) by HRM and direct sequencing revealed heterozygous mutations of the BRAF gene: 736G-C transversion in exon 6 (A246P)
for P1 and 1501G-A transition in exon 12 (E501K) for P2. Cognitive impairment, speech delay and ectodermal abnormalities as well as a distinctive
facial appearance similar for P1 and P2 were consistent with CFC syndrome characteristics. At the genetic counselling level, the management of the
risk for haematological malignancies must be discussed with parents and
clinical team. Furthermore, regarding the theoretic possibility of germline
mosaicism, prenatal diagnosis must be offered.
J05.06
Sudden infant death syndrome and unexplained intrauterine fetal
demise: role of calmodulin
407
Carotid atherosclerosis is atherosclerotic stenosis of proximal internal carotid artery (ICA), and is one of the main causes of stroke. Recent genome-wide
association studies (GWAS) revealed chromosome 9p21 INK4b-ARF-INK4a
is a novel locus for susceptibility to type-2 diabetes and coronary artery
disease. INK4/ARF transcript, p16INK4a, arrests cell cycle progression by
inhibiting the activities of CDK4/CDK6. Cell cycle regulation may be an important mechanism in vascular smooth muscle cells for atherosclerosis progression. Thus, we aimed to examine the frequency of the single nucleotide
polymorphisms (SNPs) on chromosome 9p21 in carotid atherosclerosis
(CA).
The study is composed of 50 symptomatic and asymptomatic CA patients
and 53 healthy controls. Genomic DNA extraction was performed from peripheral blood leukocytes. Real-time polymerase chain reaction (RT-PCR) was
used to analyze 4 SNPs (rs564398 A/G, rs10757274 A/G, rs2383207 A/G,
rs10757278 A/G) in CA patients and controls.
Analysis of 4 SNPs revealed a significant difference in the genotype distribution for rs564398 and rs10757278 between CA patients and controls
(p=0.027 and p=0.01). However no significant relationship was found in
genotype frequencies of rs10757274 and rs2383207 when CA patients and
controls were compared (p>0.05). There was also a significant relation in
allele frequencies of rs564398, rs2383207 and rs10757278 polymorphisms
between CA patients and controls (p=0.016, p=0.049, p=0.001). ICA stenosis
severity was found to be associated with the AA variant of rs564398 polymorphism in CA patients (p=0.01).
These results indicate that, chromosome 9p21 rs564398 and rs10757278
polymorphisms may be associated with CA. These findings need to be confirmed by further studies.
J05.09
ADIPOQ variants in patients with coronary artery disease in Turkish
population
D. Erkoc Kaya1, H. Arikoglu1, A. Asik1, S. E. Unal1, F. Iscioglu2;
1
Selcuk University, Faculty of Medicine, Department of Medical Biology, Konya, Turkey,
2
Ege University, Science Faculty, Department of Statistics, Izmir, Turkey.
Adiponectin, a hormone produced predominantly by adipocytes, is an essential modulator of insulin sensitivity and has anti-atherogenic and anti-inflammatory properties. Several studies have been performed to investigate
the association of genetic variations in the adiponectin with obesity, insulin
resistance, and type 2 diabetes (T2D), but few studies were performed in
association with coronary artery disease (CAD). Adiponectin is coded by
ADIPOQ gene located on chromosome 3q27, consisting of 3 exons and 2 introns spanning a 17-kb region. Among the variations of the ADIPOQ gene
reported, rs17300539 (11391G>A), rs2241766 (+45T>G), rs1501299
(+276G>T), and rs2241767 (+349A>G) have been most extensively studied
and are thought to be linked to CAD. In our study, the effects of these polymorphisms in ADIPOQ on CAD were investigated and 125 patients and 123
healthy controls were included. Polymorphisms were screened by PCR-RFLP
technique. Genotyping data and demographic characteristics were analyzed
by the SPSS18.0 program. p<0.05 was considered statistically significant. No
effect of these polymorphisms on CAD was detected in association analysis
under additive, dominant and recessive models (p>0.05). While Genotype
distributions were in Hardy-Weinberg equilibrium (p>0.05) in patient and
control groups, except a deviation observed in both groups for +276G>T
(p<0.05). ADIPOQ and its variants, shown having role in common genetic
background of insulin resistance, T2D and CAD in several populations, had
no effect on CAD in our society. We note that this study was based on a relatively small sample size, which limits the power to detect association. These
results need to be interpreted with caution.
408
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J05.10
Investigation of association between Leu125Val polymorphism of
PECAM-1 gene and coronary heart disease in the Iranian population
M. Bidmeshki, S. M. Seifati;
Department of biology, Ashkezar branch, Islamic Azad University, Ashkezar, Islamic
Republic of Iran.
Coronary heart disease (CHD) is a major cause of death in Iran and many
other countries. CHD is a multifactorial disease, which is probably influenced
by a combination of environmental and genetic factors. Several studies indicate that conditions leading to myocardial infraction and death can be prevented by controlling environmental factors and screening for genetic risk
factors. Atherosclerosis is the most predominant coronary artery pathology
and it seems that Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1)
plays a role in creation of atherosclerotic plaques. In this study, we have investigated the association between Leu125Val polymorphism of PECAM-1
gene and coronary artery disease in Iranian population. Blood samples were
collected from 85 healthy controls and 126 angiographically confirmed CAD
patients with stenosis in at least one coronary artery. After DNA extraction,
the PECAM-1 Leu125Val polymorphism detection was carried out by PCRRFLP method. Frequencies of GG, CC, GC genotypes were 17%, 35%, and
48% in patients and 20%, 35% and 45% in healthy controls, respectively.
Data analysis (p-value= 0.815) revealed no association between Leu125Val
polymorphism of PECAM-1 gene and coronary artery disease. Preliminary
results do not reveal an association between Leu125Val polymorphism and
coronary artery disease. This study is continuing.
J05.11
Association of rs7903146 polymorphism in TCF7L2 gene with
myocardial infarction in T2DM patients in Uzbekistan
The Coronary Heart Disease (CHD) has the similar risk factors with type 2
Diabetes Mellitus (T2DM). A polymorphism in the TCF7L2 gene has been
found to be associated with type 2 diabetes in several ethnic groups. Possible relationship between the genetic polymorphism of the TCF7L2 gene and
CHD was not clear.
We aimed to determine the association between rs7903146 (C/T) polymorphism in TCF7L2 gene with the development of myocardial infarction
(MI) in patients with T2DM in Uzbek population. We genotyped 108 patients divided into 3 groups: I - patients without CHD (n = 26, control); II patients with CHD without MI (n = 42); III - patients with MI (n = 40). All
patients were unrelated, aged over 45 years, and had disease duration over
10 years.
Results of genotyping showed that the CC genotype frequency in I group
was 15.4% and the CC genotype was present in 70% patients of III group.
The frequency of the T allele (83,8%) in patients with MI was significantly
higher compared to the group without CHD (65,4%, P <0.05, control). At the
same, we found significantly lower prevalence of allele C (16,3 4,1%) in
patients with MI compared with the control group (34,6 6,6%, P <0.05)
The results of our study suggest that TCF7L2 rs7903146 polymorphism is
significantly associated with development of MI in patients with T2DM. TT
genotype of TCF7L2 gene may be a predictor of the risk of myocardial infarction in patients with T2DM.
J05.12
GLA nonsense mutation (W162X) and cardiac involvement in
heterozygous females
Background. In the past, medical literature stated that Fabry disease affects
only male. Based on X-linked pattern of inheritance the heterozygous females are usually asymptomatic. Recent studies describe Fabry disease in heterozygous females but manifestations tend to occur at a later age than in males and are often less severe. Objectives. The aim of our study was to detect
the presence of GLA mutation in females of a family with Fabry disease and
corelate with the severity of clinical phenotype. Subjects and methods. Five
related females of the same family were enrolled and clinically assessed. Enzyme activity levels were evaluated too. Genetic testing included isolated
DNA from blood samples and sequence analysis of all coding exons and all
intron-exon boundaries of the GLA gene. Results. All five females were found
to be heterozygous for a familial pathogenic GLA mutation (c.485G>A). The
mutation caused different low levels of enzyme activity in grandmother,
mother, daughter and the two fraternal nieces of the grandmother. None of
Background and aim: Imbalance between coagulation and fibrinolysis factors is known to be a predictor of thrombosis and cardiovascular events. The
aim of our study was to compare the genotype distribution of the coagulation factors FII and FV and fibrinolysis factor PAI-1 functional polymorphisms
in patients with atherothrombotic ischemic stroke and patients with cardioembolic ischemic stroke with atrial fibrillation (AF).
Methods: 54 patients with cardioembolic ischemic stroke with AF and 60
patients with atherothrombotic ischemic stroke from Ukraine were included in this study. The genotypes of FII G20210A, FV G1691A (Leiden) and
PAI-1 5G/4G polymorphisms were determined by PCR analysis based on the
banding pattern on gel electrophoresis.
Results: No individuals with FII and FV homozygous mutations were found
in both study groups. In cardioembolic stroke patients with AF, 1 (2%) FII
and 3 (6%) FV heterozygous mutations were found. In patients with atherothrombotic stroke, 5 (8%) FII and 0 (0%) FV heterozygous mutations were
found. Among patients with cardioembolic ischemic stroke with AF, PAI-1
5G/5G, 5G/4G and 4G/4G genotypes were observed in 9 (17%), 23 (43%)
and 21 (40%), and in atherothrombotic stroke in 14 (24%), 19 (32%) and
26 (44%) patients respectively.
Conclusion: Our findings suggest that there is a tendency toward a higher
frequency of FII G20210A heterozygotes in atherothrombotic ischemic stroke patients compared with cardioembolic ischemic stroke patients with AF.
We also observed that FV Leiden heterozygotes are more frequent in cardioembolic stroke with AF. PAI-1 homozygous state is equivalent between the
two study groups.
J05.14
The importance of genetic profile for thrombophilia detection in
patients with pulmonary venous thromboembolism after operation
for anomalous pulmonary vein return malformations
G. S. Doros, A. V. Popoiu, M. Puiu, A. But, M. Gafencu;
Victor Babes University of Medicine and Pharmacy, Timisoara, Romania, Timisoara,
Romania.
Aim: To present two rare cases of pulmonary thromboembolism in vital vessels, after surgery for cardiac malformations, late discovered with positive
genetic profile for thrombophilia.
Material and methods: A 3 mo old girl presented with signs of cardiogenic
shock and myocardial iskaemia due to totally anomalous pulmonary venous
return (TAPVR) in coronary sinus. The second case was a 2 yo girl, with recurrent wheezing, discovered with right pulmonary veins draining in superior vena cava, a partial anomalous pulmonary veins return (PAPVR). Both
patients were operated.
Results: First case, two years after cardiac surgery presented with discrete
bluish discoloration of the skin, but with normal O2Sat. The pulmonary venous return was redirected to the left atrium. Angio CT detected superior
vena cava thrombosis, with reverse flow into azygos system, and complete
thrombosis of the right venous brachiocephalic trunk. Second case, was operated, redirecting the right pulmonary vein collector to left atrium. After two
years she presented with haemoptisia. Angio CT detected right pulmonary
venous trunk embolism, with pulmonary edema in the right lung. She was
treated with heparin. In both patients we detected positive genetic predisposition to thrombophylia, that changed the recommendation for aticoagulant therapy, for life long.
Conclusions: Patients with operated TAPVR and PAPVR have the risk for pulmonary thromboembolism, which is vital when the collector is redirected
to left atrium.
Genetic profile for thrombophilia is important to be searched in this type of
cardiac malformations. If positive, anticoagulant therapy is life long mandatory, to prevent thromboembolism and even death.
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J05.15
Investigation on Mitochondrial DNA deletions in Iranian Dilated
Cardiomyopathy and Hypertrophic Cardiomyopathy patients
Hypertrophic cardiomyopathy is a genetic disorder with autosomal dominant inheritance. The disorder has been estimated to occur in 0.05%-0.2%
of population. Recently mitochondrial DNA mutations have been associated
with cardiomyopathies. Mitochondria are the major site of energy production in the cell. Thus it is reasonable to assume that energy dependant tissues such as heart, brain, skeletal muscle and endocrine system are affected by
mitochondrial dysfunction. Many mitochondrial diseases arise form defects
of the mitochondrial respiratory chain. mitochondrial disorders have maternal inheritance pattern. The mtDNA mutation rate is much higher than
nuclear DNA due to lack of a repair mechanism and also having no intron.
Recent studies have reported maternally inherited, non-x linked HCMs are
associated with defects in mitochondrial oxidative metabolism. Methods:
In this study we screened 52 Iranian hypertrophic cardiomyopathys patients for mitochondrial DNA deletions. Results: Mitochondrial DNA deletions
were detected by PCR using 6 paired primers. Five different deletions were
found in 29 patients (55.8%). Eighteen patients (34.4%) showed 8.5 kb
deletion. Twelve (23%) patients had 9 kb deletion. Seven patients (13.4%)
had a 7.3 kb deletion. Eight patients (15.5%) had 4977 bp common deletion
between nt8161-nt13640, and 11 patients had 7.4 kb deletion. Multiple deletions have been found in 11 patients (21.1%). Conclusion: Mitochondrial
DNA deletions may occur as a result of aging; on the other hand mt deletions
may affect myocardium and lead to secondary hypertrophy. However, the
question regarding primary or secondary role of mtDNA deletions in hyperthrophic cardiomyopathy remains unanswered.
J05.16
Novel SMAD4 mutation causing juvenile polyposis (JP) and hereditary
haemorrhagic teleangiectasia (HHT)
T. K. Kadiyska1, M. Hachmeriyan2, A. Nossikoff3, L. Angelova2;
1
Genetic Medico-Diagnostic Laboratory Genica, Sofia, Bulgaria, 2University Hospital St.
Marina, Varna, Bulgaria, 3University Hospital Lozenets, Sofia, Bulgaria.
409
410
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Congenital Heart Defects are the most common congenital anomalies with
a worldwide 1 % prevalence and are a big part of childhood mortality and
morbidity. The etiology of conotruncal heart diseases is complex, with both
environmental and genetic causes. Hyperhomocysteinemia which is greatly
accompanied with the defects of folic acid metabolism, is known to cause
conotruncal heart anomalies. In this study we have evaluated three polymorphisms in two hyperhomocysteinemia related genes, such as Methylene
Tetrahydrofolate Reductase (MTHFR C677T and A1298C) and Nicotinamide
N-methyl Transferase (NNMT rs694539) in 79 children with conotruncal
heart disease (CHD) and 99 children without CHD. We found no association
in case and control groups for MTHFR C677T and NNMT rs694539 polymorphisms, while for MTHFR A1298C polymorphism we found a significantly
higher frequency of C allele, suggesting that C allele might be a risk factor
for CHD.
J05.21
Mutation analyses of PTPN11 and SOS1 genes in Belarusian patients
with clinical features of Noonan/LEOPARD syndrome
Noonan syndrome (NS) and LEOPARD syndrome are genetically heterogeneous autosomal dominant disorders associated with gain-of-function
mutations in various genes encoding proteins of the RAS/MAPK signaling
pathway, mainly in PTPN11, SOS1 and RAF1 genes.
8 Belarusian patients with Noonan/LEOPARD syndrome were screened for
mutations in PTPN11 gene: 6 patients with clinical features of NS (2 family
cases with mother-daughter pair, 2 sporadic cases) and 2 patients with clinical features of LEOPARD syndrome (a family with father-daughter transmission). All of the patients had cardiac diseases (pulmonary valve stenosis,
septal defect, cardiomyopathy) and characteristic minor abnormalities.
Leukocyte genomic DNA was amplified for the 15 exons and flanking intronic sequences of PTPN11 gene by PCR. The PCR products were sequenced
from both directions on an ABI3500xl autosequencer.
Four different PTPN11 mutations were identified in 7 out of 8 patients with
clinical features of Noonan/LEOPARD syndrome (4 probands, 3 relatives
available for testing). All mutations were heterozygous missense mutations:
Asn58Lys (new genetic variant c.174C>A), Asp61Gly, Asn308Asp, Thr468Met, and clustered either in exon 3 encoding the N-SH2 domain or in exons
8 and 12 encoding the PTP domain.
One out of 8 patients does not carry mutation in PTPN11 gene. Therefore,
we searched for mutations in the second frequently mutated gene SOS1 undescribed heterozygous mutation (c.797_798delCAinsAAGTA) was found
in exon 6 of SOS1 gene encoding the DH-domain. At the age of 1.6 years old
patient showed typical NS phenotype without ectodermal abnormalities.
Detection of new mutation is important for further delineation of genotypephenotype correlations.
J05.22
IL-6 gene polymorphism (-174G/C) in Romanian patients with
ischemic stroke -results from a pilot study
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Background: Antithrombin (AT) is a serine protease inhibitor that inactivates Factor IIa and Factor Xa. Patients with AT deficiency are at an increased
risk for venous thromboembolism (VTE) and pregnancy loss. Our unit is developing a study to determine risk factors in healthy women who are taking
contraceptive hormone therapy. The case that we present is a 26 years old
healthy woman without a history of thrombosis. Methods: We performed
a genetic analysis of AT Cambridge II (SERPINC1 G13268T; p.Ala384Ser)
using PCR and digestion with PvuII (Biolabs) and was confirmed by sequencing to distinguish it from the AT Cambridge I (G13268C; p.Ala384Pro). AT
plasma levels were normal (108%). Protein C, Protein S, antiphospholipid
antibodies, lupus anticoagulant, Factor V Leiden mutation, F2G20210A and
F12C46T were normal. Results: The results showed a heterozygous pattern
of AT Cambridge mutation. However, sequencing analysis revealed a new
genetic variant within exon 6 of the SERPINC1 gene, A13264T. This variant
is a synonymous mutation and causes a codon change (GCA GCT) but does
not alter the sequence of the gene product of residue Ala382. Conclusions:
This new synonymous mutation Ala382Ala was not detected in 100 healthy
control subjects, suggesting that it is a rare nucleotide variation. Although
the prevalence of this new polymorphism (A13264T) is low, its position
adjacent to the G13268 implies a possible interference with the genetic
diagnosis of AT Cambridge II (G13268T) and AT Cambridge I (G13268C).
Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, RETICS
(RD060014/0016)
J05.26
DNA methylation patterns in coronary heart disease
411
Numerous studies have relied on the idea that atherogenesis has molecular
similarities with cancer. It is possible that genomic microstructural alterations, characteristic of various
neoplasms, are also present in atherosclerotic lesions. In this study we used,
for the first time, array-CGH for detection of DNA copy number variations
(CNVs) and copy-neutral loss of heterozygosity (cnLOH) patterns in patients with coronary heart disease undergoing coronary artery bypass graft
surgery. The SurePrint G3 Human CGH+SNP 2400K microarrays were used
for DNA testing from patients white blood cells (WBC, n=5), right coronary
arteries in the area of atherosclerotic plaques (CAP, n=5), and internal mammary arteries (IMA, n=5). Polyploidy was observed in CAP of one patient. We
detected the multiple CNVs and cn-LOH in all analyzed tissues from patients
with atherosclerosis. Right coronary arteries in the area of atherosclerotic
plaques presented a higher average CNVs length and number of genes located in their vicinity in comparison with other tissues that may support the
notion of higher genomic instability. The majority of CNVs (68-91%) were
identified in disease affected tissues, suggesting that some variations might
represent somatic events. The gains in 3p21.31 (CACNA2D2), 7q32.1 (FLNC),
19p13.3 (PIP5K1C), and 21q22.3 (COL6A1) were detected in the vascular
tissues but not in WBC. We identified gain 7p15.2 (SKAP2) in all tissues that
not overlapping with any CNV regions currently reported in the Database
of Genomic Variants. CnLOH were detected in 12 out of 13 chromosomal
regions involving tumor-suppressor genes, such as SFRP1, CEBPD, RB1CC1,
DIRAS3, TUSC3 and ZDHHC2.
J05.28
The effect of endogenous opioids on apoptosis of cardiomyocytes in a
rat model of cirrhotic cardiomyopathy
I. Jahanzad1, A. Abbasi1, N. Faramarzi2, M. Khosravi3, F. Yazarlou4, M. Abbasi5, A.
Dehpour3;
1
Department of Pathology, Tehran University of Medical Sciences, Tehran, Islamic
Republic of Iran, 2Department of Cardiology, Tehran Heart center, Tehran University
of Medical Sciences, Tehran, Islamic Republic of Iran, 3Department of Pharmacology,
Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran, 4Department
of Medical Genetics, Tehran University of Medical Sciences, Tehran, Islamic Republic of
Iran, 5Department of Internal Medicine, Shahid Beheshti Medical University, Tehran,
Islamic Republic of Iran.
Background: Cirrhosis is known to be associated with various manifestation of cardiovascular dysfunction (cirrhotic cardiomyopathy). Some possible
pathogenic mechanisms has been reported and still more details should be
explored.
Aim: To explore the contribution of endogenous opioids in the apoptosis
process in a rat model of cirrhotic cardiomyopathy.
Material and Methods: Cirrhosis was induced in rats by bile duct ligation
(BDL) and resection. Cardiomyopathy was confirmed using trichrome staining for fibrosis. Naltrexone, an opioid antagonist was administered for 29
1days. Apoptosis was detected using TUNEL assay. For molecular analysis, expression of BCL2, Caspase3, Fas and FasL was explored using reverse
transcriptase real-time PCR.
Results: Left ventricular (LV) wall thickness was significantly (p<0.001)
lower in the BDL group than the sham group, either receiving naltrexone
or saline. Apoptosis density was significantly increased in BDL-saline group
(P <0.001) vs. sham-saline group. Cardiomyocyte apoptosis was significantly decreased in the BDL-naltrexone group compared to BDL-saline group
(P<0.001). There was no significant change in apoptosis density in sham
groups receiving either naltrexone or saline. BDL-saline group showed significant over-expression of BCL2 and FAS and down regulation of caspase3 by a factor of 1.44 (p<0.001) compared to sham-saline group, 1.3 (p<
0.001) compared to BDL-naltrexone group and 0.77 (p< 0.001) compared
to sham-naltrexone group, respectively. No significant change was observed
in the other 4 analysis for BCL2, caspase3 and FAS. Conclusion: Apoptosis
occurs during cirrhotic cardiomyopathy through both intrinsic and extrinsic pathways activation and endogenous opioid receptors blockade using
naltrexone decreases its amount.
J05.29
Association of CELSR2 gene with Coronary artery disease, replication
in Tehran Lipid and Glucose Study (TLGS)
M. Fallah1,2, M. Daneshpour1, A. Ebrahimi1, D. Khalili3, S. Matoo4, R. Mousavi4, F. Azizi5;
1
Cellular and Molecular Endocrine Research Center, Obesity research center, Research
412
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Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran,
Islamic Republic of Iran, 2Kawsar Human Genetics Research Center (KHGRC), Tehran,
Islamic Republic of Iran, 3Prevention of Metabolic Disorders Research Center, Research
Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran,
Iran., Tehran, Islamic Republic of Iran, 4Cellular and Molecular Endocrine Research
Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical
Sciences, Tehran, Islamic Republic of Iran, 5Endocrine Research Center, Research
Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran,
Iran., Tehran, Islamic Republic of Iran.
Human blood vessels vary in their predisposition to develop atherosclerosis. The internal mammary arteries (IMA) are commonly used as the
conduit to bypass major coronary artery stenosis, and have shown greater
long-term resistance to atherosclerosis as compared to saphenous vein (SV)
grafts. To explore differential DNA methylation profile in IMA and SV will
help to further elucidate the mechanism of graft atherosclerosis. Vascular
tissues were obtained from six patients undergoing coronary artery bypass
grafting. Genome-wide DNA methylation analysis in IMA versus matched
SV was performed using the Illumina HumanMethylation 27K BeadChip,
which contains 27,578 probes within 14,475 genes. Using our criteria of
pFDR-adjusted < 0.05 and a minimum median -value difference of 20%, we
identified 337 probes (290 genes) that were significantly differentially methylated. Gene ontology analysis revealed enrichment of biological processes associated with the development. There were four genes (ALDH1A3, DAB2IP, DLX5, WT1) with significant differences in methylation levels of three
and more CpG-sites located inside CpG-islands. In conclusion our analysis
lays the groundwork for further molecular studies of graft atherosclerosis
by identifying novel pathways and genes potentially involved in pathology.
This research was supported by President Grant for the Leading Scientific
Schools of Russian Federation (5096.2014.4).
J06.01
Link of the PPAR gamma and Omentin-1 gene expression in visceral
adipose tissue
M. Nikolaev1, T. Usenko1,2, E. Bazhenova2, X. Zhu2, A. Neimark2, P. Bogdanov2, E.
Baranova2, S. Pchelina1,2;
1
Petersburg Nuclear Physics Institute, St.Petersburg, Russia, St-Petersburg, Russian
Federation, 2Pavlovs State Medical University of Saint-Petersburg, St-Petersburg,
Russian Federation.
X linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disease characterized by progressive demyelination of the central nervous system, adrenocortical insufficiency and elevated levels of very long chain fatty acids
(VLCFAs). It is caused by mutations in ABCD1 gene located at Xq28. More
than 1300 mutations have been identified to date without any genotypephenotype correlation. In this study we report the mutational analysis of 3
X-ALD patients (1 male and 2 females) showing variable clinical spectrum.
In one of the female patients previously reported heterozygous p.W132X
mutation was detected. In the other female patient showed IVS5-6delC
(c.1489-6delC) and p.P543L variations in compound heterozygous state.
The male patient was found to be hemizygous p.R104P mutation that was
not reported previously. In conclusion the cases presented in this paper may
contribute to the mutation and clinical spectrum of X-ALD while defining a
novel mutation and a female case presenting cerebral symptoms.
J06.03
Nutritional variations in pcu children
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413
Introduction: At present the only method for enzyme assay and confirmation of mutations that causing splicing error in CPS 1 and OTC urea cycle
genes is liver biopsy. Therefore developing a noninvasive method for evaluation of gene expression in lymphocytes can be an efficacious method in
diagnosis of urea cycle disorders. Induction of gene expression using Histone deacetylase inhibitors is a usual method. we have assessed the OTC and
CPS 1 expression level after treatment of human lymphocyte cell line with
sodium butyrate.
Material and Methods: MTT assay was done to check cell cytotoxicity. Then
Quantitative assessment of gene expression was done at non-cytotoxic timeconcentrations using SYBR-GreenI Real-time RT-PCR method.
Results: After 48 hours, treatments more than 10 mM, lead to cell injury in
human lymphocyte cell line by increasing cell granulation and decreasing
cell adhesion. MTT assay also confirmed that 1 and 5 mM concentrations
are not cytotoxic. Sodium butyrate significantly increased CPS 1 gene expression (p-value <0.05). This increment was near the expression level in
Hep-G2 cell line specifically at 5 mM concentration after 48 hours. In addition sodium butyrate treatment induced OTC gene expression in human
lymphocyte cell line.
Conclusion: This study showed that sodium butyrate can induce and or increase expression of two urea cycle specific genes (OTC and CPS 1). The most
induction effect occurred in 5 mM treatment after 48 hours. Key words: CPS
1, OTC, Sodium Butyrate
Key words: CPS 1, OTC, Sodium Butyrate, HDACi
J06.08
Cystic fibrosis liver disease-ultrasound evaluation
Background: In cystic fibrosis an important feature is the pancreatic insufficiency expressed by steatorheea; in some cases, despite enzyme supplementation the chronic diarrhea is poorly controlled. Aim study was to
414
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evaluated the presence of cow`s milk allergy among cystic fibrosis children.
Methods: In one year, sixty seven children with cystic fibrosis, aged 1month
-3 years (19 infants), followed in our center were observed for CMPA. Cow`s
milk allergy was suspected in the presence of specific clinical manifestation(
gastrointestinal symptoms, cutaneous signs, respiratory features), in cases
with a suggestive medical history( failure to thrive, colics etc). In addition
CMPA-specific IgE( and lactoglobulin, casein) and diagnostic elimination
test + food challenge test.
Results: Among infants, CMPA was diagnosed in 47.36% (9 patients) of
children, predominantly in pancreatic insufficient children (77% -CF infant
with pancreatic insufficiency). Toddlers (1-3 years) were diagnosed in a
smaller percent, only 16.6 %( 8 children) proved to have CMPA, although
in more than 35% of toddlers, a positive history for CMPA diagnosis was
found. Cystic fibrosis patients with pancreatic insufficiency associated more
frequently CMPA(88.23%) than cystic fibrosis patients pancreatic sufficient.
The prevalence of cow`s milk allergy was important, cumulating a 25.37%
of CF patients.
Conclusion: Cow`s milk allergy was frequently found in CF children, especially associated with pancreatic insufficiency. The combined enteropathy
could influence the disese`s outcome and should be considered especially in
persistent diarrhea of CF children with correct enzyme supplementation.
J06.10
Heme oxygenase microsatellite polymorphism, oxidative stress,
glycemic control in type 2 diabetes Iranian patients
Mitochondrial diseases are highly variable; they can affect several organs
such as the heart in cases of cardiomyopathy, ears deafness, brain in case of
neurodegenerative diseases, and the pancreas in diabetes.... The common
denominator of these diseases is the dysfunction of the respiratory chain,
plays an essential role in the stimulation of insulin secretion and the regulation of blood glucose levels. This deficiency may be due to abnormalities
related to mitochondrial DNA or nuclear DNA. Indeed, the mitochondrion is
semiautonomous which 20 % of the proteins are encoded by the mitochondrial DNA, and 80% by nuclear DNA. We noted the nuclear gene POLG, involved in mitochondrial respiratory chain and responsible of mitochondrial
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DNA replication.
In this context, a Tunisian patient with mitochondrial DNA deletions, suffering from mitochondrial diabetes was studied: searching for mutations
and polymorphisms in POLG gene, by PCR using specific primers followed
by sequencing.
The results showed the complete absence of mutations in exons 13, 14, 19,
20 and 22, however, we note the presence of a heterozygous profile (10/ 11)
of the CAG repeat different cases of normal control, insertion of four bases
c.2734 +37-2734 +38 ins AGGT at intron 17 and a polymorphism described
c.3708 G> T in the heterozygous state (rs3087374) at exon 23.
In this study we want to look if there is any correlation between mutations
found at the nuclear POLG gene and mutations and deletions already found
at the mitochondrial DNA of the patient.
J06.15
Mucolipidosis II presenting with rickets-like features in a newborn
415
Obesity is one of the greatest medical challenges of the 21st century and its
prevalence is continuously increasing. It has a complex determinism that
involves genetic and non-genetic factors.
The aim of this study was to evaluate the impact of several genetic and nongenetic factors on common forms of obesity from Romania.
Unrelated obese patients (n=80) and healthy normoponderal subjects
(n=80) were selected. Biological samples and clinical information was collected after informed consent was obtained. The genetic markers tested
were Insulin -23Hph, IGF2 Apa I, SELL P213S, TGFb C-509T, HSPG BamH1
and IL6 G-174C.
Multiple linear regressions showed a relationship between obesity, cholesterol levels and triglyceridemia. The infection with Torque teno virus did
not show a significant association with obesity. Genetic polymorphisms did
not significantly alter the risk of obesity. However, statistical analysis showed an increased association between several genetic markers and teeth
damage risk (HSPG BamH1, TGFb C-509T, SELL P213S, and IGF2 ApaI) and
2D/4D ratio (IGF2 ApaI) in our subjects.
The complex interplay between genetic and non-genetic factors plays an important role in modulating obesity risk. Nonetheless, further information is
needed to accurately assess the impact of these factors on common forms
of obesity.
J06.18
The importance of NMR Spectroscopy in diagnosis of some inborn
errors of metabolism: lessons from hyperammonemia condition,
galactosemia, and alkaptonuria
Metabonomics is a powerful tool for identifying any disturbances in normal homeostasis of metabolic processes and this emerging fields, in which
a large number of small-molecule metabolites are detected quantitatively in
a single step, promises immense potential for early-diagnosis, monitoring,
and understanding the pathogenesis of many diseases. In many errors of inborn metabolism, the relationship between disease state, metabolic biomarker and genetics is easily understood, but the clinical/ biochemical findings
in some inborn errors of metabolism (IEM) are often nonspecific; an early
differential diagnosis made in a single urinary sample it gives an important
advantage. We present the spectrum of metabolites of urine from 1 year-old
girl with stroke-like episode, elevated transaminases, coagulopathy, being
first interpreted as encephalitis. Beside the clinical presentation and the
hyperammonemia, the fast results gave by urinary NMR-spectrum showing
a high concentration of orotic acid indicates the OTC (ornithine transcarbamilase) deficiency diagnosis. Beside this, we present our results and the
utility of this method for rapid diagnosis and monitoring steps for galactosemia and alkaptonuria. The level of excretion of the metabolites in these
three IEM has been well within the range of NMR detection. In the critical
care setting, IEM that were not diagnosed through the neonatal screening
should be considered as cause of acute neurologic, hepatic/ renal decline,
rapid diagnosis being essential. We demonstrate the effective use of NMRspectroscopic-profiles of urine in differential diagnosis for Urea Cycle Disorders and the possibility of management in other IEM.
J06.19
A novel mutations in Iranian family with Phenylketonuria
416
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J06.20
Phenylalanine hydroxylase (PAH) gene mutation spectrum in patients
with PKU in Karachay-Cherkessia Republic, Russian Federation
A. Stepanova1, S. Amelina2, R. Zinchenko1, A. Polyakov1;
1
Research Centre for Medical Genetics, Moscow, Russian Federation, 2Scientific Research
Institute of Biology Southern Federal University, Rostov-on-Don, Russian Federation.
Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism in Europeans. PKU is classified by the severity of hyperphenylalaninemia in patients. Phenylketonuria is caused by a high variety of mutations in the PAH gene (up to date more than 550 mutations is known).
We have studied 28 PKU-patients from Karachay-Cherkessia Republic,
Russian Federation. For screening of 10 PAH gene mutations (IVS2+5G>A,
IVS2+5G>C, p.L48S, p.E280K, p.R261X, p.R243X, p.R243Q, p.E390G, p.A403V,
p.Y414C) the multiplex system for MLPA PCR-analysis was created. Using
this method we detected mutation p.R261X on 45 (80,3%) chromosomes
and mutation p.L48S on 1 (1,8%) chromosome. The remaining uncharacterized PKU chromosomes were analyzed by scanning the 2, 4, 5, 6, 7, 10, 12
exons and intron/exon junctions of PAH gene by automated sequencing. In
addition three different mutations were found: p.R413P on 5 chromosomes
(8,9%), p.F331S on 2 chromosomes (3,6%) and novel mutation p.P211L on
one chromosome. Mutation p.P211L was not detected in 50 normal controls. A mutation detection rate 96,4% was achieved.
The p.R261X, p.R413P and p.F331S mutations account 92,8% PAH mutations detected in PKU patients from Karachay-Cherkessia Republic Russian
Federation.
J06.21
A Whole mitochondrial genome and POLG gene screening in a
tunisian patient with mitochondrial myopathy and Diabetes
M. M. M. B. Maalej, M. M. M. B. Maalej;
Laboratory of Molecular Human Genetics Faculty of Medicine of Sfax,, Sfax, Tunisia,
Tunisia.
Inherited mitochondrial diseases can be caused by mutations of mitochondrial DNA or of nuclear genes that encode mitochondrial proteins. Although
many mitochondrial disorders are multisystemic, some are tissue specific
.Mitochondria-related myopathies (MM) are a complex and heterogeneous
group of neuromuscular disorders defined by a varying degree of dysfunctions of the mitochondrial respiratory chain (OXPHOS). In the present study,
we performed the clinical, genetic, and molecular characterization of a Tunisian patient with clinical features of mitochondrial myopathy associated
with Diabetes. The analysis of the mtDNA extracted from the blood leucocytes in the studied patient revealed various reported polymorphisms in the
D-loop region, the ribosomal and transfert RNAs but also the coding genes
The detected missense substitutions, especially the 8701A>G(T59A),the
8860A>G (T112A) ,the 10086A>G(N10D),the 13105A>G(I257V) ,the
14766C>T(T7I) were reported in the literature in patients with LHON, Mitochondrial diabetes , Leigh syndrome, cardiomyopathy,muscle pathology
and unaffected individuals .Indeed, we detect the 15940delT in the MT-ATT
was previously described in association with Mitochondrial myopathy .In
addition,we carried out a mutational analysis of POLG1 gene encoding the
mtDNA polymerase gama ,to look for an evnetual implication of nuclear
gene in the mitochondrial diseases .The results of direct sequencing of all
the POLG exons show the presence of the known heterzygote variation
c.2492A>G in exon16 which substitutes the conserved amino acid Tyrosine
to Cysteine (Y831C) described in associtaion with mitochondrial myopathy
The described proband suffers from multisystemic disorders affecting neuromuscular and endocrine organs which could be caused by the association
of these variations.
J06.22
Clinical complexity of Prader Willi Syndrome- genotype-phenotype
correlations
Hyperoxaluria (PH) is an autosomal-recessive disorder of endogenous oxalate synthesis characterized by accumulation of calcium oxalate primarily
in the kidney; and leads to nephrocalcinosis and end-stage renal disease.
A wide spectrum of associated anomalies may be present (cardiovascular;
skeletal; neurological), and there are three forms of primary hyperoxaluria
in which the underlying defects and clinical onset and severity have been
identified.
Its about a 31-years-old man, with history of recurrent urolithiasis for who
chromosomal investigation was carried out to explore a male infertility of 1
year and half related to azoospermic profile at the semen level, varicocele
and microlithiasis on testicular ultrasound. Cytogenetic analysis carried out
using RHG banding, disclose the presence of klinefelter syndrome (47,XXY)
despite the normal morphotype. At the genetic counselling, the patient had
non consanguineous parents and familial history showed similar recurrent
lithiasis in his mother and brother, who was infertile since 2002.
To my knowledge; this is the first case of familial hyperoxaluria; especially
the type IIII (HP3; OMIM #613616); a rare and middle entity with mitochondrial transmission, which is associated with klinefelter syndrome. The last
is often associated with testicular microlithiasis; a rare entity; which may
be the consequence of overproduction of oxalate and the cause of infertility.
Thus, delineation of diagnostic both by genetic and histopathological screening is mandatory in order to clarify the genotype phenotype correlation.
Therefore; we insist in the comprehensive evaluation of infertility at cytogenetic (chromosomes abnormalities?) and molecular level (mosaicism?),
since its a heterogeneous and complex disease.
J06.24
Mutations of mitochondrial genome and atherosclerosis of coronary
and carotid arteries.
L. Egorova, A. Postnov, M. Ezhov, I. Sobenin;
Cardiology Research Center, Moscow, Russian Federation.
Aim. To evaluate the association between the level of heteroplasmy for mitochondrial mutations C3256T, G13513A, G14846A, G12315A in human
white blood cells and presence of atherosclerosis in coronary and carotid
arteries.
Methods. We included 130 patients (mean age 559 years, 116 men) with
coronary heart disease (CHD) verified by angiography. Control group consisted of 63 subjects without angiographic coronary artery disease. DNA
samples were obtained from whole venous blood using commercially
available kits for DNA extraction. For the amplification of fragments of mitochondrial DNA by polymerase chain reaction method followed by pyrosequencing, the corresponding primers and conditions were used. On the
basis of pyrosequencing data analysis, the level of heteroplasmy for C3256T,
G13513A, G14846A, G12315A mutations in DNA samples were calculated.
Results. The level of G13513A and C3256T heteroplasmy was significantly
higher (p=0.03; p=0.01, respectively) and level of G12315A heteroplasmy
was lower (p=0.004) in CHD patients versus control group. There was significant correlation of carotid atherosclerosis severity and levels of 3256
(r=0.49, p=0.0001), G14846A (r=0.48, p=0.0001) and G12315A heteroplasmy (r= -0.32, p=0.01). Level of G14846A heteroplasmy was higher in subjects older than 45 years. There was no any relation of mitochondrial genome mutations and smoking, hypertension, CHD family history. Presence
of hyperlipidemia was positively related to C3256T heteroplasmy (r=0.18,
p=0.01) and negatively associated with G12315A heteroplasmy (r= -0,2,
=0,005). There was positive significant correlation between lipoprotein(a)
level and G14846A (r= 0,23, =0,01).
Conclusion. We found independent positive correlation of mutations 3256T,
G13513A and G14846A with both coronary and carotid atherosclerosis.
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J06.25
Assessing the implication of genetic and non-genetic factors in type
one diabetes mellitus
C. Cristescu1, E. Rusu1, V. Cristescu1, S. Spandole2, M. Toma2, I. Radu2, D. Cimponeriu2;
1
Titu Maiorescu University, School of Medicine and Pharmacy, Bucharest, Romania,
2
Department of Genetics, University of Bucharest, Bucharest, Romania.
417
Introduction:Glycogen storage disease(GSD) is a metabolism disorder resulting in glycogen accumulation in hepatocytes and in development of the
secondary mitochondrial disfunction.
Aim:to evaluate functional activity of lymphocytes mitochondria in children
with genetically diagnosed GSD of Ia and Ib type.
Materials and methods:23 patients with GSD were examined,9 of them-with
Ia type,14-with Ib type.34 nominally healthy children composed the control
group.
The functional activity of mitochondria was estimated by
succinatedehydrogenase(SDH) activity, which was identified in the general
lymphocytes population,-in T-lymphocytes,B-lymphocytes and Nk-cells by
flow cytometry method(FC500).
Results:In patients with GSD of Ia type there were determined
mutations:.247>T, c.883C>T of G6PC gene. In patients with Ib
GSD, mutations of the gene SLC37A4:.1042_1043delCT , c.345insG,
c.411G>A,c.413G>A,c.528delG, as well as mutations non-reported
before:c.1016G>T(in 3 patients),c.817G>A(in 1 patient) were identified.
In all children with I type GSD,decrease of SDH activity in the general lymphocytes population in comparison with the norm was
revealed(p<0,02,Kolmogorov-Smirnov test).
In patients with Ib type, characterized by more severe disease course, there
was detected greater decrease of mitochondria functional activity than in
children with Ia type(p<0,05).
SDH activity analysis in lymphocytes population showed 40% decrease in
B-cells and 18 % in T-lf regarding the norm, with 20 % SDH activity increase
in Nk-cells and NkT-lymphocytes compared with the norm.
Conclusions:Disfunctions of mitochondrial apparatus, more prominent in
patients with Ib type, were revealed in patients with Ia and Ib GSD. SDH
lymphocytes activity analysis may be used as additional diagnostic criterion
for evaluation of the condition severity of children with GSD.
J07.01
The Importance of Test Variability in Acute Myeloblastic Leukemia
and the Comparison of Different Parameters in Follow-up of Minimal
Residual Disease
A. Ozturk Kaymak, . Snmez, S. ztomurcuk, N. Talayhan, E. zcan, M. Din, G.
Gnta;
Department of Medical Genetics Ministry of Health, Dr. A.Y. Demetevler Oncology
Training and Researc, Ankara, Turkey.
418
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and also from the capillary electrophoresis. From these three methods the
one which has the highest sensitivity is RT-PCR, then in the second place capillary electrophoresis comes and after that conventional PCR comes. From
the point of laboratory since the sensitivities of the methods are different,
studying with several methods for the follow-up of minimal residual disease
might be required. Additionally with this case, it is attempted to emphasize
the importance of examination of all chromosomal anomalies along with
known gene mutations in newly diagnosed AML patients from the aspect of
diagnosis, follow-up and prognosis of the patients.
J07.02
BIOMED-2 Protocols: The Best Diagnostic Tool for Suspicious
Lymphoma Malignancies
B cell non-Hodgkin lymphomas (B-NHL) are generally considered as aggressive malignancies which originate from of the transformed B cell. BIOMED-2
multiplex PCR has been suggested as a gold standard method for the detection of monoclonality in the immune cell system in hemato-malignancies
and lymphoproliferative disorders. Molecular clonality (assays?) were performs based on the patterns of gene rearrangements in the immunoglobulin chains loci. During B cell malignancies, immunoglobulin recombinant
constructions are produced exclusively, and are applied as diagnostic biomarkers in B-NHL. Here, we used the BIOMED-2 protocol to reveal the diagnostic value of immunoglobulin light chains (Ig, Ig) and incomplete IGH
D-J monoclonal gene rearrangements on FFPE samples. The study was performed on 70 patients with B-NHL which were previously assessed for IGH
monoclonality and failure to clarify rearrangements. Our results revealed
positive clonality in 62 out of 70 (~89%) cases in the Ig and Ig analysis.
The samples with positive clonality included 41.4% for Ig and 47.2% for
Ig. However, our investigation on FFPE tissue revealed that EuroClonality
BIOMED-2 protocols could be considered as a valuable and reliable method
for clonality detection especially in failure of the IGH analysis. In general,
clonal Ig gene rearrangement assays are applicable for the diagnosis of lymphoproliferative disorders and are a distinguishable method for differentiating between malignant and benign lymphoma disorders. Furthermore, we
are able to implement the BIOMED-2 protocol as a routine diagnostic tool
for hemato-malignancies.
J07.03
IGH Clonality Detection in Lymphoma Malignancies of an Iranian
Population Using BIOMED-2 Protocols
Down syndrome is by far the most common best known chromosomal disorders in human. It expresses multiple systemic complications with both
structural and functional defects as a part of the clinical manifestation. Mechanism of immune changes occurring in Down Syndrome is complex and
include extra gene copy of chromosome 21 and secondary dysregulation of
numerous intercellular interactions. Recent studies suggest a role of IL-17
proinflammatory cytokine located on 6p12 chromosome in the pathogenesis of autoimmune diseases. Here we aimed to analyze IL17A gene expression in peripheral white cells and interleukin-17A intracellular expression on
CD4+ T-cells. The research was carried out on the group of 58 children aged
6 to 12 including the group of 30 children with Down Syndrome (the simple trisomy of chromosome 21 only) and the reference group of 28 healthy
children. We evaluated gene IL17A expression using real-time PCR and intracellular IL-17A analyzed by flow cytometry. We revealed significantly decreased gene expression on white cells and significantly decreased expression on IL-17A levels on CD4+ T-cells in Down Syndrome. Our data indicate
that decreased IL-17A expression may play significant role in etiology of
autoimmune diseases in Down Syndrome. Moreover, we demonstrated that
in Down Syndrome the other gene located outside the extra chromosome
21 is also affected.
J07.05
Familial autoimmune Lymphoproliferative Syndrome caused by
homozygous FAS Ligand (FASLG)mutation mimicking Familial
Hemophagocytic Lymphohistiocytosis(FHL)
The von Willebrand factor circulates in plasma in a complex with coagulation factor VIII joined by noncovalent bonds. This interaction prevents enzymatic degradation of factor VIII (FVIII) and ensures its transport to the
place of the fibrin clot formation. The factor VIII is transformed to factor
VIIIa (activated) and acts as a co-factor for Factor IX activated (FIXa), which
will continue activating next step in the blood clotting cascade. Because of
their close relationship, decreasing activity of one or another factor may be
affected, or they may also affect the other. The late generates a clinical diagnosis not very accurate, sometimes for Haemophilia A or von Willebrand
Back to index
disease. We report here a Colombian family that suffers the two diseases
according to clinical diagnosis. However, the lack of a genetic study to verify
and contrast the diagnosis made by health institutions which is based on
phenotype only, can lead to a wrong classification of von Willebrand disease
as a type of mild Haemophilia. The aim of this study was to confirm the clinical diagnosis in this family by molecular analysis. To achieve this, we identify
the presence of the most common inversions of FVIII gene in introns 22 and
1 by LD - PCR and a general scan of the gene for frameshift mutations or stop
codons through three family generations. The use of molecular techniques
to confirm the clinical diagnosis for bleeding disorders will improve adequate treatment and patient prognosis in Colombia.
J07.07
Association between vitamin D receptor gene polymorphisms and
Hashimotos thyroiditis in Serbian population: a pilot study based on
FokI, ApaI and TaqI RFLP technique
J. Djurovic1, O. Stojkovic1, C. Akurut2, F. Silan2, J. Todorovic3, G. Stamenkovic4, O.
Ozdemir2;
1
Institute of Forensic Medicine, Faculty of Medicine, University of Belgrade, Belgrade,
Serbia, 2Department of Medical Genetics, Faculty of Medicine, Canakkale Onsekiz Mart
University, Canakkale, Turkey, 3Private consulting room Thyreomedicus, Belgrade,
Serbia, 4Institute of biological research Sinisa Stankovic, University of Belgrade,
Belgrade, Serbia.
Aim: Hashimotos thyroiditis (HT) is the most prevalent autoimmune thyroid disorder caused by an interaction between genes and environmental
triggers. Intra-thyroid lymphocytic infiltration may lead to progressive destruction of thyroid tissue and consequently hypothyroidism. Many studies
in other populations have shown association between vitamin D receptor
(VDR) gene polymorphisms and various autoimmune diseases, including
HT. Methods: The study included 44 female patients (mean age standard
deviation 385.4) with Hashimotos thyroiditis and 32 healthy controls of
age-, sex- and geographically matched adult without personal history of
autoimmune and endocrine diseases. Genomic DNA was isolated from peripheral blood-EDTA, and target VDR gene was genotyped by PCR-RFLP
technique after VDR-FokI (rs2228570), VDR-TaqI (rs731236) and VDR-ApaI
(rs7975232) restriction enzymes digestion. Results: We use Arlequin 3.5
integrated software for population genetics data analysis and found significant difference in the genotype distribution of VDR FokI polymorphism
between HT patients and controls (P=0,00465). For ApaI and TaqI we found
the higher frequency of variant allele but not significantly different compared to control women (p>0,05), which is consistent with previous studies.
Conclussion: The current first and preliminary results identified the association between VDR- FokI gene polymorphism and Hashimotos thyroiditis
in the Serbian population. Results need to be supported by further investigations that define haplotypes patterns for VDR gene polymorphisms in a
larger group of HT patients of both sexes.
J07.08
A Rare Alpha-Globin Mutation is Associated with Early-Onset
Hemochromatosis with HFE C282Y Homozygosity, but not C282Y/
H63D Compound Heterozygosity
A 30 year-old Caucasian man presented with significant iron overload (ferritin 3287, transferrin saturation 87%) and was found to be homozygous for
the C282Y HFE mutation. He concomitantly had mild microcytic anemia, and
was found to have a missense mutation in HBA2 (c.242T>G). This mutation is
known to produce an unstable hemoglobin (called Hemoglobin Ann Arbor)
and cause mild, chronic hemolytic anemia in the heterozygous state.
The patients mother also carries the Ann Arbor mutation. Interestingly, she is
a C282Y/H63D HFE compound heterozygote. However, at age 55, she showed
no signs of hemochromatosis (ferritin 197, transferrin saturation 40%,).
Severe iron overload has been reported as a complication of specific erythroid
disorders (including sideroblastosis and porphyrias) in the context of HFE
homozygosity (and questionably C282Y/H63D compound heterozygosity).
However, to our knowledge, no cases of HFE mutations in combination with
alpha-globin mutations or deletions have been reported.
We propose that the instablility of hemoglobin Ann Arbor leads to increased
erythropoetic activity and enhanced iron absorption, which predisposed
this C282Y HFE homozygote to atypically early-onset hemochromatosis. The
mothers normal iron profile is consistent with the weakly-penetrant C282Y/
H63D genotype, however it is noteworthy that in contrast to her C282Y homozygous son, HFE compound heterozygosity in combination with the Ann
Arbor mutation did not result in early-onset hemochromatosis in this individual.
419
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J07.09
Large deletions of SERPING1/C1NH gene in Russian patients with
hereditary angioedema
J07.12
ITPA gene variant protects against combination treatment-induced
anemia in Ukrainian patients with chronic hepatitis C
J07.10
Single nucleotide polymorphisms in cytokines genes promoters are
associated with the susceptibility to HIV-1 infection in the Ukrainians
A. Piddubna;
Sumy State University, Sumy, Ukraine.
Background. Cytokines genes single nucleotide polymorphisms (SNPs) involved in the vulnerability to HIV infection in different population groups.
We aimed to determine whether the carriage of cytokines genes allele variants influence the risk HIV-1 infection in Caucasian Ukrainians.
Methods. We examined promoter SNPs in IL-4 (rs 2243250), IL-10 (rs
1800872), TNF- (rs 1800629) among 78 HIV-1 infected European Ukrainians (68 % male, 32 % female; age at diagnosis (33,350,76) years), 22
HIV-negative persons from the high risk infection group and 100 healthy
controls using PCR-RFLP.
Results. The dominant cytokines genes variants among HIV-1 infected
Ukrainians were major allele homozygotes that correspond to controls and
the high risk infection group (C/C IL-4 - 62.82 %, C/C IL-10 - 53.85 %, G/G
TNF- - 62.82 %). T/T IL-4 and G/A TNF- genotype variants significantly
overrepresented in people with HIV-1 (p0.01-0.05); susceptibility to HIV-1
infection does not depend on gender. IL-10 minor allele distribution showed
the difference among study groups: A/A variant was associated with the disease in men (p0.05). We found that carriage of IL-10 homozygous major
allele genotype had protector effect on risk of HIV-1 infection among male
population (p0.05).
Conclusions. The first report of cytokines genes allele frequencies in Ukrainian population shows their association with susceptibility to HIV-1 infection and suggests further research in the field of host genetic risk factors.
J07.11
Association between interleukin-1 type I receptor gene
polymorphisms and the expression level of membrane-bound
receptors
420
J07.13
Combination gene therapy with Mcl-1 and survivin siRNA inhibits HL60 malignant leukemia cell growth in vitro
The over-expression of the myeloid cell leukemia-1 (Mcl-1) gene and survivin gene are associated with the survival and progression of various malignancies including leukemia. The aim of this study was to explore the effects
of Mcl-1 and survivin small interference RNA (siRNA) on the proliferation
and apoptosis of HL-60 acute myeloid leukemia (AML) cells. siRNA transfection was performed using a liposome approach. Relative mRNA and protein expressions were quantified by quantitative real-time PCR and Western
blotting, respectively. Trypan blue assay was performed to assess tumor
cell proliferation after siRNA transfection. The cytotoxic effect of siRNAs on
leukemic cells was measured using MTT assay. Apoptosis was also detected
by the annexin V/PI double-staining method. siRNAs clearly lowered both
Mcl-1 and survivin expression levels in a time-dependent manner, resulting
in marked inhibition of cell survival and proliferation. Furthermore, siRNA
co-transfection significantly enhanced the extent of HL-60 apoptotic cells
relative to single transfection.Our study demonstrated that Mcl-1 and survivin siRNAs could exert antileukemic effects in vitro. It suggested that combinatory gene therapy targeting Mcl-1 and survivin could be used as a new
strategy in the gene therapy of AML.
J07.14
Beta-defensin targeting by using anti-cytokine antibodies as a
therapeutic approach in psoriasis
M. Ghorbani, A. Sedighi;
Islamic azad University of pharmaceutical science, Tehran, Islamic Republic of Iran.
Back to index
J07.17
Hereditary thrombophilia and pregnancy. Impact on quality of life of
patients and their families
Glanzmann thrombasthenia (GT) is a rare abnormality of platelet aggregation with quantitative and/or qualitative abnormality of IIb3 integrin that
is encoded by ITGA2B and ITGB3 genes. It is more prevalent in some Middle
East population including Iraq, Jordan and Iran. Many different mutations
including point mutations, deletions, duplications and in some cases large
deletions have been reported in these genes.
In this study we screened the responsible genes (ITGA2B or ITGB3) by
linked STRs that were followed by direct sequencing of the entire coding
region and exon-intron boundaries of the candidate gene and /or deletion
investigation by Long PCR.
From 12 families with an affected child who were referred for mutation detection to our Lab, after initial screening with STRs, 2 cases that were linked
to ITGA2B gene showed no point mutation. Investigating the region by Long
PCR revealed a homozygous large deletion in exon 2 and 3 of both unrelated
affected cases that has not been reported yet. Deletion was present in the
affected children parents in heterozygote state.
As GT is a rare disorder and direct sequencing of two genes with 45 exons
could increase the test cost, screening with indirect methods like STR can be
useful for more rapid and cost-benefit diagnosis.
J07.19
SYBR GreenI RT-PCR for Quantitative Detection of TLR4 Gene Level in
Colorectal Cancer Cells
421
Wingless-Int (Wnt) signaling pathway role in embryonic development, proliferation, survival and differentiation of hematopoietic stem cells is already
established. There is strong evidence regarding involvement of defects in
the Wnt signaling pathway in several solid tumors such as breast, colorectal
and prostate cancer. Wnt receptors promote the activation of canonical
Wnt /-catenin and downstream Lymphoid enhancer factor 1/T-cell factor
(LEF1/TCF) pathway influencing genes expression of cell proliferation, differentiation and apoptosis. Our proposal was to examine gene expression
levels of LEF 1 in peripheral blood of triple negative breast cancer (ER negative, PR negative, Her2 negative) patients by qRT-PCR in association with
immunohistochemistry and clinicopathological data in order to establish
the modulation effect of LEF 1 in triple negative breast cancer circulating
immune cells. Our study included analysis performed on peripheral blood
leukocytes collected in EDTA tubes of 16 triple negative breast cancer patients and 7 healthy donors correlated with immunohistochemistry and clinicopathological data. After analysing qRT-PCR data of the expression level
of LEF 1 gene, we obtained significant statistical results that revealed LEF 1
downregulation in peripheral blood immune cells of triple negative breast
patients compared to control healthy donors peripheral blood. There are
evidence-based data that reveals elevated levels of LEF 1 in breast cancer
cell lines but considering our analysis describes LEF1 downregulation, a
difference of expression signature may arise due to the peripheral blood immune cells different environment implication.
J07.21
Elucidating the microRNA transcriptomic footprint in chronic
lymphocytic leukemia
422
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evolution of CLL.
Moreover, GES and WES data are used to study the effect of SNVs and CNVs
on microRNA expression and processing. Interestingly, expression levels of
microRNA pairs within clusters are correlated, suggesting that structural
variations in the CLL genome might indeed comprise the loss of microRNAs
located in this region.
J07.22
Hemophagocytic lymphohistiocytosis developed in visceral
leishmaniasis may have genetic etiology
Hemophagocytic lymphohistiocytosis (HLH) is a complex immune disregulation disorder developed either by genetic defects in Perforin, UNC13D,
Syntaxin 11, STXBP2 genes or secondary to various infections, other disorders, drugs. Distinction is complicated since infections; viral in particular, can trigger genetic HLH. HLH has overlapping clinical symptoms with
visceral leishmaniasis (VL) and may remain under-recognized because of
difficulties in diagnosis and rapidly fatal outcome. Genetic HLH triggered by
VL has not been described. One-year-old girl from a consanguineous family
diagnosed as VL by showing Leishmania amastigots in bone marrow aspirates (BMA). Symptoms also fulfilling HLH diagnostic criteria were resolved
by therapy for VL initially but reappeared subsequently despite clearance of
amastigots in BMA. HLH2004 protocol therapy achieved remission however
recurrence observed afterwards. Mutation analysis in HLH genes revealed
homozygous 627delT frameshift mutation in exon 8 of UNC13D gene leading to frameshift and premature termination of translation 40 amino acids
downstream. Parents were heterozygous for this single nucleotide deletion.
Stem cell donor was not available. Until she died at age four, remissions were
followed by HLH reactivations and/or five central nervous system relapses
under continuous protocol therapy. In conclusion, HLH developed in VL may
have genetic etiology and VL may trigger underlying genetic HLH, which
may make diagnosis and therapy even more complicated and mortality rate
very high. Awareness created among clinicians might thereafter have important public health impact in lifesavings, disease surveillance and eradication especially in endemic tropical and subtropical countries. This study was
supported by TUBITAK (Project No: 105S386-SBAG 3193).
J07.23
Identification of two novel missense mutations on C2 and A3 domain
of factor VIII
S. Ghasri1, S. Sahebi1, F. Zafarghandi Motlagh1, M. Fallah2,1, S. Zeinali3,1;
1
Kawsar Human Genetics Research Center (KHGRC), Tehran, Islamic Republic of Iran,
2
Cellular and Molecular Endocrine Research Center, Research Institute For Endocrine
Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Islamic Republic of
Iran, 3Department of Molecular Medicine, Biotech Research Center, Pasteur Institute of
Iran, Tehran, Iran, Tehran, Islamic Republic of Iran.
This abstract describes the discovery of the gene underlying the Vel blood
group system, the last clinically important unresolved human blood group
system. The Vel antigen is present on red blood cells (RBCs) from all humans except rare Vel-negative individuals who can form antibodies to Vel
in response to transfusion or pregnancy. These antibodies may cause severe hemolytic reactions in blood recipients. We combined SNP profiling and
transcriptional network modeling to link the Vel-negative phenotype to
SMIM1, located in a 97-kb haplotype block on chromosome 1p36. This gene
encodes a previously undiscovered, evolutionarily conserved transmembrane protein expressed on RBCs. Notably, 35 of 35 Vel-negative individuals
were homozygous for a frameshift deletion of 17 bp in exon 3. Functional
studies using antibodies raised against SMIM1 peptides confirmed a null
phenotype in RBC membranes, and SMIM1 overexpression induced Vel expression. Genotype screening estimated that ~1 of 17 Swedish blood donors
is a heterozygous deletion carrier and ~1 of 1,200 is a homozygous deletion knockout and enabled identification of Vel-negative donors. Our results
establish SMIM1 as a new erythroid gene and Vel as a new blood group system. (Nature Genetics 2013; 45(5):537-541)
J07.25
De novo homozygous mutation of the C1 inhibitor gene in a patient
with Hereditary Angioedema
M. Yakoreva1,2, E. iglane-Shlik3,4;
1
Tartu University Hospital, United Laboratories, Department of Genetics, Tartu, Estonia,
2
University of Tartu, Institute of Biomedicine and Translational Medicine, Department
of Human Biology and Genetics, Tartu, Estonia, 3Childrens Clinic, Tartu University
Hospital, Tartu, Estonia, 4Department of Pediatrics, Faculty of Medicine, University of
Tartu, Tartu, Estonia.
Back to index
ping deletions, varying from 570 kb to 6.9 Mb. Most of them have single 3.6
Mb overlapping region (chr2: 58,747,888-62,363,205 Hg19) that contains
23 genes. Here we describe an Estonian patient carrying a 1.2Mb deletion
inside this critical region, who presented milder phenotype of the syndrome. Detecteddeletion (chr2: 61,178,171-62,376,313 Hg19) contains 14 of
23 genes from the critical region. Unlike previously described cases, our patient is of normal height, and does not have microcephaly, craniosynostosis,
structural brain abnormalities and vision or hearing problems. At the age of
4 years, he is slightly dysmorphic without characteristic facial appearance
and has moderate delay in intellectual and language development. Also, he
is slightly spastic and up to age three, he had problems with chewing solid
foods. We thus assume that genes in this deleted region (AHSA2, C2orf74,
CCT4, COMMD1, FAM161A, KIAA1841, LOC100130280, LOC339803,
LOC647077, PEX13, PUS10, SNORA70B, USP34, XPO1) could not be responsible for growth retardation, microcephaly, skull and brain abnormalities in
the patients with 2p15p16.1 microdeletion syndrome and may be candidates for developmental and language delay.
J08.02
Deletion of 3q11.2 region detected by arrayCGH in a girl with an
Angelman syndrome-like phenotype
423
The array Comparative Genomic Hybridization technology (aCGH) is intensively used in research and clinical diagnosis for the detection and identification of genome rearrangements in patients with different physical/intellectual development anomalies.
The aim of our study was to apply the aCGH technology in evaluation with
high resolution of the entire genome in pediatric patients with developmental delay (DD), intellectual disabilities (ID) and dysmorphic features (DF) for
identifying the genetic causes responsible for the
clinical observation.
Twenty patients with DD, ID and DF from Romania, aged between 4 months
and 14 years, were included in high resolution aCGH analysis with NimbleGen ISCA Plus 3x1.4M Platform (Roche). All patients were previously assessed by conventional cytogenetic analysis.
The aCGH analysis detected clinically relevant chromosomal abnormalities
in seven of the patients analyzed, as follows:
- micro-deletions ranging between 2.5 Kb-6 Mb, associated with Pitt-Hopkins syndrome (18q21.2), obesity, ID/DF (6q16.1-q16.2-q16.3), DiGeorge
syndrome (22q11.2);
- micro-duplications ranging between 12.5 Kb-1.9 Mb, associated with ID
(17q23.2), the Potocki-Lupski rare syndrome (17p12-11.2) and autism
(Xp22.11);
- a 30 MB duplication (8q13.3-q22.3), leading to the identification and exact
characterisation of a marker chromosome indicated by conventional cytogenetic analysis.
Genomic anomalies identified and characterized by aCGH provided accurate
diagnosis of unidentified or unexplained diseases suspected to have a genetic cause, contributing to appropriate clinical management of the affected
patients.
Our study demonstrated that aCGH technology can play a very important
role in identification and characterization of cryptic and/or complex chromosomal rearrangements, being a valuable tool in postnatal diagnosis.
J08.05
Novel alteration in AMPD2 gene segregates with non-syndromic
intellectual disability linked to MRT4 locus, conjointly responsible
from Pontocerebellar hypoplasia
424
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context. We conclude that our case will expand the phenotypic spectrum of
damaging AMPD2 mutations.
J08.06
Array CGH findings in 280 cases with intellectual and developmental
delay, multiple congenital anomalies and autism spectrum disorders
in Iran
R. Kariminejad, K. Najafi, A. Moshtagh, G. Abbassi, N. Sadatian, K. Najafi, A.
Kariminejad, M. Kariminejad;
Kariminejad-Najmabadi Pathology and Genetics Center, 14665/154, Tehran, Islamic
Republic of Iran.
The use of high resolution array-based comparative genomic hybridization (array-CGH) in whole genome investigation led to an unprecedented
rate of discovery of clinically relevant microdeletions/microduplications in
neurodevelopmental disorders. We report on a 12-year-old boy with facial
dysmorphic features, autism (Asperger syndrome), motor and vocal tics,
speech disorder in early childhood presenting a 9q34.11 - q34.12 deletion.
Cytogenetic investigations included array-CGH investigation (105K platform, Agilent Technologies) and FISH (BAC probes). Array-CGH revealed a
de novo 2.4 Mb deletion spanning cytogenetic bands 9q34.11-q34.12 (genomic coordinates, hg19: 131485878-133931680). Approximately 50 genes
are located within the deleted region, some of these being disease-causing
OMIM genes: AGM5, CDG1M, TOR1A, ASS1, ABL1. Deletions of genomic region 9q34.11-q34.13, proximal to EHMT1, are rarely described. Few patients with deletions similar to our case have been reported in the literature
and centralized in databases (e.g. DECIPHER). Haploinsufficiency of genes
located in this region might be responsible for the clinical findings in the
reported patients (such as autism, intellectual disability, epilepsy, movement disorders etc). Thus our case adds to the body of knowledge and will
help in establishing genotype-phenotype correlations. Acknowledgments:
Professor Jean-Michel Dupont from Cochin Hospital, Paris, France for kindly
providing the BAC-FISH probe; Project PN 09.33.02.03.
J08.08
A clinical case with autism and 22q13.3 deletion or Phelan McDermid
syndrome
B. Radeva1, M. Stancheva-Ivanova2, P. Mitev3, R. Staneva4;
1
Medical Center Childens health, Sofia, Bulgaria, 2University Hospital for Active
Treatment in Neurology and Psychiatry St.Naum, Sofia, Bulgaria, 3Medical Center
Children`s Health, Sofia, Bulgaria, 4Chair of Medical genetics, Medical University, Sofia,
Bulgaria.
Rare autosomal recessive Bardet-Biedl Syndrome (BBS) is a kind of pleiotropic ciliopathies which is more common in developing countries. BBS is characterized by symptoms including obesity, retinitis pigmentosa, polydactyly,
learning problems, hypogonadism and kidney abnormalities that vary both
within and between families. Nineteen disease-causing genes are involved
in 80% of BBS cases and code proteins localized to the cilia which each function affect different parts of a cilium. BBS2 in contribution with six most
conserved BBS genes, form BBSome complex of the cilium.
Here, we report an Iranian family with a ciliopathy disorder ascribing BBS.
Whole exome sequencing (WES) was performed for proband which revealed a novel homozygote splice mutation c.535-1 G>C in BBS2 gene that
would probably lead to skipping of exon 5. Using bioinformatics software,
Mutation Taster, predicts c.535-1 G>C as a disease causing variant. WES data
were confirmed by Sanger sequencing and co-segregation was performed
for his family.
Estimating about 8% of BBS reports, BBS2 is among one of the common
genes in BBS (same results in Iranian population-unpublished data). This
result was in accordance with our expectations. In conclusion, due to the
clinical and genetic heterogeneity observed in Bardet-Biedl syndrome WES
could be considered as the method of choice for clinical genetics practice.
J08.10
Further characterization of the 16p11.2 microdeletion phenotypes
Introduction: Recurrent rearrangements of 16p11.2 are increasingly recognized as one of the most common structural chromosome disorders. Reciprocal 16p13.1 rearrangements predispose to developmental delay. Besides,
16p11.2 rearrangements have been identified in up to 1% of autistic individuals. These deletions have also been associated with obesity. A ~550-kb
deletion is commonly reported; and a smaller deletion of an adjacent region
on 16p11.2 has also been found in patients with overlapping clinical features. The phenotypic spectrum of rearrangements in this genomic region
remains to be fully characterized. Methods: We describe two new unrelated
cases with 16p11.2 microdeletions diagnosed by whole genome oligonucleotide array-CGH. We also compare the phenotype of our patients with
other cases described in the literature. Results: One patient, referred to our
clinic with moderate developmental delay, significant behavioral problems
and unspecific dysmorphisms, has the typical ~550-kb deletion. There is
a known family history of learning disabilities. However it was not possible
to test his relatives because he was placed in institutional care. The second
patient presents developmental delay and unspecific dysmorphisms. He has
a maternally inherited 187-kb 16p11.2 deletion. His mother has mild cognitive difficulties and obesity. Conclusions: Our patients present adjacent, no-
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Introduction and Objectives: Fragile X syndrome (FXS) is a neurodevelopmental disorder affecting intelligence and behaviour. Current treatments are
unable to normalize these symptoms. We propose a combination of Ascorbic
acid and Alpha-tocopherol to improve learning abilities in young patients.
Material and Methods: 100 FX patients (50 in placebo): A) 3 to 6 years old
(N= 19), B) 6 to 17 years old (N= 64), C) older than 18 years old (N= 17).
A clinical questionnaire and neuropsychological tests was performed at the
beginning of the trial (T0) and 12 weeks later (T1). Variables: Wechsler intelligence scale for children (WISC-R), manipulative and verbal subscales
and Peabody Picture Vocabulary Test (PPVT-R). The percentage of change
were tested by U Mann Whitney Test (p<0.05)
Results: Significant improvements were detected in group B (6 to 17 years
old) with 65% potency. The percentage of change in the Verbal WISC-R scales were: 30.8 in the treated group versus 13.7 in the placebo group (P<
0.05). The percentage of change in the manipulative WISC-R scales were:
35.4 in the treated group versus 10.9 in the placebo group, P= 0.01. A significant improvement was observed in the percentage of change in the Peabody median scores: 24.4 in the treated group versus 6.8 in the placebo group
(P<0,05)
Conclusions: Clinical trials for FXS are necessary. Our results demonstrate
improvement in learning and receptive language in children after 12 weeks
of treatment with a combination of antioxidants: Ascorbic acid and Alphatocopherol.
J08.12
Submicroscopic chromosomal alterations in individuals with
idiopathic intellectual disability
425
A. Alkhateeb1, S. Aburahma2;
1
Qatar Biomedical Research Institute, Doha, Qatar, 2Jordan U of Science and Technology,
Irbid, Jordan.
Intellectual Disability (ID) is one of the most common developmental disorders and its prevalence is considered to be approximately 1-3%. ArrayBased Comparative Genomic Hybridization (aCGH), also known as molecular karyotyping, is a high resolution technique developed to scan the segmental genomic copy number variations (CNV) in the genome level. Here,
we review our experience with determining CNVs using both oligo- and
BAC-based comperative genomic hybridization arrays for patients with ID
between 2009 and 2011. In a cohort of 251 patients with ID with or without dysmorphic features, additional neurodevelopmental abnormalities,
we found 68 different chromosomal aberrations in 56 patients (22.3%). The
most common aberrations were 8p23.1 and 16p11.2 deletions. Our results
showed that aCGH is a powerfull tool to detect CNVs causing intellectual
disability and is usefull for detection novel candidate genes for neurodevelopmental abnormalities. According to the available literature, this is the
first comprehensive aCGH study of a Turkish cohort of patients in this spesific population.
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J08.16
Molecular karyotyping by array CGH in an Israeli cohort of children
with intellectual disability and autism
Joubert syndrome (JBTS) is a clinically and genetically heterogeneous disorder with autosomal recessive pattern of inheritance. The disorder is characterized by cerebellar hypoplasia, intellectual disability (ID), ataxia and
oculomotor apraxia. Other clinical features include retinal degeneration,
renal anomalies, hepatic fibrosis, and skeletal involvement. The hallmark of
JBTS is a radiological pattern at magnetic imaging resonance (MRI) named
molar tooth sign.
In 2007 Baala et al. identified a new form of Joubert syndrome designated
JBTS6 with causative mutation in TMEM67 gene. To now only 5 mutations
in this gene has been detected responsible for JBTS6.
Due to high heterogeneity of ID in Iran,ourstudy launched to find more causative genes and their mutations in Iranian families affected with autosomal
recessive IDusing whole exome sequencing (WES).One of these families has
two affected with profound ID, seizure, strabismus and renal failure in one
affected. On WES data a number of variants detected in thisfamily and with
respect to the clinical features,a novel variant in TMEM67 gene chose as candidate. Because of suspicion of JS, MRI was performed and molar tooth sign
was observed. The variant co-segregated in the family and was absent in
normal population.
Underlying causes of ID remain unknown in many cases because of clinical
and genetic heterogeneity;therefore, exome sequencing is an effective and
helpful technique in detection of de novo mutation in this type of disorders.
This approach results in more precise genotype-phenotype correlation and
clinical diagnosis.
Rett syndrome is a neurodevelopmental disorder that occurs almost exclusively in females with a frequency of 1:10,000. It is characterized by arrested
development between 6 and 18 months of age, regression of acquired skills,
loss of speech, stereotypical movements (classically of the hands), microcephaly, seizures, and intellectual disability. Most cases of Rett syndrome are
sporadic. It was established that the development of Rett syndrome due to
the presence of mutations in the MECP2 gene. To date, 315 mutations in
MECP2 gene have been identified in patients with Rett syndrome. Because of
such a significant number of identified mutations in MECP2 gene and mostly
the sporadic origin of the disease, we have developed denaturing gradient
gel electrophoresis (DGGE) method for mutant variants screening in exons
2 and 3. Using this method we have analyzed exons 2 and 3 of MECP2 gene
in 14 patients with clinical signs of Rett syndrome. In five cases abnormal
migration patterns of MECP2 exon 3 PCR products have been shown. The
results of Sanger sequencing has revealed 4 known for Rett syndrome mutations R168X (502C> T), A140V (419C> T), T158M (473C> T), R133C (397C>
T). In one case (a patient with severe Rett syndrome symptoms) we have
identified a novel mutation 472 T> G which led to substitution T158P.
J08.19
Analysis of chromosomal aberrations in patients with mental
retardation using the array-CGH and MLPA technique: a single Czech
centre experience
H. Filkov1,2, D. Blakov1, K. Kakov1,2, E. Hladlkov1, V. Vallov1,2, R. Gaillyov1, R.
Beharka1, P. Kuglk1,2;
1
Faculty Hospital Brno, Brno, Czech Republic, 2Masaryk University, Faculty of Science,
Institute of Experimental Biology, Brno, Czech Republic.
Molecular-genetic inspection of patients PKU carried out throughout 19912012, living in the Novosibirsk region, has shown that frequency R408W of
PAH gene among all mutations of this gene makes 0,653. These mutation
are revealed both in the homozygous form, and in a compound with other
rare mutations. For the purpose of identification of a range of mutations of
PAH gene with use of a complex of molecular-genetic technologies research
of exons 5-12 of PAH gene at 62 patients with PKU, among which 48,38%
had easy, 45,17% - the moderate and 6,45% heavy degree of mental retardation was carried out. As a result of the carried-out inspection (besides a
major mutation of R408W) in structure investigated exons 5-12 of PAH were
identification thirteen more rare mutations and their frequency characte-
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427
428
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are studied in many neurological disorders, but are not included in ADHD
studies as much. In this study we investigated the association of four SNPs
(rs11122330, rs6675281, rs11122319, rs1417584) in the DISC1 gene with
ADHD in Iranian population. 600 subjects composed of 300 patients and 300
normal controls were included and tetra-primer ARMS PCR technique was
used for genotyping all SNPs. We found differences in genotype distributions of rs11122319 (p = 0.01) and rs6675281 (p = 0.009) variants between
patients and controls. Our findings strengthens the role of disc1 gene as a
susceptibility locus for ADHD and indicate that rs11122319 and rs6675281
variants are strong risk factors for ADHD in Iranian population.
J09.02
Genetic analysis of Aicardi-Goutires Syndrome in an Italian cohort
Aicardi-Goutires Syndrome (AGS) is a rare genetically determined encephalopathy that may overlap with the phenotype of congenital infection.
Mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C ,SAMDH1 and
ADAR1 genes have been found in the majority of AGS cases .TREX1 gene codes for 3->5 DNA exonuclease with specificity for ssDNA. AGS2, AGS3, AGS4
patients have mutations in genes coding for the three subunits of the Human
Ribonuclease H2 enzyme, complex implicated in ribonucleotides removal
from RNA:DNA duplex. The AGS5 gene, SAMHD1, encodes for a triphosphohydrolase that converts deoxynucleoside triphosphates to deoxynucleoside
and inorganic triphosphate. AGS6 can be caused by mutations in the ADAR1
gene traslating into an adenosine deaminases acting on RNA which catalyzes the hydrolytic deamination of adenosine to inosine in ds RNA In the last
years, IRCCS C. Mondino Foundation and International Aicardi-Goutires
Syndrome Association (IAGSA) were involved in many projects. An AGS
biobank to collect serum, plasma, lymphoblastoid cell lines and PBMCs has
been established. .We recruited samples of 23 patients with clinical diagnosis of AGS and their parents.. We collect two AGS1/TREX1 case, 15 AGS2/
RNASEH2B cases, one AGS3/RNASEH2B case, one AGS4/RNASEH2C case
,one AGS5/SAMHD1 case and two AGS6/ADAR1 cases. Although one of the
screened patients did not show mutations in the mentioned genes above,
we may still consider the possibility of the presence of new mutations yet
to be identified in other genes. The research leading to these results has received funding from the European Unions Seventh Framework Programme
(FP7/2007-2013) under grant agreement number 24177.
J09.03
Evaluation of PAI-1 gene 844G/A polymorphism in Iranian patients
affected by Alzheimer and Normal Individuals
L. Mazhari;
Department of Biology, Pharmaceutical Sciences Branch, Islamic Azad University,
Tehran, Islamic Republic of Iran.
It has extensively established that PAI-1 gene is involved in Alzheimer pathogenesis. Numerous genetic risk factors have been related with Alzheimer, but no study has unraveled a possible association between Alzheimer
and PAI-1 gene 844G/A polymorphism.There are many common risk factors
such as 844G/A polymorphism that play key roles in the development of
Alzheimer disease.Polymorphisms in PAI-1 gene have been associated to
Alzheimer in some populations. However, other groups failed to replicate
this finding in other populations. Evidence suggests that PAI-1 gene 844G/A
polymorphism might play a role in Alzheimer, as a result we Studied PAI-1
gene 844G/A polymorphism common polymorphisms in Iranian with Alzheimer.Materials and methods:We conducted study including a clinically
well-defined group of 52 Alzheimer patients to test the association between 844G/A polymorphism and Alzheimer in Iranian population. In the
present case control study, the PAI- gene 844G/A polymorphism has been
investigated in 52 patients with Alzheimer and 112 healthy subjects by
using ARMS-PCR methods. Then, the data were analyzed by pasw statistics
18 (SPSS) software.Results: In patients samples the genotype distribution
for PAI-1 844G/A showed 52% , 46%, 2% for AA, AG and GG respectively.
In control samples 96% of the cases were AA,4% were AG and 0% were
GG.Conclusion:The results of this study show significant association between Alzheimer and PAI-1 gene 844G/A polymorphism in Iranian population. (P < 0.05)
Introduction and Aim: Alzheimers disease (AD) is the most common type
of dementia characterized by memory impairment and alteration of diverse
cognitive abilities. Pathologically, it is the formation of amyloid plaques and
neurofibrillary tangles in the brain. The most common form of AD is Lateonset AD (LOAD), and is usually sporadic. Although several susceptibility
genes for AD have been reported, by far the strongest genetic risk factor for
LOAD is Apolipoprotein E that located on chromosome 19q13.2 and universally recognized as a major disease susceptibility gene for LOAD. APOE has
three common alleles, APOE-2, APOE-3, APOE-4. In this study we examined
association of the APOE gene polymorphism (rs121918398) with Alzheimer disease in Iran northern population.
Methods: Study included 50 patients with AD and 50 healthy volunteers.
An informed consent was obtained from all participants. Genomic DNA was
extracted from peripheral blood leukocyte. Genotypes determined by PCR
and restriction fragment length polymorphism (RFLP). Statistical analysis
was performed using the MedCalc program for windows version 12.
Discussion and Conclusion: The prevalence of genotype frequencies of the
APOE A/A, A/G, G/G were 16%, 34% and 50% respectively, in AD subjects,
and in healthy volunteers were 10%, 64% and 26% respectively. Statistical analysis has not emerged significant difference from the comparison of
either genotype (p>0.05). There was no evidence that APOE variants were
associated with AD in this population. However our results may be different
by selecting different geographical sites or bigger size population.
J09.06
Replicative association analysis of Alzheimers disease in Russian
population of Siberian region
Alzheimers disease (AD) is a progressive neurodegenerative disorder observed in the elderly. This pathology is the most common cause of dementia and accounts for 50-80% of dementia cases. The World Health Organization estimated that currently about 25 million people worldwide have
Alzheimers disease. It is a complex disorder with genetic, environmental
and lifestyle factors. Common neurological disorders including AD are the
subject of intensive genetic research based on genome-wide association studies (GWAS). Genetic variants associated with cognitive impairments, which
are an important endophenotypes for AD, also have been revealed by GWAS.
The aim of this study was to analyze associations of 15 SNPs reported in
GWAS with Alzheimers disease in Russian population of Siberian region.
SNPs were genotyped by real-time PCR in 108 AD patients and 285 agematched controls of Russian origin. The association of rs2616984 located in
the region of CUB and Sushi multiple domains 1 (CSMD1) gene, previously
reported in GWAS by Cirulli et al. (2010), was confirmed in Russians (OR =
1,5, p = 0,01). Genetic markers in loci for VRK2, SLCO6A1, NOTCH4, TCF4,
ZNF804A, AGBL1, TLR4, RELN, ZFP64P1, KCNB2, CPVL, NRGN and NRIP1 pre-
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viously reported in GWAS was not associated with the disease in Russian
population. This work is partially supported by RFBR grant #12-04-00595.
J09.07
Association study of BDNF polymorphism Val66Met in Bulgarian
patients with Alzheimer disease
429
Cockayne syndrome is clinically heterogeneous condition in which postnatal growth delay, microcephaly and neurological dysfunction are the cardinal features. Characteristic dysmorphic findings like deep-set eyes, triangular face, aged appearance can be seen in various degrees. Photosensitivity,
demyelinating peripheral neuropathy, cataract, cachexia, dental abnormalities and sclerotic epiphyses are also the features of this syndrome. It should
be noted that brain calcifications are observed but not a must. ERCC6 and
ERCC8 mutations are responsible for the clinical findings of these patients.
Here we report three patients with Cockayne Syndrome and two novel mutations in ERCC6 and ERCC8 genes. Two of them had a mild clinical course
and very mild dysmorphic findings and the other was severely affected. Insight of these findings we discuss the phenotypic heterogeneity of this condition and aimed to underline the possibility to overlook in the absence of
some cardinal criteria and mild facial features.
J09.12
One novel SCN1A mutation in a Bulgarian patient - case report
Dravet syndrome (DS) is an early-onset epileptic encephalopathy characterized by polymorphic seizure types. At onset, they are usually induced
by fever and often present as tonic-clonic or hemiclonic status epilepticus
during the first year of life. Later, patients also manifest other seizure types, including absences, myoclonic, and partial seizures, all being refractory
to treatment. The EEG is often normal at onset, but later characteristically
shows generalized spike-wave activity. Psychomotor development stagnates around the second year of life, and affected individuals show subsequent
mental decline and other neurologic deficits. DS is caused by mutations of
the SCN1A gene in more than 80% of patients. Here we report a 1-year old
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boy with seizure onset at 5 months, presenting by series of febrile or afebrile tonic-clonic or hemiclonic seizures up to status epilepticus, some with postictal hemiparesis. Several interictal EEGs were normal or with background
slowing only. Four antiepileptic drugs had no effect and long-lasting seizures
occurred every 10-20 days. Based on clinical data, at the age 10 months the
diagnosis of DS was proposed. The molecular genetic testing of the SCN1A
gene detected a novel de novo mutation - c.3587_3588insCTTC in exon 18
of the gene. The mutation causes frameshift and the shifted mRNA is most
probably degraded through nonsense-mediated mRNA decay mechanism.
J09.13
A novel mutation in SLC1A3 gene associated with episodic ataxia
Background The FOXP1 gene, located on 3p13, has recently been involved in neurodevelopment. FOXP1 haploinsufficiency has been reported in
about 10 patients with specific facial features, global developmental delay,
intellectual disability, and speech impairment, where expressive skills are
more severely affected than receptive skills. Case description We report on
a 2-year-old Caucasian boy, born at term with relative macrocephaly. He developed neonatal cholestasis for which an extensive etiological assessment
was performed, including JAG1 (Alagille syndrome) and ABCB4 (PFIC3) gene
analysis. Ductular paucity was objectified with progressive improvement
and genetic work-up was normal. Progressively, global developmental delay
was observed. He had axial hypotonia, dyskinetic movements and is still not
able to walk. Expressive language has not yet been acquired, though receptive language appears more advanced. Growth is normal. He has frequent
respiratory tract infections, bronchial hyperreactivity and gastro-esophageal reflux. Facial features include prominent and broad forehead, bilateral
palpebral ptosis, broad nasal tip and arched palate. Hypoplastic nails are
also observed. Chromosome molecular analysis using CytoScan HD-array
(Affymetrix) revealed a de novo ~120kb heterozygous interstitial deletion
at 3p13 covering exons 7 to 11 of the FOXP1 gene. Conclusion Global developmental delay, expressive language delay, relative macrocephaly and
specific facial features are associated with FOXP1. Our patient presents with
bilateral ptosis, a sign previously reported in the literature in at least two
other patients, and could be a clue for the diagnosis when present. Whether
or not the liver involvement is an associated feature remains to be studied
in larger series of patients.
J09.16
DXS998-DXS548-FRAXAC1 represents a novel informative haplotype
at the FMR1 locus in the Iranian population
M. Shirani, S. Vallian;
University of Isfahan, Isfahan, Islamic Republic of Iran.
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Average age of onset of patients was 225.5 years. Average size of alleles of
patients and controls were 1200400 and 123.5 respectively. No difference
in methylation patterns were observed between the cases and the controls
of Indian patients. The CpG islands methylated irrespective of GAA repeat
number. However this study is limited in sample size which could be one of
the factors that may over-represent the methylation phenomenon. We plan
to further extend our study on more Friedreich ataxia patients to draw a
strong conclusion along with methylation sites at the promoter region as
they are more important for gene expression.
J09.18
Atypical parkinsonism and very early-onset dementia in a patient
carrying two GBA gene in cis mutations
Hereditary Spastic Paraplegia (HSP) comprises a group of clinically and genetically heterogeneous neurodegenerative disorders. In pure HSP, lower
limb spasticity and progressive weakness are observed. Complicated HSP
additionally include neurological and non-neurological symptoms. HSP can
be inherited in autosomal dominant, autosomal recessive, or X-linked manner. Thirty-two loci and 23 genes are associated with autosomal recessive
form of HSP (ARHSP). One patient was selected for whole exome sequencing (WES) from each of six ARHSP families from Turkey. After determining
candidate variations, segregation analyses were performed in the families
to confirm that the variation can be responsible for the HSP phenotype. In
two of the families mutations were identified in known HSP genes. First mutation is the c.4321C>T (p. A1394X) variation in Spastizin gene (SPG15) in
family H61. Spastizin is the second most common causative gene for ARHSP
with thin carpus callosum that is also observed in our patient. The other mutation is c.325_326insTGTC insertion in ALS2 gene in family H59. ALS2 gene
is responsible for juvenile amyotrophic lateral sclerosis (JALS) and infantile
onset hereditary spastic paraplegia (IAHSP). Four families were found to be
negative for mutations in known HSP genes. In two of the families, novel
HSP candidate genes were identified and segregation with the disease was
shown in the families. For the remaining two families, WES data is still under analysis. Our WES analysis gives promising preliminary data for identification of novel genes in families from Turkey and it suggests evidence for
further genetic heterogeneity in ARHSP.
431
Hereditary spastic paraplegias (HSP) - is a genetically and clinically heterogeneous group of neurodegenerative disorders characterized by progressive spasticity of the lower-limbs. The major pathological feature of HSP is a
degeneration of the pyramidal tracts. The HSP frequency in Bashkortostan
Republic (BR) is 3,5:100000. Autosomal dominant (AD), recessive (AR), or
X-linked inheritance were described. To date, 30 causative genes and 50 loci
have been identified. Mutations in the SPG4 gene are responsible for about
45% of the pure AD type of HSP and for 12-18% of the sporadic cases. SPG4
codes for spastin - AAA (ATPase associated with diverse cellular activities)
family protein. Disorder onset ranges between 1 and 63 years. Over 300 mutations have now been described, with the majority of them being located in
the AAA domain.
We examined HSP patients from BR and detected one rearrangement of DNA
sequence in 12-th exon in patients from three unrelated families. This previously not described mutation is inversion translocation, starts at c.1469.
Twelve exon codes the region that corresponds to a highly conserved ATPase domain, which is also called AAA cassette. This region is responsible for ATPase activity of the spastin protein. New polymorphism in 11-th
intron (c.1413+43_46dup (TATA)) and polymorphisms (c.1098+118 A>G
(rs 12617289) and c.1098+127 A>G (rs 12617290)) in 7-th intron were revealed in the same patients.
Large chromosomal rearrangements are frequent cause of SPG4 HSP. The
mutation we found is among them. We supposed that this mutation was
spread in BR by the founder effect.
J09.21
Two novel compound heterozygous mutations in ROBO3 gene in a
sporadic case of Horizontal Gaze Palsy with Progressive Scoliosis
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mia. During admission,under the established treatment, the sepsis was remitted; nevertheless high natrium levels were maintained. In this case HPE
was complicated by neurogenic hypernatremia. Genetic testing revealed a
normal cariotype and the uncharacteristic phenotype of the mother ruled
out autosomal dominant transmission. In the presented case, neurogenic
hypernatremia was the consequence of impaired osmoregulation of ADH
release due to malformations involving midline structures of the brain.
J09.23
Homozygosity in Huntingtons disease
Huntington disease is caused by a dominantly transmitted CAG repeat expansion mutation that is believed to confer a toxic gain of function on the
mutant protein. Huntington disease patients with two mutant alleles are
very rare. In other poly(CAG) diseases such as the dominant ataxias, inheritance of two mutant alleles causes a phenotype more severe than in heterozygotes. We identified a homozygous female patient, 89 years old and
compared clinical features with those of a group of heterozygotes. The age
of onset was within the range expected for heterozygotes with same CAG
repeat lengths. However, she showed a rather different clinical course with
emotional instability and behavioral disorders. Cerebralmagnetic resonance
imaging showed no evident atrophy, notably not in the cerebral cortex or
the striatum. Clinical examination revealedshe had chorea predominating
on the upper limbs and the trunk. She was partially unable to control the
speed and the force of her movements and mild dysarthria was noted. Severe memory deficits associated with a severe frontal deficit were found, all
symptoms suggesting Huntington disease. Mutation analysis revealed two
HTT alleles with CAG repeats of 43 and 45. This suggests that although homozygosity for the Huntington disease mutation does not lower the age at
onset of symptoms, it affects the disease phenotype and progression. These
data, once confirmed in a larger series of patients, will point to the possibility that the mechanisms underlying age at onset and disease progression in
Huntington disease may differ.
J09.24
Gene expression profile in fibroblasts of Huntingtons disease
patients and controls
Kufs disease is an adult onset neuronal ceroid lipofuscinosis, which is autosomal recessive progressive lysosomal disorders. Kufs disease are clinically divided into two types; Type A and B. Type A presents with progressive
myoclonus epilepsy, whereas type B presents with dementia and motor abnormalities. Recently, the genes responsible for Kufs disease are discovered.
Mutations in CLN6 are the major cause of recessive type A, whereas mutations in cathepsin F is the cause of type B.
In the past, we have reported a type B Kufs patient, whose parents were consanguineous marriage (Sakajiri K et al. Intern Med, 1995;34:1158-63). Here
we present the first mutational report from Japan. In this study, we performed mutational analysis of responsible genes for this Kufs patient. We found
several SNPs in cathepsin F gene, and a known but homozygous mutation
(c231C>G, pN77K in exon 3) in CLN6 gene. PCR-RFLP analysis revealed that
the mutation was not a SNP. The amino acid is perfectly conserved between
human, mouse and rat. Furthermore in silico analysis using PolyPhen2, the
mutation is predicted to be probably damaging. These data suggest that the
mutation must be pathogenic one. Since we found a known but homozygous
mutation in CLN6 gene in a type B patient, genotype-phenotype correlation
seems to be complex than we expected.
J09.28
The Association of Migraine and Endothelial Nitric Oxide Synthase
Haplotypes
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counseling center, Rouyan Alley, Keshavarz Blv, Tehran, Islamic Republic of Iran,
4
Department of Genetic, Islamic Azad University Science and Research Zanjan Branch,
Tehran, Islamic Republic of Iran, 5Cellular and Molecular Research Center, Research
Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran,
Islamic Republic of Iran.
Migraine is a complex and debilitating neurovascular disorder predominantly affecting women. It recurs as attacks of severe headache associated
with nausea, vomiting, phonophobia, and photophobia. Despite its high prevalence, migraine is a disease involving complex pathogenetic mechanisms
that remain to be clarified. As other multifactorial diseases migraine also
has a genetic basic combined with triggering environmental factors. There
is strong evidence implicating nitric oxide (NO) in the pathophysiology of
migraine. Therefore, genetic polymorphisms in the endothelial NO synthase
(eNOS) gene have been studied as candidate markers for migraine susceptibility. NO plays an essential role in the control of cerebral blood flow and
may be involved in the activation of nociceptors in the trigeminovascular system and release of vasoactive neuropeptides during neurogenic inflammatory response. There are several studies on the association of eNOS gene
variations and the risk of migraine in different populations. In the present
study, we focused on one of the most common single nucleotide polymorphisms (SNP) of the eNOS gene. The prevalence of the rs1799983 in Iranian
population has been evaluated by ARMS-PCR method. We have not found
interaction between the selected SNP and the risk of the migraine in 90 patient samples compared to 113 normal individuals.
J09.29
Plasminogen Activator Inhibitor Haplotypes Associated with Migraine
P. Ghazizadeh Tehrani1, M. Ansari2, M. Moghaddasi2, S. Ghasemi3, H. Zolghadr4, A.
Ebrahimi5;
1
Department of Genetic, Islamic Azad University Science and Research Zanjan Branch,
Tehran, Islamic Republic of Iran, 2Department of Biochemistry, Tehran university of
medical science, Tehran, Islamic Republic of Iran, 3Parseh genetic counseling center,
Rouyan Alley, Keshavarz Blv, Tehran, Islamic Republic of Iran, 4Department of Cellular
and Molecular Biology, Islamic Azad University, Tehran Medical branch, Tehran,
Islamic Republic of Iran, 5Cellular and Molecular Research Center, Research Institute
for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Islamic
Republic of Iran.
Miller-Dieker syndrome (OMIM #247200); 17p 13.3 deletion with haploinsufficiency of PAFAH1B1) is a contiguous gene deletion syndrome that is
characterized by lissencephaly, facial dimorphisms, growth restriction and
seizures. PAFAH1B1 encodes LIS1, a protein essential for neuronal migration, proliferation and survival. It participates in a complex to regulated
spindle orientation during neuronal progenitor divisions.
The patient is the first child of healthy non consanguineous parents. He
was born at full term after an uneventful pregnancy. His birth weight, head
circumference, and length were in a standard range with Apgar index of
8/10. Clinical examination at birth only showed undescended testicles as
remarkable aspect. At 4 months old male referred for evaluation because of
hypotonia, microcephaly and developmental delay. Cranial sonography sho-
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Exon
28
30
24
37
51
51
51
Mutation
c.3810_3820delCATGCAGACTC
c.4077_4078insG
c.3194_3195insA
c.5257_5258delG
c.7549C>T
c.7549C>T
c.7549C>T
Mutation Type
Deletion (new)
Insertion (new)
Insertion
Insertion
Nonsense
Nonsense
Nonsense
J09.34
Mutational spectrum of the NF1 gene in Greek patients with
Neurofibromatosis Type 1
N-methyl-D-aspartate receptors (NMDARs) play an essential role in the process of synapses formation, neuronal differentiation, brain plasticity as well
as participate in the molecular response to the neurotoxic substances action. The NMDAR dysfunction is associated with schizophrenia, Alzheimers
disease, and Huntingtons disease. The influenza virus infection is also one
of the potential negative factors influencing NMDAR function, but the molecular mechanisms of neurological complications of influenza are not well
understood. The aim of our research was to study the influence of influenza A virus infection on the expression of NMDAR subunits (NR1, NR2(A-D),
NR3(A-B)) in mice. We analyzed the expression of seven genes, coding
NMDAR subunits, in brains and lungs on the 1, 3, 5 and 10 days post infection using real-time PCR. Two groups of mice were infected with H1N1 and
H3N2 strains at 0,2 LD50 respectively. The expression of all seven NMDAR
transcripts was detected only in brains of non-infected animals. The infection with influenza A virus strain led to the changes in expression levels of
NR2A, NR2B, NR2D and NR3B mRNAs in brains. In lungs NMDAR mRNAs
were not detected. Further we are going to make the more detailed analysis not only of NMDARs expression but also of other genes involved in the
regulation of the central nervous system during influenza A virus infection
in mice.
J09.37
New insights in non-syndromic intellectual disability: a case report
A 15 years old girl with obesity, dysmorphic features (down-slanting palpebral fissures, short nasal bridge; short philtrum; micrognathia), moderate mental retardation, psychosis, dyslalia, hyperkinesis, aggressivity, bilateral hypoacusis was referred for genetic investigations. aCGH on
an 105K Agilent platform revealed arr 5p13.2(37,479,936-38,118,402)
x3,11q13.5q14.1(76,158,015-83,043,032)x1,
17q25.3(79,829,409-79,910,442)x1. Interestingly, the ~6.88 Mb deletion on 11q includes 21 OMIM
genes, among which two disease-causing entities: MYO7A, and ALG8. The
first one encodes for an unconventional myosine, a motor molecule with
structurally conserved heads moving along associated actin filaments and
highly divergent tails suggested to act as carriers of various macromolecular cargo molecules; MYO7A is known to carry heterozygous mutations in
nonsyndromic neurosensorial deafness and homozygous mutations in most
cases of Usher syndrome. The second gene encodes for alpha-3-glucosyltransferase which, when mutated to compounded heterozygosity, produces
the congenital disorder of glycosylation, a severe phenotype resulting in
multiple dysmorphisms and defects leading to early infant death. The 0.63
Mb duplication on 5p harbors only one OMIM gene, GDNF, encoding for a
specific dopaminergic protein associated with motor neuron function and
Hirschsprung disease. Finally, the 0.08 Mb deleted fragment on 17q contains
8 OMIM genes, among which pyrroline-5-carboxylate reductase 1 that causes cutis laxa disease when it losses functions. The correlations of the identified genetic defects with the observed phenotype will be discussed, as well
as their impact upon patient management.
Acknowledgements: project PN 09.33.02.03.
J09.38
Report of a novel compound heterozygous mutation in a mexican
family with Niemann Pick Type C disease
Introduction. The disease of Niemann Pick type C, is an alteration in storage lisosomal, characterized for decline neurological progressive and death
premature. He has un pattern of recessive autosomal inheritance, caused
by mutations in the NPC1 and NPC2 genes. Clinical manifestations include, mental decline, ataxia, cataplexy, dystonia, vertical supranuclear gaze
palsy(VSGP) and clumsiness. Case report. Female patient of 29 years old,
daughter of the fifth pregnancy of non-consanguineous healthy parents. Fa-
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mily history: One sister died at age of 25-years-old with the same disease.
The patient initiate at 17-years-old with, loss progressive of memory, cognity disorders, disartria, and dysphagia . At the 22-years-old, she presented
coreics and dystonics movements of upper and lower extremities and gait
problems. Phisical exploration: she had ataxia and dystonics and coreoatetosics movements and VSGP. The score performed for NPC was 201 points. The
abdominal ultrasound showed splenomegaly and the magnetic resonance
cortical atrophy. Methods. It was performed PCR and sequencing of 25 exons
of the NPC1 gene and the five exons of the NPC2 gene. This study was carried out in parents and health sister of the patient. Results: We detected two
different pathogenic mutations: [c.2604+1G>A] + [c.3630T>G], compatible
with compound heterozygous genotype. The NPC2 gene sequencing was
normal. Conclusion: This is the first case informed in Mexican population of
Niemann-Pick type C. Miglustat is the important therapy in NPC disease.
J09.39
Identification of The Human Natural Resistance-Associated
Macrophage Protein 2 (NRAMP2) Gene Polymorphism in
Schizophrenia Patients
The study tested the association between the development of PTSD and
genetic variants of ADYAP1R1 (PAC1): C/C vs C/G vs G/G in survivors to
6 April 2009 LAquila earthquake. The sample examined so far consists of
103 subjects. Cells of the buccal mucous were collected to obtain DNA and
Clinician-Administered PTSD Scale (CAPS) test is administered.
It has been conducted an analysis of variance (ANOVA) with LSD Post Hoc
test to assess differences between groups in terms of CAPS scores compared
to the three polymorphic variants. In line with the literature, the results suggest a strong role of SNP rs2267735 of PAC1 C/C genotype in the regulation
of physiological response to stress, showing as a risk factor for the development of post-traumatic symptoms after a natural disaster. Further genetic investigations are still ongoing concerning: Val66Met BDNF allele and
the S allele of 5HTTLPR , in addition to evaluation of various psychometric
scales : the Hamilton Depression Scale (HAM-D), Hamilton Rating Scale for
Anxiety (HAM-A), Mini-International Neuropsychiatric Interview (MINI),
Temperament and Character Inventory (TCI-R), Toronto Alexithymia Scale
(TAS -20) and Childhood Trauma Questionnaire (CTQ). These data will be
subsequently analyzed in order to verify other possible associations in the
development of PTSD. These data to shed light on the role of genetic factors
in the formation or modulation of the stress response.
435
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notype G/G (p=0,02; OR=1,7; CI=1,09 - 2,64) and allele G (p=0,04; OR=1,53;
CI=1,02-2,3) of SPPL2C gene (rs12373139, MAPT-region). There was no
association of PD with rs1491942 and rs1907632 in LRRK2 gene in the investigated ethnic groups.
The possible reasons for the discordance between results in different ethnic
groups and previous GWAs may include effects of population structure and
population-specific environmental interactions. Our findings suggest that
additional studies are necessary to establish the role of these loci in modifying risk for Parkinsons disease in different populations.
J09.44
Molecular basis of mechanism of gene expression modulation
induced by hypericin in genes involved in the pathogenesis of
Parkinsons disease
Parkinsons disease (PD) is the second most common and a complex neurodegenerative disorder arising from a complex interaction between genetic
and environmental factors. Determining the pathogenic gene contributed to
Parkinsons disease have always been controversial. In a large meta-analysis
study on Caucasian populations another novel PD susceptibility locus, RIT2,
on chromosome 18, was identified. A genetic variant in RIT2, rs12456492,
was found to be associated with risk of sporadic PD. In the present study,
we investigated the association of the rs12456492 variant with Parkinsons
disease in Iranian population. A total of 960 subjects (480 PD patients and
480 normal healthy controls) were included and genotyped for rs12456492
polymorphism (A/G) of RIT2 gene. Genotyping was done by tetra-primer
ARMS PCR technique. We found significant differences in distributions of GG
genotype in patients compared to controls (odds ratio = 1.60, 95% CI = 1.142.23, p = 0.005). Our findings increase the likelihood of association between
PD and RIT2 variant in Asian populations.
J09.46
Partial duplication Xq27.1 in a dysmorphic and mentally retarded girl
N. Andreescu1, S. Dumitriu1, V. Plaiasu2, M. Puiu1;
1
Victor Babes University of Medicine and Pharmacy, Timisoara, Romania, 2IOMC
Hospital, Bucuresti, Romania.
Background. Duplications of the long arm of chromosomes X have been related to mental retardation, different dysmorphic features, hypopituitarism
and short stature.
Aim. We aim to examine the relation between the partial duplication of the
Xq27 region and the presence of clinical manifestation in in a dysmorphic
and mentally retarded girl.
Methods. We report on a 3 year old girl associating facial dysmorphism, hypotonia, delayed milestones, mental retardation. Proposita is the first child
of a young couple, born at 39 weeks of gestation by caesarean section due
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J09.49
Sequence variants of PRNP gene in probable prion disease patiens
Prion diseases are a family of rare progressive neurodegenerative disorders, caused by abnormally conformed infectious proteins, called prions.
The functions of these normal prion proteins are still not completely understood. The abnormal folding of the prion proteins leads to brain damage
and the characteristic signs and symptoms of the disease. There are 4 types
of this disease: the sporadic (Jakob-Creutzfeldt disease, 85-90%), familial/
genetic (10-15%), iatrogenic (1%), and variant types. Genetic form of prion
diseases are caused by mutations in the prion-related protein gene (PRNP),
and classified based on the phenotype, mutation and neuropathological
findings. In this study, we analyzed PRNP gene to evaluate the frequency
of PRNP sequence variants and their genetype-phenotype correlation in
60 probable Prion disease patients. Genetic analysis of the PRNP gene was
performed on peripheral blood samples using polymerase chain reaction
and direct-sequencing. We found four different PRNP sequence variants
in thirty-six patients (G54S, M129V, D178N, octapeptide repeat deletion:
c.247_270del). The rate of PRNP sequence variant was 60% in our samples.
PRNP screening may be useful for genotype-phenotype correlation in Prion
disease.
J09.50
Eight new cases of PEHO or PEHO-like syndrome?
PEHO syndrome (Progressive Encephalopathy with oedema, Hypsarrhythmia and Optic atrophy) (OMIM#260565) belongs to a rare neurodegenerative disorders of unknown etiology and probable autosomal recessive inheritance. Only 50 patients have been described worldwide so far, with the vast
majority in Finland. We present eight new patients who fulfill diagnostic criteria of PEHO or PEHO-like syndrome. Patients were selected from a group
of patients with a diagnosis of Progressive encephalopathy of unknown
etiology or Cerebral palsy. In all of them onset occurred during the first
few weeks or months of life. The leading symptoms were hypotonia, poor
feeding, drowsiness, infantile spasms, seizures with hypsarrhythmia in EEG,
absent eye contact, optic atrophy, no any progress in psychomotor developmental. Dysmorphic features were peculiar and consisted of: microcephaly,
pear-shaped face, narrow forehead with prominent metopic ridge, full
cheeks, receding chin, epicanthic folds, an open mouth with a curved upper
lip, hypertrophic gums, protruding ear lobes, a short nose with anteverted
nares, edema of upper and lower extremities and tapering fingers.
In six patients cerebral, cerebellar and/or brainstem atrophy were found by
MRI. This group was named as PEHO syndrome and rest two childs without
any abnormalities in MRI as PEHO-like.
J09.51
Association of GRM3 gene polymorphic loci with schizophrenia
K. O. Kinyasheva, A. Gareeva, E. Khusnutdinova;
Institute of Biochemistry and Genetics Ufa Scientific Center of RAS, Ufa, Russian
Federation.
437
Sleepwalking and night terrors are sleep disorders classified into the group
of excitement Parasomnias. These disorders generate a considerable decree
in the individuals quality of life. However the inheritance patterns for these
438
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disorders are poorly known and there is a lack of knowledge of the responsible genes for these disorders. We report a three-generation Colombian family with 18 affected individuals, showing a dominant inheritance pattern
with reduced penetrance. There is only one study that previously reported
the same inheritance pattern in an American family demonstrating linkage
with a region between the SNP type markers rs728331 and rs286819 (Licis,
et al. 2011). However, there are some mayor differences between the reported family and ours. Onset prevalence and sex-affected ratio are the mayor
phenotypic distinctions. We propose that we may have a new condition that
is caused by different genes.
J09.55
The clinical phenotype of Polish patients with SPG11 gene mutations preliminary description
I. Stepniak, A. Sulek, E. Elert-Dobkowska, W. Krysa, M. Rajkiewicz, M. Rakowicz, W.
Lojkowska, J. Zaremba;
Institute of Psychiatry and Neurology, Warsaw, Poland.
Metals are crucial for synaptic transmission, enzyme activity, proteins folding/conformation. Copper, zinc, iron and manganese are all localized to
various subcellular compartments and their regulation is controlled by a
diverse range of co-factors and chaperones. Loss of homeostasis of some
of these elements is present in neurological disorders including Alzheimer,
Parkinson and Huntington diseases, amyotrophic lateral sclerosis and Friedreich Ataxia. Metals are also key factors for ATP generation and to control
oxidative damage in mitochondria.
We selected a cohort of spinocerebellar ataxia (SCA, n=20) and Ataxia-Telangectasia (A-T, n=20) patients, and we measured copper, zinc, iron, manganese and selenium levels in their blood samples, using atomic absorption
spectrometry.
We found a significantly reduced manganese levels in SCA patients and increased copper levels in A-T patients compared to controls (p<0.01). Cu and
Mn have essential roles in mitochondria: the former is a component of complex IV; manganese has a central role in maintaining antioxidant enzyme
function and it is the superoxide dismutase co-factor (MnSOD; SOD2).
We evaluated in patients vs. controls LCLs mRNA levels of catalase-CAT,
glutathione peroxidase-GPX1 and SOD2, enzymes involved in antioxidative
response. We found SOD2 and CAT up-regulation in A-T patients and a SOD2
down-regulation in SCA patients.
We considered SOD enzymes activity and showed a reduction of MnSODactivity in SCA LCLs, whereas Cu/ZnSOD-activity was halved in A-T LCLs.
These data, although preliminaries and restricted to a small number of
samples, suggest a role for metals in ataxias and may represent biomarkers
of pathology.
J09.58
Screening of TARDBP in Iranian amyotrophic lateral sclerosis (ALS)
patients
Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder characterized by dysfunction and degeneration of both upper motor neurons in the
cortex and lower motor neurons in the brainstem and spinal cord. Genetic
analysis of familial ALS (FALS) pedigrees has led to the identification of at
least 21 loci and 18 ALS-causing genes. After C9ORF72 and SOD1, mutations TARDBP gene are the most frequent cause of disease in FALS families.
TARDBP encodes transactive response DNA-binding protein-43 (TDP-43).
In addition to its prominent role in ALS, TDP-43 is important because of its
potential involvement in the etiology of various neurodegenerative diseases.
TDP-43 is a major component of aggregates of misfolded proteins in neurons of ALS, frontotemporal dementia, Alzheimers and Parkinsons disease
patients. As C9ORF72 and SOD1 have already been screened in cohorts of
Iranian ALS patients, we here screened TARDBP in 90 unrelated patients.
Fifteen were FALS and 75 were sporadic ALS (SALS) cases. The coding exons
of the gene were sequenced by the Sanger protocol. A single missense mutation was observed in exon 6 in one FALS patient; no putative disease causing
variation was found among the SALS patients. Although the sample size was
relatively small, it appears that the frequency of Iranian ALS patients and
specifically FALS patients (6.7%) who harbor mutations in TARDBP is similar to frequencies reported for other populations.
J09.59
Genetic alterations of postsynaptic NMDA receptor related complex
are associated with autism spectrum disorder
Autism spectrum disorder (ASD) is an early childhood neurodevelopmental disorder characterized by a significant genetic aetiology and prevalence
Back to index
currently estimated at 1/100. Several biological pathways have been highlighted, particularly the excitatory synapse, affected by copy number variations (CNV) and enrichment in mutations in genes belonging to the NMDA
receptor complex (NRC). We performed a global genetic study of 100 French
families including at least 1 individual with autism, who were included in
the research project (ClinicalTrials.gov NCT01770548).
Our project aimed to evaluate both the contribution of CNV in ASD, and
secondly the presence of mutations in the NRC complex. For each family,
we firstly performed a high-resolution pangenomic comparative genomic
hybridization (CGH) analysis with 1M CGH Agilent Array format to identify
rare or de novo CNVs. In parallel, a high-throughput targeted sequencing of
216 genes mostly belonging to the NRC complex (179) was carried out with
the SureSelect Agilent strategy in order to assess the contribution of gene
mutations in our cohort.
The first results of our study allowed us to characterize candidates and likely pathogens genetic alterations in at least 10% of patient. We have found
mutations or CNV in genes encoding receptors (NLGN4X, GRM5), and in cytosolic (UPF3B), nuclear (MACROD2), and synaptic (NXPH3) proteins. They
also emphasized that the NRC is targeted both by mutations and CNV, inherited or de novo.
These results underscore the fundamental role of this multiprotein network
in the process of neuronal communication and learning and its pathophysiological impact in autism.
J09.60
The study on the effect of fibrillation inhibitory compounds on
the depolymerization of amyloid fibrils of alpha-synuclein using
fluorescent-labeled protein.
Despite numerous studies about Eating Disorders (EDs) their etiopathogenesis is currently debated and poorly understood. Studies carried out on
families and twins have highlighted genetic factors role; molecular genetic studies have identified the candidates genes potentially involved in EDs
etiology. To date research has not produced conclusive results, nevertheless
genes of serotonergic and dopaminergic neurotransmitters systems seem
to be promising candidates. Among these important candidate genes in
the EDs susceptibility, various studies have evaluated the possible role of
the 1438 G/A polymorphism within the 5-HT2A. In this study 202 Eating
Disorder patients (EDp) and 150 control subjects were analyzed for distribution of the 5-HT2A receptor promoter polymorphism and we shown
that the AA genotype is more frequent in patients (2=6.69; p=0.01) than
in control suggesting an association of 1438 G/A polymorphism within the
5-HT2A with EDs, in agreement with the past literature. Moreover within
our healthy sample subjects with a AA genotype reported higher Body Dis-
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J09.64
Linkage of a locus for autosomal recessive familial spastic paraplegia
to chromosome 8q24
Hereditary spastic paraplegia (HSP) is characterized by insidiously progressive lower-extremity weakness and spasticity. HSP being a group of genetic
disorders, they follow general inheritance rules and can be inherited in an
autosomal dominant, autosomal recessive or x-linked recessive manner. The
mode of inheritance involved has a direct impact on the chances of inheriting the disorder. This neuronal degeneration is thought to be caused by mutations at specific genes. Genetic mapping has identified at least 52 different
HSP loci, designated SPG (Spastic Paraplegia) 1 through 52. We studied An
Iranian family with autosomal recessive HSP with three affected and four
unaffected siblings of two consangeous parents. Clinical, neurophysiologic,
and neuroradiologic studies were undertaken. Genetic linkage analyses were
carried out with polymorphic DNA markers. The candidate gene within the
linked region was sequenced in order to verify the observation. Homozygousity mapping showed linkage to 8q24. As a result the first candidate gene for
screening was SPG8 also known as KIAA0196 because this gene is located in
the linked region and is one of HSP related genes. The KIAA019 contains 29
exons. Precise screenings of all 29 exons of the gene with their boundaries
were carried out using sequencing but all patients were negative for SPG8
mutations. In conclusion, these data confirm the presence of another SPG
gene on chromosome 8q24 related to autosomal recessive HSP.
J09.62
Red blood cells hemolysis influences the level of total but not
oligomeric plasma alpha-synuclein in Parkinsons disease patients
and controls
440
J09.65
The role of estrogen receptor alpha (ESR1) and oxytocin receptor
(OXTR) genes in personality traits variation
Estrogen and oxytocin integrate many processes including social behaviors, cognitive function and activity in the autonomic nervous system. We
aimed to assess the main, haplotypic and GxE effect of ESR1 (rs9340799,
rs2077647) and OXTR genes (rs4686302) on personality traits variation.
We recruited 1018 healthy individuals (68% women) of Caucasian origin
(Russians-409, Tatars-290, Bashkir-130, Udmurts-189) from Russia (mean
age: 19.812.65 years) without any history of psychopathologies subjected
to personality assessment (TCI-125). Genotyping of SNPs was performed
using PCR-RFLP. Statistical analysis was conducted with PLINK v.1.07, Haploview 4.1 followed by correction for multiple testing under FDR-procedure. Subsequent haplotype analysis revealed an association of ESR1 A*C-haplotype (rs9340799, rs2077647, D=0.76) and A*T-haplotype (PFDR=0.008)
with high and low self-transcendence (ST) (PFDR=0.042), respectively, in
Bashkir group. GxE analysis revealed the model ESR1*ethnicity explained
variations in ST (PFDR=0.036) and self-directedness (SD) (PFDR=0.036).
Accordingly, Udmurts carrying rs9340799 G-allele demonstrated decreased SD and Bashkirs with rs2077647 C-allele reported increased ST.
Moreover, association of OXTR rs4686302 A-allele and lower Persistence
(PFDR=0.031) was observed only in non-smoking group. Additionally, OXTR
rs4686302* ethnicity model determined variations in SD (PFDR=0.021):
Russian individuals with rs4686302 A-allele scored higher on this trait. Reported findings suggest that ethnicity and smoking status modulate the
association between ESR1 and OXTR gene polymorphisms and personality
traits. Thus, ESR1 and OXTR genes might be responsible for mental health
and neuroprotection, since character traits (ST and SD) are predictive for
personality disorders development. Study was supported by Russian foundation for humanities grant ( 13-06-00583a).
J09.66
Exome sequencing reveals two compound heterozygous DDHD2
mutations in a non consanguineous Italian family with ARHSP-TCC
DNA variation at the FGF20 gene has been associated with Parkinsons disease (PD). Specially, single nucleotide polymorphism (SNP) rs12720208 in the
3 untranslated region (3 UTR) was linked to PD-risk through a mechanism
that would implicate a differential binding to microRNA-433 (miR-433). In
this study, we genotyped the rs2720208 SNP in a total of 480 PD patients
and 480 healthy controls from Iran. We found significant differences in allele and genotype frequencies between patients and controls (Fisher exact
p<110-7). The results obtained in this study revealed that the rs12720208
(C/T) polymorphism is a strong risk factor for late-onset PD in Iranian population.
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J09.69
Evidence of Dysbindin gene promoter hypermethylation in peripheral
blood lymphocytes as a potential biomarker in major psychosis
M. Bahar1, M. Noruzinia2, N. Beyraghi3, Z. Rezaei4;
1
Tarbiat Modares University, Bahar Medical Laboratory, Tehran, Islamic Republic of
Iran, 2Tarbiat Modares University, Tehran, Islamic Republic of Iran, 3Shahid Beheshti
University of Medical Sciences. Taleghani General Hospital, Psychiatric Department,
Tehran, Islamic Republic of Iran, 4Behavioral Sciences Research Center of Psychiatry
Department of Shahid Beheshty Medical University, Tehran, Islamic Republic of Iran.
441
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to affected individuals and absent in the unaffected sibling were sought. Disease status in the family linked to a homozygous region on chromosomes
2. The DYSF gene that associates with LGMD type 2B was positioned within
the linked region. DYSF encodes dysferlin, a protein believed to be involved
in the maturation of new muscle fibers and in repairing damage to muscle
cells. Sequencing of DYSF exons in the proband of the Iranian revealed a
novel insertion-deletion that is the likely cause of LGMD in the family. The
phenotypic features of the patients harboring the mutation are uncommon
because of prominent proximal presentations. To date, over 400 mutations
disease in DYSF have been reported. The novel mutation reported here represents only the second mutation observed among Iranian LGMD patients.
J10.03
A multi-exonic RYR1 deletion identified in individual with confirmed
malignant hyperthermia
I. Valaskova1,2, S. Dudova1,2, Z. Svestkova1, R. Gaillyova1,2;
1
University Hospital Brno, Medical Genetics Dpt., Brno, Czech Republic, 2Masaryk
University, Faculty of Medicine, Brno, Czech Republic.
DMD gene duplications detection and analysis of deletions at female relatives of the patient has not yet been carried out in Russian patients. Thus, the
aim of this work is the development of methods of quantitative analysis of
DMD gene duplications and deletions and determining the frequency of duplications in the selection of patients with DMD/BMD from Russia. To perform this task, we have developed a quantitative method based on specific
ligation reaction (MLPA). Using this method, we analyzed 237 patients with
Duchenne/Becker muscular dystrophy, which didnt reveal deletions of hot
exons. Duplications have been detected among 37 patients. Deletions not in
hot exons have been found among 9 patients. Thus duplications are found
in 6% of cases, which is supported by the literature data. In addition, this
method allows detecting deletions / duplications in female genome, which
allows to determining mutations in DMD gene in carrier.
J10.05
A Novel POMT2 mutation defined in a girl with Walker Warburg
syndrome.
Introduction Spinal and bulbar muscular atrophy (SBMA) is slowly progressive and adult-onset motor neuron disease caused by a CAG repeat expansion in the AR gene. Few data are available for clinical characteristics
and genotype-phenotype correlation in Korean SBMA patients. Methods
Forty consecutive patients diagnosed by genetic testing were included in
this study. The age of onset was defined as the time of the patients initial
recognition of symptoms and disease duration was represented by the time
taken from onset of symptoms to genetic diagnosis. The severity of symptoms was assessed by nine ADL milestones. Rate of disease progression
was expressed as a number, differences of ADL scores between at onset and
diagnosis/disease duration. Results The median age at onset and diagnosis
was 44.5 and 52.5 years, respectively. The median disease duration was 5.00
years, median rate of disease progression was 0.2 score/year and median
number of CAG repeats in the AR gene was 44. The number of CAG repeats
showed significant inverse correlations with the age of onset of muscle weakness and the age of onset of any symptoms. Interestingly, a statistically significant correlation between rate of disease progression and age at onset
was observed while an inverse correlation between rate of disease progression and disease duration was noticed. Discussion This study reaffirms the
inverse correlation between the age at disease onset and the number of CAG
repeats. It is of note that the rate of disease progression is influenced by age
at disease onset in Korean SBMA patients.
J10.07
Molecular analysis of mutations smn1, smn2 and naip genes by
multiplex ligation-dependent probe amplification (mlpa)
B. Kamaliyeva, A. Borovikova, A. Nagimtaeva, A. Satieva, G. Abildinova;
National Research Center Maternal and Child Health, Astana, Kazakhstan.
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J10.08
Definition breakpoints in the dystrophin gene for heterozygous
carrier revealing
Duchenne muscular dystrophy - a disease caused by the presence of deletions, duplications and point mutations in the dystrophin gene (DMD), destroying one to several exons. An important aspect in the diagnosis of the
disease is to determine the break points for the diagnosis of carriers of the
abnormal gene. Objective: to develop a method of determining breakpoints
of deletions of the intron of DMD gene to identify heterozygous carrier. Material research was genomic DNA of two patients suffering from Duchenne
muscular dystrophy and their parents. DNA was extracted from whole blood
using a kit Wizard Genomic DNA Purification Kit (Promega). Diagnosis
of Duchenne muscular dystrophy was carried out using the MLPA, followed
by capillary electrophoresis. Design of primers was performed using the
program Primer 3 (http://primer3.ut.ee/). Determination of break points
in the region of exons 45-50 was performed using PCR RT, the 7300 Applied
Biosystems (USA). MLPA- analysis revealed a deletion of a segment encompassing exons 45-50 of the gene. The first break point occurred between
exons 44-45, the second - between exons 50-51. Using genomic databases
(http://www.genome.ucsc.edu) defined size between exons: 44-45 includes
~ 250,000 nucleotides 50-51 includes ~ 45 500 nucleotides. Using several
sets of primers, these areas have been divided into several parts, followed
by PCR RT, to identify the region deletions. At the last stage of the experiment break point was determined by sequencing. As a result, this study
developed a method to identify heterozygous carrier in preclinical diagnosis
of Duchenne muscular dystrophy.
J10.09
Novel pathogenic mechanism of a mid-intronic mutation in DMD gene
SUMMARY: The proximal spinal muscular atrophy (SMA) is a severe autosomal recessive neuromuscular disease characterized by degeneration of
alpha motor neurons in the spinal cord, resulting in progressive proximal
muscle weakness and paralysis. 95% SMA patients have homozygous deletion of SMN1 gene. The remaining 5% cases are caused by compound heterozygous mutation: a SMN1 deletion on the one allele and a subtle mutation
on the other allele.
OBJECTIVE: To identify the intragenic mutations in SMN1 gene in the 11 unrelated SMA patients with the one copy of SMN1 gene.
METHODS: MLPA was carried out to measure the copy number of SMN1 and
SMN2 genes. The subtle mutation analysis of SMN1 gene was performed by
direct sequencing.
RESULTS: We have identified seven mutations in eight patients with proximal SMA.(Tab.1) Mutation c.824G> C (p.Gly275Ala) has been met twice.
In six of eight cases mutations are inherited from the father.
Mutations are well correlated with the type of disease.
It has been found no mutations in three patients. Possibly, in these cases,
mutations are located outside the investigated sequences, f.e. at promoter
region. Or these patients are carriers having another SMA-like disorder.
CONCLUSION: Analysis of subtle mutations in SMN1 gene is essential to genetic counseling in non-deleted SMN1 gene SMA families.
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Type SMA
I
II
I
III
II
II
I
I
III
I
I
Genotype
1T / 2C
1T / 3C
1T / 2C
1T / 3C
1T / 2C
1T / 3C
1T / 2C
1T / 2C
1T / 4C
1T / 2C
1T / 2C
Mutation
cDNA
Protein
c.43C>T
p.Gln15X
c.684dupA
c.815A>G
p.Tyr272Cys
c.821C>T
p.Thr274Ile
c.824G>C
p.Gly275Ala
c.824G>C
p.Gly275Ala
c.835-2A>T
c.836G>T
p.Gly279Val
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Exon/
Origin
Intron
E1
de novo
E5
F
E6
F
E6
F
E6
F
E6
F
I6
F
E7
M
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Table 1. The results of current study (T, C - telomeric and centromeric copies of
SMN gene).
J10.11
Phenocopies as a possible cause of molecularly unconfirmed cases of
Emery-Dreifuss muscular dystrophy
T. Adyan1, G. Rudenskaya1, E. Dadali1, O. Ryzkova1, O. Groznova2, D. Vlodavets2, V.
Fedotov3, A. Polyakov1;
1
Research Center for Medical Genetics, Moscow, Russian Federation, 2Moscow Scientific
Research Institute of Pediatrics and Child Surgery, Moscow, Russian Federation,
3
Regional Clinical Hospital, Genetic Counseling Department, Voronezh, Russian
Federation.
The recognition of microdeletion/duplication syndromes have been facilitated by the development of array comparative genomic hybridization techniques. One of the microdeletion/duplication syndromes recently identified
is 17q22 microdeletion syndrome which has been defined in 2013. This
syndrome presents with intellectual disability, conductive hearing loss, facial dysmorphism and skeletal anomalies. Only nine patients have been reported previously. The common deleted region includes NOG gene which is
essential for joint formation and the phenotype assosiated with NOG-related symphalangism spectrum disorder is characterized by symphalangism,
joint contractures and conductive hearing loss.
Here, we present a new case with 17q22 microdeletion syndrome. She was
refered to genetics department due to facial dysmorphism, long and tapering fingers and preauricular skin tags. On physical examination at the age of
seven, she showed narrow forehead, midfacial hypoplasia, hemicylindrical
nose, short and flat philtrum, thin border of upper lip, joint contractures
and proximal symphalangism. She had severe intellectual deficit. Since she
displayed symphalangism, NOG gene was sequanced and no mutation was
revealed. Further testing by array-CGH showed a de novo 3,1 MB deletion on
17q22. The deleted region contains 36 genes, including NOG gene. The case
will be discussed in view of the literature.
444
Back to index
J11.02
TripleX Syndrome with short stature: a case report
Triple X syndrome (47,XXX) is a sex chromosomal aneuploidy characterized by tall stature, microcephaly, hypertelorism, epichantal folds, congenital abnormalities and motor and language delays. We present a rare phenothype of the syndrome: a 10 years-old girl admitted to our hospital for
short stature. She was born from non consanguineous parents at term of
uneventful pregnancy weighting 2500 gr and measuring 47 cm. The girl
was 117.7 cm (height SD score -2.87)< to the parental target eight of 154.5
cm, with a weight of 19Kg (SD score -2.13) and head circumference of 45.5
cm (below the 3rd percentile). The girls growth rate was of 4-5 cm annually. The genitalia were of normal female phenotype with Tanner 1 stage
of breasts. In addiction she presented clinodactyly, ogive palate and mild
cognitive and speech delays. Karyotype was 47,XXX; FISH (probe CEP-X) on
200 interphase nuclei confirmed cytogenetic data. There was no evidence of
growth hormone deficiency. Laboratory tests showed low insulin-like growth factor-1 (IGF-I). Bone radiographic imaging of the left carpal demonstrated a difference between bone age and chronological age of 2 years. Blood
routine test, liver and kidney function, blood glucose and insulin, thyroid
function were normal. The ultrasound examination of heart, uterus, ovary
and urinary system, as well as cranial magnetic resonance imaging (MRI)
were normal. A possible explanation might be the aploinsufficiency of the
short stature-homeobox-containing gene (SHOX gene) that is present in the
pseudoautosomal region of sex chromosomes. This study performed in our
patient is yet ongoing.
J11.03
De Novo 4p deletion and 4q duplication in a female dysmorphic child
M. Ozdemir1, H. Onur Kucuk1, E. Simsek2, N. Dzkale1, B. Durak Aras1, H. Aslan1, O.
Cilingir1, S. Artan1;
1
Eskisehir Osmangazi University, Faculty of Medicine, Department of Medical Genetics,
Eskisehir, Turkey, 2Eskisehir Osmangazi University, Faculty of Medicine, Department of
Pediatric Endocrinology, Eskisehir, Turkey.
We would like to address a case regarding a patient who suffered from multiple malformations. The symptoms include small kidneys, epilepsy, dystrophy, hyponatraemia, immunodeficiency with decreased lymphocyte number, most of which are well known in medical literature, however some of
them are unique like hypocalcaemia and pulmonary hypertension. Despite
preliminary tests, which include GIEMSA banding and FISH, we were not
able to detect the genetic alterations behind these symptoms, which is why
we expanded the routine method to array CGH. We used Agilent Human Genome G3 Sureprint 8x60K Microarray to detect the cytogenetic deletions in
We report ok a 5 years old boy with a prenatal ultrasound diagnosis of biventricular hydrocephalus. He was born with a birth weight of 2580 g, a
length of 46 cm and a head circumference of 33 cm. He did not show muscular hypotonia but his psychomotor development was delayed: he sat at the
age of 11 months and he walked independently at the age of 17 months. He
spoke single words at the age of 12 months but he could not make complete sentences until 3 years of age. Brain MRI showed colpocephaly, thinned
corpus callosum and pons, hypertrophy of massa intermedia. His EEG was
normal. Neurological evaluation showed muscular atrophy and hypotonia,
joint laxity and mild intellectual disability. On the clinical examination at
5 years of age, the patient had a weight of 15 kg (3th centile), a height of
105 cm (10th centile) and a OFC of 47 cm (<<3th centile). The examination
showed failure to thrive, microcephaly, triangular face, downslanting palpebral fissures, sparse eyebrows, large and posteriorly rotated ears, muscular
atrophy and hypotonia, ligamentous laxity, scapular winging and bilateral
flat foot . Array-CGH showed a 2.5 Mb microduplication of 2q37 and a 5.3
Mb microdeletion of 6q27 generating a de novo derivative 6 chromosome:
this rearrangement is likely to be the result of a translocation involving the
distal region of long arm of chromosome 2 and the distal region of long arm
of chromosome 6. Array-CGH showed also a maternal 714 Kb microduplication of Xq26.2.
J11.06
A case report with de novo interstitial deletion in 7q21
Back to index
Genomic rearrangements of 8q distal bands have been associated with several clinical entities, such as Langer-Giedion Syndrome. We report a unique
familial case with complementary chromosomal aberrations involving the
same distal 8q region, detected on fetus (deletion) and mother (mosaic duplication). Chromosome analysis on chorionic villi at 13 weeks of gestation
for increased Down Syndrome risk after maternal serum screening revealed
46,XX,del(8)(q?23q?24.2) karyotype. The 8q deletion was defined as interstitial by subtelomeric and painting specific FISH probes. The deletion size
was investigated by microarray and parental karyotype was performed.
Microarray result was arr[hg19] 8q24.11q24.3(119,142,459-144,825,972)
x1 with a 25 Mb deletion. The involved region included 74 OMIM genes
and no recurrent microdeletion syndromes. Maternal karyotype was mos
46,XX,dup(8)(q?24.1q?24.3)[22]/46,XX[28]; the mosaic interstitial duplication was confirmed also by FISH and microarray, which disclosed the same
fetal breakpoints. Only two cases have been described with pure 8q24 duplication characterized by microarray (Concolino et al., 2012;Wheeler, 2010)
and none in mosaic condition. Mother had hydrocephalus at birth, short stature, facial dismorphisms, psychomotor and mild cognitive delay. Fetal ultrasound investigation was normal. Similar deletions have never been reported.
We speculated about possible clinical consequences evaluating the role of
genes mapped in the deleted/duplicated region, such as KCNK9, associated
with Birk-Barel mental retardation dysmorphism sindrome (#612292); in
brain tissues, it is expressed only from maternal allele and this might have a
possible role in maternal and newborn phenotype. We have also suggested
a possible mechanism of formation of this unique familial case
J11.09
Application of array CGH technique for postnatal identification of
constitutional abnormalities
445
Chromosomal imbalances are the major cause of developmental delay combined with dysmorphic facial features. Many of these imbalances are caused
by submicroscopic deletions or duplications that are undetectable at the
level of traditional cytogenetic analysis. Array-based comparative genomic
hybridization (array CGH) is a powerful and high-resolution approach for
detection of DNA copy number variants (CNVs).
We report a 7 month old boy with mild dysmorphic features, developmental
delay, excessive skin grooves of limbs and hypoplasia of the corpus callosum.
We have used genomic array CytoChip Oligo (BlueGnome, Cambridge, UK),
format 2x105K, version 1.1. and BlueFuse Multi software, version 2.2. The
2x105K array detects 100 Kb imbalances on the backbone and has tiling of
20 probes over 137 OMIM disease loci. Array CGH- analysis revealed cryptic
deletion of 15q11.2 region spanning 4,68 Mb and an amplification spanning
19,31 Mb of 13q12.11-13q13.3. The CytoChip results were confirmed with
MLPA. The influence of the known genes in the imbalanced regions and their
correlation to the phenotype will be discussed.
J11.11
The BBS12 gene mutation is cause of Bardet Biedl syndrome in two
individuals from south west Iran
446
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Many genetic conditions are related to changes in particular genes on chromosome 22. We present three unrelated cases diagnosed in our Genetics
Department with rare chromosomal abnormalities involving the structure
of this chromosome.
Cat-eye syndrome is characterized by a recognizable clinical picture, although the variability of the physical features is enormous. A female infant,
one month old, was referred with following major malformations: hypertelorism, narrow palpebral fissures, cleft palate, bilateral preauricular pits,
bilateral coloboma of the iris, anal atresia with a fistula from the rectum to
the vagina, complex heart malformation. Cytogenetic evaluation identified
an additional marker and by FISH examination has been confirmed the involvement of chromosome 22.
Phelan-McDermid syndrome/22q13 Deletion Syndrome is a genetic syndrome caused by disruption of the SHANK3/ProSAP2 gene on the terminal
end of chromosome 22. A newborn male with neonatal hypotonia, mild dysmorphic features and large, fleshy hands was suspected with chromosomal
abnormality. G-banded chromosome analysis identified terminal deletion
involving 22q13.3. Parental karyotypes were normal. FISH analysis confirmed deletion of SHANK3 gene.
Microdeletion 22q11.2 syndrome associated with XXY karyotype (Klinefelter syndrome). This double chromosomal abnormality is a rare finding in
clinical practice. A case with DiGeorge phenotype was confirmed by conventional cytogenetic testing and FISH analysis, but the results revealed also
XXY karyotype.
There is a great variability of clinical phenotypes in chromosomal disorders:
some of clinical findings are distinct and others are very common. Clinical
skills combined with standard and molecular technologies could offer a definite diagnosis and proper genetic counseling.
In the current study 52 patients were selected for array CGH analysis after implementing stringent criteria (a combination of congenital malformations, dysmorphic features and behavioral disorders, and patients with
multiple abnormalities and the presence of at least one major anomaly). A
combination of methods was applied - cytogenetic analysis, FISH method,
oligo DNA microarrays.
The results of analysis of microarrays revealed definite etiology in 9 out of
52 patients tested. Fifteen pathological aberrations were found in them. All
pathological findings were validated by FISH analysis. Genotype/phenotypic correlations between different patients were confirmed. In addition, the
majority of the patients tested (41 patients) showed 124 normal variations
in the number of copies and 108 variations of unknown clinical significance
(34 patients). Analyses of the type and distribution of the different variations was performed and the clinical significance of variants of unknown
nature was discussed.
Our results show the advantages of high resolution microarrays for clinical
diagnosis of patients with congenital malformations associated intellectual disability. The results also highlight the need for extensive population
studies revealing the molecular nature and clinical significance of different
CNVs and the creation of detailed maps of variations in the Bulgarian population. This would facilitate greatly the precise interpretation of specific
genomic imbalances in clinical aspect and would ease the widespread introduction of microarrays in diagnostic practice not only for postnatal diagnosis of individuals with developmental delay and dysmorphism, but also for
prenatal genetic diagnosis.
Acknowledgements: Grant 02/76-21.12.2009, National Science Fund, Bulgaria.
J11.16
Duplication on chromosome 17 and deletion on chromosome 20 in a
patient with craniosynostosis
R. Pogue1, F. A. Marques1, R. S. Heredia2, J. F. Mazzeu3, M. O. Cardoso1;
1
Universidade Catlica de Braslia, Braslia, Brazil, 2Secretaria de Sade do Distrito
Federal, Braslia, Brazil, 3Universidade de Braslia, Braslia, Brazil.
We present molecular cytogenetic analysis of a boy with syndromic craniosynostosis. He was born to healthy parents after an uncomplicated pregnancy. At birth he presented with saggital craniosynostosis and seizures.
Further examination revealed complications including a cardiac malformation. At eight years and two months the patient showed neuropsychomotor
and behavioral anomalies. G-banding showed an apparently normal male
karyotype. A DNA sample was taken for chromosome microarray analysis (CMA) using the Affymetrix 750k CytoScan array. The results showed
terminal alterations of chromosomes 17 and 20. Specifically, there was
a duplication on the long arm of chromosome 17 (chr17:78,952,204-81,060,886), together with a deletion of 1.4 Mb of long arm of chromosome 20
(chr20:61,643,144-63,003,805). The deletion on chromosome 17 includes
more than 80 genes and miRNAs, while the deletion on chromosome 20 includes more than 50 genes and miRNAs. The data are indicative of an unbalanced translocation of part of chromosome 17 to chromosome 20. Partial
trisomy of chromosome 17q has been previously reported in the literature,
and the patient reported here shows phenotypic overlap with the previously reported spectrum, including psychomotor delay, craniofacial assymetry
and other dysmorphisms. Previous reports also suggest that severity of the
phenotype associated with 17q partial trisomy may be associated with partial monosomies of other chromosomes, and this also appears to be the case
with the current patient.
J11.17
Atypical presentation of 17q21.31 microdeletion syndrome
H. M. Kathom;
Department of Clinical Genetics, University Pediatrics Hospital, Medical University Sofia,
Sofia, Bulgaria.
The combination of developmental delay and dysmorphic features is frequently caused by different chromosomal imbalances. Many of these imbalances are result of submicroscopic deletions or duplications that are
impossible to detect by conventional karyotyping. One of the best analysis
which can be used in such cases is the array-based comparative genomic
hybridization (array CGH) as it is a high-resolution approach for detection
of DNA copy number variants (CNVs).
Back to index
We report a 15-month-old boy with dysmorphic features, mild developmental delay, congenital glaucoma and partial coronal craniostenosis. We have
used genomic array CytoChip Oligo (BlueGnome, Cambridge, UK), format
2x105K, version 1.1. and BlueFuse Multi software, version 2.2. The 2x105K
array detects a loss of 231 Kb that overlaps 3 HGNC and 1 OMIM gene.
OMIM disease: Chromosome 17q21.31 deletion syndrome (610443). The
maximum overlap between an ISCA pathogenic region of type loss and this
region is 30%. 56% of the region is covered by significant polymorphisms
of type loss (DGV: 56%, ISCA: 0%). The CytoChip results were confirmed
with MLPA. The influence of the known genes in the imbalanced regions and
their correlation to the phenotype will be discussed. The above mentioned
features do not correlate with the typical presentation of Koolen-De Vries
syndrome as aspected from the aCGH results.
J11.18
5p13.3p13.2 duplication associated with severe intellectual disability,
congenital malformations and chromosome instability manifested as
aneuploidy
I. A. Demidova1,2,3, S. G. Vorsanova1,2,3, Y. B. Yurov1,2,3, I. Y. Iourov1,2,4;
1
National Research Center of Mental Health, RAMS, Moscow, Russian Federation,
2
Institute of Paediatrics and Paediatric Surgery, Ministry of Health, Moscow, Russian
Federation, 3Moscow City University of Psychology and Education, Moscow, Russian
Federation, 4Department of Medical Genetics, Russian Medical Academy of Postgraduate
Education, Ministry of Health, Moscow, Russian Federation.
447
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We report a female patient with growth retardation who was referred to our
clinic from pediatric service. She was the first child of 41 year old mother
and was born at 31 weeks with 1640 gr birth weight. Her mother and father
was healthy and have normal karyotypes. On physical examination, shorth
neck, shorth philtrum, high arched palate, hypertelorism, simple ear, sacral
diple, macrocephaly and hypothenia were detected. Cardiological evaluation was normal. In the cytogenetic analysis 47,XX,+mar was detected. We
performed chromosomal microarray using an Affymetrix 750K SNP array
platform. The application of array offered a precise characterization of the
marker chromosome plus additional findings. The most significant finding
confirming the conventional analysis was a 10,145 kbp duplication on chromosomal location15q11.2-13.3. Additional unconfirmed findings above the
cut off values involvingthe sites containing OMIM genes were: A 837 kbp
gain within 4p15.32, and a 1066 kbp loss on the X chromosomes short arm,
p22.33. We observed that the patient features and array CGH results were
compatible. As a result, array CGH can be used to define a better karyotype
phenotype correlation in patients with dysmorphic featues which can not be
expained by known syndromes.
J11.21
The clinical phenotype in a familial deletion 18p syndrome
Deletion 18p syndrome is characterized by dysmorphic features, growth deficiencies, and mental retardation with a poorer verbal performance. About
1 in 50,000 babies is born with a deletion of 18p. Most reports suggest that
18p deletions affect girls more often than boys. Until now, few families have
been described with limited clinical description. Women with del(18p) are
fertile and seem to have a normal miscarriage rate. We report transmission
of deletion 18p from a mother to his son. The proband is 8 years old and
has short stature, dysmorphic features, polymorphous dyslalia and moderate mental retardation. The mother also presents dysmorphic features, mild
mental retardation and has better verbal abilities than his son. Our report
presents a mother with del(18p) syndrome having two miscarriage and no
analysis was performed on the fetus. Chromosome analysis from the proband and their mother revealed the same chromosomal deletion: 46, XX,
del(18)(p11.2).
This report sheds new lights on the familial del(18p) syndrome. Cognitive
performance may be more variable than previously suggested within the
same family. Management needs to be handled by a multidisciplinary team
and includes speech therapy, hormonal (if necessary) and psychological
care. Patients have an essentially normal life expectancy but will need to
attend regular medical visits. Genetic counselling for these patients should
take into account these new data, especially in regard of a wider variability
of intellectual outcomes and better verbal performance.
J11.22
A rare case with De Novo Isochromosome 18p Syndrome
Tetrasomy 18p is a very rare chromosomal disorder which affects males and
females equally. The small extra metacentric marker chromosome results
from a spontaneous mutation early in embryonic development in most of
the cases. Here we report a de novo supernumerary i(18p) in a 8 months
old female dysmorphic child. The case was delivered by Cesarean section at
the 38th weeks of gestation with anthropometric parameters <-2 sd. Other
significant features of the case were growth retardation, neonatal feeding
problems, microcephaly, seizures, hearing loss, strabismus, refractive errors, high arched palate, and constipation. An additional metacentric marker chromosome was revealed by conventional cytogenetic analysis. The
chromosome constitutions of the parents were normal. Based on physical
features of the case, FISH analysis specific to chromosome 18 were performed and tetrasomy 18p was diagnosed. The clinics of the patient was compared with the previously published cases.
J11.23
Clinical and genetic examination of syndromic limb defects
D. Cpn, G. C. Cozaru, A. F. Mitroi, M. Achie;
Constana County Emergency Hospital, CONSTANTA, Romania.
448
J11.24
A de novo marker chromosome derived from 15q in a patient with
growth retardation: genotype-phenotype correlation
J11.25
Recurrent pregnancy loss and familial marker chromosome: case
report
19-year-old female patient with recurrent pregnancy loss referred to Department of Medical Genetics from Department Obstetric and Gynecology of
Afyon Kocatepe University. Her physical evaluation was normal. Biochemical and hormon test results were normal. They had second cousin marriage.
There was an ectopic pregnancy and a 10 week intrauterine dead history
in their 6 years marriage. The karyotype of the abort fetus was 46,XY. The
family history was not specific, but postnatal exitus was reported in two of
her sisters. The karyotype of the propand was detected as 47,XX+mar[108].
The marker chromosome was regular and had same structure in every metaphase plate. Centromere was not detected by C-banding. Physical examination of the Probands husband was normal and his karyotype was 46,XY.
The mother of the proband had 46,XX[71]/47,XX,+mar[29] karyotype and
father had 46,XY karyotype. The proband has 7 sisters and two of them had
regularly the same marker chromosome in their karyotype, the rest had normal karyotype. Sisters who had marker chromosome have postnatal exitus
history in their pregnancies. Fluorescence in situ hybridization-FISH report
is expected in order to detect the origin of extra chromosomal structure. In
our case report, we aimed to evaluate the effect of marker chromosome in
recurrent pregnancy loss ethiology.
J11.26
The Case Of Michel Aplasia In Russian Family With Congenital
Sensorineural Deafness: Results Of Temporal Bone Ct-Images Analysis
L. A. Klarov1, N. A. Barashkov2,3, F. M. Teryutin2,3, L. A. Klarova4, A. V. Solovyev3, V. G.
Pshennikova2,3, G. P. Romanov3, N. A. Solovyeva2,3, E. E. Fedotova5, N. V. Luginov1, K.
E. Savvinova3, N. N. Gotovtsev3, A. M. Rafailov3, A. N. Alexeev6, L. U. Dzhemileva7, O. L.
Posukh8,9, E. K. Khusnutdinova7,10, S. A. Fedorova2,3;
Partial deletion of chromosome 3p is a rare disorder with variable chromosomal breakpoints and consequently phenotypes. Although most of these
deletions involve the 3p terminus, interstitial deletions may also give rise to
features of the syndrome.
We report the case of male patient with mental retardation and minor dysmorphic features and a 7.4Mb deletion of 3p26.3p26.1 [arr 3p26.3p26.1
(61,891 7,527,372) 1], encompassing the genes CHL1, CNTN6, CNTN4,
CNTN4-AS2; IL5RA, TRNT1, CRBN, LRRN1, SETMAR, SUMF1, ITPR1, EGOT,
LOC100507582, BHLHE40, ARL8B, EDEM1, MIR4790 AND GRM7. We compare the clinical phenotype of this patient to previously reported cases of
3p syndrome.
Microarray technologies are increasingly becoming the tool of choice to accurately determine the underlying genetic cause and resulting phenotype
in patients with mental retardation and multiple anomalies. aCGH allows
to precisely determine the length and breakpoints in order to better understand a childs future development and needs.
In the present case molecular karyotyping has characterized a 3p deleted
region with haploinsufficiency of neurodevelopmental genes associated
with cognitive deficit and mental retardation, which may help to identify
genes important to growth and development that contribute to the deletion
3p syndrome phenotype and aid in better understanding the molecular basis of the 3q syndrome.
J11.28
16p13.11 microduplication: a case report
Back to index
Immune deficiency may be a rare feature in chromosomal disorders. A 31year-old man with immunodeficiency, intellectual disability and facial anomalies was admitted to the genetics department. The patient was born to
nonconsanguineous parents. He had congenital hypothyroidism and intellectual disability. During childhood, he had recurrent oral aphthous lesions,
ear infections and pneumonia. Investigations revealed IgA, IgG and IgM deficiencies with a normal lymphocyte count. Magnetic resonance imaging of
brain showed hyperintense lesions in peripheral white matter. On physical
examination, he had short stature, and dysmorphic facial characteristics including flat nasal bridge, low-set ears, epicanthic folds, and short philtrum.
Facial dysmorphic features, intellectual disability and immunodeficiency
may suggest some dysmorphic syndromes including 22q11.2 deletion, ICF
and others. Karyotype analysis revealed a partial chromosome 18p deletion:
46,XY,del(18)(p11.1).
Chromosome 18p partial deletion is one of the most common deletion syndromes. The estimated frequency is 1 in 50,000 live-born infants. The cause
of this disorder is deletion of short arm of chromosome 18 or sometimes
deficiency in a ring 18 chromosome. IgA deficiency, central nervous system
abnormalities such as holoprosencephaly, and mild to severe intellectual
disability usually accompany.
J11.30
A report of partial monosomy of distal 5p and partial trisomy of distal
19q in a family with Charcot-Marie-Tooth disease
O. Liaugaudiene, A. Utkus, Z. Ciuladaite, E. Preiksaitiene;
Vilnius University Santariskiu Klinikos, Centre for Medical Genetics, Vilnius, Lithuania.
We present two sisters with mild developmental delay/ intellectual disability and additional clinical features. The younger girl, 21 months of age,
had hypotonia, short stature, microcephaly, hip dysplasia, delayed teeth
eruption, bilateral epicanthus, wide nasal bridge, short philtrum, short
neck, clinodactyly of fifth finger, low serum calcium and parathormone.
Clinical features in her sister, 11 years of age, were similar, but in contrast,
she had macrocephaly, and additional skeletal anomalies: kyphosis, pectus
carinatum, contractures of elbows and knees, right clubfoot. Positive family
history of Charcot-Marie-Tooth disease was revealed (patient`s mother had
duplication of CMT1A region, patient`s grandfather had clinical symptoms
of CMT). Testing for PMP22 duplication was performed, and it was positive for the older sister. Karyotype of both sibs established by GTG banding
was 46,XX, der(5)t(5;19)(p15.3;q13.2). Subsequent chromosome analysis
in parents revealed maternal balanced translocation t(5;19)(p15.3;q13.2),
which was later confirmed by FISH. The same translocation was detected
in grandmother.
Both del5p and dup19q are described in literature as pathogenic imbalances. 5p terminal deletion causes cri-du-chat syndrome (mewing cry, microcephaly, epicanthus, depressed nasal bridge, fifth finger clinodactyly, hypotonia). 19q13.2qter duplication is associated with wide range of congenital
anomalies and dysmorphic facial features, including growth retardation, microcephaly, heart defects, hypoplasia of the gallbladder, renal anomalies, hypertelorism/flat nasal bridge, dysplastic ears, downturned mouth corners,
clinodactyly. Synthesis of del5p15.3 and dup19q13.2 symptoms determines
the clinical phenotype of our patients. Additional skeletal malformations in
older sister are caused by CMT. This report provides clinical characterization of previously unreported chromosomal rearrangement.
449
Individuals with 7q36.1-qter microdeletion may have a wide range of clinical manifestations including developmental delay, craniofacial abnormalities (agenesis/hypoplasia of corpus callosum, microphthalmia, choanal
atresia/stenosis), skeletal anomalies (absent sacrum), heart defects (atrial
septum defect, patent ductus arteriosus, pulmonary segmentation defects),
obesity, urogenital system anomalies (ectopic/supernumerary kidneys, hydronephrosis, micropenis), seizures and behavioral changes. We present the
clinical features of a 3-year-old boy with mosaic7q36.1-qter microdeletion.
Our patient presented with unspecified developmental delay and cognitive
impairment with speech delay. Mild facial dysmorphism was noticed, such
as upslanted palpebral fissures, prominent nasal bridge and narrow palate.
Another phenotypic finding was tapered fingers. Additional features included unilateral cryptorchidism, low testicular volume and childhood obesity.
He had numerous motor mannerisms including body rocking, facial posturing, self-touching. Cytogenetic and molecular cytogenetic FISH analysis revealed a mosaic male karyotype with a terminal deletion of the long arm of
chromosome 7 in 90% analyzed blood cells. The karyotype is described as
46,XY,del(7)(q36.1q36.3)/46,XY. Both parents have normal karyotypes. No
family history of congenital anomalies or mental retardation was referred.
Our findings, therefore, suggest that the phenotypic consequences are very
variable. We came to a conclusion, that a post zygotic terminal deletion occurred in this boy, after performing FISH analysis, which is a powerful tool
in low quantity mosaic cell line detection. Further investigation by arrayCGH is needed to identify chromosomal breakpoints and the exact deleted
region.
J11.32
Mowat-Wilson phenotype in two patients with normal ZEB2 gene
study: an example of somatic mosaicism?
Multiple pterygium syndromes include a group of multiple congenital anomaly disorders characterized by webbing (pterygia) of the neck, elbows or
knees and joint contractures . Males had small penis and scrotum or cryptorchidism; females had aplasia of the labia majora and small clitoris. This
syndrome included fusion of cervical vertebrae, scoliosis, flexion contraction of fingers, and rocker-bottom feet with vertical talus and facial dysmorphism with long face, high-arched palate, small mouth, and retrognathism.
450
Back to index
The multiple pterygium syndrome is phenotypically and genetically heterogeneous. It is also called as Pterygium Colli syndrome, Escobar syndrome
or Pterygium syndrome. Multiple pterygium syndrome is a rare syndrome.
This condition may behave sometimes as a dominant, but there clearly appears to be a recessive pterygium syndrome. n this case, described a 9 year
old female patient with pterygia of the neck, axilla and popliteal, flexion
contraction of fingers, camptodactyly and rocker-bottom feet, short neck,
kyphoscoliosis, downslanting palpepral fissres, low-set ears, high-arched
palate, low-set hairline, aplasia of the labia majora. To provide molecular
verification of Multiple pterygium syndrome, direct sequencing of CHRNG
gene is planned.
J11.34
Novel mutation in exon 13 of the TCOF1 gene in the patient with
Treacher-Collins syndrome
A 7 years old girl was referred to our genetics lab. The first and only child of
non- consanguinuous parents, she was delivered at 9 months by C-section.
Her birth weight was 2690 grams. She was diagnosed with renal stones at
6 months. She had bilateral deafness and underwent surgery. At examination she had normal intelligence and development. Her facial features include epicanthal folds, hypertelorism, blue eyes, arched eyebrows, high nasal
bridge and short philtrum. She had bilateral simian crease, camptodactyly of
fourth finger of right hand and syndactyly of second and third toes.
Her karyotype was normal. Whole genome Oligo Array Comparative Genomic Hybridization was performed using CYTOCHIP ISCA 4X44K whole genome oligo array version 1.1 and was analysed using BlueFuse Multi software.
A 3.09 Mb deletion of 2q36.1q36.2 was detected.
The deletion spans nucleotides 222 Mb to 225.1 Mb covering 10 OMIM genes and 13 refseq genes. We compare phenotypic and genotypic findings
of other overlapping cases. The deleted region in our patient includes the
EPHA4 and PAX3 genes that have been formerly implicated in frontonasal
dysplasia.
J11.38
Six patients from four unrelated Tunisian families with Peters plus
syndrome harboring the same splice site mutation in the B3GALTL
gene
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altered the open reading frame of the mutant transcript and generated a
PTC within exon 9. This finding potentially elicits the nonsense mRNA to
degradation by NMD.
All these data confirm an important role of the B3GALTL gene test that provides diagnosis confirmation and improves genetic counseling for the families.
J11.39
PITT-HOPKINS Syndrome: A new case of intragenic deletion detected
by array-CGH
Pitt-Hopkins syndrome is a rare syndromic mental disorder, mainly characterized by severe intellectual disability with stereotypic movements,
typical facial gestalt (deep-set eyes, broad beaked nose, wide mouth with
bow-shaped upper lip and widely spaced teeth), childhood-onset hyperventilation and seizures.While PTHS appears to be a recognizable clinical
entity,it seems to remain underdiagnosed principally when characteristic
dysmorphic features are less typical due to similarities with other known
genetic syndromes (Rett, Angelman).PTHS is an autosomal dominant
condition caused by haploinsufficiency of the TCF4 gene on 18q21.2. The
molecular abnormalities identified in more than 100 patients include 40%
of point mutations, 30% small deletion or insertion and 30% of partial or
total gene deletions. We report on a 25-year old boy with severe intellectual
disability,absent language, ataxic gait, anxiety and agitation, dysmorphic features and smiling appearance.Due to the lack of the hyperventilation and the
presence of atypical dysmorphisms, we decided to start genetic investigations by array-CGH (Bluegnome LTD, 44K-feature whole genome). The result
was a loss of 103Kb on 18q21.2 spanning from 53,045,402 bp to 53,149,037
bp (hg19) within the TCF4 gene. This gene has 20 exons (the first and the
last non coding), with several isoforms reported. Our deletion includes
exons 4-6(NM_001083962) and is predicted to result in a frameshift. At our
knowledge, this is the second one described in literature involving these 3
exons. Reporting our case we want to contribute to the phenotype-genotype
correlation in Pitt-Hopkins syndrome, mainly in those cases with a small
intragenic deletion and a less typical phenotype.
J11.40
Genotype-phenotype correlation with ring chromosome 11
451
Treacher Collins Syndrome (TCS) is a rare clinical entity which associates essentially craniofacial dysmorphism and deafness. Symptoms can sometimes
be severe being life-threatening when choanal atresia exists. Our objective
is to look for the TCOF1 (5q32-q33.1) mutations most frequently reported
in the literature in Tunisian patients affected by TCS. We were interested
in the study of the mutations in 11 of the 24 exons of the gene TCOF1 by
the method of direct sequencing in 7 patients among whom 4 family cases.
These exons are reported as hotspots of mutations. The confirmation of TCS
diagnosis by molecular biology was possible for 3 of our 7 patients. The sequencing allowed to identify a new mutation not reported in the literature
which was found in 2 cases from the same family (a father and his daughter).
An already described mutation was found in 1 sporadic case. Eight polymorphisms were found, among which five are known. The mutations found in
our patients occur in repetitive sequences which support the hypothesis of
polymerase errors. No mutations were detected in exon 24 which account
for 20 % of already reported cases. The molecular study of the TCS allows
the confirmation of the diagnosis, the orientation of the clinical follow up
and the prenatal diagnosis in the severe forms.
J11.44
Co- occurence of tetrasomy 12p and trisomy 12q
K. Bodurolu, M. Demirel, E. Kl, G. E. Utine;
Hacettepe University Faculty of Medicine, Ankara, Turkey.
452
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Trichorhinophalangeal syndrome type II (TRPS2; OMIM 150230, LangerGiedion syndrome, LGS) is a contiguous gene deletion syndrome located
on 8q23.1-q24.1. Depending on the size and position of the heterozygous
deleted region, the genetic defect principally involves TRPS1, RAD21 and
EXT1 genes. TRPS2 presents characteristic clinical findings with multiple
cartilaginous exostoses and frequently, intellectual disability. In the present
study we analyzed a female with TRPS2 by Oligo-SNP array and detected a
de novo 8q23.3-q24.12 microdeletion of 5.464 Mb on 8q23.3- q24.12 involving seven OMIM genes (CSMD3, TRPS1, EIF3H, RAD21, SLC30A8, MED30
and EXT1). In addition, UTP23, MIR3610 and exon eight of SAMD12 were
deleted. The final cytogenetic result was 46,XX, arr [hg19] 8q23.3-q24.12
(113,858,753-119,323,017) x1 dn. Parental testing of all procedures showed
normal results. Our patient had genital anomalies with normal intelligence
not previously reported. The analysis at molecular level of delete region in
TRSP2 is imperative to understand the pathogenesis of the disease
J11.48
Congenital absence of the portal vein in a child with Turner Syndrome
. N. ahin1, S. al2, S. Yalti1, P. Bayndr3;
1
Acbadem Bodrum Hospital, Mula, Turkey, 2Acbadem Bodrum Hospital, Mugla,
Turkey, 3Ege university pediatric radiology department, zmir, Turkey.
Congenital absence of the portal vein in a child with Turner Syndrome Introduction: Turner Syndrome is a disease caused by a total or partial loss
of an X chromosome in women, showing a frequency of 1 per 2500-4000
live-born females. (1,2) Characteristic features of the disease are short stature, gonadal dysgenesis, webbed neck,cubitus valgus and congental cardiovascular anomalies. Congenital absence of portal vein, however, is rare, and
there are no systemic reviews detailing the prevalance of the absence of the
portal vein in patients with Turner Syndrome. 13 year old girl was admitted
to emergency service for high fever. At the physical examination her respiratory rate was 60/min, she had toxic appearence. She had webbed neck and
short stature.She had a shilded chest with no breast tissue and prepubertal
nipples. She had splenomegaly. Laboratory tets revealed high transaminases. Chromosomal studies showed monosomy pattern compatible with
Turner Syndrome (45XO). Echocardiography was consistent with aortic
coarctation and bicuspid aortic valve. Patient underwent dynamic biphasic
computerized tomography(CT) imaging to investigate the causes of liver disease. CT revealed the absence of portal vein. She had grade II esophageal
varices in the upper gastrointestinal endoscopy. The final diagnosis was a
case of Turners Syndrome with absent portal vein, portal hypertension and
hypersplenism.
References:1. Gravholt CH. Epidemiological, endocrine and metabolic features in Turner Syndrome. Eur J Endocrinol 2004; 151:657-87.
2.Bondy CA, Bakalov VK. Investigation of cardiac status and bone mineral
density in Turner Syndrome. Growth Horm Res.2006;16:S103-8.
J11.49
An ~1,6 Mb interstitial deletion on Xp22.31 in a patient with
psychomotor developmental delay, microcephaly, epilepsy, ichthyosis,
and spastic tetraplegia.
N. Bezniakow, A. Kutkowska-Kamierczak, M. Bartnik, B. Winiowiecka-Kowalnik, B.
Nowakowska, E. Bocian, E. Obersztyn;
1. Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland.
We present a 6-year-old boy, born at 41 weeks of gestation to non-consanguineous, healthy parents. The pregnancy was complicated by respiratory
infection in the first trimester of gestation. At birth, body weight and length
were normal, and head circumference was 33 cm (3 centile). The boy was
evaluated at genetic counselling unit at the age of 3 years because of severe
psychomotor retardation, absent speech, epilepsy, spastic tetraplegia and
severe microcephaly (- 4,04SD). Generalized ichthyosis was observed. There
were no dysmorphic features or structural malformations. Brain MRI showed thin corpus callosum without other abnormalities. Results of oligonu-
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Monosomy 9p syndrome also known as Alfi syndrome is a rare genetic disease characterized by mental retardation, developmental delay, facial dysmorphism and various type of feminization in male.
We report two cases of Alfi syndrome in siblings.
One patient (9 year old girl) was initially diagnosed with dysgerminoma of
the right gonad and gonadoblastoma of the left gonad. Following symptoms
were identified: severe mental retardation, general development delay, generalized hypotonia, contractures of all joints including talocrural, knees,
and phalanges as well as complete gonadal dysgenesis. Floating movements
of eyeballs and epilepsy were also observed. The external female genitalia
were normal. Histological analysis of gonads revealed sclerosis and no follicles as well as absence of cilia on the surface of salpinx.
Cytogenetic analysis of peripheral blood cells identified male karyotype
with unbalanced translocation 45,XY, der(9)t(9;21)(p22;q11),-21. Deletion
of fragment 9p22-pter was confirmed by serial FISH analysis.
Patients sister (5 year old girl) had identical symptoms except for sex reversal and karyotype 45,XX,der(9)t(9;21)(p22;q11),-21.
Family history was remarkable for unidentified genetic disorder with mental retardation and some chromosome 21 abnormality in mother and severe
genetic disease in maternal brother. Most probably abnormal karyotype in
this family was inherited through female line.
J11.51
Aspects Of The Early Neurogenetic Diagnostics Of Klinefelter
Syndrome
Background: In the study are analyzed peculiarities of clinical manifestations and cytogenetic features in Klinefelter syndrome, which is a sex chromosome abnormality. The aim of research is to help achieve an early neurological diagnosis and initiation of measures to improve the development
of children.
Material and methods: A group of 84 boys with Klinefelter syndrome was
investigated during medical genetic counseling in the Center for Reproductive Health and Medical Genetics, presenting phenotype selection criteria
such as: developmental anomalies of the external genitalia - peno-scrotal
hypospadias, micropenis, small testes, cryptorchidism, cranio-facial dysmorphism, waist high and disproportionate, hypogonadism, gynecomastia,
mental retardation, psychosocial problems.
Results: Homogeneous form or trisomy 47, XXY (27 cases - 84.5%) was
the most common chromosomal abnormality diagnosed in the 32 patients
with Klinefelter syndrome, followed by mosaic form (47 XXY/46, XY: 1
case - 3.1%), polysomy X-Y (48, XXYY: 1 case - 3.1% and pentasomia - 49,
XXXXY: 1 case - 3, 1%) and variants of structural abnormalities of autosomal
chromosomes (47, XXY, inv (5): 1 case - 3.1%), associated with robertsonian translocation (47, XXXY, Rob (13:14): 1 case - 3.1%). Most patients with
KS had been diagnosed in puberty (23 cases - 71.9%), 6 patients (18.7%)
were diagnosed at prepubertal, and only 3 patients (9.4%) were diagnosed
during early childhood.
Conclusion: Neurogenetic diagnosis during early ontogenetic development
and cytogenetic analysis (karyotyping, Barr test) is necessary for better investigation of children with suspection on SK to confirm the clinical diagnosis and timely provide genetic counseling.
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Background: Efficient stabilization of genetic diseases spreading in population of Moldova means, first of all, to improve prophylaxis of congenital
malformations in pregnant women. The role of genetic counseling in the
prevention system of genetic diseases is analyzed in the study. The main
prophylaxis measures and prenatal diagnosis methods applied to pregnant
women of risk group are identified by authors.
Materials and methods: Retro-prospective study of investigation included
8937 pregnant women who have asked for medico-genetic counseling in
CRHMG, in 2008-2012. Group I: 4473 pregnant women from medium and
high risk group. Group II: 4464 pregnant women from low risk group.
Results: Two researched groups were comparable in age, gestation period,
degree of genetic risk. The age of women in genetic risk group was from 17
years to 44 years (average age 26,1 5,3 years). Prenatal diagnosis contributed to the identification of severe fetal pathologies to 478 pregnant women, which constituted 5,4% of total number of investigated cases. Amniocentesis with study of the fetal karyotype has allowed the identification of
numerical and structural chromosomal abnormalities to 67 patients (3,0%).
Abnormalities of the central nervous system are the most common in the
structure of the serious fetal pathologies (1,4%), followed by abnormalities of the cardio-vascular system (0,87%), osteomuscular system anomalies (0,51%), abnormalities of renal system (0,64%) and digestive system
(0,6%).
Conclusion: Genetic counseling and prenatal diagnosis methods (fetal ultrasound, biochemical screening, karyotyping) helps to reduce up to 50% of
the frequency of chromosomal abnormalities and congenital malformations
to newborns.
J11.53
The results of clinical and molecular analysis of data of patients with
branchio-oculo-facial syndrome
T. Meshcheryakova1, R. Zinchenko2, S. Zhilina1, T. Vasileva2, N. Petrova2, A. Petrin1, G.
Mutovin1, T. Kozhanova1;
1
Scientific and Practical Center of medical care for children, Moscow, Russian
Federation, 2Research Centre of Medical Genetics of the Russian Academy of Medical
Sciences, Moscow, Russian Federation.
Branchio-oculo-facial syndrome (BOFS, OMIM #113620) is a rare autosomal dominant disorder, characterized by branchial cleft sinus defects, ocular anomalies, a dysmorphic facial appearance including lip/palate cleft or
pseudocleft and associated with mutations in TAFAP2A gene. Clinical features analysis and DNA-testing are performed in 7 BOFS patients. 3 patients
present three main clinical BOFS features: cervical cutaneous aplasia linear loci, ocular anomalies, orofacial cleft, 4 patients are symptomatic with
ocular anomalies, orofacial cleft and accessory BOFS signs. 5 patients have
lacrimal duct stenosis, one has distopy of lacrimal point in upper lip area.
Orofacial clefts are observed in all 7 patients (2 - upper lip pseudocleft, 2
- upper lip cleft, 3 - upper lip/palate cleft). 2 patients have developmental
abnormalities of auditory ossicles, 1 - atresia of external acoustic meatus, 4 anatomically narrow acoustical ducts. Conductive hearing loss is diagnosed
in 5 BOFS patients. Sequensing of TFAP2A gene coding region revealed missense- mutations in 4 BOFS patients. One patient has p.Arg251Gly mutation
previously described, 3 patients from 2 families have novel mutations: p.
Arg213Ser and p.Val280Asp. Novel mutations are not detected in healthy
members of the families.
J11.54
Establishment of national birth defect surveillance in Lebanon:
preliminary data
454
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with major BDs from hospitals across Lebanon were reported. Incidence of
BDs was calculated using data provided from the MOPH. Results: 464 infants born with a major BD were reported. The overall rate was 18.2/1000
live births. The most common were musculoskeletal defects (4.2%), mainly
clubfoot (24.3%), central nervous system defects (4.01%), mainly spina bifida (40.2%) and cardiovascular defects (3.29%), with septal defect accounting for 29.7%. Facial/neck defects represented only 0.63%. Among infants
with spina bifida, 8.3% of the mothers were on folic acid before pregnancy
while 36.1% started it after conception. Among infants with clefts, 64.1%
of the mothers reported smoking exposure during pregnancy. 30.8% of all
mothers were overweight/obese before pregnancy. Parental consanguinity
was reported in 33.4% of the cases; among those, 53.2% were first cousins.
Conclusion: Ascertaining the prevalence and determinants of BDs through
proper surveillance would lead to preventive measures contributing to a
measurable reduction of BDs in our country.
J11.55
Arthrogryposis and perisylvian polymicrogyria: report of an
emerging phenotype
E. Savickaite1, D. Serapinas1,2;
1
Department of Pulmonology and Imunology, Lithuanian university of Health sciences,
Kaunas, Lithuania, 2Mykolas Romeris university, Vilnius, Lithuania.
Backround: Prader Willi syndrome (PWS) - a rare genetic disease that manifestates in 1:10000 or 1:25000 births. The aim of the study was to evaluate
when first starting to emerge Prader Willi syndrome symptoms and how
they are distributed according to sex.
Methods: In this retrospective analysis data were collected from 29 outpatients, who had clinical symptoms of PWS. The patients weight and height
were evaluated at birth and the first visit to the doctor at the time (weight
and height procentile evaluated), as well as the face, extremities, breast development and other changes.
Results: All patients were applied for the obesity to the doctor, when average
age was 3.23 years. Comparison of average age between boys and girls showed no statistically significant difference .The almond -shaped eyes were
33.3% of girls and 47.4% boys. Thin upper lips 33.3% of girls and 36.6%
boys. Short limbs were 50.0% of girls and 57.9% boys. Tapering fingers
22.2% of girls and 26.3% boys. Sandal gap 11.1% of girls and 15.8% boys.
A comparison of the other symptoms (sucking reflex absence, epilepsy, stigma) dependence on sex, a statistically significant relation has not been established. These symptoms occurred in 66.7% girls and 47.4% boys.
Conclusions:
Average ages of patients was 3.23 years, when they first applied for the obesity to the doctor. Weight on first visit to the doctor does not depend on birth
Background: Colorectal cancer is the third most commonly diagnosed cancer in both men and women. Progressive loss of cell cycle control is an important feature on the colorectal cancer. p21 (CDKN1A/CIP1/WAF1), one of
the cyclin-dependent kinase inhibitors, plays a key role in regulating the cell
cycle. The aim of this study was to investigate associations of the CDKN1A
gene polymorphisms (rs3176336,rs1801270,rs762624) with risk of colorectal cancer (CRC) in an Iranian population.
Methods: The study subjects were 150 cases of colorectal cancer and 150
controls for any polymorphisms. Genomic DNA was extracted using standard salting out method. Genotypes were determined using the polymerase
chain reaction and restriction fragment length polymorphism (PCR-RFLP)
method.
Results: A significant relation was found between rs1801270 in the CDKN1A
gene and colorectal cancer. The distribution of AC genotypes among sporadic CRC patients was more frequent than that in the control group (P value =
0.003).We found no significant difference between studied polymorphismS
and colorectal cancer .
Conclusion: to our knowledge this is the first study on association of CDKN1A \polymorphisms with CRC risk in Iranian population. Our findings indicated that there is association between rs 1801270 and risk of colorectal
cancer
J12.003
The 1303CA mutation of solute carrier family member 2 gene in lung
cancer
As an essential element for human life, iron is widely involved in many important metabolic processes, such as DNA synthesis, electron transport,
and oxygen delivery. Numerous studies have found a positive correlation between iron storage and risk of cancers such as hepatic cancer, renal
cancer and lung cancer(LC). Solute carrier family-member-2(SLC11A2) is
the only transmembrane iron transporter known to be involved in cellular
iron uptake. Mutations in SLC11A2 gene are associated with hypochromic microcytic anemia with iron overload. One of SLC11A2 gene variants,
1303C/A, occurs in the coding region of SLC11A2 and results in an amino
acid change from leucine to isolecine. Due to the role of SLC11A2 in iron
regulation, 1303C/A mutation in SLC11A2 gene (rs144863268) was investigated in 100 subjects (LC patients: n=50; healthy-controls: n=50) by
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456
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Background:
The primary endpoint of this study was evaluation of the expression of genes related to the resistance to the most common anticancer drugs in muscle invasive bladder carcinomas.
Materials & Methods:
Gene expression analysis of the 168 genes from two panels for Cancer drug
resistance and metabolism (PAHS004) and Cancer Drug Targets (PAHS507z)
was performed. A total of 47 transitorial cell bladder cancer samples of stage pTa, pT1, pT2, pT2b and lymphoepithelioma-like pT2a were investigated. Gene expression analyses of the individual samples as well as the pool
samples (pTa, pT1 and pT2) in comparison to negative and positive (nonmalignant lesions) controls were carried out.
Results:
The pool analysis revealed statistically significant difference (p<0,0001)
in the expression level between muscle invasive and non-invasive bladder
tumors. More than 10 times higher expression is observed for AKT1, AURKA, CSTB, EGFR, ERBB2, ERBB4, HDAC and MDM2 genes which are targets
for current and trial anticancer drugs. More than 5 times up-regulation was
established for ABCC1, ABCC3, ARNT, CYP1A5, CYP3A5, EPHX1, MVP, PPARG
and TXN genes. These are involved in the multi-drug resistance and metabolism of cyclosporine, steroid hormones, PAHs as well as anticancer drugs
Vincristine, Thiopurine and Taxol.
In the fourteen tested individual samples we found an up-regulation for
AHR, AR, CCNE1, CLPTM1L, CYP1A1, CYP3A5, MVP and TOP2B genes. In the
78,5% from them the expression of the PPARG gene, which activity is influenced by retinoid, was elevated.
Acknowledgements: Contract 03/48, 12.12.2011 of the Ministry of
Education and Science, Bulgaria.
J12.012
Matrix Metalloproteinase and Tissue inhibitor of metalloproteinase
gene polymorphisms involved in bladder cancer development
The aim was to investigate MMP1 (rs494379, -519 A>G), MMP9 (rs3918242,
-1562C>T; rs17576, 2660A>G), MMP12 (rs2276109, -82 A>G), MMP2
(rs2285053, -735C>T), TIMP3 (rs 9619311, -1296T>C) polymorphisms and
bladder cancer susceptibility in population of The Republic Bashkortostan
(Russian Federation). A total of 307 bladder cancer patients and 271 controls
were genotyped by PCR- RFLP. It was found 2660A> G MMP9 association with
bladder cancer development in the dominant model (p=0.044, OR=1.45, 95
% CI (1.01-2.07)) and MMP9 2660G/-1562C haplotype significantly more
common in patients (34.52 % vs. 21.17%, OR=1.90, 95 % CI (1.22-2.97))
compared with control. It was defined MMP2 -735C>T significant association with bladder cancer development in the overdominant model (p=0.0009,
OR=2.21, 95 % CI(1.38-3.53)). -1296T>C TIMP3 associated with disease in
additive model (p=0.016, OR=1.44, 95 % CI(1.07-1.95)). No association with
bladder cancer was observed for -519 A>G MMP1 and -82 A>G MMP12. It
was found -735C> T MMP2 was significantly associated with the development of bladder cancer as an invasive (OR=1.93) and non-invasive forms
(OR = 2.17). Analysis of gene - environment interactions showed a statistically significant interaction between MMP2 and MMP9 polymorphisms
with the status and smoking index (p=0.014 and p=0.017, respectively) in
dominant model. Thus we can assume MMP9, MMP2, TIMP3 polymorphisms under investigation make a definite contribution to the bladder cancer
development.
The investigation was partially supported by RFBR 14-04-97006 r_
povolzye_a, 14-06-97003 r_povolzye_a, 13-04-00287 A; RFH 13-06-00101.
J12.013
Bladder cancer risk associated with XPD polymorphism in the
Belarusian population
Bladder cancer (BC) is the fourth most common cancer among men in Europe, and the annual incidence rate is 1000 in Belarus. Among risk factors,
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cigarette smoking and occupational chemical exposure lead to accumulation of DNA damage contributing to carcinogenesis. In this context, polymorphism of error-free excision repair genes seems to affect the cancer risk.
Polymorphism of XPD Asp312Asn, XRCC1 Arg399Gln, OGG1 Ser326Cys,
ERCC6 Met1097Val was studied in the group of BC patients (336 individuals) as compared to the control group (370 volunteers). The allelic variants
were determined in DNA samples using the PCR-RFLP method. Minor allele
frequencies in Belarus were close to those in Caucasians and significantly
differed from their frequencies in Asians. Analysis of polymorphisms in a
single gene revealed increased BC risk in carriers of heterozygous genotype
Asp/Asn of XPD gene that was pronounced in the elderly (OR95%CI=3.34
[1.35-8.28] p=0.009). The combination of this genotype with homozygous
genotypes Ser/Ser of OGG1 or Met/Met of ERCC6 also increased cancer
risk. Analysis of interaction between four genes showed that carriers of
combinations Asp/Asn, Arg/Arg, Ser/Ser, Met/Val and Asn/Asn, Arg/Arg,
Cys/Cys, Met/Val possessed more predisposition to cancer (OR95%CI=3.20
[1.47-6.95] p=0.003 and OR95%CI=9.97; p=0.1 respectively). Thus, XPD312
polymorphism was predominantly associated with BC risk in Belarus.
J12.014
Cell free DNA quantification in urine of patients with bladder
urothelial carcinoma
Aim of the study Cell free DNA (cfDNA) represents one of the new bladder
cancer markers. Comparision of total urine cfDNA values of the control
group and of the patient one was the aim of our study. Patients and methods
Naturally voided second morning urine samples were collected from 99 individuals and 3 different groups of individuals were compared: (16 healthy
volunteers (HV), 20 control patients with urological diagnoses different
from bladder cancer (CP) and 63 patients with bladder cancer (BCP) (pT0
in 5 cases, pTa in 21, pT1 in 17, pT2 in 8, pT3 in 6, pT4 in 6 cases, both LG
and HG in each stage group). Results: The comparison showed statistically significant difference between HV and BCP (p value = 0,000049). There
were no statistical differences between the CP and BCP (p value = 0,372059)
or between CP and HV groups, as well (p value = 0,046749). Patients were
then divided into two groups according to their cancer stage. The group 1
(pTa patients, patients with another urological diagnoses and healthy volunteers) and the group 2 (patients with stages pT1 and more). Comparing these two groups by Mann-Whitney test we have shown significant difference
with p value < 0.001. Conclusion: pTa patients significantly differ from pT1
ones and more regardless to the cancer grade. Patients with stages pT1 and
more have significantly higher ucfDNA levels than other individuals. Acknowledgements: Supported by the Internal Grant Agency of the Ministry of
Health of the Czech Republic no. NT12417.
J12.015
BRCA1 Gene Polymorphisms and chromosomal aberrations as a risk
of borderline ovarian cancer patients in Tamil Nadu population, South
India
S. Arun, K. Sasikala;
Human Molecular Genetics laboratry, Department of Zoology, School of Lifescience,
Bharathiar University, Coimbatore, India.
Ovarian cancer is the leading cause of death due to gynaecological malignancies among women. The aim of the study was to analyse the BRCA1 gene
polymorphisms and major chromosomal aberration in the borderline ovarian cancer (BOC) patients. A population-based, case control study of ovarian
cancer was performed in Tamil Nadu. Inferred consent forms were obtained
from 34 BOC patients individually (Case & Control). We have examined for
BRCA1 Q356R amino acid changing polymorphisms. Cytogenetic analyses
were carried out using 3 mL of blood (100 metaphase). The major chromosomal aberrations were gains from chromosome arms and deletions of
1p, 12q, 14q, 15q, 16p, 17p, 17q, 19p and 19q sites in BOC patients. The
correlation between family history, age, infertility, and diet - body weight
were selectively evaluated and analysed. The most significant chromosomal
abnormalities like deletions were recorded in BOC patients compared to
the control subjects. Findings reveal that the patients above 60 years of age
were at high risk. Thus the study concludes that the BRCA1 gene polymorphisms and chromosomal aberrations play a key genetic role along with the
risk factors for BOC patients.
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particularly in the homologous recombination (HR) pathway for doublestrand DNA repair (2). BRCA1/BRCA2 germline mutations are related to an
increased risk of developing HBOCs, therefore the carrier identification is
crucial especially before the onset of the disease. Furthermore, testing for
BRCA gene mutations allow to improve the clinical management of highrisk patients and of their mutation carriers family members. However, the
correct interpretation of variants with uncertain significance or of novel variants still remains a problem for genetic counseling.
Here we describe a novel intronic variant identified in a HBOC patient and
involved in the BRCA1 gene splicing regulation. This variant is predicted to
be deleterious according to Human Splice Finder and NetGene2, causing the
loss of a canonic donor splice site at position +2 in the intron 21 of BRCA1
gene. Variant effects were experimentally verified on patient cDNA by PCR
amplifications using different primers pairs ad hoc designed. Our results
indicate that intron 21 is completely retained in the transcript RNA. Despite other studies are needed to confirm the role of this splice variant, our
preliminary results strongly support the pathogenicity of the novel found
mutation.
1. Pruthi S, et al. Mayo Clinic Proceedings, 2010;85:1111-20.
2. Venkitaraman AR. Cell, 2002;108:171-82.
J12.019
Polymorphisms in the FGFR2 and miR146 genes are associated to
increased risk in familial breast cancer in young women.
Solid tumors are characterized by considerable interindividual and intratumoral genetic heterogeneity. High genetic variability of breast cancer (BC)
probably manifested in the presence of various types of structures of infiltrative component (morphological structures): tubular, trabecular, solid,
alveolar and discrete groups of tumor cells. It may be assumed that different
types of morphological structures are manifestations of phenotypic diversity, which is based on a set of specific cytogenetic differences. The aim of
this study was to investigate intratumoral cytogenetic variation in BC and
its relationship with the morphological subtypes of breast cancer. Material
for analysis was taken from single patient with breast cancer. We isolated
five morphological structures from two regions of breast cancer with laser
microdissection (PALM, Carl Zeiss, Germany). Analysis of chromosomal aberrations was performed using Agilent microarrays (SurePrint G3 Cancer
CGH + SNP Microarray Kit, 4x180K). The number of identified unbalanced
chromosomal aberrations in the various structures ranged from 24 to 270.
While the least amount of rearrangements found in tubular structures and
the largest - in solid. The cluster analysis results indicate that the samples
Breast cancer is the most common malignancy in women and affects approximately 1 out of 10 females. Risk of developing breast cancer is strongly
influenced by genetic factors. Germ-line mutations in BRCA1 and BRCA2
genes are the primary cause of up to 5-10% of breast cancer incidence. To
reduce increased risk of developing cancer or to increase the likelihood of
early detection, carries of BRCA1 or BRCA2 mutations are offered surveillance programs and effective preventive medical interventions. This study
aimed to investigate the contribution of BRCA mutations to breast cancer
cases in Uzbekistan. 67 patients with breast cancer as well as the sex and age
matched control group of healthy individuals (n=103) were included in this
study. By means of real-time allele-specific PCR we analyzed DNA samples
of these groups for the presence of BRCA1 5382insC, BRCA1 4153delA, BRCA1 185delAG, BRCA1 300T>G and BRCA2 6174delT mutations. 3 unrelated
samples (4.5%) were found to be positive for the heterozygous 5382insC
mutation representing a possible founder mutation in the Uzbek population. In the investigated group of patients we didnt find BRCA1 4153delA,
BRCA1 185delAG, BRCA1 300T>G and BRCA2 6174delT mutations. All investigated mutations were not found in any DNA sample of control group. The
presented data confirm contribution of BRCA1 5382insC mutation to breast
cancer development in Uzbek people and taking into account a high disease
penetrance in carriers of BRCA1 mutation, it seems reasonable to suggest
inclusion of 5382insC mutation test in screening programs for breast cancer
prevention in Uzbekistan.
J12.022
Preliminary study of BRCA1/BRCA2 mutations in Bulgarian women
with breast cancer
The mutations in BRCA1 and BRCA2 genes are the most significant genetic
determinants of predisposition to develop breast cancer (BC). There is still
insufficient data about BRCA1/2 mutations in Bulgarian BC population. It
was a preliminary study aiming to investigate the frequency of five common
deleterious point mutations (reported before in Bulgarian patients) and
large genomic rearrangements (LGR) in BRCA1/2 genes in a target group
(defined by preliminary prepared selective criteria) of Bulgarian women
with BC.
The list of patients diagnosed with BC was taken from the Cancer Registry
(2009-2013) of University Hospital, Pleven. One hundred and seventy five
women with BC were interviewed and pedigree was constructed for each of
them. The patients were classified into ten categories, according to personal,
disease and family history. Based on the selective criteria we defined a target group of 79 Bulgarian women with BC. They were screened for five deleterious point mutations (5382insC; C61G, in BRCA1 6174delT, 9326insA;
9908delA in BRCA2) by DNA sequencing and for LGR by Multiplex Ligation
Probe Amplification. Among the screened women we found: one deleterious
mutation (5382insC in BRCA1) with frequency 1.2%; two polymorphic variants in BRCA2 gene - rs4987117 in exon11N and rs9534262 in exon17
with frequency 2.7% and 36% of the patients, respectively. None LGR was
detected.
In conclusion, the insufficient results of our study suggest that the strategy
for genetic screening in Bulgarian patients requires complete analysis of the
BRCA1/2 genes and stringent compliance to the internationally accepted
BCLC/NCCN criteria.
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J12.023
Mutation Analysis of ESM-1 Gene in Breast Cancer Patient Tissues
Breast cancer is the second common type of cancer in the world. In developed countries, breast cancer is one of the leading causes of women deaths.
In recent years, many genes have been identified related to breast cancer.
But underlying molecular mechanisms have not been clarified yet. Endocan, (ESM-1: endothelial cell-specific molecule-1) soluble proteoglycans, is
secreted by endothelial cells. Expression of ESM-1 gene is regulated by the
cytokines. The expression increases with the stimuli of VEGF, TGNF-, LPS
and IL-1 in endothelial and epithelial cells. The ESM-1 gene function has
an important role in tumor angiogenesis and growth. It has been shown to
involved in pathogenesis of lung, liver, gastric and breast cancer. ESM-1 gene
is reported to have an effective role in cell proliferation, cell migration, invasion, and metastatic spread of cancer cells in recent studies.
In this study, we analyzed ESM-1 gene mutation in breast cancer from paraffin-embeded tissue samples of breast cancer patients. All three exons for
ESM-1 gene mutation were sequenced by Sanger Sequencing.
Our results showed a homozygous silent mutation (CAG>CAA) in the second
exon at codon 118. Another case displayed the same mutation heterozygously. In nine cases, the polymorphism TTG>CTG was evaluated as being
remarkable in the third exon of poly-A region which maybe the miRNAs binding site.
J12.024
Association of estrogen receptor- A908G (K303R) mutation with
breast cancer risk
Genetic mutations in premalignant breast lesions may have a role in malignancy progression or influence the behavior of subsequent disease. A point
mutation in estrogen receptor- (ER-) as A908G (Lys303Arg) was originally involved to hypersensitive to estrogen breast hyperplasia. We detected
this mutation among Iranian women with invasive breast cancer. A population-based case-control study was conducted in 150 newly diagnosed invasive breast cancer and 147 healthy control individuals controls to screen for
presence of the ER- A908G mutation by using single-strand conformation
polymorphism (SSCP) analysis and 33Pcycle DNA sequencing. We detected
the 10.7% ER- A908G mutation in the form of heterozygote genotype only
among cancer patients (2=22.752, P=0.00). The allelic frequency of mutant
allele AGG in codon 303 was significantly (2=29.709, P=0.001) higher in
patients with the family history of breast cancer (28.9%) than those without the family history of breast cancer (1.9%). Our data suggest that ER-
codon 303 mutation is correlated with various aspects of breast cancer in
Iran. ER- genotype might represent a surrogate marker for predicting breast cancer developing later in life.
Keywords: Breast cancer, mutation, estrogen receptor, PCR-SSCP, lymph
node metastasis
J12.025
The association between G-2548A polymorphism of leptin gene and
breast cancer risk
S. Rostami, l. kohan;
Arsanjan Branch, Islamic Azad University, Department of Biology, Arsanjan, Islamic
Republic of Iran.
Introduction: The Breast cancer is the most common cancer in woman and
the second cause of death in women between 35 to 55 years. Risk creation
the breast cancer is in time life woman 12/5% (one of the eight cases) and
risk of death from breast cancer 3/6% (one case of twenty eight case). Leptin hormone is secreted from adipocytes which is involved in the regulation
of body weight and serum levels are correlated with breast cancer risk. Genetic polymorphisms G-2548A promoter region of leptin gene expression
and hormone secretion rates of faty tissue will be affected. The purpose of
this study is to investigate the relationship between G-2548A polymorphism
in the leptin gene and breast cancer risk.
Material and methods: We here carried out a case-control study that included 50 patients and 50 healthy subjects. We examined the genotype distribution of Leptin promoter G-2548A polymorphism, using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)
approach,to investigate the possible role of this SNP as a risk factor in breast
cancer.
459
Breast cancer is the most common cancer in women worldwide. The evaluation of the expression of estrogen receptors and progesterone and HER- 2
has become essential for the treatment of patients bearing breast cancer
since it provides information of prognostic and therapeutic order. The purpose of this study was to evaluate the expression of hormone receptors
and Her-2 protein in breast cancer , study the correlations between them
and with other prognostic factors, compare our results with those in the
literature and evaluate our immunohistochemical technique . This is a prospective study of 130 cases of breast cancer diagnosed at the Laboratory of
Pathology of the University Military Hospital and Regional Oran ( HMURO )
since the year 2006 until 2009. The evaluation of the expression of hormone
receptors and HER2 by immunohistochemistry was performed . ERs are expressed in 64% of cases, PR in 71% of cases and HER2 in 31 % of cases. The
correlation between the percentage and intensity of marking the RE and RA
was statistically significant . On PR , they were correlated with age but not
with tumor size or lymph node status with . HER2 was associated with histological type . Our results are generally consistent with those in the literature and our immunohistochemical technique has proved its reliability. The
development of molecular study and collaboration with the various departments of our University and Regional Military Hospital of Oran ( HMURO )
means help understanding some of our patients .
J12.027
Analysis of microRNA expression in breast cancer tumor:
identification of cluster co-expressed miRs.
The investigation of 14 microRNA (miR-10b, miR-21, miR-31, miR-155, miR29a, miR-195, miR-192, miR-182, miR-125b, miR-145, miR-221, miR-222,
miR-34a, miR-335) expression levels in a sample of breast cancer tumors
in relation to the normal breast tissue was performed. All patients of the
sample were not subject to radiation, neither to chemotherapy. The expression level of miR-182 most often was increased - in 81% of cases. In 59% of
cases there was over expression of miR-182 - more than 10 times. The expression of miR-31, potential metastasis suppressor, was decreased in 54%
of cases. There was identified cluster co-expression 5 microRNA: with increased expression miR-10b, miR-21, miR-155, miR-34a and miR-335. The
cluster includes 33% of studied tumors. Frequency over expression of these
miRs among tumors in the cluster was 4 times higher compared to other tumors (p < 0.00001). The transcriptional regulation analysis shows that 4 microRNAs of cluster are transcriptional targets of p53 and RelA-NF-kB. All the
tumors in the cluster were characterized by infiltrating lobular and mixed
variants (infiltrating lobular combined with infiltrating ductal) breast cancer and were positive in the expression of estrogen and progesterone receptors. The cluster includes 60% of all tumors with local metastases in lymph
nodes (i.e., metastases occur approximately 3 times more often than in the
other tumors of the sample). It allows assuming existence of the association
co-expressed in this cluster microRNA with pathomorphological features of
the tumors, in particular, with a predominant presence of metastases.
460
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J12.028
Breast carcinomas with negative estrogen and progesterone receptor
status and the expression of various potential useful biomarkers
M. Frangou, E. Plemenos, D. Karandrea, E. Boutou, D. Vlachodimitropoulos;
Athens University, Athens, Greece.
In primary breast cancer, hormonal receptor status is very useful in predicting response to endocrine therapy and prognosis. Negative ER, PR cases
have limited therapeuting options, so it is very helpful to identify biomarkers that can be used as targets for the new targeting therapies, or can be
used for prognostic value. We studied 623 tissue samples from operable primary breast adenocarcinomas and 101 of them had negative receptor status. From these 101 cases 52.3% was c-erbB2 positive vs 27.3% of the total
amount, and the HER-2 gene was amplified in 36.6% vs 16.8% of the total
cases. p53 was expressed in the 59.5% of the negative ER cases vs 26.6% of
the total amount. The expression of the stem cell markers was almost identical in positive and negative ER cases, except of cd24 which was expressed in
25%vs80% respectively (p0.02) of the cancer cells of this group. According
to our results the positivity of c-erbB2 is higher in negative cases and many
of the negative c-erbB2 immunohistochemically cases show amplification of
the gene, proposing that herceptin may be a benefit in these cases. The high
percentages of the p53 and cd24 expression shows that this group of cases
tends to have poorer prognosis, as cd24 is expressed in the more primitive
mammary stem cells. A CISH or FISH test is recommended for this group,
whatever the c-erbB2 result of the protein is, in order to give the oncologist
more detailed information about the biological behaviour of these cancer
cells.
J12.029
Study of Her-2/neu and TOP2A Expression in Familial Versus Sporadic
Breast Carcinoma
S. A. Hammad1, A. M. S. El-Gerzawy1, M. M. S. Al Ansary2, I. L. Hussein3, A. M. Mohamed1,
F. M. El-Kasem4, S. G. Abd-Allaha1;
1
National Research centre, Cairo, Egypt, 2Department of Clinical and Chemical
Pathology, Faculty of Medicine,, Cairo, Egypt, 3Departments of Pathology and dMedical
Oncology, National Cancer Institute, Cairo University, Cairo, Egypt, 4Pathology and
dMedical Oncology, National Cancer Institute, Cairo University, Cairo, Egypt.
Genetic predisposition is one of the risk factors that increase the incidence
of breast cancer. Her-2/neu gene is considered the most frequently amplified oncogenes and is accompanied with amplification or deletion of TOP2A
gene. Our study aims to compare familial breast cancer (FBC) patients with
sporadic breast cancer (SBC) and to evaluate the hormonal status, immunostaining for Her-2/neu, and Her-2/neu and TOP2A copy number alterations
by fluorescence in-situ hybridization (FISH). Twenty two patients with invasive breast cancer were involved: 12 positive for the criteria of FBC. Sections
of formalin-fixed paraffin embedded blocks were subjected to immunostaining for Her-2/neu (Hercep test), estrogen receptors, progesterone receptors and FISH for Her-2/neu and TOP2A genes copy number alterations. Pathological parameters and immunohistochemical markers proved that FBCs
were more aggressive than SBC. TOP2A gene amplification was observed in
25% of FBCs compared with 20% of SBC patients. Co amplification of both
Her-2/neu and TOP2A genes were observed in 8.3% of FBC patients and
in 20%SBCs. In contrast, Her-2/neu amplification was observed in 25% of
FBCs compared with 10% of SBCs. Importantly, TOP2A amplification without Her-2/neu gene amplification was observed in 16.7% of FBCs but was
not observed among SBC patients.
In conclusion TOP2A gene amplification occurs both in FBCs and SBCs but
more frequently among FBCs independent of Her-2/neu amplification. This
study highlights the importance of TOP2A gene amplification in diagnosis of
FBC. In addition, a combined approach using immunohistochemical analysis
and FISH can optimize Her-2/neu testing for breast cancer patients.
J12.030
Burkitt lymphoma: Unfamiliar presentation of a familiar disease
Recently, Hematology has been witness to great strides in genetic advancement. Lymphoma is now regarded as heterogeneous genetic disease. The
genetic aberrations not only aid diagnosis, but also dictate choice of therapy,
therapeutic response and portend prognosis. This is such a case exampling
application of cytogenetics. A 4 year frail child presented with abdominal
pain and distension of 15 days duration. Ultrasonography and Magnetic
Resonance Imaging revealed bilateral renal mass. Provisional clinical diagnosis of Bilateral Nephromatosis / Neuroblastoma was offered. Preliminary guided Fine Needle Aspiration showed monomorphous population of
vacuolated lymphoid cells suggestive of malignant lymphoma - Burkitt type.
Introduction: Testis-specific genes which has normal expression in restricted tissues, contains a subgroup demonstrating expression in various malignancies known as Cancer-Testis Antigen (CTAs). Considering testis as an
immune privileged organ, aberrant expression in cancers could cause spontaneous humoral and cell-mediated immune responses. Breast cancer is a
promising target for vaccination and immunotherapy using CTAs. Methods
and material: We analyzed the expression of 4 CTAs, PEPP-2, ODF4, ACRBP
and SPATA19 in 40 samples of invasive ductal carcinoma (IDC), adjacent
normal tissue and 10 fibroadenomas, as well as MCF-7 and MDA-MB-231
cell lines using RT & Real Time RT-PCR. Results: ACRBP was expressed in
normal tissue. SPATA19 expression was not significantly different in normal
and cancer tissues. ODF4 and PEPP-2 were expressed in 62.5% and 22.5% of
IDC samples respectively. Real Time results showed increased expression of
ODF4 and PEPP-2, 2.96 (p<0.001) and 3.31 (p<0.01) times in IDCs samples,
respectively in comparison with testis tissue. Both genes were up-regulated
in MCF-7 and MDA-MB-231 cell lines compared with normal testis sample.
Comparing the expression of ODF4 & PEPP-2 in MDA-231 with MCF showed 1.758 & 1.77 (p<0.001) times up regulation in MDA-MB-231, respectively. Conclusion: ODF4 and PEPP-2 could be potential cancer biomarkers.
Therefore, they can be used for active immunotherapeutic interventions.
Expression of ODF4 and PEPP-2 in malignant tissues and their absence in
benign and normal tissues make them putative cancer biomarkers. We showed ACRBP expression in normal breast tissue, so its application as cancer
biomarker or in immunotherapeutic approaches is under question.
J12.032
CAT C262T, GSTM1, GSTT1 and CML risk
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Loss of the Y-chromosome (-Y) is observed in peripheral blood of healthy elderly men, however it may be found also in bone marrow cells of males with
different subtypes of hematological malignancies. Therefore it is unclear
whether -Y occurs as normal age-related event or it is a marker of neoplastic
clone. The aim of our study was to review molecular cytogenetic findings
in bone marrow cells of patients with oncohematological diseases and to
assess the role of the Y-loss during malignant process.
We found loss of the Y-chromosome in bone marrow samples of 142 males
(median age 72 years, range 21-89 years) with myeloid or lymphoid disorders. The most frequent diagnosis was myelodysplastic syndromes and acute myeloid leukemia (54 patients). All samples were analyzed by conventional G-banding and by interphase FISH. Cut-off level for FISH was established
at 10% and the extent of cell clones with Y-loss varied from 12 to 98%. Most
of the patients (131) showed a clone with -Y at the time of diagnosis. In 114
cases Y-loss was the sole abnormality and in 17 patients it was associated
with autosomal structural and/or numerical aberrations. In 11 patients -Y
clone was detected during the progression of the disease.
As quoted in the literature (Van Dyk, 2001) the presence of >75% cells with
-Y is associated with the malignant disease. In our cohort, this was found in
25 patients (18%) and therefore such findings may be considered as related
to malignity.
Supported by MHCR 00023736, RVO-VFN64165, GACR P302/12/G157/1.
J12.035
Polymorphism of XRCC1, XRCC3 and XPD genes and risk of chronic
myeloid leukemia in a Romanian population
461
Renal cell carcinoma is the most common neoplasm affecting the adult kidney. One of the most frequent events in clear cell renal cell carcinoma (ccRCC) is inactivation of Von Hippel-Lindau gene (VHL). A series of studies
recently demonstrated that a number histone modifying and chromatin
remodeling genes, including SETD2, have mutation in ccRCC. Furthermore,
SETD2 gene is within a 50-Mb region on chromosome 3p that encompasses
VHL and is deleted in ~90% of ccRCC.
The goal of the study was to investigate inactivation of VHL and SETD2 genes by mutations. We studied 105 DNA samples of tumor tissues and normal
renal parenchyma in ccRCC patients from Bashkortostan Republic of Russia,
using PCR, SSCP and direct sequencing. Mutations in VHL gene were found in
tumor tissues with the frequency of 21,9% (23/105). We detected 22 mutations in 23 ccRCC patients, including 8 point mutations (35%), 13 deletions
(60%) and 1 insertion (5%). Ten somatic mutations hadnt been described
in literature previously. VHL inactivation through sequence alterations in
tumor DNA didnt differ by histopathologic characteristics or occupational
exposure. Analysis of SETD2 gene was performed in 50 ccRCCs DNA samples
and matched normal tissues. According to literature, mutations in SETD2
gene are detected in 4-15% cases of ccRCC. In our research SETD2 gene
mutations were not found. This may be due to an insufficient number of
samples in the investigated cohort. Further studies of chromosome 3 tumor
suppressor genes in ccRCC tumorigenesis and validating the clinical impact
of these novel mutations are required.
J12.037
Familial inversion accompanying unbalanced karyotype in a case with
CML
A. Acar, M. Balasar, E. Tuncez, P. Tasdemir, N. Somuncu;
Necmettin Erbakan University, Meram Medical Faculty, Department of Medical Genetics,
Konya, Turkey.
%90-95 of patients with CML have t(9;22)(q34;q11) Philadelphia chromosome. Other abnormalities besides the Ph chromosome are observed as %7
in chronic phase and %40-70 in blast crisis. Generally additional abnormalities are translocations and deletions, the inversion rate is just %2. Inversions
constitute %10 of all structural chromosome aberrations and generally had
no phenotypic effects. In inversion carriers, depending on which chromosome or segment are involved, the risk of reproductive wastage increase.
Here we reported a 43 years old female patient with Ph chromosome and
additive chromosomal abnormalities which has been diagnosed in CML. In
the analyses performed using BCR-ABL1 FISH probe into the bone marrow
interphase cells, %12 of the cells were normal, %20 had classical fusion, in
%68 of the cells increased ABL signals were present giving rise to complex
rearrangements. In the molecular genetic analysis the BCR-ABL1 was positive (IS:0.75924).
The karyotype of bone marrow cells was 46,XX,?add(16)(q24),inv(18),
add(21)(q22),Ph(+).C and G-banded metaphases of peripheral blood had
16qh+ and inv18, however add21(q22) and Ph chromosome which are observed in the bone marrow cultures were not encountered. The patient had
7 pregnancies but she had only 2 healthy children. Her father and 1 child
also had 16qh+ and inv18. It was concluded that only Ph+ and add 21(q22)
was present in the leukemic clones of the patient and that 16qh+ and inv18
was inherited.
As a result, it was concluded that evaluation of the contribution of inversions identified in haematologic malignancies to unbalanced karyotypes and
carcinogenesis should be made wisely.
462
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J12.038
The comparison of cytogenetic & fluorescence in situ hybridization
study on bone marrow & peripheral blood cells in 30 chronic
myelogenous leukemia patients
BACKGROUND: Chronic myeloid leukemia (CML) is a myeloproliferative disease. The cytogenetic hallmark of CML is philadelphia chromosome (Ph).
The aim of this study was to diagnose assumed CML patients, to monitor CML
patients under Imatinib therapy on the bone marrow and peripheral blood
samples by cytogenetic and fluorescence in situ hybridization (FISH), and
also to compare the results on both specimens. MATERIALS & METHODS:
Chromosome banding and FISH analysis was performed on 30 suspected
CML patients on the bone marrow (BM) and peripheral blood (PB) specimens. RESULTS: The comparison of FISH and karyotyping in 30 patients on
BM and PB specimens, respectively showed that 9 (30%) and 8 (26.66%) of
them were Ph+, and only (18.18%) of Ph positive patients showed atypical
patterns. In comparison between BM-cytogenetic and PB I-FISH, BM-cytogenetic was more reliable than PB I-FISH to detect Ph. CONCLUSION: Our
data demonstrate FISH analysis is a rapid, reliable and sensitive technique.
Our study in comparison between BM and PB, showed BM cant replace by
PB, even in detecting by FISH.
J12.039
Translocatioon t(7;11)(p15;p15) in a patient with chronic
myelogeneous leukemia (CML) Ph-positive
Cancer is a leading cause of death worldwide and accounted for 7.6 million deaths (around 13% of all deaths). Gastric cancer is the fourth and fifth
most common cancer in men and women, respectively. The northern and
northwestern regions of Iran are high risk areas for gastric cancer.
Gastric cancer carcinogenesis refers to accumulation of genetic alteration of
multiple genes such as oncogenes, tumor suppressor and mismatch repair
genes. C-MYC proto-oncogene is one of the most frequently activated oncogenes, and is estimated to be involved in 20% of all human cancers.
To evaluate MYC copy number and its protein expression, CISH and IHC ana-
Background
The life-time risk for colorectal cancer (CRC) in the Swedish population is
approximately 5 % with a mortality second only to that of lung cancer. It
is estimated that 5 % of all CRC develop in individuals with a Mendelian
predisposition for the disease, for example Lynch syndrome (LS), familial
adenomatous polyposis (FAP), MUTYH-associated polyposis (MAP), juvenile
polyposis syndrome (JPS), Peutz-Jeghers syndrome (PJS) or PTEN hamartomatous tumor syndrome (PHTS). However, diagnose-selective genetic
screening fails to detect a disease-causing mutation in the majority of patients with suspected hereditary CRC.
Aim
The SWedish Extended genetic analysis of hereditary colorectal Neoplasia
(SWEN) is a prospective national study involving researchers and clinically
active staff at the cancer genetics clinics in Sweden with the overall aim to
improve genetic testing and the care of patients and families with genetic
susceptibility for CRC.
Material and Methods
Six hundred adult patients with clinically suspected Mendelian CRC of any
type are offered mutation screening of the 11 genes associated with LS, FAP,
MAP, JPS, PJS, PHTS and additional selected candidate CRC susceptibility
genes. Genetic and clinical data for each family is registered in a national
database followed by genotype-phenotype correlation studies.
Current Status
The study is ongoing since February 2014 and inclusion of patients will continue for at least three years.
Significance
The results from the SWEN study should improve genetic testing, personalized risk estimation and effectiveness of surveillance programs for individuals with hereditary predisposition for CRC.
J12.042
Microsatellite instability analysis in sporadic colon cancer patients in
Croatia
T. Cacev, V. Musani, S. Kapitanovic;
Rudjer Boskovic Institute, Zagreb, Croatia.
Colorectal cancer is a result of accumulation of genetic and epigenetic alterations associated with the transformation of normal colonic epithelium to
colon adenocarcinoma. Two major pathways involved in colorectal carcinogenesis, the suppressor and the mutator pathway have been identified, with
different clinical behaviors and response to chemotherapy. Approximately
15% of sporadic colorectal carcinomas (CRC) arise from mutator pathway
which is characterized by microsatellite instability (MSI) caused by deficient
mismatch repair system (MMR) and defects in MMR genes.
In this study we have examined the incidence of MSI using the NCI Bethesda
panel of microsatellite markers (Bat-25a, Bat-26, D2S123, D5S346 D17S250)
in 200 sporadic CRC patients. Analysis was performed using ABIPRISM 310
genetic analyzer. The sample was denoted as MSI high (MSI-H) if two or
more of the markers demonstrated instability. The sample was denoted as
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463
The aim of our study was to evaluate the effectiveness of EGFR gene mutation detection in diverse materials from 707 NSCLC patients using the two
ultra-sensitive allele-specific real-time PCR assays in the routine diagnostic
procedure.
629 samples were analyzed by PNA-LNA PCR clamp, 164 samples by CE-IVD
certified allele-specific PCR assay and 86 samples by both methods. PNALNA PCR clamp products were analyzed by Sanger sequencing to confirm
rare mutations. NSCLC specimens were characterized by tumor cells content and type of fixation [Table].
62/707 (9%) specimens were positive for EGFR mutations: 32 detected in exon 19, including 4 rare deletions and p.R748K substitution (c.2243G>A); 28 p.L858R (c.2573T>G) mutations, 1 rare variant of
p.L858R (c.2572_2573delinsAG) and 1 double mutation p.L858M+p.L861Q
(c.2572C>A; c.2582T>A) detected in exon 21. There were 71% women in the
EGFR+ group. 98% of EGFR+ were adenocarcinoma samples, thus mutation
frequency among adenocarcinoma patients was 9.8% (61/617 patients). Ultrasensitive PNA-LNA PCR clamp assay and allele-specific PCR CE-IVD test
presented high results conformity with 95.3% overall percent agreement
(95%CI=90.9-99.8) and Cohens Kappa score of 0.9 (95%CI=0.88-0.92).
Both real-time PCR assays provided reliable and robust detection of most
common EGFR activating mutations in various clinical NSCLC specimens.
fresh-frozen tissue
84
NSCLC specimens
FFPE tissue
FFPE biopsy
299
198
cytology smear
126
J12.046
An investigation of relationships between hypoxia-inducible factor-1a
gene polymorphisms and endometrial cancers
L. .. Kafshdooz1,2, A. Dastranj3, S. Mohaddes4, T. Kafshdooz4;
1
medical university Department of Genetics, Tabriz, Islamic Republic of Iran, 2Womens
Reproductive Health Research Center Tabriz university of medical sciences, Tabriz,
Islamic Republic of Iran, 3Womens Reproductive Health Research Center Tabriz
University of Medical sciences, tabriz, Islamic Republic of Iran, 4medical university
Department of Genetics, tabriz, Islamic Republic of Iran.
Colorectal cancer (CRC) often metastasizes even at early stages that is the
main cause of death. Epithelial-mesenchymal transition (EMT) is a complex
process that is required for dissemination of tumor cells. EMT leads to the
transformation of epithelial cells phenotype to mesenchymal phenotype.
Tumors with mesenchymal subtype is associated with poor prognosis.
EMT, somatic mutations in KRAS and BRAF, microsatellite instability were
investigated in 17 tumor samples and 20 carcinomatosis nodes from 20
CRC patients with peritoneal carcinomatosis. To determine EMT program
gene expression profiling ZEB1, ZEB2, VIM, SNAIL1, CDH1 by real-time PCR
was analyzed. EMT was detected in 5 of 17 tumor samples (29%) and 17 of
20 carcinomatosis nodes (85%). These CRC samples were characterized by
high frequency of somatic mutations (11 KRAS and 2 BRAF mutation, 65%),
microsatellite stability (17 MSS and 3 MSI-L tumors) and low grade. We ob-
464
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served a high concordance between tumor and carcinomatosis node for mutations and MSI status, but not for EMT. We suppose the tumors eventually
may lose mesenchymal phenotype (mesenchymal-epithelial transition) or
intratumoral heterogeneity exists. Our data shows that the most of CRC with
peritoneal carcinomatosis undergo EMT process.
J12.048
Role of Dido1 in the progression and development of esophageal
squamous cell carcinoma
P. Naeemi Khorasani Zadeh1, M. Forghanifard2, M. Abbaszadegan1;
1
Avicenna Research institute, Mashhad, Islamic Republic of Iran, 2Islamic Azad
University, Damghan, Islamic Republic of Iran.
Background: Bone morphogenetic proteins (BMPs) are implicated in several processes during embryonic development and adult tissues homeostasis.
Many cancers are linked to either the BMPs or the molecules functioning in
this signaling pathway. Dido1 is a novel BMP-specic Smad-regulated target gene, which is studied in few cancers. However, its expression level has
not been yet elucidated in esophageal squamous cell carcinoma (ESCC). To
determine this level and its probable clinicopathological consequences, expression of the gene was analyzed.
Methods: Dido1 expression in fresh tumoral and distant tumor-free tissues from 50 esophageal squamous cell carcinoma samples was compared by
real-time polymerase chain reaction (PCR).
Results: Dido1 mRNA expression level was overexpressed in 26% of tumors,
while its underexpression was detected in 18% of ESCC samples. There
was a significant correlation between level of Dido1 mRNA expression and
increased depth of tumor invasion (P= 0.043). Furthermore, Dido1 mRNA
expression was correlated with the progressed stage of tumor cells in ESCC
samples (P = 0.04). Dido1 mRNA expression was inversely correlated with
the age of patients (P< 0.05).
Conclusions: These results indicate a relationship between Dido1 expression and depth of tumor invasion and staging. Along with the promising
evidences of its role in regulation of BMP signaling pathway which is one
of the main involved pathways in tumorigenesis, Dido1 may have a role in
progression and invasiveness of ESCC.
Keywords:Esophageal Squamous Cell Carcinoma (ESCC); Dido1 gene; Expressional analysis; Real-time PCR; BMP signaling pathway
J12.049
Impact of HES1 on depth of tumor invasion in esophageal squamous
cell carcinoma
S. Taleb, M. R. Abbaszadegan, M. Moghbeli, M. M. Forghanifard;
Division of human genetics, immunology research center, Avicenna research institute,
Mashhad Univers, Mashhad, Islamic Republic of Iran.
Extramedullary relapse (EM) of multiple myeloma (MM) is defined as infiltration of plasma cells (PC) outside of the bone marrow. EM is an aggressive
form of the disease with a adverse outcome.
Back to index
AA substitution in the extracellular juxtamembrane region and might initiate spontaneous dimerisation of the receptor, leading to factor-independent
growth and hyperresponsiveness to ligand, as suggested for similar defects
in endometrial cancer and patients
with craniosynostosis syndromes. Having in mind the low burden of the variant, we believe that K367E mutation is a late event alteration in a subclone
which may be responsible for the highly aggressive pattern of the disease.
This should be clarified by mutational burden analysis of the primary and
secondary tumors of these and other patients.
Table 1. Mutations spectrum in the analyzed patients *
patient
CC330
PIK3CA
BRAF
CC553
CC562
CC567
CC571
G1049R
(34%)
R88Q
(29%)
V600E
(28%)
TP53
R248F
(67%)
R273C
(63%)
G245D
(50%)
FGFR2
K367E
(8%)
K367E
(11%)
K367E
(13%)
KRAS
G13D
(55%)
G13D
(10%)
APC
Q1349X
(50%)
FBXW7
R465C
(70%)
R465C
(26%)
J12.053
FGFR3 and TP53 gene mutations in bladder cancer in the Belarusian
population
M. Smal1, A. Rolevich2, S. Krasny2, S. Polyakov2, T. Nabebina2, R. Goncharova1;
1
Institute of Genetics and Cytology NASB, Minsk, Belarus, 2N.N. Alexandrov National
Cancer Centre of Belarus, Lesnoy, Minsk region, Belarus.
J12.052
FGFR2 K367E mutation is frequently found with low burden in
patients with early onset aggressive colorectal cancer
We conducted next-generation sequencing using Ion AmpliSeq Cancer panel (Life technologies) which covers relevant regions across 46 oncogenes
and tumor suppressor genes on 5 primary colorectal tumors. The analyzed
patients presented similar clinical features: early onset (39-50yrs), a highly aggressive disease, distal localization, microsatellite stable tumors and a
lack of a family history of malignant diseases. The mutations found (Table 1)
generally correlate with the most abundant alterations in colorectal cancer.
A K367E mutation in exon 9 of the FGFR2 gene was found with low burden
(8-11%) in the tumors of 3 patients. This is a novel mutation that results in
465
466
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J12.057
Double knockdown of apoptotic genes and their relationship with
other mechanisms involved in tumor cell survival
V. Pileczki1, C. Braicu1, L. Pop1, I. Berindan Neagoe1,2;
1
Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania, 2Ion
Chiricuta Institute of Oncology, Cluj-Napoca, Romania.
The simultaneous inhibition of two important genes - p53 and TNF - involved in tumor cell development and evolution, with the siRNA technology,
will hopefully bring us new significant and interesting data in triple negative
breast cancer (TNBC) cell signaling. TP53 is involved in the intrinsic apoptosis pathway, and the tumor necrosis factor (TNF) is the main inducer of the
extrinsic apoptosis signaling mechanisms. In order to evaluate the impact
of TNBC dual gene knockdown, we used PCR array technology (qRT-PCR)
to analyze the most relevant genes involved in apoptosis along with other
staining assays that will unravel important and new data in the tumor cell
pathways cross-talk. Our experiments were performed on a triple negative
breast cancer cell line (Hs578T) and in relation with untreated cells that
serve as control. After analyzing the transcript quantification data we obtained statistical relevant results for 16 genes, of which 12 were up-regulated
and 4 down-regulated. By simultaneously inhibiting p53 and TNF genes on
Hs578T cells, we observed that they affect the angiogenesis mechanisms by
inhibiting the tumor cell network formation and self-degradation of cellular
components through autophagy. By integrating the expression profiles of
triple negative breast cancer untreated cells with the ones obtained from
knocking down p53 and TNF, simultaneously, we obtained a network-based
platform, that is important in finding a functional interaction between the
networks of intrinsic and extrinsic apoptosis cell signaling pathways and
other mechanisms involved in tumor cell angiogenesis, autophagy and cell
death in breast cancer.
J12.058
Genetic profiles comparative Analysis: Adult glioblastomas vs young
adult
Background : Glioblastomas (GB) are malignant astrocytic tumors, which often occur in patients aged over 55 years. However, younger age (<40 years)
was also described in several GB cases. Therefore, GB are newly divided into
2 subtypes according to patients age: young adult GB (Y.GB) ( 40 years).
Basically, clinical presentation and histopathological descriptions are similar between the 2 GB subtypes. Nonetheless, the assumption of different tumorogenic pathways was suggested. This hypothesis is supported by molecular alterations differences between Y.GB and A.GB.
Purpose: This study aims to compare the genetic profiles between Y.GB and
A.GB in Tunisian glioblastomas.
Methods: 43 samples composed by 40 A.GB and 13 Y.GB were collected during the period of 5 years from 2009 to 2013. In order to investigate discriminative genetic alterations between the 2 GB groups, tumoral DNA was
analysed by MLPA (Multiplex Ligation Probe Amplification). We used the 4
SALA MLPA probemix designed for Gliomas analysis: P370, P088, P105 and
ME011.
Results: Similarities were observed between the 2 GB groups, concerning
tumor size and locations as well as some chromosomal alterations: 10q, 17q
and 19q. Interestingly, 1p 19q co-deletion, 1p deletion and CDKN2A (p14)
were only found in A.GB group. While IDH1 mutation was more frequently
detected in Y.GB.
Conclusion: GB genetic profile variability noticed in our sample cohort
supports the hypothesis of different tumorogenic pathways between A.GB
and Y.GB. Despite of the crucial role of clinical and histopathological data
in tumor diagnosis, molecular investigations seems of a great importance
as well.
J12.059
Association analysis of rs7903146 (C>T) variant of TCF7L2 Gene with
Glioblastoma in a Turkish Population
D. Erkoc Kaya1, H. Arikoglu1, M. Taspinar2, Y. E. Guner3, F. scioglu4, H. C. Ugur3;
1
Selcuk University, Konya, Turkey, 2Yuzuncu Yil University, Van, Turkey, 3Ankara
University, Ankara, Turkey, 4Ege University, zmir, Turkey.
Glioblastoma (GBM) is the most common malignant brain tumor in the adult
population defined as grade IV astrocytoma according to severity of necrosis
and vascular proliferation. It can be seen at any age, but commonly between
the ages of 45-75. GBM is difficult to treat and presents high morbidity and
BACKGROUND
Autophagy is an adaptive and highly regulated response in unfavorable conditions such as starvation. During this process, long-lived proteins and organelles are self-eaten by double membranes vesicles, called autophagosomes,
which transport them through the cytosol to the lysosome, where they will
be degraded. The resulting macromolecules can be recycled to the cytosol
for reuse. Autophagy plays an important role in cellular development and
differentiation and it has been reported that its malfunction is implicated in
several diseases including cancer. Glioblastoma is a very infiltrating and aggressive heterozygous glioma. Its behavior is directly related to their genetic
and chromosomal alterations. Here, we have analysed common polymorphisms in autophagy genes in order to evaluate the role of these variants in
modulating glioblastoma risk.
PATIENTS AND METHODS
112 newly diagnosed patients with primary glioblastoma (grade IV) and
189 controls without cancer were included in the study. Genomic DNA was
extracted from peripheral blood using phenol/chloroform procedure and
genotyped using TaqMan 5-exonuclease allelic discrimination assays (Applied Biosystems) (table 1). Statistical analysis was performed using SPSS
software.
RESULTS
No significant differences were found in genotype distribution for ATG16L1
RS2241880 ATG2B RS3759601 and ATG5 RS2245214 between patients
with glioblastoma and control subjects. However, statistical differences in
genotype distribution for ATG10 RS1864183 were found. The A allele was
associated with increased risk of developing glioblastoma (OR = 2,788, 95%
CI 1,40-5,55; p= 0,004).
CONCLUSIONS
ATG10 RS1864183 POLYMORPHISM IS ASSOCIATED WITH SUSCEPTIBILITY TO SUFFER GLIOBLASTOMA, SUPPORTING THE IMPORTANCE OF AUTOPHAGY IN CANCER DEVELOPMENT.
J12.061
Genetic polymorphisms in GSTM1, GSTT1, GSTP1 and the
susceptibility to basal cell carcinoma in Iranian population
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Up to 75% of Hereditary ovarian carcinomas (HOC) are attributed to mutations in BRCA1 and BRCA2 genes, whereas DNA mismatch repair genes would
cause about 10% of them. Genes involved in FA-BRCA pathway will account
for an additional percentage of HOC.
Several studies identify BRIP1 (a FA-BRCA pathway gene) as a moderatepenetrance breast cancer susceptibility gene in BRCA1/2 negative familiar
cases. Truncating BRIP1 mutations have been described as cancer susceptibility alleles in families with a great ovarian cancer burden. Thus, we aimed
to assess whether BRIP1 alterations may contribute to breast and ovarian
cancer (BOC) susceptibility as c.1702_1703delTT mutation has been associated with BOC in Spanish population.
Forty-nine out of 140 families In North West-Central of Spain with family history of breast and ovarian cancer carried a pathogenic mutation in BRCA1
or BRCA2. We performed BRIP1 mutational analysis in 72 BRCA negative
families. Mutation screening was performed with High Resolution Melting
Curve Analysis (HRMA). Cases displaying abnormal HRM patterns were evaluated by direct Sanger Sequencing.
There is no evidence of pathogenicity in the identified variants. Mutation
c.508-31C>G was found in ovarian cases. Mutation c.2755T>C was carried by BOC patients. Around 60% of the samples harbors c.2637A>G and
c.3411T>C changes. Variant c.577G>A has been previously reported in a UK
study with similar frequency of occurrence.
Although clearly pathogenic mutations have not been identified in the
samples screened, BRIP1 polymorphisms may increase ovarian cancer risk,
so these findings can be used as a potential tool for improving cancer diagnosis and treatment planning.
J12.063
Analysis of polymorphisms in the EGFR pathway involved in the
response and toxicity of treatments in head and neck squamous
carcinoma
Head and neck squamous carcinoma (HNSC) is one of the most prevalent
cancers in Spain. The classic risk factors strongly associated with developing
HNSC are tobacco smoking and alcohol consumption.
The common therapies in HNSC are platinum-based chemotherapy, radiotherapy and/or monoclonal antibodies against EGFR, although the response
rates are very variable. We have evaluated the influence of some polymorphisms in different genes of the EGFR pathway and antibody dependent cellular cytotoxicity receptors (ADCC) associated with differences in toxicity
and responses of these treatments.
Genomic DNA was extracted from peripheral blood samples of 91 Spanish
HNSC patients treated with monoclonal antibodies against EGFR. The selected polymorphisms realized by QPCR with TaqMan probes were: FCGR2A
rs1801274, FCGR3A rs396991, EGFR rs28384375, rs2227983, rs17336639
and KRAS lc6 rs61764370.
Statistical analysis was performed comparing the different distribution of
the polymorphisms and patients were stratified according to treatment and
response.
We found differences in the distribution of FCGR2A A519C (rs1801274) polymorphism (p=0.012), conferring an increased risk of developing dry skin
in carriers of two copies of the C allele; OR=4.018 (1.257-12.850). Moreover,
we found that carriers of the heterozygous genotype in K-RAS lcs6 polymorphism (rs61764370) exhibit a decrease in global toxicity of anti-EGFR antibodies therapy; p=0.014, OR=0.294 (0.107-0.805).
Our results support the correlation between some germline polymorphisms
467
Non-random chromosomal aberrations are a common feature of many hematological disorders, making cytogenetic analyzes essential for the most
common abnormalities in myelodysplastic / myeloproliferative diseases,
leukemias and lymphomas.
Between March 2013 and January 2014, 77 bone marrow samples were
analyzed cytogenetically from adults aged between 23 and 82 years. Where
possible three independent cultures (direct method, 24 and 48 hours) were
established and a minimum of 20 metaphases were analyzed.
The karyotype of 70 patients was analyzed successfully. For 7 patients with
insufficient number of metaphases FISH analysis was performed using BCR/
ABL1plus probes (MetaSystems). Complementary specific FISH probes (MetaSystems) were used according to clinical hematological diagnosis for 5 patients with normal karyotype.
Among the successfully karyotyped samples, 35 cases of CML, and 3 cases of
ALL showed various homogeneous or mosaic chromosome abnormalities.
The most common abnormality was the presence of the Philadelphia chromosome (Ph), found in varying percentages (between 9 and 100%) with 2
cases having an additional cell line with double PH chromosome. One case
had a complex translocation involving chromosomes 9, 10 and 22. Other
common abnormalities were trisomy 8, isochromosome 17, autosomal hyper and hypodiploidy.
Identification of cytogenetic abnormalities by conventional karyotype is important to confirm the diagnosis and provide useful information for classification, staging and prognostic information, for choosing therapy conduct
and for signs of remission or relapse. Because some cases show a normal
karyotype by conventional banding, FISH and aCGH analysis are valuable
techniques recommended for identifying the presence of possible submicroscopic abnormalities.
J12.065
Her-2neu gene in neck squamous cell carcinomas (SCC) a potential
target for therapy
M. Neagu;
Nationale Inst. Path.Victor Babes, Bucharest, Romania.
468
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Several genome-wide association studies (GWAS) have identified loci located at 15q25 (IREB2) and 4q22 (FAM13A) associated with chronic ob-
Back to index
Muir-Torre syndrome is a clinical sub-type of Lynch syndrome which is associated with sebaceous skin lesions and keratoacanthomas in addition to
internal malignancies. As established in other Lynch syndrome-associated
cancers, these skin lesions demonstrate loss of mismatch repair (MMR)
protein expression on immunohistochemical (IHC) staining, allowing targeted germline mutation analysis of the MMR genes associated with Lynch
syndrome (MLH1, MSH2, MSH6, PMS2). However, not all lesions with loss of
MMR protein expression will result in a molecular or clinical diagnosis of
Muir-Torre or Lynch syndrome, as this protein loss may be due to a sporadic
mechanism. There is emerging literature that the specific type of sebaceous
lesion, along with its location on the body, can provide an indication as to
which lesions are more likely to have loss of MMR protein expression and
subsequent association with Muir-Torre/Lynch syndrome. In order to assess this theory within the experience of Genetic Health Queensland (GHQ),
an internal audit was performed. All patients seen at GHQ between 2005
and 2013 with a primary reason for referral of a sebaceous skin lesion were
included in a retrospective analysis. The information reviewed included
subtype and location of lesion, age of diagnosis, personal and family history
469
Although histopathological analysis is the gold standard for melanoma diagnosis, distinguishing potentially lethal melanomas from benign melanocytic nevi with atypical histopathologic features needs further analysis. Most
melanomas have chromosome copy number abnormalities. Fluorescent
In Situ Hybridization (FISH) is an applicable technique in the diagnosis of
these chromosome abnormalities and so its usability is emerging in melanoma diagnosis. The aim of this study was to evaluate the diagnostic role
of theFISHin the assessment of malign melanomas and melanocytic nevi.
Histologically evaluated most suitable formalin-fixed, paraffin-embedded
(FFPE) tissue sections of 25 malign melanoma tumors and 25 melanocytic nevi samples were analysed for copy number abnormalities of CCND1,
MYB, RREB1 and centromere of chromosome 6 but no FISH result could be
obtained from a tumor tissue. Copy number abnormalities were detected
in 20 malign tumors (83.8%) and four melanocytic nevi (16.0%) and statistically significant differences were seen between the tissues for each gene
(MYB deletion and RREB1/CEP6 p<0,05 , RREB1 amplification p<0,01 and
CCND1 amplification p<0,001). Histopathologic features of the melanocytic
nevi tissues with gene copy number abnormalities were similar to the characteristics of malign melanomas and they had had premalignant diagnosis.
The CCND1 amplification was the predominantly seen abnormality and its
importance as a marker should be evaluated in detail. The study demonstrated that copy number analysis of FFPE tumor samples by FISH is a reliable
method in the diagnosis of melanoma and the technique is also informative
to understand molecular mechanism of the melanocytic tumors.
J12.076
Prevalence of P16 methylation and prognostic factors in plasma cell
myeloma of single institution in Korea
470
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Catholic University of Korea, Seoul, South Korea, Seoul, Korea, Republic of.
Background: The primary purpose of this study was to investigate the prevalence and characteristics of p16 methylation and determine the prognostic implications of the clinical data, hematologic data and p16 methylation
changes in plasma cell myeloma (PCM). Methods: We reviewed clinical
characteristics, laboratory tests and investigated the response to combination chemotherapy, and survival time. DNA methylation of the p16 gene was
tested by methylation specific PCR and evaluated the clinical significance.
Results: A total of 103 patients were enrolled in this study. The patient median age was 59.0 years at diagnosis and male to female ratio was 1.15:1. According to the International Staging System (ISS), patients were diagnosed
as stage: I (n=17, 16.5%); II (n=41, 39.8%); III, (n=39, 37.9%); (not classified 6). Forty five (43.7%) patients and thirty six (35.0%) patients showed
abnormal karyotype and complex karyotype on chromosome study, respectively. Thep16methylation was found in 39 of 103 (37.9%) patients, but there was no significant association of p16 methylation status with other clinical, laboratory factors and survival outcome. The male gender, albumin and
complex karyotype were independent prognostic factors for overall survival
by multivariate analysis (P<0.05). Conclusions: The male gender, albumin
and complex karyotype were independent prognostic factors in PCM. And
p16 methylation was relatively common in PCM, but didnt influence a response to survival outcome.
J12.077
Analysis of oncogenic mutations in 52 MGUS patients - two case
reports of KRAS mutation
Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant condition permanently associated with a risk of progression into
malignant disease, especially to multiple myeloma (MM). While in MM, oncogenic mutations have been described in several studies, no mutation study was done in abnormal plasma cells (aPCs) in MGUS. In our study, we performed genetic analysis in 52 samples of aPCs (CD138+CD19-CD56+/-) of
MGUS patients by aCGH (SurePrint G3 CGH+SNP, 4180K, Agilent) together
with High Resolution Melting (HRM) followed by Sanger sequencing in HRM
positive cases. By HRM, we focused on DNA single nucleotide mutations in
11 hot spots of MYC, NRAS, KRAS, DIS3, BRAF, FAM46C and TP53 genes. We
found two cases of KRAS mutations p.Q61L and p.A146T with damaging
prediction effect. Both MGUS cases showed positive IGH disruption (in 56%
and 85% aPCs) analysed by I-FISH, but only the first case with p.Q61L mutation showed unbalanced chromosomal changes in genome-wide profile: hyperdiploidy (+3, +5, +9, +11, +15, +19), losses of chromosomes 8, 13 and Y, 7
segmental losses (1p34.2-p13.1, 6p23, 6q12-q27, 7q36.3, 12p12.1-p11.23,
12q12, 12q21.2-q23.3) and 4 segmental gains (6p25.3-p23, 6p23-p11.1,
6q11.1-q12, Xq21.33-q28). Interestingly, only this patient has progressed
after 6 months to MM. Genome-wide profiling of the second patient with
p.A146T mutation did not show any unbalanced chromosomal changes and
the patient is still stable for more than 16 months. In our study, we showed
that KRAS p.Q61L mutation combinated with chromosomal changes could
be potential markers of MGUS progression. Support: IGA NT13492,OPVK
CZ.1.07/2.3.00/20.0183, MH CZ-DRO (FNBr, 65269705)
J12.078
Gene polymorphisms of microRNAs in Helicobacter pylori- induced
high risk atrophic gastritis and gastric cancer
Aims: The purpose of this study was to evaluate potential associations between miRNA-related gene polymorphisms (miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608) and the presence of gastric cancer (GC) or high
risk atrophic gastritis (HRAG) in European population.
Methods: Gene polymorphisms were analysed in 995 subjects (controls:
n = 351; GC: n = 363; HRAG: n = 281) of European descent. MiR-27a T.C
(rs895819), miR-146a G.C (rs2910164), miR-196a-2 C.T (rs11614913),
miR-492 G.C (rs2289030) and miR-608 C.G (rs4919510) single nucleotide
We studied miRNA expression profiles in patients with acute myeloid leukaemia at diagnosis and in the first remission and we compared them with
profiles found in healthy controls. Plasma samples were collected from 8
patients at the first diagnosis of AML and at the first complete remission and
from 10 healthy volunteers. Isolated miRNA were transcribed to cDNA and
applied on TaqMan Human MicroRNA Array A to find the expression of 381
genes. Relative quantification and statistical analyses were performed with
Expression Suite v1.0.3 and with qbase+ v2.4. Relative quantification was
calculated using the reference gene miR-16 or the global mean. Both values
of miRNA expressions were compared between the patients and controls
using Mann-Whitney tests with multiple testing corrections. The differences
of various statistical significance were found. Among them there were 12
miRNAs (miR-125a-5p, miR-145, miR-221, miR-652, miR-744, miR-146a,
miR-181a, miR-191, miR- 27a, miR-340, miR-134, let-7d) giving the p values
< 0.01 with both applied normaliazation methods and another 4 miRNAs
Back to index
Recent studies have revealed a number of epigenetic alterations that contribute to myeloproliferative neoplasms (MPNs) pathogenesis and determine
the clinical outcome. According to the published data, mutations involving
EZH2, which encodes a histone methyltransferase, are founded in 6% cases
of primary myelofibrosis (PMF), 1% of polycythemia vera and 1-3% of essential thrombocythemia regardless of the presence of mutations in JAK2
or MPL. EZH2 mutations may be of prognostic value in MPNs at the time of
transformation to the blastic phase. The goal of our research was to determine the frequency of mutations in EZH2 in two groups of patients with different chromosomal aberrations. We examined 47 patients with BCR-ABLnegative MPNs. The first group included 20 patients with normal karyotype,
and 16 patients with the isolated chromosomal aberrations del(13)(q22),
del(20)(q12),-Y associated with favorable prognosis, and add(22)(q13),
del(1)(p32), del(6)(q15), t(10;12)(q22;p13) that are referred to as intermediate risk. The second group included 11 patients with complex abnormalities and the chromosomal aberrations of unfavorable prognosis +8, +7,
inv(7)(p11;q21). Mutations in 8,10,17,18,19 exons of EZH2 were defined by
sequence analysis. The Ile713Thr mutation in EZH2 gene was detected in
2.1% (1/47 cases). The patient with mutation had a del(6)(q15) karyotype
which is associated with an intermediate risk, and he subsequently underwent transformation from PMF to myelodisplastic syndrome in 9 months
after the disease onset. Conclusions: mutations in the EZH2 gene could be
preliminarily assessed as additional prognostic markers of unfavorable prognosis in patients with BCR-ABL-negative MPNs and with different chromosomal aberrations. Further studies are needed.
471
In multiple myeloma (MM), malignant cells retain the self-renewing potential. Majority of myeloma cells stay in the G1 phase, however after leukemic transformation in plasma cell leukemia (PCL) myeloma cells become
highly proliferative. We anticipate that complex re-setting of cell cycle
gene expression coordination during leukemic transformation creates required background for proliferation restoration. The aim of this study was
to define and describe complex re-setting of cell cycle gene expression
coordination in MM and PCL. Gene expression profiling was performed
in 7 healthy donors, 6 MM and 7 PCL patients utilizing Affymetrix GeneChip Human Exon 1.0 ST Arrays. Genesets, connected with cell cycle regulation (GO:0045786; GO:0045787) and regulation of apoptotic process
(GO:0043065; GO:0043066) together with all descended direct connected
genesets were taken for the GSEA analysis and Gene Set Differential Coordination Analysis. Comparison of PCL, MM and healthy donors revealed
coordinating expression changes between regulation of mitosis, apoptosis
and cell cycle arrest for both positive and negative regulation genes. In MM,
co-expression changes were associated with early phase of cell cycle, whereas in PCL with both - early and late phase of cell cycle. We anticipate that
expression of cell cycle positive regulators is in dynamic equilibrium with
cell cycle negative regulators. We suppose that this equilibrium serves as a
compensatory mechanism to oncogenic events. Despite compensation mechanisms activation, whole regulatory complex seems to be imbalanced by
growing oncogenic stress during MM to PCL progression. Supported by
grants NT12130, NT13190 and OPVK CZ.1.07/2.3.00/20.0183.
J12.085
MYCN oncogene amplification in advanced neuroblastoma patients
from Republic of Macedonia
472
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Over the years, (IR) has become a universal diagnostic and therapeutic tool,
making the largest man-made contribution to the population dose. Thus,
medical personnel represent the group most consistently exposed to low
doses of IR.
Many cytogenetic studies have been conducted among hospital workers exposed to IR.
The cytokinesis-blocked micronucleus (CBMN) assay is widely used, since
it represents a reliable test to assess radiation-induced chromosme damage
and its a valuable biomarker in many biomonitoring studies on human populations exposed to IR.
The aim of our study is to assess chromosomal damage in Tunisian hospital
workers occupationnally exposed to low levels of IR.
The CBMN in peripheral lymphocytes of 67 exposed workers compared to
43 controls.
The clastogenic/aneugenic effect of IR was evaluated using the CBMN assay
in combination with flurescence in situ hybridization ( FISH) with pan-centromeric DNA in all the exposed subjects and controls.
The centromere analysis performed in our study showed that MNs in hospital staff were predominantly centromere negative and the mean negative
labeled micronucleus (C-MN) frequency was significantly higher in the exposed subjects than in the controls ( 9.044.57 vs. 1.17 0.77). The
multivariate regression analysis showed that only the time of exposure ti IR
had a significant effect on the level of MNs and C-MN.
The results of the study confirm the well-known clastogenic properties of
IR.
J12.089
The detection of mutations in gene PTEN in patients with ovarian
cancer from Bashkortostan Republic of Russia
Z. Takhirova1, M. Bermisheva1, O. Urkina2, E. Khusnutdinova1,2;
1
Institute of Biochemistry and Genetics, Ufa, Russian Federation, 2Bashkir State
University, Ufa, Russian Federation.
Back to index
Pancreatic ductal adenocarcinoma (PDAC) is a rare disease, whose incidence rates are very close to mortality rates, due to its difficult and late
diagnosis. Noninvasive diagnostic biomarkers emerges as a possibility to
perform a safe diagnosis, preferably early in the disease course. Recently,
microRNAs (involved in the initiation and maintenance of tumors, and able
to discriminate the presence or absence of certain diseases) were discovered as stable molecules in circulating samples. We evaluated the expression
levels of six miRNAs (miR-21, -34a, -155, -196a, -200b and -376a) in serum
(24 PDAC and 9 healthy patients) and saliva (10 PDAC and 10 healthy patients) samples. The aim of our study was to investigate the presence of diagnostic and/or prognostic biomarkers. Expression levels were measured by
relative quantification using qRT-PCR. In serum, miR-21 and miR-34a were
significantly more expressed in samples from patients with PDAC (P<0.001
and P=0.001) and both were able to distinguish patients with and without
disease with clinically acceptable sensitivity and specificity (AUC for miR21 and miR-34a of 0.894 and 0.865). We also found a strong and positive
correlation between the expression levels of these two miRNAs in serum
(rs=0.681; P<0.001; Kappa-tests: 0.477; P=0.022). However, the mechanism
related to this correlation remains unknown. In salivary samples, generally,
miRNA levels were very low. No difference in miRNA expression levels was
seen between cases and controls. These findings suggest that the chosen
miRNAs are not adequate as diagnostic biomarkers in saliva, although these
findings require confirmation in a larger series of cases.
J12.091
Association of Platelet Derived Growth Factor-B (PDGF-B) and Human
Epidermal Growth Factor Receptor -2 (HER-2/neu) Single Nucleotide
Polymorphisms (SNPs) with Gallbladder Cancer
473
474
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matic mutations were detected in the AR gene. The rest of the markers: DD3
overexpression, GSTP1 gene promoter hypermethylation and TMRSS2-ERG
fusion were positive in fresh prostatic tissues and biopsies from all three
patients, whereas only one blood sample showed triple positive result. The
appearance of PCa specific molecular markers in blood was considered as a
predictor for a pronounced migration and dissemination of prostatic tumor
cells in circulation. The GSTP1 promoter hypermethylation is the earliest
epigenetic fluctuation, which indicates cancerous changes in the gland the
first and long-lasting marker, in the blood circulation. The molecular profile
during cancer treatment could be used to predict the individual response
Acknowledgements: the study was supported by the grants 4-D/2011
and 26-D/2012, Medical University Sofia, Bulgaria.
J12.095
Analysis of the PCA3, TMPRSS2-ERG, TERT genes expression in biopsy
samples and urine sediments as potential markers for diagnostics of
prostate cancer
Prostate cancer (PCa) is one of common tumors among men in Russia. Low
levels of vitamin D are implicated as a potential risk factor for PCa development, and vitamin D receptor (VDR) gene may be important in the onset and disease progression. In this study, SNP variants rs2228570 (FokI),
rs1544410 (BsmI), rs7975232 (ApaI), rs731236 (TaqI) in the VDR gene
were investigated in PCa patients and controls of Russian (N=122 and N=95,
respectively), Tatar (N=70 and N=170), and Bashkir ethnic origin (N=42 and
N=82) to determine whether they are associated with PC risk. We evaluated
the association between these SNPs in the VDR gene and PCa risk as well as
clinical characteristics (prostate-specific antigen level, clinical stage, pathological stage) in men (237 PCa patients who underwent a prostatectomy and
orchectomy) using logistic regression.
We did not observe significant differences for either the VDR BsmI, ApaI and
TaqI genotype and allele frequencies in PCa patients and healthy individuals. However, the frequency of the VDR *T/*T genotype of rs2228570 (FokI)
was statistically different between PCa patients and controls of Tatar ethnic
origin (OR = 1.88, 95%CI=1.22-2.58, p=0.0023). There wasnt association of
the studied VDR BsmI, ApaI and TaqI polymorphisms with clinical characteristics of PCa patients. We found linkage disequilibrium between the BsmI
*A and ApaI *C alleles (D=84%). Our study indicates that VDR FokI variant
might increase PCa risk in Tatars. However, current limitation for small cohorts might have false positive effects; therefore it should be overcome via
further large-scale validating studies.
During prostate carcinogenesis prostate tumor cells lose the ability to accumulate Zn2+ ions in high levels. The aim of this study was to investigate the
expression of four genes ZIP1, ZIP7, MT1-F and MT2 involved in the maintenance of homeostasis of zinc cations in prostate cells in tumor tissue and in
benign prostate hyperplasia (BPH) by RT-PCR and determine whether there
is an correlation between the mRNA expression of four genes and the TNM
classification, Gleason score, PSA level and age and thus evaluate its diagnostic and prognostic potential.
Isolation of mRNA from prostate cancer in 65 patients has been performed
in period 2011 - 2013. As a control group, 27 patients with BPH were used.
Statistically significant lower relative expression of MT1-F and ZIP1 genes
was detected in prostate cancer tissue than in BPH (p<0.00048, p<0.0082,
resp.). The PSA level correlated positively with the ZIP7 expression
(p<0.0099). Decreased expression of all four genes correlated with higher
age. No correlation of the gene expression with the TNM classification and
Gleason score was observed.
According to our results, it is possible to consider that the genes MT1-F and
ZIP1 may be candidate tumor suppressor genes for prostate cancer. Supported by Diana Lucina, the Ministry of Health project for conceptual development of research organization 00064203 and by grant MSM
0021620808.
J12.098
Association between the RAD50 rs17166050 polymorphism and risk
of childhood leukemia and adult cancers
M. Mosor, I. Zikowska-Suchanek, A. Dzikiewicz-Krawczyk, D. JanuszkiewiczLewandowska, J. Nowak;
Institute of Human Genetics Polish Academy of Sciences, Pozna, Poland.
The RAD50 gene encode one of the protein of the MRE11-RAD50-NBN complex involved in cellular response to DNA damage and the maintenance of
genome stability. The aim of this study was to answer the question whether the RAD50_rs171660505 polymorphism may be associated with childhood leukemia or adult cancers. We estimated the frequency of RAD50_
rs171660505 polymorphism in the group of 220 children diagnosed with
leukemias and in the 280 non-selected breast cancer (BC) patients, 175 with
a single laryngeal cancer (LC) and 115 with multiple primary tumors but
one malignancy (primary or second primary) localized in the larynx (MPTLC) and 68 multiple primary tumors localized in the head or neck (MPT) and
controls (n=504). The analysis was performed by multi-temperature singlestrand conformation polymorphism technique. We performed two molecular tests to examine any potential function of the detected the c.551+19G>A
SNP in RAD50 gene. The frequency of either the AA genotype or A allele
of RAD50_rs17166050 were significantly different in controls compared to
childhood leukemia group (ALL+AML) (P<0.0019 and P<0.0019, respectively). In MPT group, heterozygous genotype GA was significantly more frequent, even after multiple testing correction, than in controls (P=0.0205).
The cDNA analysis of AA or GA genotypes carriers has not revealed evidence
of splicing abnormality of RAD50 pre-mRNA. We measured the allelic-specific expression of G and A alleles at c.551+19G>A and the statistically significant overexpression of the G allele has been observed. This data demonstrate that some specific alternations of the RAD50 gene may be associated
with childhood ALL.
J12.099
Research of association between the SNP309 of MDM2 gene and the
occurence retinoblastoma in Algerian population
A. M. Boubekeur1, L. Louhibi1, K. Mahmoudi2, R. K. Abderrahmane1, F. Z. Moghtit1, M.
Aberkane3, N. Mehtar1;
1
University of science and technology, Oran, Algeria, 2Pediatric hospital of Canastel,
Oran, Algeria, 3Faculty of medecine, Oran, Algeria.
Back to index
The RUNX1 (AML1) gene is a key regulator of hematopoiesis which is frequently rearranged in acute leukemias and myeloid malignancies. More
than 55 different translocations of RUNX1 have been described. However,
majority has been reported rather sporadically and as complex RUNX1 rearrangements they remain undefined at the molecular level. A series of 21
adult patients with uncommon structural abnormality of 21q21-q22 detected by conventional cytogenetic/multicolor FISH methods and diagnosis of
AML or MDS was collected. Three patients with no further available material
were excluded from the study. All the others were tested for involvement
of the RUNX1 using FISH analysis with Vysis LSI TEL/AML1 or LSI AML1/
ETO probe (Abbott). The split signal of RUNX1 gene was confirmed in four
patients with: 1) t(8;21;7)(q22;q22;p13); 2) t(3;21)(q12;q22); 3) der(21)
ins(21;10)(q22;q?)t(14;21)(q11;q22); 4) der(12)t(12;22)(p11.2;?)t(12;21)
(q13;?),der(21)t(5;21)(?;q21)t(5;12)(?;q13). Further FISH analyses have
been focused on the reciprocal translocation t(3;21)(q11;q22) and have
been performed with BAC probes located at 3q11-12. The breakpoint has
been specified between clones RP11-138C11 and RP11-153N1. In conclusion, four until now unreported RUNX1 alterations with the new potential
fusion partners are described. One of them was specified into 293 kbp region at 3q12. Two protein and three RNA coding genes have been mapped
at this interval. The description and identification of atypical RUNX1 fusions
on the larger cohort of patients is an important tool for understanding and
characterization of the pathogenic mechanisms of RUNX1 rearrangements.
Supported by MHCR for conceptual development of research organization 00023736, RVO-VFN64165/2012, GACR-P302/12/G157, RVOUK-/27/
LF1/1.
J12.101
SALL4 as a new biomarker for early colorectal cancers
475
476
Back to index
H. Choi1, J. Kim1, A. Kwon1, E. Han1, H. Lee2, M. Kim1, Y. Kim1, K. Han2, S. Lee3, H. Kim3, D.
Kim3;
1
Catholic University of Korea, Seoul, Korea, Republic of, 2Catholic Genetic Laboratory
Center, Seoul, Korea, Republic of, 3Department of Hematology, Seoul, Korea, Republic of.
rs1800858
rs1800859
COSM918120
rs17158558
rs2435351
rs2505530
rs2472739
rs72781242
rs2742236
Consequence
type
Synonymous
variant
Synonymous
variant
Synonymous
variant
Missense
variant
Intron
variant
Intron
variant
Intron
variant
Intron
variant
Intron
variant
Intron
variant
Phenotype (n)
Base
AA
Allele frechange change quency (%) sMTC hMTC relatives
A/G
A45A
23.15
32
12
29
C/T
G733G
G/A
33.42
40
17
45
25.16
52
23
48
C/A
C/T
G/T
A/G
A/G
C/T
G/A
V125V
R982C
-
4.34
41.05
66.66
0.85
29.86
50
80
2
56
20
26
-
24
52
91
2
69
J12.108
TP53 Arg72Pro and MDM2 SNP309 polymorphisms and colorectal
cancer risk: a West Algerian population study
R. Abderrahmane1, louhibi L, F. Moghtit1, A. Boubekeur1, F. Fodil2, M. aberkane1, N.
Mehtar1;
1
Universit des Sciences et de la Technologie dOran, Oran, Algeria, 2Universit des
Sciences et de la tTchnologie dOran, Oran, Algeria.
The tumor suppressor gene TP53 and its regulator MDM2 are both key players involved in multiple pathways including apoptosis, cellular transcriptional control, and cell cycle regulation. Common germline polymorphisms
in these genes may affect colorectal cancer susceptibility. An arginine-to-
Back to index
proline substitution at codon 72 in the p53 gene is reported to decrease apoptotic potential, while a thymine-to-guanine polymorphism at nucleotide
309 (named SNP309) of murine double minute 2 MDM2 gene increases its
transcription. These two polymorphisms therefore may be of importance in
colorectal carcinogenesis. The relation of these polymorphisms to colorectal
cancer in the Algerian population was addressed in this study.
DNA samples from 121 controls and 116 cases were genotyped for these
two polymorphisms by PCR/RFLP then confirmed by sequencing.
Unexpectedly no significant association was found between this potential marker TP53 Arg72Pro and CRC (p>0.05). However, our findings reveal
that individuals with the MDM2 SNP309 GG genotype have a low risk of CRC
(OR=0.49; 95% CI, 0.24- 0.98, p=0.04) relative to the TT genotype and with
more significance in females (OR= 0.16; 95% CI,0.06-0.41, p<0.05). Moreover, no significant association was observed between the combined TP53
and MDM2 genotypes and colorectal cancer.
Contrary to initial expectations that the GG genotype with high MDM2 levels
will increase cancer risk, our results demonstrate that the MDM2 SNP309
GG genotype is associated with decreased risk of colorectal cancer. This is
suggesting that other mechanisms independent of increased MDM2 levels
can influence cancer susceptibility.
J12.109
Acquired uniparental disomy (UPD) involving the short arm of
chromosome 17 in patients with myelodysplastic syndromes (MDS)
and complex karyotype
Acquired uniparental disomy (UPD), which can be an important step in cancer development and progression, are found in various subtypes of haematological malignancies including myelodysplastic syndromes (MDS). The
aim of the study was to evaluate the frequency of 17p UPD in bone marrow
cells of patients with newly diagnosed MDS and complex karyotypes and to
assess correlation of 17p UPD with mutations of tumour suppressor gene
TP53 located at 17p13.1.
Bone marrow samples from 49 patients were analyzed by aCGH/SNP (BlueGnome) and UPD of 17p was found in 7 of them (14%). All cases had deletion of 5q and additional recurrent chromosomal aberrations: 7q deletion
(4x), monosomy 7 (1x), or 12p deletion involving ETV6 gene (5x). Average
extent of 17p UPD was 14-20 Mb involving TP53 gene. In all 7 cases with
17p UPD, two copies of TP53 gene were detected by FISH (Abbott). DNA isolated from bone marrow cells of 5/7 patients was NGS-sequenced (Roche)
and in all of them homozygous TP53 mutations were found.
Our study confirmed that acquired UPD 17p is a recurrent cytogenetic defect
in patients with MDS with complex karyotypes and is strongly associated
with homozygous mutations of TP53 gene. This lesion is cryptic unless analyzed by SNP array. UPD might have a fundamental role in tumorigenesis,
therefore further characterization of 17p UPD will lead to better understanding of the initiation and progression of MDS.
Supported by RVO-VFN64165, GACR P302/12/G157/1, PRVOUK-P27/
LF1/1.
J12.110
Association between polymorphism of glutathione S-transferase
genes (GSTM1) and uterine leiomyoma in Iranian population
477
478
Back to index
in blood -that have been interpreted up to now as de novo mutations occured at gametogenesis- could actually be postzygotic events.
J12.113
XPD Lys751Gln and Arg156Arg Polymorphisms and Acute Myeloid
Leukemia risk
The aim of the study was to determine the heterogeneity of AML patients
with FLT3-ITD mutation and CD7 aberrant expression.
Mutation FLT3/ITD was detected by PCR in 21/101 (20.8%) patients. The
cytogenetic analysis revealed 13 pts with normal karyotype (NK) and 8 pts
with different chromosomal aberrations. A high WBC count was found to be
significantly associated with FLT3/ITD mutation (p=0.049). CD34 expression in control group and group with FLT3-ITD was 83.3% against 90.0%
(p=0.635). We found the significant association of FLT3/ITD mutation with
aberrant expression of HLA-DR (50.0% cases with FLT3/ITD against 5.6%
without FLT3/ITD) and CD7 (100% samples with FLT3/ITD mutation and
55.6% of samples without FLT3/ITD) markers.
The expression of CD7 in the group of 31 patients is varied over the range
from 20 to 97.7% (average 70.4%). We formed 2 comparative groups: 13 pts
with CD7 expression below the average and 18 above. Patients with high
level of CD7 expression were older than patients with lower expression:
57.5 vs 44 years, respectively (p=0.011). There was no significant difference
between these two groups in respect of FAB and karyotype variants, the presence of FLT3/ITD mutation and CD34 expression.
Summary. Leukocytosis, aberrant expression of CD7, and/or hyperexpression of HLA-DR on blast cells are probable markers for the detection of
FLT3-ITD mutations in AML patients with NK. Patients with AML and CD7
aberrant expression are heterogeneous in morphological, cytogenetic and
molecular characteristics. CD7 expression in AML blasts is not an independent prognostic factor.
J12.115
Tailored Targeting of Inflamed, Regenerative, and Mutated K-RasG12D
Upregulated cDNA Microarray Gene Signatures in Pancreatic Ductal
Cells
J12.117
Common genetic variants in NEFL influence gene expression and
neuroblastoma risk
Back to index
Using genome-wide association study, we identified single nucleotide polymorphisms (SNPs) associated with neuroblastoma at the LINC00340,
BARD1, LMO1, DUSP12, HSD17B12, HACE1 and LIN28B gene loci, but these
explain only a small fraction of neuroblastoma heritability. Other neuroblastoma susceptibility genes are likely hidden among signals discarded by the
multiple testing corrections.
Eight genes were selected based on their proven involvement in neuroblastoma differentiation. Here, we tested SNPs at the eight candidate genes for
association with disease susceptibility in 2101 cases and 4202 controls. We
replicated the associations of the identified gene in an independent cohort
of 459 cases and 809 controls. Replicated associations were studied for ciseffect using gene expression, transient overexpression and cellular differentiation assays.
NEFL showed three SNPs associated with neuroblastoma (rs11994014;
Pcombined=0.0050; OR=0.88, rs2979704; Pcombined=0.0072; OR=0.87,
rs105911; Pcombined=0.0049; OR=0.86). The protective allele of rs1059111
correlated with increased level of NEFL expression and we observed significant growth inhibition upon over-expression of NEFL, specifically in neuroblastoma cells carrying the protective allele. We also demonstrated that
NEFL expression enhanced differentiation and impaired proliferation and
colony growth in soft agar of cells with protective allele and basal NEFL expression while impairing invasiveness and proliferation of cells homozygous
for risk genotype. Finally, high NEFL expression in diagnostic primary neuroblastomas was associated with better overall survival (P=0.03; HR=0.68).
Our study shows that common variants of NEFL influence neuroblastoma
susceptibility and indicates that NEFL likely has a role in disease initiation
and progression.
J12.118
Characterization of the rs2802292 SNP identifies FOXO3A as a
modifier locus predicting cancer risk in patients with PJS and PHTS
hamartomatous polyposis syndromes
Hamartomatous polyposis syndromes (HPS) are inherited conditions associated with high cancer risk and frequently carrying mutations in the LKB1
or PTEN genes. Estimation of cancer risk is crucial in order to optimize surveillance, but no prognostic markers are currently available for these conditions. Our study is based on a signal transduction hypothesis relying on
the crosstalk between LKB1/AMPK and PI3K/PTEN/Akt signals at the level
of the tumor suppressor protein FoxO3A. Of note, the FOXO3A rs2802292
G-allele was shown to be associated with longevity, improved insulin sensitivity and increased expression of FoxO3A mRNA.
Thus, we typed this polymorphism in 150 HPS unrelated patients. We found
a significantly higher risk for malignancies in female patients and TT genotype carriers compared to those having at least one G-allele. Indeed, subgroup analysis for each HPS syndrome revealed a G-allele-associated beneficial effect on cancer risk occurring mainly in males.
Our results suggest an inverse correlation between the copy number of the
protective allele (G) and the risk of cancer and might be useful to optimize
surveillance in HPS patients. Further investigations will be needed to confirm our hypothesis and to ascertain whether differences exist in terms of
therapeutic response across genotypes
J12.119
Gene expression of MMP9 and its prognostic role in patients with
gliomas
479
M. S. Aly1,2;
1
Faculty of Science, Jazan University, Jazan, Saudi Arabia, 2Faculty of Science, Beni-Suef
University, Beni-Suef, Egypt.
480
J13.02
Hemolysis to Red Blood Cell
Back to index
C. Murugaiah;
Newcastle University Medicine Malaysia, Nusajaya, Malaysia.
A. Rafailov, M. Rafailova;
Departament of Biology, Institute of Natural Sciences, M.K. Ammosov North-Eastern
Federal University, Yakutsk, Russian Federation, Russian Federation.
The evolution of the world demand will make that Algeria will develop, in
a near future; nuclear energy .This will require a competence in this field,
and particular in the evaluation effect of the radiations emitted in the event
of radiobiology accidents. The importance of the effects of ionizing radiation is to increase the risk of cancer or genetic defects. The most reliable
method of biological dosimetry, is analysis of chromosomal modifications
radiation-induced, particularly, the dicentric chromosomes, in lymphocytes
of peripheral blood. For this purpose, we established two calibration curves
Dose/effect, by the methods of cytogenetic in uniform staining. The selected
experimental conditions are the same as those used by the team of Pr J,F.
BARQUINERO (University Autonomy, Barcelona, SPAIN). The first was established like witness, from the blood of a Spanish person. As for the second,
it was conceived for the Algerian laboratory from a blood sample of an Algerian person, without history to exposure to radiations. The frequency of
dicentric chromosomes increases as dose and the statistical analysis shows
that the values obtained follow a Poisson distribution revealing thus that
the irradiations were correct and homogeneous. The two curves obtained
from the dicentric analyses, obey the quadratic linear model and the relation Dose/effect is expressed by the equation: Y = C + D + D2 .
This study has allowed us, in Algeria to establish a dose effect curve for biodosimetry laboratory, which could in the future be part of an international
biodosimetry network
J13.06
Dysgonosomies at University Hospital Hassan II Fes: Cytogenetic and
molecular aspects
K. Belhassan;
CHU FES, genetics department, Fes, Morocco.
Back to index
sults No mutations were detected within the studied FANCA gene exons.
Conclusion This is the first molecular study of FA in Egyptian patients that
proved absence of the common mutations previously reported in other
countries this may denote molecular heterogeneity in Egyptian patients.
Further studies are recommended to establish the underlying mutations responsible for Egyptian FA cases as an important step in disease control.
J13.08
Over expression of the glycoprotein P human in chemoresistance
S. Seddiki, F. El Kebir;
University of Oran, Oran, Algeria.
Uterine myoma (UM) is a benign and most common tumor that affects 2045% of women of fertile age. Previously it was shown that somatic mutations in the MED12 gene occur in most women with uterine myoma. We
analyzed exon 2 nucleotide sequence of MED12 gene from 94 DNA samples
extracted from fibroids, endometrium, myometrium cells and peripheral
blood leukocytes of 17 women with uterine leiomyoma. Twenty-four samples exhibited various MED12 gene mutations. No mutations were found in
DNA from myometrium cells and peripheral blood leukocytes. Two mutations have been identified in codon 44 from endometrial tissue sample. In 22
of the 47 samples (47%) isolated from fibroids we found different mutations of the MED12 gene. Each fibroid is characterized by its MED12 mutation. Up to five different mutations per individual patient could be identified
depending on the amount of fibroids. Altogether we identified 18 mutations
in codon 44 and 5 deletions of varying lengths. A single nucleotide substitution c.92T>A was also found within the area of alternative splicing. MED12
gene mutations were identified in 12 of the 17 women (70%). Mutations
of MED12 gene could make substantial contribution in UM progression by
modifying the activity of other genes that encode proteins involved in cell
proliferation and blast transformation.
J13.10
Serotonin effects on NDUFS2 gene expression, a schizophrenia animal
model research
A. Haghighatfard, A. Rahimpour, M. Amini fasakhodi;
Islamic Azad university-science and reasearch branch, Tehran, Islamic Republic of Iran.
481
Background and aims: Nowadays, cancers are one of the biggest concerns
of human societies. Polyphenolic compounds and antioxidants are a key factor in the prevention or treatment of various cancers. In this study, the effects of carvacrol on prostatic cancer cells line PC3 were investigated using
comet assay technique.
Methods: 130, 230, and 360 M concentrations of carvacrol were selected
according to IC50 using MTT assay on cells line PC3. Then, alkaline electrophoresis was done and 100 comet pictures were analyzed with CASP software and all results were analyzed by SPSS statistical software.
Results: The IC50 for carvacrol was determined at 360M by MTT test. Rate
of tail to head in alkaline electrophoresis at 130, 230, and 360 M of carvacrol concentrations were 15.92.1, 38.74.2, and 65.32.0 percent, respectively.
Conclusion: Carvacrol is one of the effective polyphenolic compounds in
treatment the cancers and has destructive effects in prostatic cancer cells
line PC3. The genomic destruction effects of carvacrol on cells line PC3 is
more effective in near the IC50 concentration.
J13.13
Random Aneuploidy in Amniocytes from Aneuploidic Pregnancies
482
Back to index
and 47,XXY (study group) and compared them to amniocytes from pregnancies with normal karyotypes (control group). A significantly higher rate of
random trisomy and more triploidies were observed in trisomy 21 and 18
cases. However, in the 4 cases of 47, XXY higher rates of random trisomies,
but not triploidy were observed. Monosomies appeared in both study and
control groups, which could be the result of technical problems. The observed differences in the random aneuploidy rates between the somatic and sex
chromosome aneuploidies might reflect different mechanisms of random
aneuploidy between the two types. Triploidy was significantly higher in the
somatic aneuploidies compared to the sex aneuploidy and control groups.
As previously shown in CML patients, triploidy, which occurred more often
in the study group, probably reflects an increased predisposition to develop
malignancy compared to random aneuploidy. In most aneuploidies, malignancy will develop as a result of an oncogenic event that occurs in addition
to the existing genetic instability. Occurrence of triploidy may reflect such
an event.
J13.14
Small Supernumerary Marker Chromosome Originating From
Chromosome 10 Associated With an Apparently Normal Phenotype
T. Roberta1, R. Santacroce1, A. Pansini2, M. Gentile2, M. Margaglione1;
1
Laboratory of Human Genetics, Foggia, Italy, 2Cytogenetics Laboratory, Bari, Italy.
A normal 40-years old Italian woman was referred for cytogenetic evaluation before having undergone controlled ovarian hyperstimulation, in
vitro fertilization and embryo transfer. Family history was noncontributory except for her long-term infertility. QFQ-banding revealed a karyotype
47,XX,+mar[49]/46,XX[51] with one sSMC. Studies by aCGH and FISH confirmed that the marker originated from chromosome 10, with duplication
of 10p11.21p11.1 segment. Molecular cytogenetic characterization in her
parents, sister and brother defined a normal karyotype 46,XY for father
and brother while the mother was 47,XX,+mar[61]/46,XX[39] and sister
was 47,XX,+mar[77]/46,XX[23] with sSMC derived from chromosome 10.
Chromosome 10 is rarely involved in the formation of marker chromosomes. Eight cases have been reported in literature and in six out of these, sSMCs were associated with an increased risk of abnormal phenotype. In our
case, there was an apparently normal phenotype, probably associated with
minimal involved material and gene content. Some sSMCs derived from the
same chromosome, but in spite of this, a great variation in phenotype was
observed, probably due to the degree of mosaicism that may vary in different patients as well as in different tissues in the same patient. Furthermore,
most sSMCs were ascertained in phenotypically abnormal subjects, but it
isnt always possible to correlate the phenotype with sSMC; so it is difficult
to predict the precise phenotype-karyotype correlation and phenotypic
outcome. This report shows the importance of cytogenetic characterization
in patients with comparable chromosome defects, giving the possibility of
identifying similarities in the clinical picture that will benefit the counseling
of future cases.
J13.15
AvaII-TaqI-HindIII represents a novel informative haplotype at the
-globin gene cluster: Application in carrier detection and prenatal
diagnosis of beta-thalassemia in the Iranian population
S. Givi, T. Moradi, S. Vallian;
University of Isfahan, Isfahan, Islamic Republic of Iran.
Thalassemia is one of the common monogenic disorders, with a high demand for carrier detection and prenatal diagnosis in the Iranian population. In view of the presence of a large number of mutations associated with
the disease, the polymorphic markers present in the -globin cluster region were commonly used in linkage analysis of the disease. Markers usually
show a population dependent base haplotype frequency. Among the polymorphic markers, five markers including AvaII, RsaI, HinfI, TaqI and Hind III
were genotyped in 150 unrelated healthy individuals from the Iranian population. The haplotype frequency was estimated using PHASE program and
linkage disequilibrium (LD) was analyzed by MIDAS program. Among the 31
possible haplotypes, seven haplotypes showed relatively high frequencies
5%. The haplotype AvaII-TaqI-HindIII could be suggested as an informative
haplotype for possible carrier detection and prenatal diagnosis of beta thalassemia in the Iranian population. Moreover, RsaI and HinfI (located in the
hotspot region) were not associated with the 5 sub-haplotypes or 3 subhaplotypes. The data suggested that RsaI and Hinfl markers might be excluded as strong molecular diagnostic markers in beta-thalassemia carriership
and prenatal diagnosis in the Iranian population.
Induced pluripotent stem cells (iPSC) are primary undifferentiated cells that
are able to create almost any type of cells in the body. The purpose of the
present study was production and transmission of TetO-FUW-OSKM lentiviral vector to human dermal fibroblast cells (HDFs) and investigation the
application of this vector. In this experimental study after isolation and culture of HDF cells, TetO-FUW-OSKM lentiviral vector containing the reprogramming genes (as transfer vector) and psPAX2 and pMD2.G vectors (as
packaging plasmids) were transfected to HEK-293T cell line for virus production. The supernatants of packaging cells were harvested in 48h and 72h
after transfection. Then, these viruses were transducted to HDF for reprogramming these cells. The results of this study demonstrated the successful
production of TetO-FUW-OSKM lentiviral vector and suitable expression of
the transcription factors in HDF cells after transduction. According to these
findings lentiviral vectors could be used for gene transmission and reprogramming of adult cells such HDF to generate iPS cells in future studies.
J13.18
A rare case of a 12-year-old boy with a 45,X karyotype: cytogenetic
and molecular genetic
Back to index
analysis showed the presence of an intact SRY homeobox region and the ZFY
region, instead the regions AZF a,b,c are delete.
Fluorescence in situ hybridation (FISH) with chromosome paints for chromosome Y and with subtelomeric probe for 10q, showed the presence of a
rare aberration with an unbalanced karyotype constituted by 45 chromosomes including a der(10) deriving from the Y/10 traslocation. Finally it results a monosomy of the region 10q26.3-qter and a deletion of Yp11.2-qter.
The breakpoints were, later, by molecular genetic analysis (Agilent 8x60k
array-CGH), ascertained.
The karyotype is:
45,X,der(10)t(Y;10)(q26.3;p11.2).arr
Yp11.2
(9,394,173-28,548,485)
x0,10q26.3 (131,561,314-135,404,523)x1
J13.19
Developmental role of NF-Y as an epigenetic element on regulatory
region of SALL4 gene in human embryonic carcinoma cells
Introduction
During development epigenetic mechanisms have key roles in molecular
regulation of gene transcription. Through various epigenetic mechanisms,
NF-Y act as a histone substitute protein, which precisely binds to the CCAAT
box of promoter and has a significant role in chromatin remodelling of their
target genes. SALL4, spalt-like transcription factor 4, is a coding gene with
CCAAT box, play a crucial role in maintaining the properties of embryonic
stem (ES) cells and governing the cellular fate. Embryonal carcinoma (EC)
cells derived from testicular tumors are worthwhile models for elucidating
molecular and cellular genetic and epigenetic functions involved in developmental process due to its similarities with embryonic stem (ES) cells.
Material and Method
In this study, the chromatin immunoprecipitation (ChIP) coupled with realtime PCR was performed using anti-NF-Y antibody, on chromatin extract
from a human embryonal carcinoma cell line, named NT2/NTera2, to evaluate incorporation levels of NF-Y on the regulatory region of SALL4 gene.
Results
The results of ChIP real-time PCR analysis clearly showed a considerable
incorporation of NF-Y complex on the regulatory region of SALL4 in NTera2
cells.
Conclusion
According to the presence of NF-Y protein on SALL4 regulatory region, it can
be concluded that NF-Y has a dynamic epigenetic role in regulation of SALL4,
a master gene in controlling the molecular pathway of pluripotency during
development.
J14.01
An automatic algorithm to extract mid-sagittal plan of fetus in first
trimester from 3D volumes
P. Tsai;
Department of Obstetrics and Gynecology, National Cheng Kung University Medical
College and Hospital, Tainan, Taiwan.
Background:
In first trimester, there are many important ultrasound examinations during
prenatal care. In evaluation the fetal structure in utero, the three-dimensional ultrasound is the most convenient and powerful tool. However, accuracy
image (e.g. mid-sagittal plan) is extremely important. Thus, we proposed an
automatic algorithm to extract mid-sagittal plan of fetus in first trimester
from 3D volumes.
Materials and Methods:
3D volumes with gestational ages of 11 to 13 weeks were used in the study.
We plan a registration method based on the selected feature points. After finishing the registration of image and model with affine transform, we adjusted the scale of model to obtain the optimal size, which is similar to the object in the image. The process to put the statistic shape to the testing volume
image is similar to the adoption of all training shapes in the statistic model
construction. The following step was to make the geometric information of
the statistic shape model was used to avoid the excessive distortion caused
Result:
The measurement results were compared with those manually obtained by
an expert. The experimental results show that the proposed method overcomes the difficulties and achieves good consistency between the automatic
method and manual measurements.
Discussion:
Due to the difficulties in treating highly-noise US images, the fully automatic image segmentation tool for the fetal ultrasound is lack. The proposed
483
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time to the genetic diagnostics of these disorders and may be helpful to investigate other familial genetic disorders as well.
A. Szpechcinski, R. Struniawski, M. Skronski, J. Chorostowska-Wynimko, B. PoplawskaWisniewska, W. Kupis, P. Rudzinski, K. Maszkowska-Kopij, J. Zaleska, E. Radzikowska, E.
Puscinska, R. Langfort, P. Sliwinski, T. Orlowski, K. Roszkowski-Sliz;
National Institute of Tuberculosis and Lung Diseases, Warsaw, Poland.
J14.02
Genetic diagnosis of Bardet-Biedl syndrome by MiSeq exome
sequencing
Bardet-Biedl syndrome (BBS) is a rare autosomal-recessive ciliopathy characterized by obesity, postaxial polydactily, retinitis pygmentosa, mental retardation and kidney abnormalities. At least 18 genes have been shown to
be associated with BBS, therefore genetic testing for this disease is highly
complicated. We consulted a family with strong clinical features of BBS in 2
children. While considering the labor and consumables costs for analysis of
at least 7 common BBS genes (BBS1, BBS10, BBS2, BBS6, BBS9, BBS12 and
BBS13), we opted for exome sequencing as a viable alternative to multiple
single-gene tests. Using Illumina MiSeq platform, we identified homozygous
BBS7 L656fsX673 (c.1967_1968delTAinsC) mutation in 2 affected sibs; both
parents and their healthy son were heterozygous for this allele. Presence of
an identical gene defects in non-consanguineous parents is uncommon for
BBS, however Russian population was repeatedly shown to have an unusually pronounced founder effect. We are currently examining whether the
above mutation is indeed recurrent in Russia: at the time of abstract submission, we genotyped 1817 healthy donors and identified 1 (0.06%) additional carrier of BBS7 c.1967_1968delTAinsC.
We conclude that even medium-throughput next generation sequencing
platforms may facilitate clinical diagnosis of genetically heterogeneous disease.
J14.03
Detection of Borna Disease Virus in peripheral blood cells of a
number of obese patients in Iran via Nested RT-PCR
R. Sonboli;
Mazandaran University ,babolsar, Babolsar, Islamic Republic of Iran.
BRCA1 and BRCA2 are two well-known genes in the background of hereditary cancer syndromes. There is also evidence that several other genes
play an important role in the pathogenesis of these type of diseases. Latest
population-scaled studies showed that certain mutations in different genes
could cause as high risk elevation as BRCA2 mutations. In this study we present a reliable and cost-effective method to analyse the risk assessment of
different types of cancer. Using Haloplex, a novel sequence capture method
combined with benchtop non-optical next-generation sequencing we were
able to achive short turn around time with the screening of 16 genes that
could be associated with an increased risk of breast, ovarian and other types
of cancer. The analysis of this 16-gene set can explain the inherited background of almost 30% of hereditary and familiar cases of breast and ovarian
cancers. Thus, it opens up a high-throughput approach with fast turnaround
484
J14.05
The clinical utility of cell-free DNA analysis for non-small cell lung
cancer diagnostics and radical therapy effectiveness monitoring
Objective: The presence of cell-free DNA (cfDNA) in plasma/serum of nonsmall cell lung cancer (NSCLC) patients demonstrates promising clinical implications as the minimally-invasive liquid biopsy for diagnostic, prognostic
and predictive applications. To date, we performed quantitative evaluation
of cfDNA in plasma of NSCLC patients and non-malignant controls. Methods: Plasma cfDNA concentration and integrity index (DII) were measured by real-time PCR in 60 NSCLC patients (stage I-IIIA), 100 patients with
chronic respiratory inflammation (COPD, sarcoidosis, asthma), 15 patients
with non-malignant lung nodules (hamartoma, fibrosis, tuberculoma) and
40 healthy volunteers. Results: NSCLC patients presented significantly higher plasma DNA levels (8.0 ng/ml) than patients with chronic respiratory
inflammation (3.4 ng/ml) and healthy controls (2.3 ng/ml; p<0.0000), but
not than patients with non-malignant lung nodules. The ROC analysis provided 90% sensitivity and 80.5% specificity in discriminating NSCLC from
healthy individuals, while 56% specificity and 90% sensitivity in distinguishing NSCLC from any non-NSCLC subjects (p<0.0001). Similarly, the mean
DII in NSCLC (4.0) significantly differed from healthy control values (1.0;
p=0.000), but not from DII in chronic respiratory inflammation (3.7) and
non-malignant nodule group (4.0). During 3-6 month follow-up plasma DNA
level were significantly reduced in relapse-free NSCLC patients (2.8 ng/ml),
while in relapsed subjects were higher than at baseline. Conclusions: The
plasma DNA quantification, though insufficient for routine NSCLC detection,
is still superior to diagnostic accuracy of conventional serological markers.
Epi-/genetic alteration analysis might improve the diagnostic power of cfDNA. Long-term post-operative plasma DNA level follow-up might prove promising in monitoring of radical NSCLC therapy.
J14.06
Chitotriosidase as a biomarker for the diagnostic approach of
lysosomal storage disorders in Colombia
N. Pacheco-Fernndez, A. Uribe;
Universidad de los Andes, Bogota, Colombia.
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Introduction: EGFR signaling pathway has an important role in the development of head and neck squamous cell carcinoma (HNSCC). Therefore any
mutation that affects its components, may influence the course and management of the disease. The amplification of the EGFR gene is such significant
event that is present in HNSCC. We compared the efficacy of a targeted detection of the EGFR gene amplification with the paralogue ratio test (PRT) to
results obtained with the array comparative genomic hybridization (aCGH).
Methods: Samples of genomic DNA extracted from tumors of 50 patients
with various stages of HNSCC were first analyzed with the aCGH method. An
oligonucleotide pair that maps inside the amplified EGFR region was selected from a collection at www.prtprimer.org and used for PRT analysis.
Results: The aCGH analysis identified EGFR amplification in 7 samples, with
EGFR gene copy number ranging from 3 to 6 copies. Subsequent PRT analysis also identified all 7 EGFR amplifications and it produced concordant
results in all remaining samples which were without EGFR gene amplification.
Conclusion: Paralogue ratio test can be adopted for a rapid, targeted detection of gene amplification, such as EGFR amplification present in HNSCC,
The method may be applicable as a screening tool and for conformation of
aCGH results.
J14.11
Molecular evaluation of carbapenemases and Metallo beta lactamases
production among Gram-negative bacteria with carbapenem
resistance
H. Ghafarzadeh1,2, S. Nobary1, S. Zeinali2, F. Shahcheraghi1;
1
Department of microbiology, Institute Pausteur of Iran, Tehran, Islamic Republic of
Iran, 2Kawsar human genetics research center, Tehran, Islamic Republic of Iran.
Emerging multidrug resistant (MDR) microorganisms among hospital isolations have been limited the effective drugs to treat or prevent bacterial
infections. This study was performed to determine the rates of antibiotic
resistance in Gram-negative isolates from clinical samples and to identify
carbapenemases and Metallo beta lactamases genes variation in the strain.
Identification and assessment of gram negative bacteria with Carbapenem
resistant sensitivity to 11 antibiotic was done through biochemistry and
disk diffusion methods, respectively. 11 isolates were checked by PCR for
identification of blaVIMI,II blaIMP blaSPM blaGES blaNDM blaKPC
blaOXA-48 genes MHT and DDST tests were used assessment for Carbapenemases and Metallo beta lactamases production in these strains. The identified genes were confirmed by sequencing technique.
In this study, a total of 134 isolates were studied, including 57 Escherichia
coli (E.Coli) strain, 26 Klebsiella strain, 21 Acenitobacter strain, 17 Pseudomonas strain, 8 Citrobacter strain, 3 Proteus strain, 2 Enterobacter strain.
Confirmatory tests showed that 44 strains were EDTA positive and 17
strains were H-Test positive. A PCR based screening revealed the presence of
the blaIMP-1 gene in 4 isolates. E.coli showed the highest level of resistance
to imipenem. Most of positive H-test (41.17%) and positive DDST (39.13%)
strains were Acenitobacter.
Owing to the presentation of blaIMP-1 gene in Klebsiella, Citrobacter, Acenitobacter and E.coli and feasibility of horizontal gene transfer among bacteria, but also due to the importance of Metallo beta lactamase production
strains in hospital, change in antibiotic prescription policies are required.
J14.12
Improved methods for preparing high quality genomic libraries from
challenging FFPE tumour samples for use on Illumina sequencing
systems
R. Sanches-Kuiper, H. McGarvey, A. Jackson, T. James, O. Mead, S. McArthur, S. Berri, S.
Humphray, V. Smith;
Illumina Cambridge Ltd., Essex, United Kingdom.
The research community needs robust and rapid sample preparation work-
485
E. Paladi1, M. Sprincean1,2;
1
Institute of Mother and Child, Chisinau, Moldova, Republic of, 2State University of
Medicine and Pharmacy N. Testemitanu, Chisinau, Moldova, Republic of.
Background: Congenital pathology is the leading cause in Republic of Moldova of infant morbidity and mortality. Invasive techniques of prenatal diagnosis (amniocentesis) are very important in efficient medico-genetic counseling for determining caryotipe, used in diagnosis of many chromosomal
abnormalities (Down syndrome, Patau and Edwards syndromes etc.).
Object of study. Efficient diagnosis and prevention of chromosomal abnormalities and congenital malformation can be assured only by medico-genetic, pedagogical, psychological theory and practice as wide interdisciplinary
knowledge on genetic diseases and hereditary pathologies.
Methods. In this research were used a number of practical methods such
as gathering of anamnestic data, conversation, questioning, observation,
psychological and psycho-pedagogical testing as well as a complex of cytogenetic and molecular-genetic methods. Clinical-genealogical examination
of pregnant women from genetic risk group was provided at the beginning
of the study.
Results obtained. The diagnosis of chromosomal abnormalities and congenital malformation was provided during medico-genetic counseling and
during sessions of qualified psychological assistance, to 74 pregnant women
of risk group. Results of the study can be used in family planning and counseling for prophylaxis of congenital pathology as well as to increase the level
of awareness of people of risk group on prevention of genetic diseases.
Conclusions. This study stated the necessity of implementation in Moldova
of a diversity of medico-genetic strategies during counseling of pregnant
women of risk group, for improve the efficiency of diagnosis of chromosomal abnormalities and congenital malformation, which can be achieved by
close cooperation between specialists of many fields as genetic physicians,
psychologists, pedagogues.
J14.14
Seroprevalence Of Helicobacter Pylori In Children In A Rural Area
T. Sabbi;
Pediatric Unit, Rome, Italy.
486
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We and others have shown that the BCR-ABL mRNA expression is higher
in the CD34+ cellular population, representing an enrichment of BCR-ABL+
cells in the progenitor fraction or a higher expression of the fusion gene. Primitive progenitor BCR-ABL+ cells isolated from CML patients will maintain
their functionality after Imatinib treatment. Therefore, CML progenitor cells
appear to be more insensitive to Imatinib than more mature cells suggesting that TKIs are unable to inhibit the function of the primitive leukaemic
stem cells. During the assessment of minimal residual disease (MRD) by RTqPCR, some of the early leukaemic progenitors of malignant cells with significantly higher levels of BCR-ABL transcript cannot be differentiated from
other groups of homogeneous cells. This fact may cause a failure to perform
personalised monitoring of MRD; while the sensitivity and reliability of the
assay are directly affected by bulk measurements where the number of normal cells that do not express the target genes. In this study, K562 cells were
grown in suspension and a small fraction of these cells was noted to have
adhered to the plastic dish after the removal of the media. These adherent
cells (K562/Adh) were passaged serially. The FACS separated individual
K562 cells into PCR wells with a fixed amount of qPCR buffer. Optimized
amplification RT-qPCR was performed on single cells to reveal the nature
and significance of cellular heterogeneity with the measurement of BCRABL levels in K562 cells was investigated.
J14.17
Novel methods for phenotyping mice pups during the first few hours
after birth
E. Durand;
PhenoPups SAS, Evry, France.
Mice are the preferred species to study the biological function of mammalian
genes through mutation but there are still specific challenges to solve in order to characterize their phenotype. As early lethality is common in genetically engineered mice, an important proportion of homozygous mutant mice
cannot be phenotyped. When the corresponding heterozygous mice have no
phenotype, the effects of this mutation remain unknown. Unfortunately, studies in newborn mice face major difficulties due to the small size of the pups
During last years the next generation sequencing (NGS) technologies have
significantly enhanced the discovery of causative genetic alterations and
identification of mutations in rare diseases. The diseases with locus heterogeneity are still a challenge in clinical practice and sometimes patients
remain for years with unknown genetic status.
In order to accelerate the establishment of proper genetic status of patients
with rare diseases we analyzed exons of 552 genes underlying rare monogenic recessive disorders using TruSight Inherited Disease Sequencing Panel
(Illumina). As model we used diseases with clear unequivocal phenotype
(CF, DMD) and included in the study patients in whom routine DNA testing
was insufficient to obtain genetic diagnosis. The study included 4 patients:
2 with CF with identified only one mutation, 1 with DMD without deletions/
duplications in DMD gene and 1 with polymalformative syndrome including
Dandy-Walker anomaly, AVSD, hypoplastic genitalia, MR and facial dysmorphism. NGS revealed CFTR: p.Gly1349Asp as second pathogenic mutation in
one patient with CF and DMD: p.His2921Arg in patient with DMD. Both mutations are reported for first time in patients of Bulgarian descent. In one patient with CF NGS revealed only one causative mutation and MLPA showed
a large deletion encompassing exons 18-20 of CFTR gene. A homozygous
in-frame deletion p.Ser372del in MKS1 gene was found in the patient with
polymalformative syndrome.
The NGS technology has the potential quickly and cost-effectively to point
out disease causing mutations in patients with rare genetic diseases and
Back to index
Prader -Willi syndrome (PWS) is a complex, multisystem disorders of genetic origin due to lack of paternally active genes on critical area on chromosome (15) (q11-13) . PWS is almost always sporadic and is the most
common syndromal cause of human obesity with an estimated incidence of
1 in 25,000 Material and methods:Conventional G-banding, FISH using an
SNRPN Prader-Willi /Angelman chromosome region probe by AppligeneOncor and methylation specific-PCR studies were conducted on 23 patients
(13 males and 10 females age ranged from 6.5 months to 20 years selected
according to suggested criteria proposed by Gunay- Aygun et al, 2001. Aim:
This study aims at confirming/ excluding PWS in clinically suspected patients through a sensible strategy of investigations on the cytogenetic level,
FISH technique and molecular level using DNA methylation tests for phenotype/ genotype correlation, early diagnosis and proper counseling. Results:
On the cytogenetic level, 10 out of 23 cases showed deletion of 15q (1113). FISH confirmed the deletion in in 6 cases with mosaicism in one case
(50%); detected deletion in 4 cases with normal cytogenetic findings, and
excluded deletion in 13 cases. Molecular methylation studies conducted on
17 patients (6 cases spared upon request) confirmed diagnosis in 10 cases
detected 1 new case and excluded PWS in 6 cases. In conclusion: we reinforce the necessity of analysis of DNA methylation within the chromosome
15q 11-13 region, which is an important tool for the correct diagnosis and
for detecting etiology among children presenting with neonatal hypotonia,
mental deficiency and obesity.
J14.22
Procalcitonin Gene Expression is a Real Biomarker of Aging Process
M. A. Soylemez;
Marmara Medical School, Istanbul, Turkey.
Objective: Inflammation is the single greatest precipitator of aging and agerelated diseases such as, diabetes, heart disease, alzheimer, arthritis. Inflammation, which takes place on a cellular level, is triggered by a wide variety
of factors such as the oxidative stress, hormonal changes and eating a proinflammatory diet (Hyperglycemic diet). The procalcitonin gene has promoter sites for NF-kappa and AP-1, factors induced under inflammatory
conditions. Hyperglycemia is associated with oxidative stress and elevation
of advanced glycation end products (AGEs). AGEs interacts the receptor for
487
Sex chromosome abnormalities represent a special challenge in the analysis of SNP-array data. Data analysis is complicated by the complex structure
of the sex chromosomes (PAR regions, interfering homologous regions and
only one of each sex chromosome in males). At present, no guidelines can
help interpretate the analysis of sex chromosome abnormalities identified
by SNP array.
In this study we analysed SNP-array data from 25 samples with known sex
chromosome abnormalities.
We present the results from this study and present a general guideline for
SNP-array interpretation of sex chromosomes. Hopefully this will aid others
in the interpretation of sex chromosome abnormalities identified by SNParray.
J14.26
NextGen Sequencing: a tool for deciphering the BRCA1/2 and TNBC
relationship
Although during recent years science and technology have generated a great
deal of progress in medicine, breast cancer still is the most common malignancy and the leading cause of death by cancer in women. Triple negative
breast cancer (TNBC), characterized by the absence of Estrogen and Proge-
488
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Some of the major reasons for high throughput sequencing being more and
more frequently applied in cancer research and cancer diagnostics today,
are the significant improvements in accuracy, throughput, and speed. In the
past, sequencing was clearly the bottleneck but the most time consuming
step currently is the data analysis - more precisely the identification and
characterization of variants in tumor samples. Whole exome and genome
sequencing protocols are commonly used and allow for the identification
of SNPs and InDels in protein coding regions. However, both methods fail to
show which allele variants are actually expressed in the tumor tissue. Contrariwise transcriptome sequencing (RNA-seq) reveals the expressed alleles
and can in addition provide insight into the transcript expression levels, expressed isoforms, and instances of fusion transcripts. Here, we illustrate the
benefits of combining the analysis of whole exome sequencing and RNA-seq
using CLC Cancer Research Workbench. This software suite includes algorithms for quality control, read mapping, variant calling, and RNA-seq analysis, as well as numerous tools for the comparison and annotation of variants.
The analysis is carried out with ready-to-use workflows and the results are
visualized in a track based genome browser. In the present work differences
found between the variants identified in exome and in RNA-seq data from
uveal melanoma samples will be highlighted.
J14.28
Molecular-genetic analysis of predisposition to ovarian cancer among
women of Uzbekistan
S. Nurmatova1, B. Adylov1, S. Turdikulova1, D. Nabiyeva2, M. Isamuhamedova2;
1
Educational-Experimental Centre for High Technologies in Tashkent, Tashkent,
Uzbekistan, 2Tashkent Institute of Postgraduate Medical Education, Tashkent Regional
Oncological Dispenser, Tashkent, Uzbekistan.
Ovarian cancer is the 9th most common cancer, with an estimated 22240 new
cases in 2013. Most women arent diagnosed until the cancer has spread,
leading to a poor five-year survival rate of 43%. Only 10-15% of malignant
epithelial ovarian tumors are genetically determined.
The most significant genes associated with ovarian cancer include BRCA1,
BRCA2, CHEK2. These genes produce tumor suppressor proteins which help
repair damaged DNA and play a role in ensuring the stability of the cells genetic material. When either of these genes is mutated, DNA damage may not
be repaired properly. As a result, cells are more likely to develop additional
genetic alterations that can lead to cancer.
This study aimed to investigate the contribution mutations of BRCA1, BRCA2 and CHEK2 genes to ovarian cancer cases in Uzbekistan.
66 patients with ovarian cancer were included in this study. By means of
real-time allele-specific PCR we analyzed DNA samples of above mentioned
group for the presence of 5382insC mutation in BRCA1, 6174delT - in BRCA2 and IVS2+1G>A - in CHEK2. 6 unrelated samples (9.1%) were found to
be positive for the heterozygous 5382insC mutation representing a possible founder mutation in the Uzbek population. We didnt find 6174delT and
IVS2+1G>A mutations. The presented data confirm contribution of BRCA1
5382insC mutation for ovarian cancer development in Uzbek people and taking into account a high disease penetrance in carriers of BRCA1 mutation,
it seems reasonable to suggest inclusion of 5382insC mutation test in screening programs for breast cancer prevention in Uzbekistan.
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Twin studies indicated that heart rate variability (HRV) and particularly its
high frequency (HF) spectral component show significant heritability, and
their genetic variance is amplified by mental stress. This genetic association study investigated the influence of the brain-derived neurotrophic factor
(BDNF) Val66Met polymorphism and the triallelic serotonin transporter
gene promoter (5-HTTLPR) polymorphism on state anxiety and time and
frequency measures of HRV reactivity to mental stress. In comparison to
BDNF Val homozygotes, the Met allele carriers showed larger cardiovagal
withdrawal during stress. The triallelic 5-HTTLPR did not significantly influence any of the psychophysiological measures. Although BDNF Val66Met
only explained a small part of the variance of cardiovagal withdrawal during stress, this polymorphism may be part of a complex genetic pathway
underlying the comorbidity of autonomic dysfunctions and emotional vulnerability.
489
BRCA1 and BRCA2 genes are suppressor of malignant tumor and predictive markers sensitivity to platinum-drugs. Malignant transformation and
carcinogenesis accompanied by alters the expression of tumor-suppressor
genes. Loss of any of these genes expression is associated with positive response on treatment by platinum-drugs. Tumor and normal tissue samples
of colon among 30 patients with colorectal cancer were investigated. High
level (3 and more time) of BRCA1 and BRCA2 expression was found in 22/30
(70%) tumors and 24/30 (80%), respectively. As well high expression of
both genes was detected in 19 tumors. The expression of BRCA1 was not
changed in 7 of 30 (23%) tumors and in one tumor was decreased in 7
times. The expression of BRCA2 was not changed in 4 of 30 (13,3%) tumors
and was decreased (in 7 times) in two tumors. The expressions of both the
genes were not altered in two cases. However BRCA1 and BRCA2 expression
was not a coordinately decreasing in neither case. We obtained information
about change BRCA1 and BRCA2 expression in tumor for Russian patients
with colorectal cancer. These data can improve the therapy for colorectal
cancer patients by platinum-drugs.
J15.04
The effects of hypericin on p53 and ADAMTS gene expression in
MCF-7 breast cancer cell line
490
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Our aim was to investigate the effects of hypericin which is obtained from
the plant Hypericum perforatum on the expression and the regulation of
ADAMTS8 and ADAMTS9 genes in MCF7 breast cancer cells and on the viability of these cells. MCF7 cells were cultured and were separately exposed
to 2, 10 and 50 l/mL of hypericin. After 24 hours, RNA was isolated from
these cells and converted to cDNA. The expression levels of ADAMTS8 and
ADAMTS9 genes were evaluated using the real-time RT-PCR. XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt) cell viability assay was used to determine cytotoxicity. ADAMTS9
expression in MCF7 cells were increased 1.8 and 3.6 fold with the use of 2
and 10 l/mL of hypericin, respectively; and decreased 0.7 fold with the use
of 50 l/mL of hypericin. There was no significant change in the ADAMTS8
expression. Rapid cell death was observed in the cancer cells when hypericin
was used at a dose of 50 l/mL. The increase in ADAMTS9 expression can
be a useful factor in the prevention of possible metastasis in breast cancer
and for the occurrence of a tumor suppressive effect. Hypericin increases
the expression of ADAMTS9, therefore, it may show its antitumoral and antiapoptotic effects by means of ADAMTS9.
J15.06
Effects of tomatine in breast cancer
Metastasis in breast cancer is still a complicated issue and matrix metalloproteinase family has a significant role in metastasis showing the link
between MMP members and tumor development. Tomatine is a secondary
metabolite synthesized by tomatoes and has a role in natural defenses
against plant fungi, viruses and bacteria. Besides, it is known to disrupt cell
membranes and be a strong inhibitor of human cancer cells. In this study, it
was aimed to evaluate the effect of tomatine on cytotoxicity and apoptosis
on MCF-7 cell line. Firstly, IC50 dose of tomatine was measured as 7.07 M
by using xCELLigence system. Further, it was shown that tomatine induced
apoptosis is 3 times greater than control cells with by Annexin V labeling on
Flow Cytometry . Additionally, miRNA expression profiles of breast cancer
cells were determined by qRT-PCR and 22 downregulated and 15 upregulated miRNAs were found. Active and zymogen forms of MMP-2 and MMP-9
were detected by zymography method and the activations of MMP2 and
MMP9 were decreased significantly. In conclusion, these results show the
suppression of matrix metalloproteinase activity on metastasis.
J15.07
Aberrant miRNA Expression by Ellagic Acid Alters Apoptosis and Cell
Cycle Regulation in Breast Cancer Cells and Breast Cancer Stem Cells
Breast cancer has still been the most important subject with other cancer
types. This cancer is the most common and second leading cause of death
among women. It is known that various plant extracts, including ellagic
acid, have anti-proliferative and pro-apoptotic effect on breast cancer cells.
However, miRNAs which are associated with tumor initiation for estrogen
dependent breast cancer are suppressed by ellagic acid treatment. In this
study, it was aimed that ellagic acid could induce apoptosis related mechanism in breast cancer cells and this induction could be trigged by altered expression profiles of miRNAs. Cytotoxic effects of ellagic acid on MCF-7 breast
cancer cells and breast cancer stem cells were examined by WST-1 reagent
test. Apoptosis and cell cycle analysis were detected by flow cytometry ana-
The significance of genes for tumor cell viability and understanding of the
cell genetic features under which high apoptotic effect of gene silencing will
observed - both are important in development of targets. We were searching
of genes, silencing of that will increase sensitivity of colorectal cancer cells
to oxaliplatin. The panel of genes was formed using of expression data bases and publications analysis. The expression gene profile was determined
in dependence on the dose and the time of oxaliplatin action for two colorectal cancer cell lines: HT-29 and HCT-116 p53-/-. The over expressed and
demonstrating dose dependence under different period of incubation genes
have been identified. It was interesting that the identified genes - c-IAP2 and
Livin - were the same for both cell lines, although the lines are different genetically. The silencing of genes was performed by small interfering RNA
(siRNA). Joint silencing of the gene found has led to a substantial (70-80%)
suppression of the cell viability and increase of 5 -10 times the sensitivity of
cancer cells to small oxaliplatin doses (5 - 10 M). Common for HT-29 and
HCT-116 is mutation in TP53 gene. Possibly, this feature is responsible for
the similarity of response to oxaliplatin genetically different cancer cells. In
conclusion, there were found the genes silencing of that by siRNA gives the
possibility to reduce approximately of 10 times the standard dose of chemotherapeutic drug with reaching a high apoptotic effect on genetically different colorectal cancer cells.
J15.09
Analysis of CYP2E1 gene using multiplex HRMA. A study in the Polish
patients under sevoflurane anaesthesia
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Faculty of Dentistry, University of Medicine and Pharmacy Iuliu Hatieganu, ClujNapoca, Romania, 2Research Center for Functional Genomics, Biomedicine and
Translational Medicine, Iuliu Hatieganu University of Medicine and Pharmacy,
Cluj-Napoca, Romania, 3Research Center for Functional Genomics, Biomedicine and
Translational Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, ClujNapoca, Romania, Cluj-Napoca, Romania, 4Department of Functional Genomics and
Experimental Pathology, Institute of Oncology Prof. Dr. I Chiricuta,, Cluj-Napoca,
Romania, 5Department of Immunology, Faculty of Medicine, Iuliu Hatieganu University
of Medicine and Pharmacy, Cluj-Napoca, Romania.
1
491
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RT-PCR. No significant change was detected in the expression levels of those genes in NaF group. The expression levels of BAD did not significantly
change in all study groups as well. The ratio of pro-apoptotic BAX to antiapoptotic BCL-2 was greater than 1 which demonstrates that those fluoride
agents cause induction of apoptosis. In conclusion we suggest to protect the
soft tissues during the application of the fluoride agents, to be careful for
patients not to swallow the fluoride agents and to avoid unnecessary usage
of fluoride in clinics..
J15.13
Implication of HLA B27 and ITPA polymorphisms in treatment
response of HCV infected patients
Homozygous
Mutant
Mutant
Heterozygous
Homozygous Heterozygous
wild type
homozygous
homozygous
AC
wild type CC
CA
AA
CC
AA
76
23
1
88
12
0
A allele
87.50%
C allele
94%
frequency
(0.875)
frequency
(0.94)
C allele
12.50%
A allele
6%
frequency
(0.125)
frequency
(0.06)
The HLA-B27 allele frequency (6%) obtained in our study is similar to corresponding data reported in literature for Caucasian populations (8%). Up to now,
we found neither association between the ITPA markers and Pegylated Interferon-Ribavirin treatment induced anemia, nor between the presence of HLA-B27
allele and spontaneous viral clearance. To find any correlation between these two
genetics human factors and response to Pegylated Interferon-Ribavirin therapy,
more HCV-infected patients will be included in our ongoing study.
Acknowledgement: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNDI-UEFISCDI, project number
88/2012, PN-II-PT-PCCA-2011-3.2 (GO).
J15.14
Cytogenetic monitoring of response and karyotype evolution in lowrisk MDS patients treated in the leMon5-study
492
J15.15
The effect of Centaurea lydia on MDM2 gene expression in human
acute lymphocytic leukemia cells via transfection of miR-150 by
MATRA
Chronic myeloid leukemia (CML) is a malignant disorder of the haematopoietic stem cell arisen from the reciprocal translocation between the breakpoint cluster region (BCR) gene on chromosome 22, and the Abelson (ABL)
murine leukemia virus gene on chromosome 9, t(9;22)(q34;q11), resulting
in the formation of Philadelphia chromosome which has constitutive tyrosine kinase activity, leading to leukemogenesis. Resveratrol is a naturally
occurring phytoalexin with apoptotic and growth inhibitory effects in leukemic cells. MicroRNAs play a pivotal role in normal hematopoiesis. In this
study we aimed to evaluate the cytotoxic and apoptotic effect of resveratrol
in CML cells by questioning miRNA expression levels associated with CML
progression. K562 cells were treated with the IC50 dose (100 M) of resveratrol for 72 hours. Cytotoxicity analyses were conducted by WST-1 analysis.
Apoptosis was evaluated by AnnexinV-enhanced green fluorescent protein
(EGFP) and by ApoDIRECT In Situ DNA Fragmentation Assay Kit. The RTqPCR is used for miRNA expressions analysis. miRNA expression levels were
evaluated by using miScript miRNA PCR Array. miRNA expression levels was
analyzed by using miScript miRNA PCR Array Data Analysis. Log2 transformation was applied. Significant increase in miR-181 family was observed in
K562 cells treated with resveratrol according to control. Resveratrol up-regulated miR-181a, miR-181b, miR-181c and miR-181d expressions as 8.96,
6.42, 10.15, 17.19 fold according to the control cells, respectively. Resveratrol upregulated tumor suppressor miR-181 family in K562 cells through
induction of apoptosis and this effect can be used for alternative detection
of chronic myeloid leukemia progression.
J15.19
Zoledronic acid inhibits glioma cell growth and induces apoptosis by
up-regulating miR-22 expression
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The aim of the study was to evaluate the effect of zoledronic acid on the expression of 84 miRNAs. In our experiments, U87-MG cell line (Human glioblastoma-astrocytoma) is used as an in vitro model of human glioblastoma cells
to investigate the cytotoxic and apoptotic effect of ZA towards glioma cells.
U87-MG cells were treated with 25 M (IC50) ZA during 72 hours. Apoptosis
assays were performed by using ApoDIRECT In Situ DNA Fragmentation Assay. The RT-qPCR is used for miRNA expressions analysis. miRNA expression
levels were evaluated by using miScript miRNA PCR Array.
Results showed that IC50 dose of ZA induced apoptosis 4.25 fold when compared to control cells that untreated with ZA. Also IC50 dose of ZA of miRNA
expression results showed that; mir-22 expression level was upregulated
3,08 fold according to control group .
In conclusion, these novel findings showed that ZA can be important in prognosis of glioma and it is necessary to question whether it can be used as a
drug candidate in glioma treatment with further research on miR-22 and its
target gene expression.
J15.20
Nicotine and Cotinine Dependent Regulation of P-glycoprotein
Expression in Caco-2 Cells
Introduction: The use of chemotherapy in node-negative, ER-positive breast cancer has changed dramatically since the introduction of OncotypeDX
to determine systemic recurrence risk based on tumour genomic signature.
Aims: This study aims to 1. Document longitudinal changes in chemotherapy use 2. Assess the impact of new evidence on local protocol Methods:
A cohort study was undertaken, including consecutive patients with early
node-negative, ER-positive breast cancer diagnosed between 2006 and May
2013. Data was collected regarding clinico-pathological features, OncotypeDX use, recurrence score, and chemotherapy use. All therapeutic decisions followed multidisciplinary discussion, with adherence to guidelines and
consideration of trial protocol and OncotypeDX recurrence scores. Results:
The study group included 476 consecutive patients, of whom 240 (50%)
underwent OncotypeDX testing, 96 as part of TAILORx trial. OncotypeDX has
been used in 84%(n=125) patients since being made available for use in the
public sector (see table).
Time Period
2006-2007
2007-May 2010
Oncotype
OncotypeDX test Oncotype Not
Available on
availability
Available
Trial only
Number of
55
199
patients treated
OncotypeDX use
0 (0)
96 (48)
(n(%))
Chemotherapy
34 (62)
101 (51)
Use (n(%))
19 (26)
33 (45)
October
2011-May
2013
Oncotype in
Clinical use
149
125 (84)
48 (32)
493
Breast cancer (BC) represents a heterogeneous disease in which many different genetic, epigenetic and environmental factors are involved. The aim
of our study was to investigate the association of DNA methylation of tumor
suppressor genes and histopathological profiles of BCs. The methylation
status of 24 tumor suppressor genes was determined in 124 BCs using MS
MLPA001 kit. Methylation of more than 15% was regarded indicative for
gene hypermethylation. SPSS statistics was used for statistical analysis. A total of 103 BCs (87.5%) showed a methylation of at least one tumor suppressor gene. Seventeen of the 24 analyzed genes showed methylation. The most
frequently methylated genes were RASSF1 (66.1%), APC2 (41.9%), CDH13
(35.5%), GSTP1 (25%), DAPK1 (15.3%), and CASP8 (8.1%). Triple negative
BCs showed positive association with ESR1 (p=0.006), BRCA1 (p=0.006)
and TIMP3 methylation (p=5x10-5) and negative association with RASSF1
(p=0.003) and GSTP1 methylation (p=0.02). Similar associations showed the
BCs with BRCA1ness profiles, determined by MLPA analysis. Estrogen (ER)
and progesterone (PR) positive BCs were positively associated with RASSF1
(p=0.001 and p=0.003, respectively) and DAPK1 methylation (p=0.018 and
p=0.027, respectively). ESR1, CDH13, BRCA1 and TIMP3 were less frequently methylated in ER+ tumors. RARB1 and CD44 showed positive association
with tumor size (p=0.008 and p=0.006, respectively). Herceptine, p53 and
Ki-67 positive BCs were more frequently non-methylated at any of the analyzed genes (p=0.05, p=0.022 and p=0.008 respectively). In conclusion, our
study shows that DNA methylation is a frequent event in BC and that different genes are methylated in BCs with different histopathological features.
J16.02
Investigation of the promoter hypermethylation in ILC and IDC of the
breast
Backround: IDC and ILC are the most common invasive breast cancer tumor
types. About 80% and 10% of invasive breast cancers are infiltrating ductal
494
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Colorectal cancer (CRC) is the 3rd most common cause of cancer death in
the world.Introducing a suitable method for screening and early detection
of CRC will be one of the most important strategies. The presence of cancer-specific DNA methylation biomarkers in the body fluid of CRC patients
will provide a simple, non-invasive screening test for colorectal cancer. This
study was designed to evaluate methylated SPG20, ALX4, ITGA4 genes as a
novel epi-panel for screening and detection of CRC. In the first step 90 blood
samples were collected from diagnosed cases as colorectal cancer and 40
samples were collected from healthy volunteers as control from Baqiatalah
University of Medical Sciences, Tehran, Iran.DNA from plasma samples was
extracted by using Qiagene DNA purification protocol following Bisulfite modified DNA was amplified by using specific primers for Methylation Specific
PCR(MSP).Finally the MSP evaluation was performed on the patients and
control groups.The results up to now showed that ITGA4, SPG20, ALX4 were
hypermethylated in between 30 to 50% of CRC and polyp patients whereas
normal samples were rarely methylated. These candidate markers can be
considered for further evaluation in screening or diagnostic applications for
G. Belaaloui;
Faculty of Medicine. Hadj Lakhdar University, Batna, Algeria.
Ionizing radiation (IR) imposes risks to human health and the environment.
Taking into account the fact that Kazakhstan is one of the worlds leading
uranium producer, and considering the magnitude of the damage from Semipalatinsk nuclear test site, the study of radiation effects on genes has
always been a priority. It is known that LIG3 and XPA genes may serve as
biomarkers for sensitive methods of dosimetry. The purpose of this study is
to determine the impact of the IR on gene expression in uranium industry
workers of Stepnogorsk city, Kazakhstan.
Peripheral blood samples (n=20, n=19) were collected from workers who
were exposed to different radiation doses (50mSv, 20mSv). To examine gene
expression profiles of radiation exposed workers quantitative real-time PCR
were used. We used B-actin gene as an endogenous control for our study.
The expression of LIG3, XPA genes were analyzed and compared between
two groups.
Gene expression analysis showed statistically significant difference in expression of DNA repair genes (LIG3, XPA) between two groups exposed to
different doses of radiation. The greater the delivered dose of IR, the greater
the expression level of DNA repair genes.
Altered expression profiles in this study can be used as biomarkers of dosimetry for uranium industry workers. This strategy on risk assessment
should be considered in the construction of industrial safety and health of
workers occasionally exposed to IR.
J16.07
Whole genome sequencing of Mycobacterium tuberculosis in
Kazakhstan: first sequence results of two clinical isolates
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Bladder cancer is the one of the most common cancers worldwide and
O6-Methylguanine DNA Methyltransferase (MGMT) promoter methylation
might be contributing to clinicopathological features of the disease. We analyzed formaline-fixed, paraffin-embedded bladder tissue samples from 101
patients with urothelial carcinoma. All samples were analyzed by real-time
methylation-specific PCR. MGMT promoter methylation was detected in 15
(14.9%) of the samples and was not significantly different between any of
the groups, although the number of high grade tumors with MGMT promoter methylation was twice the number of low grade tumors. Methylation of
MGMT in high grade tumors might play a role in aggressive proliferation of
the tumor cells and a poor prognosis.
J16.09
PRKCDBP downstream hypermethylation and its influence on gene
expression in squamous intraepithelial lesions and carcinomas
Protein kinase C delta binding protein (PRKCDBP) is a potential tumor suppressor although molecular basis of its action has been poorly understood.
PRKCDBP gene is inactivated in colorectal, lung and breast cancer by genetic or epigenetic alteration which leads to down-regulation of PRKCDBP
expression. The purpose of this primary study was to examine PRKCDBP
hypermethylation in 7 CpG sites (assays predesigned by QIAGEN) by pyrosequencing and to find out the expression in squamous intraepithelial lesions and carcinomas. A total of 51 cervical specimens were divided into
groups with a diagnosis of normal cervix (n=21) and squamous intraepithelial lesion or carcinoma (n=30). Downstream hypermethylation was found
significantly hypermethylated in two CpG sites in intron (p=0.0033; 95%
CI: -3.26 to -0.69 and p=0.0015; 95% CI: -4.07 to -1.03) and one in exon 2
(p=0.0222; 95% CI -5.06 to -0.41) when compared to group with normal
cervix. PRKCDBP gene expression was also reduced in group with cervical
lesion or carcinoma. Downstream hypermethylation has been reported as
widespread phenomenon with different effect on the gene expression and
promoter hypermethylation. These results could indicate the potential impact of downstream hypermethylation on reduced PRKCDBP expression during cervical carcinogenesis.
J16.10
Short runs of homozygosity as an underappreciated mechanism for
imprinting disorders
495
Runs of homozygosity (ROH) are epigenetic changes encompassing relatively large genomic loci and presenting as stretches of consecutive homozygous genotypes scattered through the genome. Usually, ROH exceeding 5-10
Mb in length are only considered to have an effect on phenotype. On the
other hand, heterozygosity losses of a single imprinted gene can produce an
epigenetic change leading to imprinting disorders. Therefore, such epigenetic changes have not necessarily to encompass large genomic loci. Studying
ROHs in 115 children with intellectual disability and congenital malformations by Affymetrix 2.7M SNP/array, we have found that 1-Mb-long-ROHs are
associated with imprinting disorders. In a case of atypical Angelman syndrome a ROH at 15q11.2 (size 1.16Mb) was detected. Furthermore, two cases of
atypical Beckwith-Wiedemann associated with ROHS at 1115.515.4 (size:
1.36Mb and 1.15Mb) were identified. In addition, a long ROH at 7p14.2p12.1
(14.95Mb) affecting GRB10 (an imprinted candidate gene for Silver-Russell
syndrome) was detected in a child with severe growth retardation, microcephaly and intellectual disability. Three children (2.6%) demonstrated
overall ROH burden typical for offspring of a consanguineous relationship.
Retrospectively, consanguinity was confirmed by genealogical analysis.
Thus, our observations show that 3.5% of intellectual disability cases can
be attributed to ROH. Moreover, ROHs can be as short as 1Mb, which are
usually overlooked, when SNP/array assays or other methods for epigenetic analysis are applied. We conclude that these epigenetic mutations are a
relatively common mechanism for imprinting disorders. Supported by the
Russian Federation President Grant (MD-4401.2013.7).
J16.11
The review od epigenetic modification in human thyroid cancer
Thyroid cancer is the most common endocrine malignancy worldwide. However, there is not a definite treatment for advanced thyroid cancer, which
comprises poorly differentiated, anaplastic and metastatic or recurrent
differentiated thyroid cancer that do not response to radioiodine; treated
patients had not a complete response. Biological therapies have been proposed on the basis of the recognition key oncogenic mutations and these
treatments could be effective in stabilizing progressive disease. Epigenetic
abnormalities are present in almost all cancers and together with genetic
variations led to tumor progression. Epigenetic modification is mostly occur
in genes, which play role in the control of cell proliferation and invasion such
as RASSF1A, PTEN, DAPK, RAP1GAP, and specific genes of thyroid are mostly
epigenetically silenced in thyroid cancer. Rap1, a member of RAS family of
small GTPase has been implicated in regulation of mitogenic and oncogenic pathways in thyroid, and its CpG island methylation is reported in some
kind of tumors. In addition, miRNAs play important role in cell differentiation, proliferation and survival. They considered as the regulators of gene
expression at posttranscriptional level. Deregulation of miRNAs expression is suspected to be an important regulator of tumor development and
progressive. Epigenetic modification pattern is different not only between
normal and tumors tissues, but also between different stage of malignancy
and between primary and metastatic tumor. Therefore these variations may
become useful novel biomarkers for diagnostic and treatment tumors.
J16.12
Epigenetics, maternal responsibility and neurological development
K. Hens;
Maastricht University, Maastricht, Netherlands.
It is believed that epigenetics can partly explain the development of neurological conditions. As epigenetic changes often happen in utero, maternal
behavior may affect brain development. This raises questions regarding the
responsibility of the pregnant woman. To which extent is she responsible for
the neurological status of the future child? Should she, for example, avoid all
stress, if this is shown to lead to molecular changes increasing the chance of
the child to develop ADHD later in life? Should she ensure that she takes optimum nutrition for development of synaptic plasticity of her fetus? With of
496
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this talk I demonstrate first that epigenetics complicates the question of the
responsibility of the pregnant woman. To which extent does the increasing
knowledge about epigenetics influences in utero further complicate this responsibility, if even behavior of years before the conception may influence a
fetus? Is this an individual responsibility or also a collective one? Second, by
using the examples of autism and extreme intelligence, I demonstrate that ,
although discussions about parental responsibility towards future children
have centered on duties to prevent suffering or to maximize welfare or the
chance of a good life, the distinction between prevention and enhancement
is difficult to maintain in the context of neurological conditions. By analyzing arguments from the neurodiversity movement, whose members often
insist that their neurological condition is not a disease, but an integral part
of their identity, I shall demonstrate that there is a need for a revisiting of the
concept of maternal responsibility.
J16.13
Anchored Assembly: Comprehensive variant detection using NGS data
J. Bruestle, B. Drees, Ph.D.;
Spiral Genetics, Seattle, WA, United States.
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rian families with clinical symptoms referred to those of the disease. DNA
was extracted from the periferal blood with salting out method. Polymerase
chain reaction (PCR) and triplet repeat primed PCR (TP-PCR) were used to
confirm the diagnosis.
Results: From 63 analyzed patients SCA was established in 16 patients from
15 out of 50 kindreds (7.5%) . We have determined that SCA1 is found in 5
(2.5%) and SCA2 in 10 (5%) of all families. From all genetically confirmed
patients 66% were diagnosed with SCA2 and 33% with SCA1.
CONCLUSIONS: The frequency of SCA2 is substantially higher than that of
SCA1, 3, 6 and 7 in patients with SCA from Bulgarian families, unlike the
worldwide investigations showing that SCA1 and SCA 3are reported to be
most common. TP- PCR proved to be reliable method for routine diagnosis
J17.03
CCR5del32, CCR2-64I SDF-1 3A mutations of chemokine receptors
genes in West-Ukrainian population
H. Makukh, M. Tyrkus, H. Akopyan;
State institution Institute of Hereditary Pathology of Academy of Medical Science of
Ukraine, Lviv, Ukraine.
Ukraine has one of the highest speeds of HIV-1 infection spread in Europe
but Western region is the most trouble-free: 7 compare to 100 - 300 cases
of HIV per 100 thousand inhabitants in other regions. A speed of epidemic
depends on correlation between susceptible and resistant individuals in
single population.
The purpose of study to set up the frequency of allelic variants of genes,
which lead to higher resistance to HIV-1 among people of the Western region of Ukraine.
It was used the DNA samples of 200 healthy people (50% men and 50%
women). The PCR products were digested with the restriction enzymes and
subjected to agarose electrophoresis.
Heterozygote mutations CCR5del32 was detected in 17.0% and homozygote
CCR5del32 in 0.5% people with same frequency in both sexes. Heterozygous mutation CCR2-64I was revealed in 14.5% people and homozygotes in
1.5%. This mutation was found in women almost twice often than in men.
Heterozygote mutation SDF-1 3A in studied group was found in 30.5% of
people, homozygote - in 3% of all with same frequency in both sexes.
The CCR5del32 was combined with CCR2-64I (1 sample), with SDF-13A
(8), CCR2-64I with SDF-1 3A (9) and combination of homozygotes mutation
CCR2-64I and heterozygotes mutation SDF-1/3A (1 person). The number of
people who do not have any protective genotype is 33%.
Thus, the frequency of mutations in chemokine receptors gene in people of
Western region of Ukraine is indicate about the high genetic resistance to
HIV-1 infection compared with other ethnic groups.
J17.04
Determination of allele frequencies, duplication and mutation of 17
Y-STR loci in UAE population
H. B. Turkiah, S.H.Sanqoor, M. Jarrar, S. Behl, S.Gaur, M.S.Nazir;
University of Modern Sciences, Dubai, United Arab Emirates.
Determining the allelic frequencies and an inconsistency of the transmitted alleles of Y-STR haplotype among different populations is crucial for
the correct interpretation of DNA profile matches in paternity and kinship
analysis. In this research, STR allele duplications, mutations and the allelic
frequencies of the 17 Y-STR loci in the UAE population, will be determined.
Cheek swabs samples will be collected randomly from 200 unrelated male
individuals. Donors will be UAE citizens, whom ancestors have long-lived in
the country. DNA will be extracted using Genomic DNA Isolation Kit, (NORGEN Biotek CORP, Canada). The quality of the extracted DNA samples will be
assessed using agarose gel electrophoresis (AGE). DNA quantification will
be carried out using Quantifiler Duo DNA Quantification Kit followed by
multiplex PCR amplification using AmpFlSTR Y-filer PCR Amplification
Kit (Applied Biosystems, USA). Capillary electrophoresis (CE) of amplified
multiplex PCR will be performed using an ABI 3130 Prism Genetic Analyzer (Applied Biosystems). The data obtained from capillary electrophoresis
will be analyzed and the allelic frequencies of Y-STR loci will be determined
using a population-genetics-software called ARLEQUIN. This study will
provide an additional information to the framework of variation involving
17 Y-STR loci in forensic case work samples as well as a further contribution
to the Y-STR database for UAE male population. This study will also help
to determine allele duplications and mutations plus an inconsistency of the
transmitted alleles appeared in the UAE population.
497
Background and Purpose: We studied the -globin gene genotypes, hematologic values, and transfusion dependency of patients with hemoglobin H
(HbH) disease in Khuzestan Province, Southwest of Iran.
Methods: We detected alpha globin gene mutation by Using gap-polymerase
chain reaction (gap-PCR) and direct DNA sequencing.
Results: We identified 44 patients with hemoglobin H disease. Of these patients, 15 (34%) had deletional form of HbH disease, 29 (66%) had different
form of nondeletional HbH disease. The most frequently observed HbH genotypes were --Med/ -3.7 in 14 patients (31.8%), poly A6/ poly A6 in
nine patients (20.5%),and poly A4/ poly A4 in five patients (11.3%).
Some persons with HbH disease required blood transfusion, whereas some
others with the same alpha globin genotype were not transfusion-dependent.
Conclusions: Our data show diversity in the genotype and clinical presentation of deletional and nondeletional HbH disease. This discrepancy between
similar genotypes with different phenotypes may be due to other modifying
factors. Therefore, we cannot describe a general conclusion regarding the
need for blood transfusion in the patients with HbH disease
J17.06
Prevalence of alpha-1-antitrypsin deficiency among Polish patients
with lung or liver disorders
R. Struniawski1, B. Poplawska-Wisniewska1, A. Szpechcinski1, P. Kuca1, M. CzajkowskaMalinowska2, J. Kozielski3, P. Sliwinski1, K. Gorska4, P. Korczynski4, R. Krenke4, J.
ChorostowskaWynimko1;
1
National Institute of Tuberculosis and Lung Diseases, Warsaw, Poland, 2Regional
Center of Pulmonology, Bydgoszcz, Poland, 3Silesian Medical University, Zabrze, Poland,
4
Medical University of Warsaw, Warsaw, Poland.
Background: In Poland, the overwhelming majority of individuals with alpha-1-antitrypsin (AAT) deficiency still remains undiagnosed. We estimated
the AAT gene frequency and prevalence in a large cohort of Polish chronic
lung or liver disease patients eligible for AAT testing.
Methods: Blood samples were collected prospectively from 419 respiratory patients (COPD, emphysema, bronchiectasis, asthma) and 281 patients
referred for liver transplantation due to cirrhotic and non-cirrhotic chronic
disease. AAT serum concentration was measured by nephelometry and PIphenotype identified by isoelectrofocusing. The PI*S and PI*Z alleles were
confirmed by real-time PCR; rare phenotypes were identified by DNA sequencing.
Results: 53 (12.6%) lung disease patients and 18 (6.4%) liver disease patients demonstrated AAT deficiency phenotypes. Calculated frequencies expressed per 1000 were for PI*Z 46.6 (95% CI: 32.3-60.8), PI*S 20,3 (95%
CI: 10.8-29.8) in respiratory patients and PI*Z 19.6 (95% CI: 8.1-31), PI*S
10.7 (95% CI: 2.2-19.2) in liver disease patients. The AAT gene prevalence
calculated by Hardy-Weinberg equilibrium were: 1/1.16 for MM, 1/26 for
MS, 1/2429 for SS, 1/11 for MZ, 1/530 for SZ and 1/462 for ZZ in respiratory patients and 1/1.07 for MM, 1/48 for MS, 1/8773 for SS, 1/26 for MZ,
1/2393 for SZ and 1/2610 for ZZ in liver disease patients.
Conclusion: Our results show relatively high frequency of AAT deficiency
among Polish patients with chronic obstructive respiratory disorders. Estimated frequency for PI*Z and PI*S allele in respiratory group was about
two-fold higher than in liver disease patients and four-fold higher than estimated prevalence in healthy Polish population.
J17.07
The epidemiology of anorectal malformations in Russia in 2000-2012
J. Vydrych1, A. Asanov1, N. Demikova2;
1
I.M. Sechenov First Moscow State Medical University, Moscow, Russian Federation,
2
Research Clinical Institute of Pediatrics of Pirogov Russian National Research Medical
University, Moscow, Russian Federation.
498
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Results. Among participating registries total number of ARM cases was 970
with prevalence of 1.84 per 10,000 births. Most of them (85.26%) are isolated and 14.74% cases are combined with other anomalies. Among isolated
cases 47,76% were cases of congenital anomalies of anus (CAA) without
fistula (code Q42.3), 33,25% - CAA with fistula (Q42.2), 11,12% - congenital anomalies of rectum (CAR) without fistula (Q42.1) and 7,87% - CAR
with fistula (Q42.0). Among the combined cases CAA without fistula were
42%; CAA with fistula - 25,1%; CAR without fistula - 21%; CAR with fistula - 11,9%. The male affected more frequently than female (1,6:1). Among
infants with all kinds of ARM are more common the infants with the low
birth weight.
Conclusions. On average ARM affected 1 in 5435 births. There are no changes in prevalence of ARM during research period. The regular monitoring of
congenital malformations allows the study of the epidemiological characteristics of even the rare birth defects.
J17.08
Status of RANK and MMP9 gene polymorphism in beta-thalassemia
patients of Indian origin
M. M. Singh, K. Singh, S. R. Phadke, S. Agarwal;
Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India.
-thalassemia is an autosomal recessive, commonest hematological disorder found in India. Regular transufusion to sustain life in thalassemia major
leads to iron overload. The Cardiac complications and bone deformities are
very well documented as outcome of regular transfusion in thalassemics. To
evaluate the genetic predisposition for these two important clinical complications, RANK and MMP9 gene prevalence in transfusion dependent cases have been studied. Receptor activator of nuclear factor (RANK) is one
of the proteins in regulation of osteoclastogenesis via RANK/RANKL/OPG
pathway. In a recent GWAS study RANK was found to be associated with
osteoporotic fractures and variations could alter the expression so the two
polymorsphisms were studied in beta thalassemia patients. Seventy-five
patients of thalassemia major were selected and the genotyping of RANK
(G -> A) polymorphism was performed in thalassemia major patients. High
prevalence of G allele [86%] was observed compared to 13% prevalence of
A allele. Second RANK (insdel C) polymorphism showed 88.6% of insertion
cases while remaining were deletions. Matrix metallopeptidase 9 (MMP9)
plays a pivotal role in early atherosclerosis, vascular remodeling and development of atherosclerotic lesion. The potentially functional MMP-9 gene
polymorphism may contribute to the susceptibility of acute coronary syndrome (ACS). This study includes -1556C>T polymorphism of MMP9 gene in
-thalassemia major patients where 75.3% allele C was found in thalassemia
major patients. The present study reports the higher prevalence of wildtype
alleles of RANK (+34901G>A, +35966insdelC) & MMP9 (-1556C>T) polymorphism in Thalassemia patients of Indian origin.
J17.09
FTO (rs17817449 and rs9939609) mutations in a Romanian obese
children population
O. Marginean, C. Duicu, V. Moldovan, A. Crauciuc, F. Tripon, C. Banescu;
University of Medicine and Pharmacy Tg. Mures, Romania, Tg. Mures, Romania.
One of the major problems facing the world is the pollution of the environment and the study of its effect on the rate of mutation and inheritance
rights. Petroleum products and petroleum industries affect pollution.
Aim: To evaluate the effects of prolonged exposure to chemical mutagens on
chromosome population Zhylyoi district of Atyrau region, living in ecologically unfavorable regions.
Material for cytogenetic analysis: the culture of peripheral blood lymphocytes from 71 residents living in the area with the development of the oil
industry. Culturing lymphocytes, cooking preparations, coloring G-method
performed by standard methods. The control group consisted of 25 people
in an ecologically clean region.
The results of the study. We studied the frequency and spectrum of chromosomal aberrations in the groups studied. Found a higher incidence of
aberrant cells (1.75 per 100 cells) and the total frequency of chromosomal
aberrations (1.9 per 100 cells) in affected individuals compared with controls (p <0,05).
Frequency paired fragments were 0,101,99 per 100 cells, dicentric
0,01,0,23, ring chromosomes 0,140,38 frequency of stable chromosome
aberrations 0,021,17 100 cells in people living in Zhylyoi area. The frequency of chromatid type aberrations was 0,870,01 100 metaphases and
is represented mainly by single fragments. Findings suggest the influence of
chemical factors on the chromosome of people living close to the oil companies and petroleum industries.
Conclusion. Cytogenetic studies of the population will be used to define a
group of families of high risk for the prevention of genetic disorders in
ecologically unfavorable regions.
J17.11
Association between C282Y and H36D mutations of the HFE gene with
hepatic cirrhosis in Lithuanian population
S. Juzenas1, L. Kucinskas1, J. Sumskiene2, V. Petrenkiene2, J. Kondrackiene2, I. Valantiene3,
J. Skieceviciene1, L. Kupcinskas1;
1
Institute for Digestive research, Lithuanian University of Health Sciences, Kaunas,
Lithuania, 2Department of Gastroenterology, Lithuanian University of Health Sciences,
Kaunas, Lithuania, 3Institute for Digestive Research, Lithuanian University of Health
Sciences, Kaunas, Lithuania.
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largely determined by overweight and obesity. Obesity in menopause women occurs as a result of genetic background, hormonal changes and acquired changes in eating behavior and physical activity. Evidence showed
that the endogenous cannabinoid system (ECS) plays a role in obesity and
related metabolic disorders. To investigate the association between CNR1
and CNR2 gene polymorphisms and metabolic risk factors in the Chinese
postmenopausal women, five hundred and fifteen post-menopausal women
were recruited. Anthropometric measures (body mass index (BMI), waist
circumference (WC)), blood pressures, and metabolic parameters were
determined. Four SNPs from CNR1 gene and 1 SNP from CNR2 gene were
selected for genotyping on these post-menopausal women. We found that
post-menopausal women carrying TT+TC genotype of CNR1 rs2023239 had
increased risk of metabolic syndrome (p=0.047). We also found two SNPs
were significantly associated with pre-hypertension or hypertension (OR=
1.72, 95% C.I.: 1.12-2.63 for rs806381 and OR= 1.77, 95% C.I.: 1.00-3.11 for
rs1049353, respectively). We further observed that CNR2 rs2229579 was
significantly associated with hyper-triglycerides in our post-menopausal
women (OR= 1.85, 95% C.I.: 1.06-3.23). Haplotype analysis also revealed
that those women carry the CGTA haplotype of CNR1 gene were less likely
to develop pre-hypertension or hypertension (aOR= 0.67, 95% C.I.: 0.470.96). Our findings provide initial evidence that the CNR1 gene variants may
contribute to pre-hypertension or hypertension and CNR2 gene variant may
predict hyper-triglycerides in post-menopausal women in Taiwan.
J17.13
A study on the association of ERCC1 Asn118Asn polymorphism and
colorectal cancer risk in West Algerian population
Colorectal cancer (CRC) is one of the most common causes of death due to
cancer in both men and women throughout the world. It has been suggested that sporadic CRC is most likely caused by lowpenetrance genes, including those involved in DNA repair mechanisms. Furthermore, the accumulation of DNA damage may contribute to colorectal carcinogenesis. Many
epidemiological studies have explored the association between ERCC1 Asn118Asn (C/T, rs #3177700) polymorphism and colorectal cancer risk in
various populations, its role in colorectal carcinogenesis in Algerian population has not been established. Therefore, we conducted this case-control
study in a West Algerian population to assess the potential role of this genetic polymorphism on the risk of colorectal cancer. Peripheral blood samples
of 90 sporadic CRC patients and 100 normal controls were collected, DNA
extracted genotyped using pyrosequencing technique. The distribution of
genotypes of ERCC1 Asn118Asn genotype and allele frequencies among CRC
cases was not significantly different from those among controls (P>0,05).
The variant genotype combinations did not show any significant association
with CRC susceptibility risk suggesting that the ERCC1 codon 118 polymorphism does not convey moderate increase in susceptibility to CRC in West
Algerian population. This is the first study on ERCC1 gene polymorphism
in our population suggesting that the Asn118Asn polymorphism may not
be associated with the colorectal cancer risk in West Algerian population.
Further research with a larger sample size is needed to reveal more information about this polymorphism and the appearance of colorectal cancer in
our population.
J17.14
Cystic Fibrosis in Arab populations: Identification of novel mutations
and complex allele in Egyptian and Lebanese Patients
A. EL-Seedy1,2, R. Farhat2, M. Pasquet2,3, H. Shafiek4, T. Morsi4, M. EL-Komy1, A.
Megarbane5, A. Kitzis2,3, V. Ladeveze2;
1
Department of Genetics, Alexandria University,, Alexandria, Egypt, 2Universit de
Poitiers - Ple Biologie Sant, Gntique de Maladies Rares, Universit de Poitiers,
Poitiers, France, 3CHU de Poitiers, Poitiers, France, 4Department of Chest diseases,
Alexandria University, EL-Sultan Hussein St.-Azarita,, Alexandria, Egypt, 5Unit de
Gntique Mdicale, Facult de Mdecine, Universit Saint-Joseph, Beirut, Lebanon.
499
Introduction: Cystic Fibrosis (CF) [MIM # 219700] is the most common autosomal recessive genetic disease, in Caucasian populations [1].
In Algeria, no information is available about the incidence of CF. In the Maghreb, there are few data about the molecular basis of CF, probably due to
an under diagnosis.
This study evaluated the effectiveness of the ELUCIGENE CF30 Kit in a sample of Algerian CF patients.
Patients and methods: Subjects: Twenty four (24) unrelated CF patients
were recruited from the Pneumology and Allergology Department of the
specialized Hospital Center in Canastel Oran (Algeria).CF diagnosis was
based on a clinical findings and repeated positive sweat chloride tests ( >60
mmol/l).
DNA extraction and genotyping
- 10 ml of whole blood was collected from all participants in EDTA tubes.
- Genomic DNA was extracted using standard Salting Out procedure [2].
- Mutations were explored by the ELUCIGENE CF 30 Kit (Tepnel Diagnostics,
Oxon, United Kingdom) which is base
d on a PCR/ARMS technique.
In screening the twenty four CF patients (48 chromosomes) for 30 mutations available in ELUCIGENE CF30 kit, only five mutations were found:
- c.1521_1523delCTT (F508del).
- c.579+1G>T (711+1G>T).
- c.1624G>T (G542X).
- c.3909C>G (N1303K).
- c.1652G>A (G551D).
All the mutations found were validated.
The Elucigene CF30 detected 5 mutations in our sample, which is about
16.66%.
The detection rate seems low compared to European populations. Conclusion: The most interesting is to develop in collaboration with ELUCIGENE
one specific Maghreb Kit and inclued mutations which frequency higher
than 2%.
J17.16
Notification of death from cystic fibrosis in Brazil during 30 years
from 1981 to 2010
The aim of this work was to evaluate the notification of cystic fibrosis (CF)
as primary cause of death in Brazil, from 1981 to 2010, and to compare with
developed countries. The Brazilian data was obtained from SIM-Data/SUS
and the American from the CDC WONDER. The Brazilian median age at death
(MAD) was 3.9 years in 1981 and 12.4 years in 2010 for typical severe CF.
In 1994, the Brazilian MAD was much lower (7.2y) than that (21y) of seven
developed countries reported by Forgarty et al, 2000. We found no increase
in MAD in Brazil from 1981 to 1985; from 1986 to 2010 it showed a threefold increase. From 1981 to 1990, there were few deaths over 25 years of
age in Brazil, mostly concentrated in younger age groups. During the sample
period there was an increase in deaths in higher age groups in Brazil, which
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may reflect better patient survival rates due to increased knowledge of the
disease, with repercussions for its diagnosis and treatment. We also observed an increase in the reported cases of CF deaths per 100.000 inhabitants
in Brazil over the years, possibly due to the better knowledge of the disease
and consequently more accurate death notification, since this increase is not
typical of a genetic disease. For the USA we observed a decrease in notified
CF deaths, despite the population increase which may be a reflection of a
smaller number of people born with CF, due to appropriate genetic counseling and prenatal diagnosis.
J17.17
SERPINA1 gene polymorphism frequency in clinically confirmed
cystic fibrosis patients
In this work, were typed in 100 unrelated individuals (sex ratio 60:40) from
South Romania eight DNA markers. Aim was to analyze the genetic variability and to establish the relation between this region and other European
populations. We establish Hardy-Weinberg equilibrium, gene diversity, genetic distances and tree topology by PHYLIP 3.68 package. Polymorphisms
frequencies were similar to the mean frequencies calculated for the whole
set of populations included in the study. The mean value of Ht was 0,201,
and for Hs was 0,18. The most affiliated population with our lot are Italy,
Spain, Poland, Germany, Greece and Turkey the most distant population are
United Kingdom, Sweden, Croatia and Slovacia.
J17.19
A new variant of Ehlers-Danlos syndrome with inborn errors of
mucopolysaccharide metabolism in the mother and son
E. V. Bugaeva, Y. B. Grechanina, E. Y. Grechanina;
KSMGC, Kharkiv, Ukraine.
Asthma is a complex respiratory disease, which can be caused by environmental factors and genetical predisposition. It is one of the most widespread
diseases in the world; it has a high presence in most ethnic group. The lowaffinity IgE receptor (FcRII /CD23) encoded by the FCER2 gene (Fc fragment of IgE) plays important role in the regulation of IgE responses and
inhaled corticosteroids (ICS) therapy in asthma. Corticosteroids influence
FCER2 expression and FcRII receptor function. The intronic rs28364072
polymorphism (T2206C) of FCER2 gene associated with elevated IgE levels,
severe asthmatic exacerbations and decreased gene expression. The variation in the FCER2 gene contributes to variation in ICS treatment response
in asthmatics. Our aim was to investigate the ethnic differences, allele and
genotype frequencies of intronic variant of FCER2 in average Roma and
Hungarian population. We examined 458 Roma (206 males, 252 females,
mean age: 46.418.4) and 397 Hungarian subjects (222 males, 175 females;
37.8 mean age12.6 years) with PCR-RFLP method. We found more than
twofold increased homozygous CC genotype frequency in Hungarian group
compared to Roma samples (5.8% vs. 2.8%, p<0,05). The C allele frequencies were similar in each group (24.8% in Romas and 24.6% in Hungarians).
The current study demonstrated that unfavourable variants can diversely
occur in Hungarian and Roma individuals. Genetic test of FCER2 variant
are likely necessary to assess the outcome of asthma treatment. Homozygous 2206C allele carrier Hungarians have higher chance for insufficient
response to corticosteroids compared with Roma subjects due to genotype
higher risk frequency. This research was supported by TMOP-4.2.3-12/1/
KONV-2012-0028.
J17.22
Common MEFV gene mutation profiles in Familial Mediterranean
Fever patients in Canakkale Population
Objective:In the current results it was aimed to find out of the current stu-
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Tatars - are the second sized ethnos of the Russia. The load and genetic
diversity of monogenic hereditary disorders (HDs) in three major ethnographic groups of Tatars from Tatarstan Republic were analyzed (Kazan
Tatars-3 Districts, Mishars-2 Districts and Teptyars-3 Districts. The size of
the investigated populations was more than 270,000 inhabitants (213,000
Tatars). The total population was examined by standard protocol of medical
genetic research elaborated in laboratory of genetic epidemiology, Research
Centre for Medical Genetics. About 3500 HDs of OMIM could be identified by
this protocol. Clinical investigations were performed by neurologists, ophthalmologists, orthopedic, otolaryngologys, dermatologists, pediatricians
and clinical geneticists, focused on diagnostic of HDs. Genetic diversity of
HDs in the investigated population consisted of 256 disorders (1597 affected): 135 AD, 97 AR and 24 X-linked recessive. Genetic differentiation of load
of Mendelian HDs between populations of different ethnographic groups
was found. The average prevalence rates were 1:172 persons in Kazan Tatars; 1:120 persons in Mishars and 1:150 persons in Teptyars. Variation of
prevalence of all HDs in districts was from 1:350 persons to 1:85 persons.
Significant differences in the load and diversity of HDs was found between
groups Kazan Tatars- Mishars and Teptyares - Mishars.
501
Aimes: Different alleles within the same gene can cause a similar variant
phenotype. Previously published studies showed the allelic heterogeneity of
GJB2 gene as main genetic cause of isolated congenital hearing-loss phenotype. The proportional distribution of the different mutations within GJB2
gene varies in different ethnic groups. The aim of the present study was to
provide a complete and updated spectrum of mutations in GJB2 and GJB6
gene and to identify the most prevalent mutations in Romanian population.
Material and Methods: To overcome our aims, we used clinical data from 80
unrelated persons with congenital hearing-loss and performed ARMS-PCR
and DNA sequencing techniques for detection of known mutations and identification of mutations within GJB2 gene. Analysis of del(GJB6-D13S1830)
and del(GJB6-D13S1854) was performed by multiplex PCR.
Results: Most prevalent mutation was c.35delG (40.0%) in both homozygotic and heterozygotic forms. The second mutant allele was W24X (8.75%)
also founded in homo- or heterozygotic forms, followed by c.-23+1G>A originally named IVS1+1G>A and R127W mutations with lower frequencies.
Conclusions: The study reveals c.35delG mutation as most prevalent one,
absence of GJB6 gene deletions, genetic background of congenital hearingloss in local population and supports improvement of genetic counselling
services.
J17.27
Alleles analysis of GSTA1 and GSTP1 genes in the Polish population
502
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to their genetic variability which may affect enzymes activity. The aim of our
study was to determine the alleles GSTA1*A/*B, GSTP1*A, *B, *C distribution in the Polish population. We performed our analyses on the DNA of 160
subjects from the Polish population. In the GSTP1 gene we have genotyped
two polymorphisms (I105V and A114V) with pyrosequencing method and
the GSTA1 gene was screened for the changes in the promoter region using
sequencing. The detected variants were subjected to the haplotype analysis. We found alleles GSTP1*A (c.313A, c.341C), *B (c.313G, c.341C) and *C
(c.313G, c.341T) with 65.3%, 23.8% and 10.9% frequency, respectively. As
a result of GSTA1 gene promoter sequencing we detected 8 SNPs located
in sites: G-52A, C-69T, C-115T, A-513G, T-567G, G-631T, A-1066T, G-1142C,
G-1245A. The allele GSTA1*B, associated with the decreased enzyme activity
was observed in our study with frequency of 43.1%. Finally, 4 haplotypes
have been determined in 160 Polish individuals. Our study demonstrated
that the distribution of the alleles GSTA1*A/*B and GSTP1*A, *B, *C in the Polish population corresponds to data for Caucasians. Furthermore, we found
novel SNPs, excluding three well known changes (G-52A, C-69T, T-567G),
which are linked to the alleles GSTA1*A/*B affecting enzymes activity.
J17.28
Genome-wide association analysis identifies a locus on DMD
(dystrophin) gene for power athlete status in Russians
Karachays live compactly in 4 regions of Karachay-Cherkessia (Russia). Karachays are one of the Turkic-speaking North Caucasian peoples. Values of
Wright random inbreeding (a quarter of the sum of squares of frequencies
of surnames) are counted on the basis of distribution of adults surnames
for rank of district population and Village Council population. Both of
thees assessments are necessary for carrying out various types of analysis.
District population rank Fst values are: 0.0035 (Malokarachaevsky district),
0.0014 (Prikubansky district), 0.0016 (Ust-Dzhegutinsky district), 0.0015
(Karachaevsky district). The average-weight values Fst for Village Council
population rank are counted separately for rural and urban populations
(where urban present) and make: 0.0056 (Malokarachaevsky, rural), 0.0033
(Prikubansky, rural), 0.0042 and 0.0010 (Ust-Dzhegutinsky, rural and urban), 0.0084 and 0.0016 (Karachaevsky, rural and urban), thus the general
assessment of the average-weight Fst value for these areas without division
into urban and rural people make: 0.0056, 0.0033, 0.0024, 0.0059, respectively.
J17.32
An early manifestation of LBSL syndrome, case description
E. P. Zdubskaya;
KSMGC, Kharkiv, Ukraine.
Background :Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation is an autosomal recessive disease. Mutation
in gene DARS2 is associated with this syndrome. The gene is located on the
long arm of chromosome 1 (1q 25.1), the disease is associated with deficiency of mitochondrial aspartyl - tRNA synthetase.
Care report: 1 year and 5 month old boy T., with static kinetic and psychospeech development delay, excess body weight (16kg). The child is from I
pregnancy against the background of threatened miscarriage. Delivery
at 36-37 weeks by cesarean section. Birth weight - 3300g. At the time of
examination we paid our attention at the decreased muscle tone, muscle
strength tendon and periosteal reflexes are not observed, nystagmus, ataxia.
ENMG - the neuropathic type of changes, myopathic syndrome. MRI of the
brain - using T1, T2 and FLAIR- modes - white matter lesions of the brain
and cerebellum. The karyotype - 46,XY. Amino acid levels, blood lactate are
unchanged. Partial analysis of the DARS gene by sequencing: in 5 gene locus
- mutation c492+2T-C in the heterozygous state.
Conclusion: The disease manifests in the age of 3-15 years. Cerebellar ataxia, spastic tetraparesis and cognitive impairment develop. Before the onset
of the disease, psychomotor and speech development corresponds to age,
movement disorders develop further, patients become disabled by the se-
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cond to fourth decade of life. In our case, the disease manifested in the heterozygous carrier by one year of life and was accompanied by obesity.
J17.33
Association analysis of haplotypes of the leptin gene promotor region
with BMI in Roma population
M. Zajc Petranovi, . Tomas, A. Barei, A. Ivankov, S. Petri, M. Perii Salihovi, N.
Smolej Narani, B. Janiijevi, T. kari-Juri;
Institute for Anthropological Research, Zagreb, Croatia.
Leptin, an adipocyte-derived protein, plays a key role in regulating energy intake and consumption. Increased circulating leptin levels, commonly
found in obesity, indicate a failure to signal satiety and halt the progression
of obesity. Aim of this study was to investigate the association of single nucleotide polymorphisms (SNP) and haplotypes of the leptin gene promotor
region with body mass index (BMI) in Roma (Gypsy), a population known to
be vulnerable to developing obesity presumably due to both their life-style
and genetic background.
We sequenced 1500 bp region of leptin gene promotor in the sample of 377
Roma individuals from Croatia and identified 9 polymorphic sites. Haploview software was used to assess linkage disequilibrium (LD) among polymorphic loci as well as the Unphased version 3.0.13 software for haplotype
association analysis.
All nine SNPs were grouped into single LD block due to their proximity. None
of the single markers (rs1349419, rs146378188, rs13245201, rs185230264,
rs791614, rs12535708, rs10487506, rs11770725 and rs12535747) showed individual statistically significant association with BMI. However, two
haplotypes, A-A-G-G-A-C-G-T-C and G-A-A-G-G-A-A-T-A, showed significant
association with BMI (p=0.005061 and p=0.03214). Both p-values remained
significant after 1000 permutation tests.
Our data showed that haplotype association analysis provided advantage
over individual SNPs analysis. Haplotypes spanning the leptin gene promotor region were found to be significant predictors of BMI in Croatian Roma.
J17.34
Germline variants of ARID5B as possible risk factors for childhood
acute lymphoblastic leukemia in Latvia
M. Kreile, L. Piekuse, D. Rots, A. Zarina, E. Cebura, Z. Kovalova;
Riga Stradins University, Riga, Latvia.
503
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system potentially associated with viral infection. RIG-I-like receptors genes
(RIG-I, IFIH1 and LGP2) have been hypothesized as involved in the viral etiology of MS, since they are implicated in viral recognition. Previous studies
have produced conflicting results regarding the association between IFIH1
gene polymorphisms and MS. Here we examined the effects of 13 single nucleotide polymorphisms (SNPs) in the RIG-I, IFIH1 and LGP2 genes on MS in
the German population. The study comprised 716 patients with MS and 706
healthy control individuals. Genotyping was performed using RFLP, TaqMan
genotyping assays and high-resolution melting (HRM) analysis. There were
no significant differences (p>0.05) between MS patients and healthy individuals for any of the investigated SNPs as well as haplotypes derived from
these SNPs. Further, no differences were observed between healthy individuals and MS subgroups stratified according to disease characteristics. Our
results suggest that variation in the RIG-I, IFIH1 and LGP2 genes does not
exert a major influence on MS risk.
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J17.38
Associations between MX2 polymorphism and the acquisition of HIV
and co-infections in Caucasian intravenous drug users (IDUs)
Noonan syndrome is an autosomal dominant disorder with an estimated incidence of 1 in 1 0002 500 live births. It is characterized by short stature,
typical facial dysmorphia and congenital heart defects. This disorder results
in almost 50% of patients from protein-tyrosine phosphatase, non-receptor
type 11 (PTPN11) mutations, that lead to gain of function in the non-receptor protein tyrosine phosphatase (SHP-2) into the signaling RAS- mitogen
activated protein kinase (RASMAPK) pathway.
Till now, mutational screening of PTPN11 has been carried out in different
populations. Thus, the aim of this study was to screen the PTPN11 mutations
in series of Moroccan Noonan Syndrome patients.
Twenty-three patients were recruited in HASSAN II university hospital of
Fez within the last five years. We have extracted genomic DNA from Blood
samples. Then, we used PCR and bidirectional direct sequencing of the PTPN11 hotspot exons reached by mutations for 23 Moroccan children.
We detected 5 heterozygous missense mutations in 23 sporadic patients
(21.7%). Those patients share the characteristic facial traits of Noonan syndrome. Most of them suffer from pulmonic stenosis.
The present study allowed identification of mutations clustered in hotspot
exons of PTPN11 gene in Moroccan Noonan syndrome cohort, and enabled
us to give an appropriate genetic counseling to the mutation-positive patients.
J17.42
Role of the Neuregulin 1 gene(NRG1) in multiple sclerosis
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system, characterized by inflammation, demyelination and axonal injury, probably caused by environmental factors in genetically susceptible
individuals.
Neuregulin 1 (NRG1) is involved in neuronal specification and migration,
gliogenesis, neuron-glial signalling. Viehover et al. (2001) found that the
expression of Neuregulin is dramatically reduced in MS lesions and hypothesized that the absence of this protein contribute to the paucity of remyelination in the disease. For these reasons, the NRG1 gene could represent a
good candidate for a research on patients with immuno-related and demyelinating disordes. We focused our attention on it, aiming at evaluating its
role in Southern Italy by means of a case control study.
We examined 226 patients with MS and 200 healthy controls. Genotyping
was performed by amplification and subsequent digestion with the restriction enzyme CviQI.
We found significant differences of allele and genotype distributions
(p=0.011 and p=0.05, respectively) between cases and controls. To our
knowledge, this is the first study performed on NRG1 in patients with MS.
On the basis of the preliminary data obtained in our population, we could
hypothesize an involvement of the gene in the susceptibility to the disease.
In particular, the presence of the T allele, under-represented in the patients,
seems to be a protective factor against MS. However, due to the small number
of participants, the confirmation of the role of NRG1 requires further studies
extended to a larger sample of subjects and also to other populations.
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J17.43
Variability of Genes Associated with Obesity in Populations of Russia
and Neighboring Countries
I. Khitrinskaya, V. Kharkov, V. Stepanov;
Institute for Medical Genetics, Tomsk, Russian Federation.
The prevalence of obesity and related diseases reached a high level throughout the world. The role of genetic factors in the disease development has
been proved by numerous studies on twins and adopted children by testing
candidate genes predisposing to obesity. Genome-wide association studies
(GWAS) is one of the advanced methods used to detect mutations associated
with overweight. We selected 20 SNPs associated with obesity with high
level of significance from GWAS-catalog. Allele frequencies for these SNPs,
were analyzed in 14 populations of Russia (Yakuts, Buryats, Tuvans, Komi,
Mordovians, Russians, Khanty, Kets, Chuvashes, Kabardinians, Karachai,
Ossetians, Tatars, Udmurts and Karelians) and neighboring countries (Kazakhs, Uzbeks, Kyrgyz, Megrelians and Moldavans). Data on 7 native nonadmixed populations from HapMap and 1000 Genomes project were also
included into analysis. Correlation analysis of allele frequencies and genetic
diversity with geographic (latitude, longitude, elevation) and climatic (mean
annual temperature, average temperature in January and in July, temperature range, maximum and minimum temperature and precipitations) parameters was performed. Allele frequency of 16 from 20 studied SNP correlated with climate and geography. 5 SNP showed correlation with latitude
only (rs5762430, rs1805081, rs7138803, rs2531995 and rs11042023), 6
- with latitude and climate (rs1704198, rs9299, rs10182181, rs6110577,
rs999943 and rs7603514) and 5 with climate only (rs3101336, rs7784447,
rs259067, rs2275848 and rs7474896). The revealed relationships between
genetic and geo-climatic parameters may indicate the presence of positive
selection on genes associated with obesity and adaptive changes in the gene
structure in human populations during human dispersal out of Africa.
J17.44
The common polymorphism Val109Asp in the omentin gene
is associated with daily energy intake in the Central-European
population
Omentin was originally first described in 2003 and was reported to be expressed specifically in human omental adipose tissue. So far, very little is
known about the relationship between the genetic variability of the omentin gene and pathophysiology of obesity. The aim of the study was to investigace two common polymorphisms in the omentin gene (rs2274908 and
rs2274907) and dietary composition and anthropometric parameters of
obesity in the Central European population.
Material and methods.The total of 495 subjects were included into the study
that were further dividend into the non-obese, obese and morbidly obese
cohorts. Dietary habits were established using the 7-d food records and
selected anthropometric parameters were measured.
Results: There were significant differences in genotype distributions of
rs2274907 between the obese and morbidly obese cohorts (Pg = 0.01). In
the multivariate modelling, the rs2274907 polymorphism expressed independent prediction role for the daily energy intake, independently on the
age and gender distribution (p = 0.03); the TT genotype being associated
with the lowest average energy intake (7877 2780 J/day) and the AA genotype with the highest daily intake of energy (8764 2467 J/day). Significant
association of the rs2274907 were also observed for the daily consumption
of fat and proteins.
Conclusion: This is so far the first study to investigate the polymorphisms
in the omentin gene in a large population cohort of obese and non-obese individuals. Based on our results, the rs2274907 polymorphism is associated
with the daily energy intake as well as daily intake of fat and protein.
J17.45
Associations between polymorphisms in PAX9 gene and congenital
missing teeth
505
PMS2 gene product is involved in DNA mismatch repair process. Besides the
gene, there are several pseudogenes in the human genome, showing high
sequence homology. To describe and distinguish sequence polymorphism
in highly homologous loci which could be co-amplified in PCR assays, we
have sequenced short 243 bp fragment containing co-amplifying regions
chr7:6026862-6027104 (part of PMS2 exon 11) and chr7:6776854+6777096
(part of PMS2CL pseudogene) in 50 DNA samples. In 14 DNA samples, additional long fragment encompassing region chr7:6026862-6027488 (part
of intron 10 and exon 11 PMS2) has been sequenced. The same heterozygous
positions in both fragments, together with peak height differences in heterozygous positions within the short fragment allowed us to determine their
location (gene/pseudogene) and to calculate MAF estimates. Altogether 6
variable positions were identified and most of them had been registered as
SNPs in PMS2 in public databases (NCBI). Among them were rs111255573
(C/T) in intron 10 (MAF=0,036) and rs15020462 (A/G, Gly460Asp) in
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Chile is an upper middle income country, has an infant mortality rate of 7.7
per 1000 births in 2011. Birth defects (BD) are an important public health
problem, account for 35% of the infant mortality, being the second leading
cause after prematurity. Aware of that Chilean Ministry of Health has desi-
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507
Spinocerebellar ataxias (SCAs) represent a clinically, genetically and pathologically heterogeneous group of rare hereditary untreatable neurodegenerative disorders, which affect the cerebellum and its connections. As
far as we known, there are more than 30 subtypes reported in literature.
In Europe, its estimated that one to three per 100 000 individuals suffer
from a SCA subtype, but this number could vary among different ethnic and
geographic groups. Epidemiological data on SCAs is considered scarce and
fragmentised, with only their relative frequency within a population usually
being better known. The goal of this study was to systematically compile and
update the available information regarding prevalence of SCAs, as well as
several indicators, namely number of published reports, expert centres, diagnostic tests, patients organizations, available biobanks, as well as performed and ongoing clinical trials. Data were collected considering literature
reports from 2000 until 2013, and using several databases, such as Orphanet and EuroGentest. Updating SCAs current data could be an important tool
for needs assessment in specialized molecular diagnosis resources, planning
future health and community services and better resources allocation, being
helpful to evaluate if the available resources are in accordance with the real
requirements in different European countries.
J17.56
The SHBG gene polymorphism (rs12150660) is associated with elite
power athlete status and muscle mass
E. S. Egorova1, L. J. Mustafina2, I. I. Ahmetov2,1;
1
Kazan State Medical University, Kazan, Russian Federation, 2Volga Region State
Academy of Physical Culture, Sport and Tourism, Kazan, Russian Federation.
Testosterone regulates muscle mass and strength, bone mass, fat distribution and the production of red blood cells. Sex hormone-binding globulin
(SHBG) is the key protein responsible for binding and transporting of testosterone. SHBG regulates its bioavailability and therefore its effects in the
body. Polymorphism at the SHBG gene locus (rs12150660 G/T) has been
associated with testosterone concentrations. Since individuals with the TT
genotype have higher serum testosterone concentrations in comparison
with carriers of the G allele (data from GWAS), we hypothesized that the
carriage of the T allele may give some advantage for strength and power
performance. The aim of the study was to investigate the association between the SHBG G/T polymorphism, athlete status and muscle mass. A total
of 363 Russian athletes and 130 controls were genotyped using RT-PCR.
Muscle mass was measured by body composition analyzer Tanita MC-980.
The frequencies of the T allele in power-oriented athletes (n=143, 20.3%;
P=0.7462), endurance-oriented athletes (n=220, 15.0%; P=0.2054) and a
whole cohort of athletes (17.1%; P=0.5078) were not significantly different
from controls (18.8%). However, the frequency of the T allele in elite poweroriented athletes (n=65, 26.2 vs. 12%, P=0.0061) was significantly higher
as compared with elite endurance-oriented athletes (n=58). Furthermore,
correlation analysis showed positive association between the T allele and
muscle mass among non-elite female athletes (n=8, P=0.0072, r= 0.8729).
Although more evidence is needed, one might suggest that the SHBG gene
G/T polymorphism is associated with power athlete status.
J17.57
Extreme Carrier Frequency Of The Splice Site Ivs1+1g>A Mutation In
Gjb2 Gene In The Eastern Siberia Is Comparable To Carrier Frequency
Of The Sickle Cell Anemia In Africa
This study presents data on the carrier frequencies of the splice site mu-
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The aim of the present research was: to study the polymorphism of mitochondrial DNA and 10 X-linked microsatellites (DXS8378, GATA172D05,
DXS7132, DXS9898, DXS7423, DXS8377, DXS101, DXS6809, DXS6789,
HPRTB) in population of Kazakh.
Genetic and geographic analysis of the genetic structure of Kazakh population using data of the frequencies of mtDNA haplogroups was conducted. The
high degree of intensity of gene exchange between the Kazakh population
and frontier populations of Russia was identified on the north-west, north,
northeast and east of Kazakhstan. The phylogenetic tree was constructed
for Kazakhs on female lineage and was detected position of the studied population between ethnic groups in Europe and Asia.
Ten STR loci demonstrate the high level of genetic diversity in Kazakhs. The
average genetic diversity in a combined sample is quite high (mean diversity
for 10 STRs, H=0.768). Rare alleles were detected for DXS8378 (7 repeats),
DXS8377 (57, 59, 60), DXS101 (31), DXS6789 (12) loci. All rare alleles were
Recently, scientists are redirecting their researchers to find the genetic origin of diseases; stroke is part of them. Polymorphisms of the lipoprotein
lipase gene have been extensively studied in different populations finding
variations in results among societies. Showing that the HindIII and PvuII
polymorphisms can act as risk markers for the development of cerebrovascular disease by increasing levels of tiacilgliceroles and decreasing HDL.
However, Ser447X can be a protective marker causing an increase of HDL
levels and reducing triglycerides levels. The aim of the study was to analyze
if there is an association between the presence of polymorphisms in the LPL
gene (HindIII , PvuII and Ser447X ) with development of ischemic stroke in
Colombian population. Sample size was 347 stroke patients (clinical diagnosis and x-ray CT) and 347 healthy subjects. PCR -RFLP technique was used to
detect Ser447X , HindIII and PvuII polymorphisms in the LPL gene. Results
were analyzed by Stata12 and Arlequin software. As results, allele and genotypic frequencies of the studied polymorphisms did not show a statistically significant difference between the cases and controls. In the present
research was not found any association between any of the LPL gene polymorphism and stroke in the population sample used. These results suggest
that in the Colombian sample used, LPL gene polymorphisms are not genetic
markers for the development of stroke
J17.61
Recurrent spontaneous abortion - importance of testing the
thrombophilic mutations. The experience of Genetic Center, Romania
on 627 consecutive cases
M. S. Militaru1,2, A. Trifa1,2, R. Popp1, I. V. Pop1, C. Andrei2, C. Gug3, M. Militaru4, M.
Stefanut2;
1
University of Medicine and PharmacieIuliu Hatieganu, Department of Medical
Genetics,, Cluj-Napoca, Romania, 2Genetic Center, Cluj-Napoca, Romania, 3University of
Medicine and PharmacieVictor Babes, Department of Medical Genetics,, Timisoara,
Romania, 4University of Medicine and PharmacieIuliu Hatieganu, Department of
Pediatrics 2, Cluj-Napoca, Romania.
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J17.62
Association between TLR8 and TLR9 gene polymorphisms and
Pulmonary Tuberculosis
M. Taheri, S. Hashemi-Shahri, G. Bahari, M. Hashemi;
Zahedan University of Medical Sciences, Zahedan, Islamic Republic of Iran.
Hungary was a part of the Ottoman Empire between the 16th and 17th centuries for about 150 years. It is also known that Roma, most significant ethnic group of Hungary, migrated through Turkey in the 12th century on their
way to Europe. Genetic relationship of Turks with Hungarians and Roma
living in the Carpathian Basin was investigated based on genome-wide autosomal single nucleotide polymorphism (SNP) data. Computational methods
were carried out with pruned datasets containing 111,928 SNPs. In order
to understand Turk, Hungarian and Roma relationship in context to other
European and Middle East populations, principal component analysis was
applied. Our collection of 27 Roma samples, 20 Turkish and 20 Hungarian
samples were merged with 20 Stanford-HGDP populations and 13 populations from the Caucasus obtained from public domain. In order to infer population relationships in a model-based manner, clustering algorithm based on
maximum likelihood estimation method was applied, also with 11 HapMap3
populations. The admixture events detected, based on these methods, were
investigated with formal tests of admixture. The date of admixture event
was inferred based on the exponential decay of admixture linkage disequilibrium. Our results show that extents of Hungarian genetic elements in Turks
are significant, and admixture between Turks and Hungarians occurred during the 17th century. The proportion of Hungarian ancestry in Turks was 2
times higher than the share of ancestry from several groups of the Caucasus
region, Balkars, Druze and Kurds. Interestingly, contribution of Roma genetic elements in Turks is difficult to detect.
J17.64
Association of Polymorphism Gene for TGF-B1 with the Forming of
Kidney Scars among the patients with vesicoureteral reflux (VUR) in
children
D. Jugovic, L. Brankovic, R. Milicevic, D. Radulovic, P. Miljkovic, T. Jevtovic - Stoimenov,
M. Despotovic;
Pediatric Clinic, Nis, Serbia.
509
The characteristic feature of the Kazakh nomadic society was the presence of
a hierarchically organized and widely branched tribal-clan structure called
Shezhire, which reflected complex system of ethno-social organization. In
the context of the Shezhire, Kazakh populations are divided into three ethno-territorial association of tribes called Zhuz (Great, Middle, and Small
Zhuzes) and a group of aristocratic tribes (Tore, Kozha, Sunak).
This study aims to compare Y-chromosomal polymorphism of three Kazakh
Zhuzs and group of aristocratic tribes (total sample size N= 1407). We analyzed 40 SNP and 17 STR Y-chromosomal markers. Summary statistics were
calculated using Arlequin 3.5. Neighbor-joining tree was constructed by the
program MEGA 5.0. Multidimensional scaling plot was drawn by the software package Statistica v.7.1.
Population pairwise FST values were calculated from the Y-chromosomal
haplogroup frequencies to assess the genetic similarity among studied
groups of Kazakh tribes. The most distant ones were the tribe of Sunak and
the Small Zhuz (0.393), whereas the shortest distance was found between
the tribe of Tore and the Great Zhuz (0,021). These genetic distances are
associated with the geographic distances between studied populations. The
distribution of Y-chromosomal haplogroups is strongly correlated with the
tribal-clan structure of Kazakhs. Presence of certain haplogroups at high frequency at particular tribes is in favor to the hypothesis that many tribes go
back to one biological founder, confirming the link between Kazakh family
tree Shezhire with the genetic composition.
J17.66
A mitogenomic phylogeny of haplogroups U2e and U3: revealing the
phylogenetic signals for population expansions in the Slavs prehistory
B. Malyarchuk1, M. Derenko1, T. Grzybowski2, M. Perkova1, G. Denisova1, A. Litvinov1, U.
Rogalla2, K. Skonieczna2;
1
Institute of Biological Problems of the North, Magadan, Russian Federation, 2Institute of
Forensic Medicine, Nicolaus Copernicus University, Bydgoszcz, Poland.
To resolve the phylogeny of some uncommon and poorly studied West Eurasian mitochondrial DNA (mtDNA) haplogroups, we sequenced 32 U2e and
19 U3 complete mitogenomes of Central and Eastern Europeans (Czechs,
Slovaks, Poles, Russians, Ukrainians and Belarusians) and re-analysed the
available at the present time data on 74 U2e and 80 U3 complete mtDNAs.
Molecular dating suggests that the coalescence time estimates are ~21 and
~35 thousand years (ky) for haplogroups U2e and U3, respectively. Detailed
analysis of about 500 Slavic complete mitogenomes belonging to different
haplogroups allowed us to identify a number of lineages that seem specific for Central and Eastern Europe (U3b1b, U4a2a1, U5a2a1c, U2e1b1a,
U2e1b1, U3a1a, H5a1f, U5a1a1a1, U5a1c1, U2e2a1a, U4a2a, H5a2, U2e2a1d
and U5a1b1b). These subhaplogroups consist of similar haplotypes revealed
in different ethnic groups of modern Slavs, thereby proving the existence
of ethnolinguistic community of Slavs through DNA testing. Evolutionary
age of Slavic-specific subhaplogroups is calculated to approximately 3.9 ky
(from 2.3 to 5.9 ky, according to the mutation rate proposed by Soares et al.
(2009) for the entire mtDNA molecule). This indicates that the ancestors of
modern Slavs inhabited areas of Central and Eastern Europe from the times
of Bronze and Iron Ages, i.e. earlier than it was estimated on the basis of
archaeological, historical and linguistic data. This study was supported by
Russian Foundation for Basic Research (grant 14-04-00131) and the Program of Presidium of Russian Academy of Sciences (grant 12-I-P30-12).
510
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J17.67
Incidence of alpha globin gene defect in the Lebanese population: a
pilot study
Background: Inherited hemoglobin disorders are the most common monogenic defects described worldwide. It is well established that Mediterranean
and Arab populations are at high risk for thalassemias in general, and for
-thalassemia in particular. Prenatal as well as premarital screening programs in countries with high prevalence have already been founded. In this
study, we aim at assessing the incidence of alpha thalassemia deleterious
alleles in the Lebanese population. Methods: DNA was extracted from 200
newborns dried blood cards remaining from routine neonatal screening
at the American University of Beirut Medical Center. DNA samples were
screened for the 21 most common -globin deletions and point mutations
reported worldwide, through multiplex Polymerase Chain Reaction (PCR)
and Reverse-Hybridization technique. Results: The carrier rate of -thalassemia in our sample population was 8% which is higher than that reported
from Jordan (2-4%). This finding is comparable to Mediterranean countries
(Israel: 5-9%, Greece: 7%, Adana-Turkey: 7.5%) but lower than that reported in other Arab countries (UAE: 16.5%; Oman: 38.6%; Saudi Arabia: 50%).
Two mutations were detected: the -3,7del single gene deletion (75%) and
the non-gene deletion 2 IVS1 [-5nt] (25%). These mutations are common
worldwide. Interestingly, the - 4.2 and MED mutations, particularly common in Arab and Middle Eastern populations, were absent in our survey
Conclusion: This study is the first dedicated to investigate -thalassemia
genotype incidence in Lebanon. Data obtained demonstrates a high carrier
rate in a relatively, highly consanguineous population. These results may impact premarital and newborn screening policies in our country.
J17.68
5-HTT genotype and sexual behavior traits in healthy female subjects
F. Cieri1,2, M. Sergi1, M. Balsamo2, M. Franzago1, L. Donofrio1, A. Grilli2, L. Picconi2, M.
Tommasi2, A. Saggino2, V. Gatta3, L. Stuppia2,3;
1
Department of Neuroscience and Imaging, Chieti, Italy, 2Department of Psychological
Sciences, G. dAnnunzio University, Chieti, Italy, 3Functional Genetics Unit, Center of
Excellence on Aging (CeSI), Chieti, Italy.
Several mutations in the IL12B, IL12RB1, IFNGR1, IFNGR2, STAT1, and IKBKG genes lead to the development of rare syndrome of atypical familial mycobacteriosis (OMIM # 209950). We set out to test whether rare variants
and polymorphisms in these genes can predispose to tuberculosis. These
genes were studied in two Siberian populations of Russians (304 patients,
Analyses of large autism datasets have provided statistical and functional evidence for the role of rare point mutations (ORoak, 2012, Sanders,
2012; Yu, 2013) and transmitted and de novo copy number variants (CNVs)
(Morrow, 2008; Pinto, 2010; Levy, 2011; Sanders, 2011), and offer crucial
insights into the diverse genetic mechanisms that can lead to Autism Spectrum Disorders (ASDs). Here we present CNV analysis for a cohort of 183
consanguineous families with one or more children affected with ASD. We
provide new insights into the genetic architecture of ASDs as our cohort is
uniquely enriched for recessive loss of function variants. We follow up findings and draw comparisons with additional large ASD and control datasets: the Simons Simplex and the Autism Genetic Resource Exchange (AGRE)
collections (2,670 affected individuals; 9681 total individuals). Comparing
across these cohorts, we demonstrate that ascertainment can lead to selection of different underlying genetic mechanisms causing ASDs. These differences are reflected in metrics such as the affected male:female ratio and
the relative contribution of de novo CNVs versus inherited homozygous deletions. Specifically, we find that de novo CNVs play a significant role in nonconsanguineous families with a single affected child (p=0.04), but a lesser
role in multiplex families, and they are no more common in ASD cases than
controls in multiplex consanguineous families. In contrast, we present the
strongest statistical evidence (p=0.013) to date that homozygous deletions,
are a major contributor to ASD disease burden in consanguineous families,
contributing to as much as 5-10% of cases.
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J17.72
Cost-effective designs for genotype imputation in sequencing based
genome-wide association studies
Genotype imputation is now an essential tool in genetic studies to systematically infer missing and untyped genotypes. Accordingly, progressively larger reference panels are being constructed based on whole-genome
sequencing in various populations. Developing general guidelines for optimally cost-effective imputation designs, however, requires evaluation of
performance issues that include the relative utility of population-specific
compared to general/multi-population reference panels; genotyping with
various array scaffolds; and effects of different ethnic backgrounds. Here we
compared the effectiveness of a Sardinian specific (SardSeq) panel, derived
from whole-genome sequencing of 2,120 Sardinians, to the 1000 Genomes
Project (1000G) reference panels, using combinations of two genome-wide
and three custom arrays as baseline genotype, in both Sardinians and other
Europeans. In Sardinians, the SardSeq panel provided better coverage and
genotype imputation accuracy than the 1000G panels, particularly for low
frequency and rare variants (mean squared correlation with true genotypes
0.95 vs 0.68, respectively, at variants with MAF >1% and <5%). In particular,
when imputing with the SardSeq panel, even gene-centered custom arrays
interrogating a smaller number of variants (on the order of 200,000, as the
Illumina CardioMetaboChip) provided highly informative content across
the entire genome. Notably, in Sardinians a combined panel including both
the SardSeq and the 1000G sequencing data did not provide substantial improvement. By contrast, we showed that a combined panel could be advantageous in other Europeans. We expect our results to be useful in planning
future studies and in current sequencing efforts that are not part of the 1000
Genomes Project.
J17.73
Association of single nucleotide polymorphism in SLC30A8 gene with
aging in Tehran lipid and glucose study
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, acting primarily via the GABA receptors. Multiple lines of
evidence indicate that dysfunction of GABA B receptors may be involved in
511
A. Ulgen1, W. Li2;
1
Eastern Mediterranean University. Mersin 10-Turkey, KKTC. Famagusta., Turkey,
2
Robert S Boas Center of Genomics and Human Genetics, Manhasset, NY, United States.
512
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J18.01
Chromosomal microarray analysis in paediatric patients with
cognitive impairment/behavioural abnormalities/epilepsy and
congenital anomalies: well-known syndromes, novel syndromes,
parental contribution. A clinical perspective
Out of 171 consecutive myopathic index cases recruited from the Italian National Registry for FSHD and carrying of a D4Z4 reduced allele (DRA) with
4-8 repeats, we identified 148 subjects completely fulfilled the clinical diagnostic criteria for FSHD and 23 subjects that presented myopathic features
suggestive of an alternative diagnosis. In this group we observed a wide clinical heterogeneity, including 1) involvement of muscles that are not usually
affected 2) sparing of muscles that are typically affected in FSHD, 3) non
autosomal dominant inheritance, 4) additional clinical features. Molecular
analysis of the 4q35 subtelomeric haplotype did not reveal any molecular
element enabling us to differentiate these patients from classical FSHD cases. Further investigations in two patients with overlapping syndromes allowed us to identify additional mutations, respectively a heterozygous CAV-3
gene mutation and a heteroplasmic transition of mitochondrial tRNALeu.
In an elderly woman the muscle biopsy resulted suggestive for the diagnosis of inflammatory myopathy with inclusion bodies. Three cases presented
a bent spine syndrome: histological and immunohistochemical analysis of
muscle biopsies failed to detect other pathological conditions and additional
genetic testing are ongoing. Collectively, our analysis highlight the necessity
to re-evaluate the significance and the predictive value of DRA, not only for
research but also in clinical practice. Further clinical and genetic analysis of
FSHD families will be extremely important for studies aiming at dissecting
the complexity of FSHD. This approach will favor correct diagnosis and genetic counseling.
J18.05
Constructing a family tree. Frequent mistakes and new needs
M. O. Mkheidze;
Academy for Postgraduate Teacher Education, St.Petersburg, Russian Federation.
Analyzing more than 1500 pedigrees some frequent mistakes were shown:
a) the absence of a horizontal line for an offspring, b) symbol location between generation lines, c) displacement of children birth order, d) wrong
numbering of generations, e) incorrect numbering of symbols in each generation. Human infertility is an important problem. 10 to 15 percent of couples all over the world are infertile. Assisted reproductive technology (ART)
is modern effective therapy for overcoming infertility. To draw a pedigree
with ART it is necessary to use adequate standard genetic symbols that are
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absent so far. The author offers to discuss the following symbols to construct
a family tree with ART: 1) bold symbols of biological parents, 2) a broken
line to image germinative cell donation, 3) a symbol of substitutive, ersatzmother can be denoted by a circle that contains a pregnancy symbol inside.
All particular facts of medical history and different kinds of medical treatment ought to be in legend. Some family trees that are drawn with different
symbols are compared. Symbols suggested are founded by the author.
J18.06
Legislative and ethical peculiarities of human genetic data protection
D. Serapinas1,2, D. Lekarauskaite2;
1
Department of Pulmonology and Imunology, Lithuanian university of Health sciences,
Kaunas, Lithuania, 2Mykolas Romeris university, Vilnius, Lithuania.
Genetic science related to the individual as the main subject of the research
is exposed to a wide range of ethical and legal issues. From the developments in genetic science other science have evolved, thanks to which the
modern world is able to protect the genetic information of data, and to receive the sanction while ignoring the laws of such data. However, there are still
many problems related to the protection of personal genetic information,
such Regulatory standards, the inviolability of an individual, the assurance
of freedom and privacy of information.
Genetic discrimination is strictly forbidden by international conventions
and declarations (Convention on Human Rights and Biomedicine, Additional Protocol to the Convention on Human Rights and Biomedicine, concerning Biomedical Research, Directive 95/46/EC of the European Parliament),
which means that discrimination on grounds of personal genetic information (diseases, abnormalities) is not available in all areas, including employment and insurance. However, individuals face some problems unable to get
a job due to the publicity and information disclosure to their employers, or
increasing the amount of insurance premiums. It is essential to protect genetic rights, because of that any form of discrimination against a person on
grounds of his genetic heritage is prohibited and intervetion in the health
field or disclosure of such information may only be carried out after the person concerned has given free and informed consent to it.
J18.07
The impact of genetic counselling on prevention of mental
retardation in south of Iran
Introduction: Mental retardation has high prevalence in south of Iran because of consanguineous marriages. As uncurable nature,prevention is the best
way.This study was designed to evaluate the impact of genetic counseling
on prevention of mental retardation. Methods: This case control study was
done between 2010-2013 and 120 women with mental retarded children
were participated.All of them had another pregnancy after their mental retarded child.60 of them had pregnancy after genetic counseling (case group)
and 60 women had pregnancy without doing genetic counseling (control
group).The study data was analyzed using SPSS19. RESULTS: In case group
mean maternal age was 334.9 and mean age of mental retarded children
was 7.84.5 years.In control group it was 345.2 for mothers and 102.9
for mental retarded children.Genetic counseling before pregnancy was protective factor for having mental retarded child.(Odds Ratio 4.261, 95% confidence interval 1.312-13.834). 71.7% of parents in case group and 55% in
control group had consanguineous marriages.Screening tests in pregnancy
were done in 78.3% of mothers in case group and 21.7% in control group.
Down syndrome was the most common cause of mental retardation in both
groups. 3.3% of mothers in case group were referred for abortion because
of abnormal amniocentesis. Conclusion: Genetic counseling is effective in
prevention of mental retardation.
J18.08
Hereditary Hyperferritinemia Cataract Syndrome in two Italian
patients.
We describe two unrelated Italian patients with a combination of congenital nuclear cataract and hyperferritinemia without iron overload. Affected
patients had normal serum iron, transferrin saturation, and transaminases,
but high serum ferritin levels, 1819 and 823 ng/ml, respectively. The first
patient was referred to us with the suspect of thalassemia trait. Only asking
a detailed clinical story, a previous surgical intervention (at the age of 15)
513
V. Radoi1,2, L. Bohiltea2;
1
Life Memorial Hospital, Bucharest, Romania, Bucharest, Romania, 2UMF Carol Davila,
Bucharest, Romania.
514
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J18.11
Genetic counseling for a pregnant woman with Marfan syndrome
Marfan syndrome is well known autosomal dominant disorder of connective tissues and is caused by mutation of FBN1. Most cases are inherited from
parents but 25% cases are caused by de novo mutation.
A pregnant lady aged 27 with Marfan syndrome was referred to genetic outpatient at 10 gestational weeks. She visited to us with her younger brother.
Her main concern was the risk of Marfan syndrome for her baby, however
she did not realized that neonatal presentation of severe and rapidly progressive disease in multiple organ systems. She knew she could take genetic
test, but she was not into it and prenatal test, either.
She admitted maternal ward from 25 weeks because of her heart condition.
She needed keeping rest and monitoring with cardiovascular team. At 27
weeks, Valsalva sinus expanded to 43mm and complained slight dyspnea
gradually. At 31 weeks, we decided to perform Caesarean section because
the circulating plasma volume usually increases at 30-32 weeks in pregnancy. She delivered a girl weighed 1532g with Apgar score
5/5. The girl is now 2 years old and does not show any symptoms.
Generally, pregnancy should only be considered after appropriate counseling from a medical geneticist or cardiologist familiar with this condition.
However this lady did not have any counseling before pregnancy because
the cardiologist asked her not to be pregnant. It is important to consider
how we could share the genetic information with female patients who is not
allowed to conceive, but might happen to be pregnant.
J18.12
The application of the Illumina NextSeq500 sequencing platform to
whole human, exome and RNA sequencing
K. Hall1, V. Smith2, M. Wang3, C. Rogert4, B. Crane4, J. Moon4;
1
Illumina Ltd., Cambridge, United Kingdom, 2Illumina, Cambridge, United Kingdom,
3
Illumina, San Diego, CA, United States, 4Illumina Inc., San Diego, CA, United States.
Here we will present data and analyses obtained on the Illumina NextSeq500 sequencing platform with whole human genome resequencing of
clinically relevant samples, together with data from indexed exome and RNA
samples.
Further developments by Illumina of their sequencing by synthesis chemistry together with advances in instrumentation design have enabled the
production of the first desktop high throughput sequencing system.
The NextSeq500 is capable of producing up to 120G of high quality data with
400 million tags in <29 hours from paired 150 cycle runs. Shorter paired
and single 75 cycle runs are also possible together with the availability of
mid throughput configuration delivering 40G from 130 million tags with
paired 150 cycle reads.
The system has a number of innovative features including the use of:
Miniaturized optics
Novel 2 channel chemistry
Dry flowcells
Simplified workflow with reagent cartridges
Connectivity to BaseSpace for cloud analysis or an onsite version enables
simple secure storage and manipulation of data. Data can be analyzed using
a variety of tools relevant to the sample such as BWA/GATK or the Illumina
iSAAC pipeline for DNA samples. RNA data can be analyzed by the Tophat
and Cufflinks applications.
J18.13
Silver-Russell syndrome - novel (epi)genotype-phenotype
correlations
Silver-Russell syndrome (SRS) is a congenital imprinting disorder with hypomethylation of ICR1 on chromosome 11p15.5 or maternal uniparental disomy of
chromosome 7 (mUPD7), characterized mainly by severe pre- and postnatal
growth retardation, relative macrocephaly and a small triangular face.
Retrospective and prospective assessment included 77 SRS patients (36 females and 41 males), at the mean age of 4.75 years (range 0.1-18.5 years), with
the molecular confirmation of ICR1 hypomethylation in 63 (82%) and mUPD7
in 14 (18%) subjects.
Anthropometric measurements were performed using standard techniques
Floating-Harbor syndrome (FHS; OMIM #136140) is a rare genetic condition characterized by short stature, delayed bone age and speech development, and typical facial dysmorphism. The face of FHS is the most distinctive
aspect for making the right diagnosis; otherwise it can be difficult. The typical FHS phenotype consists of triangular facial shape, deep set eyes with
long eyelashes and low set, large, often posteriorly rotated ears, nose narrow at the root broadening to the tip, low hanging columella, large nares,
short philtrum and wide mouth with thin upper and everted lower lip.
Whole-exome sequencing revealed heterozygous truncating mutations
(nonsense or frameshift) in the SRCAP gene in several affected persons
(Hood at al., 2012). The mutations are de novo, some of them are recurrent
and all are tightly clustered within the final exon, with the exception of a
recently identified stop mutation in the penultimate exon 33 (Kehrer et al.,
2013).
Here we present two patients with typical FHS facial features, although they
slightly differ one from the other. In the first patient a nonsense mutation
c.7330C>T (p.Arg2444*) was detected. This is a recurrent mutation described in several other patients. The parents of the patient did not harbour
this mutation, thus supporting its de novo origin. A frame shift mutation
c.7257_7258delAA (p.Gln2419fs*23) was detected in the second case. To
our knowledge this mutation has not been described yet. Phenotype / genotype correlation is further discussed.
Supported by 00064203 and CZ.2.16/3.1.00/24022.
J18.15
Two supernumerary marker-chromosomes in a healthy woman: a
case report
P. Tesner, R. Pourova, J. Drabova, E. Kocarek;
Department of Biology and Medical Genetics, Charles University - 2nd Faculty of
Medicine and University hospital Motol, Prague, Czech Republic.
Presence of supernumerary marker chromosome (SMC) is a rare chromosomal abnormality with broad-spectrum of possible clinical consequences.
Most carriers are healthy people, but some SMCs could be associated with
infertility, especially in males. They are also more frequent in intellectual
disability and cases with various congenital defects. Genetic counselling in
cases of prenatal detection of SMCs is rather challenging since prognosis
differs widely depending on the SMC chromosomal origin and presence of
specific segments. Thus, every new published case is valuable for decisionmaking.
Here, we report a case of the 33-year-old healthy woman referred to clinical geneticist as parent of the deaf child. She was cytogenetically examined
due to chromosomal aberration in the pregnancy of her mother. In her peripheral blood lymphocytes two SMCs in a mosaic form were detected by
various FISH methods. Our analysis revealed that the first SMC is derivative
chromosome 7 containing Williams-Beuren region (7q11.23) which belongs
to considered dosage-sensitive area of this chromosome but was clinically
silent. Besides the larger SMC we also recognized a smaller SMC, where due
to its low frequency and size it was impossible to determine its origin (likely
from constitutive heterochromatin). Our observations are compared with
similar case reports.
Supported by the GAUK-264811 and TACR-TA01010931.
J18.16
Mind as epigenetic modifier in mood disorders
Back to index
D. Serapinas1,2, A. Serapiniene2;
1
Department of Pulmonology and Imunology, Lithuanian university of Health sciences,
Kaunas, Lithuania, 2Mykolas Romeris university, Vilnius, Lithuania.
D. Serapinas1,2, D. Bekasn2;
1
Department of Pulmonology and Imunology, Lithuanian university of Health sciences,
Kaunas, Lithuania, 2Mykolas Romeris university, Vilnius, Lithuania.
The following objectives have been set: to frame the concept and the requirements for informed consent, to analyse the legal acts of the Republic of
Lithuania covering the issues regarding informed consent, to analyse types
of and indications for paternity testing and to name legal and ethical problems arising from application of the informed consent.
The concept of informed consent is based on the principle of autonomy. The
obligation of doctors to inform patients is inseparable from the requirement
to receive informed consent. The two parts are mandatory for any medical
procedures and interventions. The main requirements for the informed consent include rationality, sufficient and clear information, free will and the
form of consent conforming to the legal acts. However, the informed consent
is not an absolute requirement as the patient has a right to remain uninformed. Additionally, under certain circumstances it might be impossible to
inform patients or to receive consent from patients or their duly authorised
representatives. The main problems of the informed consent in paternity
testing, by outlining two stages of the process: conveyance before testing
and interpretation of the results with its effectivenes.
515
Introduction: An important point of the genetic consulting is fixing a correct diagnosis, which most often requires the appointment and conduct of
genetic research. Because of the hereditary nature of diseases and predispositions in our clinical practice when working with patients may result moral
and ethical, psychological, social and legal cases and problems. Goals: The
aim of our study is to investigate and analyze peoples personal position on
the moral and ethical aspects of medical genetic consulting and conducting
of genetic research. Materials and methods: In the study were included in
general 140 people, aged from19 to 61 years, dominated by people aged
between 20 and 22 years. The study was conducted on complete and voluntary and anonymous principles. Discussion: Significant contingent of
respondents are positive about the conducting of genetic testing and genetic consultation. Most of them think that good, empathic attitude of the
physician to the patient and the strict observance of medical confidentiality
is very important. For 57% of respondents application of genetic test is a
serious stressful situation, associated with a number of psycho-emotional
and moral ethical factors. Therefore, consistent with the characteristics of
the patients presentation and interpretation of the results of the conducted
genetic tests is crucial for the beneficial effect from the counseling itself.
Conclusion: Genetic counseling requires abilities and approach to communicate, establishment of confidence and partnership between doctor and
patient, correct presentation and interpretation of genetic information with
respect for autonomy and the right of personal choice of the patient.
J18.20
Teaching workshops of Medical Genetics to students
R. Dragotoiu;
Medical and Pharmacy University Carol Davila, Bucuresti, Romania.
Today the major guide for educators in establishing their teaching goals
and objectives is Blooms Taxonomy of the Cognitive Domain revised by
Krathwohl. From only knowing the specific concepts, students should then
develop more and new skills, so that they could analyze, synthesize and even
critically evaluate their own work or their colleagues.
During each workshop that I taught from October 2013 to January 2014 students had to apply their knowledge to either create an original presentation
(short story) or solve different problems. 100 first year students from the
Medical and Pharmacy University Carol Davila answered 20 questions at
the beginning of their training and corrected these during the last workshop, to prove that they have learned fundamental genetics. The answers
were then corrected by a peer and in the end by the teacher. Workshops
were intended to strengthen students knowledge by means of creative thinking and also a lot of comparative approaches. Because I replaced the frontal
teaching with group or individual activity, during workshops students had
to apply the concepts of the lectures to explain a certain practical situation,
showing they were able to use under new circumstances what they had previously learned.
In the paper I discuss the comparative results and the degree to which students discovered and corrected their own mistakes, being thus aware of
their personal improvement in knowledge besides connecting with peers
and learning to work in groups.
The study demonstrates the achieved ability in understanding and interpreting genetic information of the medical students.
J19.1
Predictive genetic testing in families with growth hormone deficiency:
an Indian experience
A. Sharma, S. Birla;
All India Institute of Medical Sciences, New Delhi, India.
516
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the impact of the disorder and socio-economic concerns which are a decisive factor in opting for treatment. Families with one child affected with GHD
were ardent to discern the recurrence in subsequent pregnancies based
on which were willing for termination. The solution to this problem in developing countries is predictive genetic testing enabling early detection for
treatment and prenatal diagnosis for prevention. Timely genotyping can be
provided if the genetic spectrum is known in the population as variations in
mutation type and prevalence are documented worldwide. Equipped with
this knowledge identification of common mutations will be easier and faster
reducing anguish, improving risk perception, providing quality life and thereby eliminating the burden of morbidity.
J19.2
Population genetic carrier screening for cystic fibrosis, fragile X
syndrome and spinal muscular atrophy: Exploring experiences of
carriers identified through the VCGS Reproductive Genetic Carrier
Screening program
With advancing genetic technologies, carrier screening for multiple inherited conditions can now be offered within the population. This research
aimed to explore how women experience undergoing carrier screening for
three common inherited conditions; cystic fibrosis (CF), spinal muscular
atrophy (SMA) and fragile X syndrome (FXS), through the Victorian Clinical
Genetics Services Reproductive Genetic Carrier Screening program. Adopting a qualitative approach using phenomenology as the theoretical framework, the study utilised in-depth semi-structured interviews, which were
transcribed verbatim. The transcripts were coded using thematic analysis
to identify emerging themes. Eight female participants took part: five received a carrier result for SMA and three for CF. The majority of participants
were pregnant during screening and described the decision to have the test
as straight forward. Participants experienced emotional responses such
as anxiety and stress whilst waiting for their partners test result and also
completed online research to find out more about the relevant condition during this time. Participants were in favour of population carrier screening,
preferably offered prior to conception. The findings of this study elucidated
that genetic counsellors (GCs) play an essential role within this program to
adequately support couples after they receive a carrier result given the varying consent processes undertaken prior to screening. The implementation
of Internet resources and GC facilitated guidance to access reliable online
information is crucial to help empower couples and assist the coping process. Improving awareness of the availability of population carrier screening
within the community will also help improve knowledge levels and facilitate
preconception screening.
J19.3
The utility of incidental findings seen through the eyes of parents
with children in genetic research
The use of new generation sequencing technologies has added complexity to the issues surrounding the return of research results to participants.
Guidelines are in flux in both the research and clinical settings. In ongoing
scientific and bioethical discussions about a possible duty to return incidental findings (or not), the criteria of clinical utility plays a central role.
Some authors have been arguing for a multi-dimensional concept of utility,
including utility as perceived by patients as well (Foster et al., 2009, Grosse
et al., 2010, Bollinger et al., 2012). But what does the concept of utility of an
incidental finding actually entail from a research participants standpoint?
Our presentation aims to shed light on this issue, drawing on semi-structured in-depth interviews conducted in Qubec (Canada) with parents of
children undergoing oncology treatment and participating in research.
Following an overview of the concept of clinical utility under professional
guidelines, we will analyse the multifold reasons as revealed behind the willingness of parents to know their childs incidental findings in four case-scenarios. Parents expressed very practical reasons, even though they do not
necessarily rely on risk factors or on the clinical actionability of results.
Finally, we propose some limits to the adoption of this broader concept of
utility by parents in the hopes of informing bioethics guidelines on return of
research incidental findings.
9q21.3: P08.57-S
9q22.1q22.31 deletion: P09.002-M
9q34.11-12 deletion: J08.07
A
galactosidase A: P06.59-S
-thalassemia: P07.41-S
A2ML1: C21.3
AAAS gene: P11.012-M
AARS2: C15.6
Aarskog-Scott Sydrome: P03.02-M
AAT: J17.06
AATD: J17.06
ABCA1: J05.01
ABCA4: J02.20
ABCB1: P15.30-M
ABCB11: P03.33-S
ABCB4: P03.33-S
ABCC6: P04.55-S
ABCC8 gene: P05.29-S
ABCD1: J06.02
ABCG1: J05.02
abdominal aorta aneurysm: P05.02-M
Abdominal aortic aneurysm (AAA):
J05.03
abdominal aortic aneurysm: P05.03-S
aborted material: J01.65
abortion: J01.01
absence of nails: J08.22
Acadian/Cajun: P17.81-S
ACADVL: P10.17-S
Access genetic testing: EP10-M
access: C13.2
accountability: P14.58-M
ACDMPV: P03.06-M
ACE gen: J06.16
ACE I/D allele: J17.01
ACE: J02.01, J17.58
aCGH: EPL8.6, J01.02, J08.04,
J09.37, J11.17, J12.008, J12.020,
P08.12-M, P08.55-S, P09.003-S,
P09.128-M, P11.011-S
achalasia: C04.3
Achalasia-Addisonianism-Alacrima:
P11.012-M
Acinar Cell carcinomas: P12.005-S,
P12.106-M
ACM: P05.12-M
ACO2: C12.5
ACP5: P04.61-S
Acrocallosal syndrome: P11.013-S
Acromegaly: J12.006
acroosteolysis: P04.33-S, P11.014-M
ACS: P11.020-M
ACTA2: J05.04
ACTB: P11.022-M
ACTG2: P11.098-M
ACTN1 gene: P07.01-S
ACTN3: J17.58
acute coronary syndrome: P15.07-S
acute liver failure: J03.28
Acute Lymphoblastic Leukaemia:
P12.051-S
acute lymphoblastic leukemia: J12.044
Acute lymphoma leukemia (ALL):
P12.001-S
Acute Myeloblastic Leukemia: J07.01
acute myeloid leukemia: J12.004,
J12.106, J12.113, P07.02-M,
P12.002-M
acute phase response: P05.50-M
Acute Promyelocytic Leukaemia:
J12.005
ACVR1: J04.10, J04.11, P04.01-S
ADA2: PL2.1
ADAM23: P09.010-M
Adams-Oliver syndrome: P04.02-M
ADAMTS and p53: J15.04
ADAMTS-8 and ADAMTS-9: J15.05
adaptation: P17.20-M
ADD1: J02.01
Additional chromosomal abnormalities:
J12.005
adducins: P08.65-S
adenoma: J03.19
ADHD: C11.1, J09.01, P09.002-M
adipocytes: J06.12
adipogenesis: P06.51-S
Adiponectin: J01.87, J05.09
Adipose tissue: J06.01
Adiposity: P05.04-M
ADLD: P09.070-M
admixed population: P05.41-S
admixture: J17.63, P17.01-S
Adolescent: P18.02-M, P18.03-S
Adoption: EP45-S
ADPKD: P03.03-S, P03.04-M
ADRB1: P15.06-M
ADRB2: P15.06-M
adrenocortical tumor: P11.027-S
adrenoleukodystrophy: J06.02,
P06.47-S
ADSCA: J17.02
Adult Genetics: P16.31-S
advanced maternal age: EP28-M
adverse drug reaction: P15.01-S
advocacy: EPL5.6
AFA syndrome: P11.015-S
AFF2: P08.09-S
Age of mutation: P10.09-S
age of onset: P09.126-M
age of woman: J01.02
ageing: P08.63-S
Age-related macular degeneration:
P02.01-S
agilent array: P11.123-S
aging process: J14.22
Aging: ES4.2, J16.05, J17.73,
P16.55-S, P17.02-M
AGK gene: P06.53-S
agnathia-otocephaly: P09.124-M
AGO genes: P08.04-M
agranulocytosis: P15.01-S
Agreeableness: P17.66-M
AGT protein: J03.32
AGXT gene: J03.32
AHR: J12.006
Aicardi-Goutires Syndrome: J09.02
AIDS: J17.03
AIS: P01.105-S
Aizel Wassila: J06.03
ALADIN protein: P11.012-M
Alagille syndrome: P11.016-M
albinism: P02.02-M
Albright hereditary osteodystrophy:
P03.36-M
alcoholism: J17.75
ALDH3A2: P04.58-M
Alfi syndrom: J11.50
Algerian population: J17.15
Algerians: P17.76-M
ALK: P12.003-S, P12.141-S, P14.01-S
alkaptonuria: J06.18, P06.01-S
Alkyl mercury chloride compounds:
J13.04
ALL: J12.007, P12.059-S
Allele drop out: P08.44-M
Allele frequencies: J17.04, P17.93-S
alleles: J17.27
Allele-specific expression: C20.1
allelic drop-out: P14.02-M
allelic expression: PL2.4
Allergy: P17.03-S
All-trans retinoic acid: J12.005
ALMS1: P02.03-S
Alox12B: J04.39
ALOX5AP: P05.60-M
alpha 1-antitrypsin deficiency: J04.02
alpha 1-antitrypsin: J03.01
alpha- and beta-thalassemia and
fragile X syndrome: P01.003-S
alpha and non-alpha globin clusters:
P17.47-S
Alpha Haemoglobin Stabilizing Protein:
P14.03-S
alpha thalassemia: J17.05, J17.67,
P08.08-M
alpha-1 antitrypsin deficiency: J17.06,
P03.05-S
alpha-1 antitrypsin: J03.21, P13.04-M
alpha-galactosidase A: P09.053-S
alpha-synuclein: J06.27, J09.60
Alport syndrome: C19.6, J03.02,
J03.03, P03.49-S, P11.017-S,
Back to index
P17.85-S
Alport: J03.30
aLQTS: P05.05-S
ALS/ PDC: P09.100-M
ALS: P09.004-M, P09.154-M,
P10.01-S
ALS2: J09.19
Alspac: P17.04-M
alternative splicing: P06.51-S,
P09.088-M, P13.31-S, P13.35-S,
P16.72-M, PL2.2
Alu-polymorphism: J17.29
Alveolar capillary dysplasia: P03.06-M
Alzheimer disease: J09.07, P09.005-S
Alzheimer: J09.03, P09.009-S
Alzheimers disease: J09.04,
P09.006-M
Alzheimers disease: J09.05, J09.06,
P16.01-S
Alzhemers disease: P09.008-M
amenorrhea: P01.004-M
AML: J12.008, J12.080, J12.100,
J12.114, P12.004-M
AML-M2: J12.009
Amniocytes: J13.13
AMPD2: J08.05
amplicon design: P14.47-S
Ampliseq: P14.28-M, P14.85-S
Amyloid Precursor Protein: P09.007-S
Amyotrophic lateral sclerosis: J09.58,
P09.138-M
an early manifestation: J17.32
analytical and intuitive: S06.1
Anaplerotic therapy: P09.066-M
ancient mitochondrial DNA: P17.06-M
Anderson-Fabry disease: J06.04
anDNA: P17.07-S
Androgen receptor: P10.33-S
anembryonic pregnancy: J01.83
Anemia: J07.12
aneuploidies: J14.20, P01.117-S
aneuploidy screening: P01.126-M,
S13.1
aneuploidy: J01.86, P01.013-S,
P01.021-S, P09.008-M, S05.2
aneurysms: P05.07-S
angioedema: P15.01-S
Angiogenesis: J01.37, P01.094-M
angiogenin: P09.009-S
angiotensinogen: P05.06-M
animal model: J13.10, P09.010-M
ANKRD11: P11.088-M
ANKRD26: P07.03-S
Ankylosing spondylitis: J04.03
annotation: P16.02-M, P16.03-S
ANO5: P10.02-M
anomaly of the hand: P04.73-S
anomaly: J11.13
Anophthalmia/microphthalmia:
P02.04-M
anorectal anomalies: J17.07
Anorexia Nervosa: C11.2
antenatal diagnosis: P01.071-S
antenatal screening: EPL2.3
anterior cruciate ligament rupture:
J04.04
Anti Mllerian Hormone: P01.005-S
anticipation: EP17-S, P09.043-S
Anti-cytokine: J07.14
ANTI-EGFR ANTIBODIES: J12.063
Anti-EGFR therapy: P12.114-M
antiepileptic medication: P09.098-M
Antifolate chemotherapy: J12.056
antihypertensive drugs: P15.02-M
antioxidants: J08.11
antithrombin: J05.25
ANTRX2: P04.29-S
anxiety: J15.01
aortic aneurysms/dissections:
P05.59-S
aortopathies: P05.07-S
AP1: P15.39-S
AP-1: P17.21-S
AP4-deficiency syndrome: P11.062-M
APC gene: J12.069, P12.005-S
APC/MUTYH: J12.051
APC/MYH germline mutations:
517
518
B
-globin gene: J13.15
B3GALT6: P04.21-S
B3GALTL gene: J11.38
b3gnt7: P16.39-S
bacterial artificial chromosome:
P12.130-M
Balanced chromosomal
rearrangement: P14.06-M
balanced translocations: P11.021-S
Balkan Endemic Nephropathy (BEN):
P03.10-M
Balkan endemic nephropathy:
P03.09-S
Band-like calcification: P09.025-S
Baraitser-Winter Syndrome:
P11.022-M
Bardet Biedl Syndrome: J11.11,
P02.06-M
Bardet-Biedl syndrome: J14.02,
P11.023-S, P11.024-M, P11.025-S,
P11.026-M
Bardet-Biedl: J08.09
Barth syndrome: P06.02-M
Bartter syndrome: EP05-S
Bashkortostan Republic: J09.43
batch effect: P17.70-M
BBS 7 genes: P11.023-S
BBS1: P11.024-M
BBS12 gene: J11.11
BBS12: P11.025-S
BBS2: J08.09
BBS9: P04.14-M
BCAP31: C15.1
B-cell lymphoma: P16.16-M
bcr2: J12.004
BCR-ABL1: J12.106
BDNF: J09.07, P17.09-S
BDV: J14.03
Becker muscular dystrophy (BMD):
P10.03-S
Becker muscular dystrophy: EPL4.1
Beckwith Wiedemann Syndrome:
P11.027-S
Beckwith-Wiedemann syndrome:
P11.028-M, P11.029-S, P11.073-S,
P16.06-M, P16.07-S, P16.08-M,
P16.37-S
behavioural: P11.026-M
Behets disease: J04.07
benchmarking: J16.15
benign chromosomal imbalances:
J18.15
benign joint hypermobility syndrome:
P04.04-M
Bernard-Soulier syndrome: P07.20-M
Beta thalasemia: P07.04-M
Beta Thalassemia: J01.06
beta-catenin 1: P08.21-S
beta-catenin, TCF1, LEF1: P12.137-S
Beta-defensin: J07.14
beta-galactosidase: P06.03-S
beta-propeller protein-associated
neurodegeneratio: P08.53-S
Beta-thalassemia: J01.46, J17.08,
P01.010-M, P15.05-S
BETAXOLOL: P15.06-M
bevacizumab: J02.14
BFLS: P08.16-M
Biallelic mutations: P12.082-M
Bicuspid aortic valve: J05.04
Biobank: P17.10-M
Biobanking: P18.04-M, P18.11-S
biobanks: P18.05-S, P18.41-S
biochip: J14.31
biodosimetry: J13.05
Bioethical and humanistic expertise
and auditing: J18.02
bioethics: C22.2
Bioinformatic analysis: P02.06-M
Bioinformatics predictions: P13.43-S
bioinformatics resource: P16.11-S
bioinformatics: C06.6, J14.27,
P09.131-S, P16.09-S, P16.10-M,
P16.29-S, P16.48-M, P16.51-S
biomarker: J09.69, J14.05, J15.02,
P09.059-S
Back to index
CAPN10: P08.14-M
CAPN3: P10.16-M
CAPS: P07.05-S, P07.43-S
carbapenemas: J14.11
Cardiac arrhythmias: P05.21-S
Cardiac channelopathies: J05.06
cardiac malformation: P11.056-M
cardiac surgery: P15.22-M
Cardiofaciocutaneous syndrome:
P11.103-S
cardiology: S09.3
Cardiometabolic diseases: P17.53-S
cardiometabolic phenotypes: P06.05-S
Cardiometabolic: C04.6
Cardiomyopathy: J05.07, P05.22-M,
P05.23-S, P05.24-M, P05.53-S,
P05.61-S, P16.15-S, P17.34-M, S09.1
cardiomyopatrhy: P14.12-M
Cardiovascular development: S15.3
cardiovascular disease: J05.25,
P05.14-M, P05.15-S, P17.15-S, S09.2
cardiovascular malformation:
P05.54-M
Cardiovascular: P14.55-S
Carnitine Palmitoyltransferase 1A:
P17.25-S
Carotid atherosclerosis: J05.08
carrier frequency: P06.09-S
carrier screening: J01.07, J19.2,
P18.08-M
carrier status: J01.81
Carrier: EP40-M, P14.75-S
Carriers: EP38-M
Case - control study: J01.25
case report: J01.58, P09.048-M
case-control design: P17.57-S
Catalase: J12.032
cataract: P01.022-M
cathepsin C gene: J04.34
cathepsin F: P09.069-S
Caudal regression syndrome:
P11.031-S
CBC Electrophoresis: J01.06
CBCD: P09.031-S
CBS 844ins68bp: J06.11
CCHS: P09.032-M, P09.033-S
CCR5: J07.15
CD25: P07.06-M
CD44: J12.033
CD7: J12.114
CD96: P11.109-S
Cdcp2: P16.39-S
CDG1a: P06.06-M
CDH1: J12.102, P12.073-S
CDH10 gene: P09.151-S
CDK5RAP2: P08.60-M
CDKL5: P09.034-M
cdkn1a.: J12.002
CDKN2A: P12.087-S
CdLS: C05.5, C16.4, C16.5, P14.13-S
CDPX2: P01.011-S
CDY1: J01.62
CE mark: J14.30
CECR1: P07.26-M, PL2.1
celiac disease: J03.07, P14.14-M
Cell Cycle: P12.086-M, P13.32-M
cell death and apoptosis: J15.10
cell free DNA testing: C01.2,
P01.012-M
Cell free DNA: J12.014, P01.104-M,
P14.63-S, S13.1
Cell Free Fetal DNA: P01.013-S,
P01.065-S
Cell monolayer model: J03.23
cell-free DNA: J14.05, P01.014-M,
P01.038-M
Cell-free fetal DNA: C01.1, J01.08
Cellular Stress: C20.6, P13.05-S
CELSR2: J05.29
Central serous chorioretinopathy:
J02.02
centromere: P12.004-M, P13.22-M
Centromere-micronucleus assay:
J12.088
CEP290: C12.4
Cerebellar ataxia: P06.04-M,
P09.072-M
Back to index
P16.17-S
chromosomal ring: P13.06-M
Chromosomal translocation: P13.07-S
chromosome 10: P11.124-M
chromosome 12: P11.068-M
chromosome 13: P01.041-S
chromosome 14: P01.073-S
chromosome 15: P11.033-S
chromosome 17: J12.109, P08.49-S
chromosome 22: J11.14
Chromosome 3p: J11.27
Chromosome 8: P13.18-M
Chromosome 9p21: J05.08
chromosome abnormalities:
P12.002-M
chromosome abnormality: P01.031-S
Chromosome instability: J11.18
chromosome marker: J01.57
Chromosome microarray analysis:
J11.16, P11.048-M
chromosome microarrays: EPL3.5
chromosome mosaicism: P18.46-M
chromosome polymorphisms: J01.11
chromosome translocation: P13.45-S
chromosome Y: J12.034
chromosome: J11.13, J13.03,
P13.24-M
Chromosomes 4 and 11: P13.07-S
chromothripsis: P13.09-S, P13.38-M,
P16.16-M, P16.17-S, S10.1
Chronic hepatitis C: J07.12
chronic intestinal pseudo-obstruction:
P03.12-M
chronic kidney disease: P17.16-M
Chronic lymphocytic leukemia: J14.08
Chronic Myelogenous Leukemia:
J12.032
Chronic Myeloid Leukemia: J12.035,
J15.18, P12.037-S, P14.18-M
chronic non spherocytic haemolytic
anemia: P13.27-S
chronic obstructive pulmonary disease:
J12.071
chronic periodontitis: J04.38
Chronic phase chronic myeloid
leukemia: P12.038-M
Chronic renal failure: P16.23-S
CHSY1: J04.41
ciliopathies: P09.068-M, P11.080-M
ciliopathy: P04.08-M, P11.081-S
Cipher: J16.03
circadian rhythm genes: P01.101-S
circadian rhythm: P17.17-S
circulating miRNAs: P14.14-M
circulating tumor cells: S17.3
circumferential skin creases: P04.09-S
circumferential skin folds: P04.09-S
Cirrhosis: J17.11
Cirrhotic cardiomyopathy: J05.28
CISD2: P11.151-S
CISH: J12.040
citrin deficiency: P06.07-S
citrullination: P07.31-S
Civilian control on human genetics:
J18.02
Classes of mosaicism: ES5.1
CLCN1 gene: P10.21-S
CLCN2: P09.036-M
clear cell renal cell carcinoma:
J12.036
Cleft lip and palate: P11.034-M,
P11.035-S
cleft lip with or without cleft palate:
P04.43-S, P13.10-M
Cleft lip: P11.036-M
Cleft palate: P11.036-M
cleidocranial dysostosis: J01.13
Clinical care: P18.31-S
Clinical charaterization: P09.123-S
Clinical diagnosis: P12.132-M, PL2.3
Clinical exome: C07.6
Clinical Genetics and Dysmorphology:
P11.037-S
clinical molecular genetics: P18.09-S
clinical performance: J14.15
clinical phenotypes: P11.123-S
clinical risk: P12.107-S
519
520
Back to index
Cyp2D6*4: P15.11-S
CYP2E1 gene: J15.09
CYP3A5, CYP3A4, MDR-1: P15.36-M
Cyprus: P18.20-M
cysloporine: P15.30-M
Cystic fibrosis: EPL4.3, ES7.2, J03.12,
J06.08, J06.09, J17.14, J17.15,
J17.16, J17.17, J19.2, P13.15-S,
P14.23-S, P14.24-M, P14.48-M,
P14.51-S, P17.22-M, P18.34-M
Cytogenetic analysis: J13.16
cytogenetic anomaly: J01.74
cytogenetic assay: J12.015
Cytogenetic: J11.19, J12.043
CYTOGENETICS: J12.007, J12.030,
J13.05, J14.08, J15.14, P13.16-M,
P14.25-S
cytokine genes: J03.18
cytokines genes: J04.13
Cytokines: J07.10, P05.32-M,
P09.023-S
CytoScan HD array: P08.07-S
Czech population: P01.046-M
D
D4ST1: P04.23-S
D4Z4 Reduced Allele: P10.12-M
Damage: P14.15-S
Danon disease: P06.14-M
data analysis: P14.78-M
data base: P17.19-S
Data exchange: C13.5
data mining: P17.19-S
data sharing: P18.13-S
database: P14.10-M, P16.75-S
data-sharing: P18.16-M
DCM: J05.15
DCSTAMP: P04.07-S
DD/MCA: P13.47-S
DD3 overexpression: J12.094
DDCH: C15.1
DDD Study: P11.114-M
DDHD2: J09.66
De lange-like: P08.31-S
de novo homozygous mutation: J07.25
De novo microdeletion: P01.112-M
de novo mutation: C18.2, C20.2,
P01.112-M, P09.130-M, P12.093-S
de novo rearrangements: P13.17-S
de novo: P03.18-M, P11.031-S,
P14.78-M
deafness, non-type 1 diabetes:
P06.56-M
deafness: C12.3, P02.09-S, P02.49-S,
S15.1
decision making: EP17-S, S06.3
Decision-making in human genetics:
J18.02
decreased PAPP-A: P01.116-M
deep sequencing: P12.076-M
Del 9p: P08.32-M
Del 9q: P08.32-M
del14q24.1q24.3: P11.051-S
del18q: P11.099-S
del5p15.33-31 dup5p15.31:
P11.130-M
deletion 10q terminal: P09.080-M
deletion 17q21.31: J11.17
deletion 19p13: P11.054-M
deletion of 9p: J11.50
deletion: J03.05, P02.19-S, P05.11-S,
P05.42-M, P09.135-S, P10.01-S,
P11.052-M, P11.053-S, P11.102-M,
P13.18-M
deletions at 7q33-q35 and 11p13:
P13.12-M
deletions: P10.03-S
Dementia with Lewy Bodies:
P09.110-M
dementia: J09.06
demyelination: P09.071-S
de-novo mutation: P10.35-S
DEPDC5: P09.054-M
Depolymerization: J09.60
Derivative chromosomes: P11.055-S
DES: J05.07, P10.08-M
Desbuquois dysplasia type 2: C10.3
dysferlinopathy: J10.01
Dysgonosomies: J13.06
Dyshormonogenesis: P06.13-S
dyslexia: C09.5, C15.5
dysmorphic features: P09.044-M
Dysmorphism: J11.19, J17.41,
P11.056-M
dysmorphology: P11.082-M, P14.13-S
dysphagia: P17.83-S
Dysplasias: P01.108-M
dystonia: P09.045-S, P09.062-M
Dystrophic Epidermolysis bullosa:
P04.71-S
dystrophies: P10.30-M
E
EAAT1: J09.13
Early Detection: J16.04
early onset obesity: P11.062-M
early rheumatoid arthritis: P07.16-M
earthquake: J09.40
East Asia: EPL5.4
Eastern Siberia: EP33-S, J11.26
Eating Disorders: J09.61
eating: EPL1.1
EB Centre CZ: P18.17-S
EBV Viral Integration: P16.73-S
ectodermal dysplasia: EPL2.6
ectopic calcification: P04.19-S
EDA gene: P04.27-S
EDS: EP49-S
educating physicians: C13.6
education level: C03.5
Education: P15.15-S, P18.21-S,
P18.40-M
Educational attainment: C11.1
Edwards syndrome: P01.028-M,
P01.029-S
efficacy: P18.19-S
EFMR: P09.114-M
EFTUD2 gene mutation: P11.093-S
EGA: J14.09
EGFR gene: J14.10
EGFR Kras: P14.79-S
EGFR mutations: J12.045
EGFR: J15.16, P12.141-S, P14.34-M
Ehlers Danlos syndrome (EDS):
P04.69-S
ehlers danlos syndrome: P04.04-M
Ehlers-Danlos syndrome: J04.09,
J17.19, P03.43-S, P04.21-S,
P04.22-M, P04.23-S
Ehlers-Danlos: P04.20-M
EHMT2: P08.24-M
EIF2S3: P08.47-S
electrical status epilepticus during
slow-wave sle: J08.24
Electrocardiogram: C14.2
Electronic pedigree: EP29-S
Ellagic Acid: J15.07
ELSA: P18.15-S
ELSI: C22.3, P18.16-M
ELUCIGENE CF30 Kit: J17.15
embryo: P01.031-S
embryofetal: C01.3
Embryonic stem cell: J01.18
Embryonic stem cells: J13.11
EMC1: P02.38-M
EMD, LMNA, FHL1: J10.11
Emery-Dreifuss muscular dystrophy:
J10.11
emotion: J15.01
Emotional distress: EP22-M
EMSA: EP47-S
encephalopathy: P06.45-S, P09.047-S
Endocan: J12.023
Endocrine system: J01.30
Endometrial Carcinoma: J12.001
endometrial carcinoma: J12.046
Endometriosis, Height, Educational
attainment: J17.70
Endometriosis: J01.19, J01.82, J17.20
Endometrium: J01.35
endosomal Na+/H+ exchanger (NHE6):
J08.24
Endothelial Nitric Oxide Synthase:
J09.28
Back to index
endothelin: C10.4
endothelium-dependent vasodilation:
P09.028-M
enhancer: C17.4, C20.3
enlarged vestibular aqueduct:
P02.29-S
eNOS Glu298Asp: J01.20
eNOS: P05.31-S
enrichment comparison: P14.82-M
Ensembl: P16.40-M
Enteric nervous system: C05.4
Entrobacteriaceae: J14.11
entropy: P13.44-M
enzyme activity: P06.16-M
EOGT: P04.02-M
EPCR Gene: J06.05
EPHA1: P08.25-S
epidemiology: J17.07, P17.49-S,
S09.2
epidermal barrier function: P04.03-S
epidermolysis bullosa congenita:
P18.17-S
Epidermolysis bullosa: ES5.2
Epigallocatechin gallate: J15.10
Epigallocathechin-3-gallate: J15.11
Epigenetic Biomarker: J16.04
Epigenetic changes: J16.10
Epigenetic therapy: P16.28-M
Epigenetic: J01.85, J13.19, J16.11,
S16.2
epigenetics: J09.69, J16.05,
J16.12, J18.16, P16.23-S, P16.25-S,
P16.60-M, S12.1, S12.2
epigenome: S12.3
Epilepsy and ID: S03.3
Epilepsy: C18.3, J09.12, J09.26,
P08.35-S, P08.36-M, P09.038-M,
P09.048-M, P09.049-S, P09.050-M,
P09.105-S, P13.41-S, P14.27-S,
P16.56-M
epileptic encephalopathies: P09.115-S
epileptic encephalopathy: P09.051-S,
P09.052-M, P09.105-S
epimutation: J01.86
episodic ataxia: J09.13
epithelial ovarian cancer: P16.29-S
Epithelial-mesenchymal transition:
J12.047
EQA: P14.16-M
eQTL: C06.1, C06.2
eQTLs: C11.5
equivocal findings: P14.66-M
ERBB1, MYC, Her2/neu, and Top2A
oncogenes: J12.103
ERCC1 Asn118Asn: J17.13
ERCC6: J09.11
ERCC8: J09.11
erroneous disease genes: C12.3
ERT clinical research trials: EPL4.2
erythroid progenitors: P07.07-S
ESCC: J12.048, J12.049
escobar syndrome: J11.33
ESE site: J10.09
ESM-1 gene: J12.023
esophageal atresia and choanal
atresia: P11.093-S
Esophageal Atresia: P11.042-M
Esophageal squamous cell carcinoma:
J12.033, P12.052-M, P12.053-S,
P12.135-S
ESRD: J03.30
essential hypertension: P05.32-M
essential tremor: P09.106-M
estrogen receptor alpha: J09.65
Estrogen Receptor: C19.4, J12.024
estrogen: P15.16-M
ESX1: P01.032-M
ethical, legal and social issues:
P18.12-M
ethical, social and policy issues:
P18.05-S
ethical: S13.3
ethics: C01.6, C22.4, EP04-M,
EPL2.5, EPL3.4, EPL7.1, EPL8.3,
J16.12, J18.06, J18.20, P16.36-M,
P18.11-S, P18.13-S, P18.18-M
Ethnogene: P15.25-S
521
522
Familial Hypercholesterolaemia:
EP12-M
Familial Hypercholesterolemia:
J14.30, P06.17-S, P17.43-S
Familial inversion: J12.037
familial syndrome: J11.21
familial translocation: P01.059-S
familiar cardiomyopathy: P05.40-M
Family analysis: P16.64-M
family based design: P17.80-M
Family Communication: EP45-S
Family design: P16.76-M
family experiences: EPL2.4
family genomics: P03.21-S
Family members: P18.27-S
Family planning: EP08-M
family studies,PND: J19.1
Family System: EPL6.1
family: EP37-S, J18.05
Family-based association study:
J17.50
family-based design: P17.59-S
family-based samples: P17.73-S
family-provider interactions: EPL2.4
FANCA gene: J13.07
FANCA: P11.064-M
FANCF: P11.065-S
FANCM: P12.057-S
Fanconi anemia gene: S15.2
Fanconi anemia: J13.07, P11.059-S,
P11.064-M, P11.065-S, P14.31-S
Fas Ligand: J07.05
Fas/Fasl: P12.036-M
FBLN5 gene: J01.45
FBN1: P04.36-M, P05.47-S,
P05.48-M, P16.57-S
FCER2: J17.21
FCGR3B: P16.22-M
FDR: P17.32-M
Febrile convulsions (FC): J09.14
Female carriers: EPL9.5
female fetuses: P01.011-S
fetal aneuploidy: P01.012-M
Fetal ascites: J01.21
Fetal Balanced Translocation:
P01.089-S
Fetal deletions / duplications:
P01.064-M
Fetal demise: P11.030-M
fetal karyotype: J01.22, J01.72
fetal sex determination: P01.036-M
fetal sex: J01.08
fetal sexing: EPL2.1
fetus karyotype: J01.74
Fetus: J01.64, J01.88
FFEVF: P09.054-M
FFPE tissue: P12.058-M
FFPE: J14.12
FGD1: P03.02-M
FGF10: P11.021-S
FGF16: P04.24-M
FGF2: P12.119-S
FGF20: J09.68
FGF23: P06.18-M
FGFR1 mutation: P11.072-M
FGFR2 gene: P01.025-S
FGFR2: J12.052, P11.049-S
FGFR3: J12.053, P12.009-S
FH: EP12-M
FIBP: C16.3
fibrodysplasia ossificans progressiva:
J04.10, J04.11, P04.01-S
FII: J05.13
filaggrin: J04.05, P04.03-S
final consultation: EP30-M
First Trimester Combined Screening:
J01.23
first trimester: J14.01
first-tier testing: J14.07
FISH: J01.24, J11.02, J12.038,
J12.064, J12.065, J12.075, J13.13,
P11.006-M, P11.033-S, P13.24-M,
P13.25-S, P14.01-S, P16.16-M
FKBP14: P04.20-M
Floating-Harbor syndrome: J18.14
FLT3-ITD: J12.114
Fluorescence in situ hybridation
(FISH): J13.18
Fluorescence in situ hybridization
(FISH): J11.25
fluoride: J15.12
fluorouracil: P15.35-S
fmf: J03.11, J17.22, P03.14-M,
P07.10-M, P18.24-M
FMR1 gene: J01.25, J01.26
FMR1 premutation: P01.081-S
FMR1: P01.034-M, P08.27-S,
P14.32-M
FMR2: P01.034-M
FOCAD: P12.075-S
focal segmental glomerulosclerosis:
P03.15-S
focus group interview: EP14-M
foetal pathology: P01.033-S
FokI polymorphism: J09.41
FokI RFLP: J04.06
FokI, ApaI and TaqI RE: J07.07
Folate: P13.20-M
Follicle stimulating hormone receptor:
P01.035-S
Food preferences: P17.26-M
Fosl1 gene: P13.07-S
founder effect: P06.23-S, P06.50-M
founder mutation: J04.01, P09.076-M,
P09.117-S
foveal hypoplasia: C12.6
FOXF1: P03.06-M
FOXG1 gene: P09.055-S
FOXG1 syndrome: P09.055-S
FOXG1: P09.125-S
FOXK1: J11.55
FOXO3A: J12.118
FOXP1: J09.15, P08.26-M
FOXP3: P07.06-M
fractures: J04.24
Fragile X associated Premature
Ovarian Failure: P01.081-S
Fragile X premutation: J01.49
Fragile X syndrome: C20.5, EP50-M,
J08.11, J09.16, P13.26-M
Fragile X: P08.27-S, P08.28-M
Fragile-X Syndrome: P09.056-M
framework: P15.13-S
Free fetal DNA: P01.036-M
frequency: P01.124-M
Friedreich Ataxia: P09.057-S
Friedreichs Ataxia: J09.17
Frontonasal dysplasia: P11.066-M
FSHB: P01.037-S, P15.17-S
FTL: J18.08
FTLD: P09.058-M
FTO gene: P09.044-M, P17.27-S
FTO mutation: J17.09
FTO: P17.62-M
Functional genomics: P16.34-M,
S04.1, S04.2
Functional linear model: P17.73-S
functional: P16.02-M
Functional-enrichment analysis:
P16.34-M
fusion gene: P12.010-M, P12.059-S
fusion transcripts: P12.076-M
fusion: P14.42-M
FV: J05.13
FYB: P07.37-S
FZD4: J02.03
G
G6PC: P06.22-M
G6PD mutation: P13.27-S
GABA: J17.75, P09.077-S
GABBR2: J17.74
galactosemia: J06.18
galactosialidosis: P06.03-S
Gallbladder cancer: J12.091
gamma globin: P07.36-M
gastric cancer: J12.054, J12.055,
J12.078, P12.060-M, P12.061-S,
P12.062-M
gastroenterology: EPL6.6
gastrointestinal tumors: P12.129-S
Gaucher disease: P06.19-S,
P06.20-M, P09.060-M
Gaucher: J06.27, P09.059-S
Back to index
genotype-phenotype correlation:
J06.22, J10.06, P03.49-S, P11.024-M
genotyping: EP27-S, J04.26, J09.04,
P07.21-S, P12.123-S
germline chromothripsis: P13.12-M
Germline mosaicism: P10.41-S
Germline mutation: J12.074
germline mutations: P12.032-M
germline predisposition: P12.041-S
GEUVADIS: S19.2
GFER: P06.21-S
GH1,GHRHR ,PROP1,POU1F1 genes:
P03.17-S
ghrelin: P16.39-S
giant cell tumor: P04.25-S
Gipsy: P15.33-S
girls: P09.150-M
GJA3 gene: J02.17
GJB1: J09.67
GJB2 gene: J17.57
GJB2 mutation: P02.08-M
GJB2: EP33-S, J02.04, J17.26,
P02.15-S, P02.16-M, P02.25-S
GJB6: J02.05, J17.26, P02.09-S,
P02.15-S, P02.16-M
Glanzmann syndrome: J07.18
Glaucoma: P02.32-M
GLCCI1: P15.04-M
Gli2: P17.30-M
glioblastoma multiforme: J12.068,
P12.067-S
glioblastoma: J12.058, J12.059,
P12.063-S, P12.064-M, P12.065-S,
P12.066-M
Glioblastomas: J12.060
glioma cell: J15.19
glioma pathogensis: P12.075-S
glioma: J12.119, P12.068-M,
P12.069-S, P13.31-S
glomerular disease: P03.15-S
Glucocerebrosidase: P06.19-S
glucocorticoid receptor: P15.22-M
glutamate receptor: P02.37-S
Glutathione S transferase: J12.032
Glutathione S-transferase: J01.32
Gluten: P07.12-M
Glycaemic traits: C14.5
Glycogen Storage Disease type 1:
P06.22-M
Glycogen storage disease type III:
P06.23-S
glycolipid: S18.1
Glycoprotein IIIa: J05.18
Glycoprotein P: J13.08
glycosylation: P11.040-M
glycosylphosphatidylinositol anchor:
S18.1
GM1-gangliosidosis: P06.03-S
GNAI3: P11.020-M
Goldenhar syndrome: P11.068-M
Goldenhar: P11.067-S
Gold-nanoparticles: P14.34-M
Golgi apparatus: P08.20-M
Goltz syndrome: P11.069-S
gonadal mosaicism: P01.041-S,
P01.111-S, P18.44-M
GoNL: C14.2
Gorlin syndrome: C08.4
GPC3: P01.106-M, P11.070-M
GPC4: P01.106-M
GPHN: P13.11-S
GPI pathway: P08.43-S
GPM6A: P08.29-S
GPR98: J02.16
gr/gr microdeletion: J01.56
gramd1b: P17.55-S
gray platelet syndrome: P07.13-S
GRCh38: P16.40-M
GREM1: P04.26-M
GRHL3: P17.60-M
GRHPR: P03.35-S
GRM3: J09.51
gross gene rearrangement: P06.02-M
Growth defect: P11.009-S
Growth Disorders: P16.38-M
Growth hormone defeciency: J19.1,
P03.17-S
Back to index
523
524
Hydrops: J05.19
hydroxyprostaglandin dehydrogenase:
P04.16-M
hyperacetylation: J01.62
hyperammonemia: P06.45-S
Hyperhomocysteinemia: J06.11
hypericin: J09.44, J15.04, J15.05
hyperkeratosis: P02.09-S
Hypermobility: P11.084-M
hypernatremia: J09.22
Hyperphosphatasia with Mental
Retardation syndrome: C03.3
Hyperprolactinemia: J01.36
hypertelorism: P11.111-S
Hypertension: P05.31-S, P05.39-S
hypertophic cardiomyopathy (HCM):
P05.40-M
hypertrichosis: P11.039-S
hypertriglyercidemia: P05.41-S
Hypertrophic cardiomyopathy: C04.5,
P05.42-M, P05.43-S, P05.44-M,
P05.56-M, S09.1
Hypertrophic Myocardiopathy:
P05.45-S
HYPOACUSIA: P02.15-S, P02.16-M
hypochromic microcytic anemia:
P07.41-S
Hypoglossia-hypodactyly: J11.36
hypohidrotic ectodermal dysplasia:
P04.27-S
Hypomagnesemia: P03.20-M
hypomyelination: P11.119-S
hypophosphatasia: P01.039-S
Hypothyroidism: P03.37-S, P17.39-S
hypotonia: P10.06-M
Hypotonia-cystinuria syndrome:
P09.119-S
Hypotrichosis: P04.64-M
Hypsarrhythmia: J09.50
I
I172N mutation: J06.06
Ichthyosis: J04.42, J17.25, P04.58-M,
P04.64-M
ICL repair: S15.2
ID gene family: J01.37
ID: C03.4, P08.50-M
IDH1: P12.069-S
Idiopathic basal ganglia calcification:
J09.53
idiopathic epilepsy: P09.010-M
idiopathic ID: J08.12
idiopathic mental retardation: J09.25
idiopathic short stature: C20.3
Idiopathic ventricular fibrillation:
P05.46-M, P14.38-M
IDS: P06.37-S
idursulfase enzyme replacement
treatment: P06.61-S
idursulphatase gene: P06.38-M
I-FISH: J12.034
iFISH: P12.122-M
IFITM5: P04.47-S
IGF: P17.68-M
IGF1R: P11.002-M
IGF2: J01.61
IGFBP2: J12.068
IGHMBP2: P10.14-M
IHC: J12.040, J12.065
IL-1 : J09.14
IL-1: J07.11
IL10: P12.088-M
IL11RA: J04.12
Il17A gene: J07.04
IL1R1: P16.05-S
IL1R2: P16.05-S, P17.40-M
IL1RI: J07.11
IL22: P07.17-S
il6: J04.27, J05.22
Illumina: J09.32
imatinib resistance: P14.39-S
IMMP2L: P09.142-M
immun deficiency: J11.29
Immune System: P09.023-S
immunmodulation: J07.20
Immunochip: C14.6, P17.14-M
Immunofluorescence: P09.029-S
Immuno-genetherapy: P12.052-M
immunohistochemistry: J12.026
Immunotherapy: P12.043-S,
P12.089-S
impact on parents and children:
EPL4.2
impact: EP28-M
imprinted genes: P01.110-M
Imprinting disorder: J08.13
Imprinting disorders: J16.10
imprinting: J01.61, P04.31-S,
P11.141-S, P16.47-S
in situ: P14.40-M
in vitro fertilization: J01.28, J01.67
In vitro neurogenesis: P09.149-S
In vitro studies: C14.5
Inbreeding coefficient: P17.74-M
inbreeding: J17.30
incidental finding: C22.4, EPL6.4
incidental findings: C13.4, EPL3.4,
J19.3, P14.91-S, P18.25-S, P18.26-M,
PL3.4
Incidental Variants: C02.5
incontinentia pigmenti: P04.28-M,
P08.52-M
increased nuchal translucency:
P01.019-S, P01.040-M
increased sensitivity with CES: C07.2
Indian population: P12.102-M
Indian populations: P17.48-M
Indigenous Australians: EPL5.3
Indigenous population: P17.31-S
Individualized medicine: EP10-M
Indonesia: P03.19-S
Induced Pluripotent Stem Cells
(IPSCs): P09.149-S
induced pluripotent stem cells: C03.6
infantile epilepsy: P09.114-M
Infantile Systemic Hyalinosis:
P04.29-S
Infection susceptibility: P07.11-S
infertile males: J01.12
infertility: J01.10, J06.23, P01.004-M,
P13.16-M
inflamation: J04.27
inflammatory Bowel Disease: C02.3,
C14.6, P03.21-S, P13.21-S
inflammatory genes: P12.102-M
inflammatory mediators: J04.13
Influenza A virus: J09.36
Information requirements: P18.02-M,
P18.03-S
information: P18.33-S
Informed choice: EPL8.4
informed consent: EP35-S, EPL2.3,
J01.28, J18.19, P18.03-S
inguinal hernia: P04.30-M
Inherited cardiac conditions: EP25-S
Inherited cardiac diseases: P01.087-S
Inherited cardiomyopathy: J05.17
Inherited eye diseases: P02.17-S
Inherited macrothrombocytopenia:
P07.01-S
Inherited Retinal Dystrophies:
P02.18-M
Inherited thrombocytopenia: P07.18-M
Inherited thrombocytopenias: ES1.2
Inhibitor: J15.22
insertional translocation: P04.19-S
insulin deficiency: P11.135-S
Insulin resistance: P03.40-M
integrated system: J03.15
Intelectually diasbility: P08.32-M
intellectual Disabilities: P13.29-S
Intellectual disability genes: P16.30-M
intellectual disability/developmental
delay: P08.19-S
intellectual disability: C03.1, C03.2,
C18.1, J08.03, J08.04, J08.15, J08.16,
J08.18, J08.25, J11.18, P08.03-S,
P08.04-M, P08.08-M, P08.15-S,
P08.21-S, P08.24-M, P08.25-S,
P08.29-S, P08.31-S, P08.33-S,
P08.34-M, P08.35-S, P08.36-M,
P08.37-S, P08.38-M, P08.39-S,
P08.40-M, P08.55-S, P08.70-M,
P08.71-S, P08.77-S, P08.78-M,
Back to index
J04.33
Large CNV: P09.002-M
Large cryptic genomic rearrangements:
P11.091-S
Large deletions: J07.09
large rearrangements: P14.57-S
LARS2: P02.31-S
lasso, elastic nets: P17.69-S
late onset BMD: P10.03-S
late onset diseases: PL3.3
late onset neurodegenerative diseases:
EP18-M
late onset: J06.26
Late replication: P13.48-M
Latency-Associated Peptide Domain:
P04.65-S
laterality: C09.5
LBSL syndrome: J17.32
LDLR: P06.17-S, P17.43-S
Learning disability: C10.6, P08.43-S
Learning sidabilities: J08.10
Lebanese population: P08.34-M
Lebanon: J11.54
Leber congenital amaurosis: J01.39,
P02.19-S, P02.20-M, P02.21-S
LEF1: J07.20
left-sided ventricular outflow tract
obstruction: P05.54-M
Legal framework: P18.27-S
legal issues: C22.5
Leigh Syndrome: P06.26-M
Leigh-like disease: P06.25-S
LEMD3: J04.17, P04.35-S
Lenalidomide: J15.14
Lenticonus: P11.017-S
Lenz-Majewski Syndrome: C16.2
LEOPARD syndrome: J05.21,
P11.092-M, P11.121-S
LEP PPARG2: J09.63
LEPR gene mutations: J03.17
leptin gene promotor region: J17.33
leptin: J12.025
Leptin-melanocortin pathway:
P06.31-S
Leri-Weil Dyschondrosteosis: J04.16
Lri-Weill dyschondrosteosis:
P04.57-S
Leri-Weill syndrome: P01.045-S,
P11.132-M
let-7c: P01.054-M
letter: EP37-S
Leukaemia: J14.16
leukemia: J07.21, J17.34, P11.071-S,
P12.076-M, P14.41-S
Leukocyte adhesion deficiency:
P07.19-S
leukodystrophy with vanishing white
matter: P09.071-S
Leukodystrophy: P09.001-S,
P09.036-M
Leukoencephalopathy: C15.6, J17.32
Levo-dopa: P09.112-M
Lewy Body Dementia: P09.061-S
LFS: P12.128-M
LGMD: P10.02-M, P10.16-M,
P10.17-S
LHX1: P16.58-M
Li Fraumeni Syndrome: EP23-S
library: P14.54-M
lifestyle risk factors: EP26-M
Li-Fraumeni syndrome: P12.077-S
Li-Fraumeni: P12.050-M
limb defects: J11.23
Limb girdle muscular dystrophy:
P10.19-S
limb reduction defect: P04.32-M
LIM-domani protein: P10.18-M
limiting PCR: P14.22-M
linear mixed model: C11.3
linkage analysis: J02.19, J09.16,
J13.15, P09.072-M, P17.71-S
linkage: P09.049-S, P09.050-M
lipid metabolism: J01.15, J17.23
Lipidomics: S18.3
Lipids traits: P17.31-S
lipids: J06.16
Lipodystrophy: J04.19
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526
microsatellite: J01.53
microsatellites: P01.003-S
microsattlelite: J06.10
Microtia: P02.47-S
microvesicle: P01.044-M
MID1: P11.111-S
MIDD: P06.30-M
mid-intronic mutation: J10.09
MIF: P17.48-M
MIGLUSTAT: J09.38, P09.103-S
Migraine: J09.28, J09.29, J17.76,
P16.76-M
milk allergy: J06.09
MILLER-DIEKER: J09.30
Milroy syndrome: J05.19
mind: J18.16
Minimal Residual Disease: J07.01
minors: P18.37-S
miR-137 gene: J03.14
miR-15/107: P09.006-M
miR-150: J15.15
miR-155: P15.08-M
miR-181 family: J15.18
MIR184: J02.08
miR196a2: P01.100-M
miR-22: J15.19
mir-23: P09.070-M
miRNA polymorphism: P17.53-S
miRNA: J12.080, J15.11, P01.057-S,
P05.50-M, P09.091-S, P12.070-M,
P12.091-S
miRNA-433: J09.68
miscarriage: J01.40, J01.69,
P01.031-S
miscarriages: J01.86
MiSeq: J05.17
Mismatch repair system: P12.093-S
missense mutations: P05.56-M
MITF: P12.087-S
mitochondria: C12.5, P05.25-S
mitochondrial alanine tRNA: C15.6
Mitochondrial anomalies: P09.013-S
mitochondrial complex I deficit:
P06.33-S
mitochondrial diabetes: J06.14
mitochondrial disease: P06.09-S
mitochondrial disfunction: J06.28
mitochondrial disorders: C17.2
Mitochondrial DNA: J12.081, J17.66,
P01.049-S, P09.058-M
mitochondrial gene: P01.107-S
mitochondrial mutations: J06.24,
P12.094-M
Mitochondrial Myopathy: J06.21
mitochondrial: P06.21-S
MKS1: J14.19, P11.081-S
MLCRD: P05.51-S
mlh1: ES4.1, J12.120, P12.083-S
MLH3 C2531T: J01.42
MLL2 gene: P11.083-S
MLL2: P09.082-M, P11.086-M
MLPA, aCGH: J08.12
MLPA, IHC, FISH, RT-PCR: J12.104
MLPA: J08.19, J10.07, J10.08, J11.12,
J11.20, J12.085, P02.44-M, P03.01-S,
P04.57-S, P08.50-M, P10.10-M,
P11.006-M, P11.049-S, P11.057-S,
P13.28-M, P17.41-S
Mmachc: P06.08-M
MMIHS: P11.098-M
MMP: J15.06
MMP3 rs679620 variation: J04.04
MMP9 gene: J17.08
MMP9 mRNA expression: J12.119
MMPs and TIMP3: J12.012
MMR mutation: P18.23-S
MNGIE: C17.3
Moderate degree of Hearing Loss:
P02.23-S
modifier genes: P09.126-M
MODY: P03.16-M, P03.27-S
Molar pregnancy: P01.123-S
Moldavian: J17.01
molecular and in silico analyses:
P13.15-S
molecular basis of disease: S04.3
Molecular Combing: P14.37-S
Back to index
mt-tRNA: P12.094-M
MUC1: P03.25-S
Mucolipidosis II: J06.15
mucopolysaccharide metabolism:
J17.19
Mucopolysaccharidosis type II:
P06.37-S, P06.61-S
MUCOPOLYSACCHARIDOSIS:
P06.34-M, P06.36-M
mucopolysacharidosis: P06.38-M
Muir-Torre syndrome: J12.073
Mllerian aplasia: P16.58-M
multicenter database: P12.012-M
multicystic: P03.23-S
multidisciplinary assessment:
P05.27-S
multidisciplinary team: J01.60
Multidrug resistance: J15.20
multifactorial disease: J02.09,
P04.56-M
Multi-family discussion group: EPL6.1
Multi-infarct dementia: P09.084-M
multi-locus defects: P16.47-S
Multilocus methylation defects at
imprinted genes: J01.31
Multilocus Methylation Defects:
P16.07-S
multi-phenotype associations:
P06.05-S
multiple anomalies: J11.05
multiple congenital anomalies
syndrome: C16.3
multiple congenital anomalies:
P11.043-S
Multiple Endocrine Neoplasia type 2A:
J12.083
Multiple Endocrine Neoplasia:
P18.30-M
multiple epiphyseal dysplasia: J04.21
multiple fetal malformations:
P01.073-S
multiple malformation: J11.04
multiple myeloma: J12.084
multiple primary malignancies:
P12.025-S
Multiple primary tumours: P12.097-S
multiple pterygium: J11.33
multiple scelerosis: P17.54-M
Multiple sclerosis: C02.1, J09.31,
J17.37, P09.085-S, P09.086-M,
P09.087-S, P09.088-M, P09.101-S,
P15.26-M, P15.27-S, P16.59-S,
P17.55-S
Multiple Testing: P17.32-M
Multiplex Families: P17.56-M
Multiplex HRMA: J15.09
multiplex hyperlipidemia: J06.16
mUPD7: J18.13
Muscle wasting: P10.24-M
muscular distrophy: P10.26-M
muscular dystrophy limb girdle: J10.02
muscular dystrophy: J10.04, J10.05,
P10.40-M, S16.2
MUSK: P10.14-M
Mutation analysis: P04.67-S,
P05.18-M, P06.44-M
mutation records: P17.18-M
mutation screening: P14.50-M
mutation signature: S10.2
Mutation spectrum: P03.17-S
mutation update: P04.18-M
mutation verification: P16.70-M
mutation: C04.4, C19.2, J01.06,
J01.84, J03.02, J04.05, J05.25,
J06.19, J09.33, J11.34, J12.077,
J12.083, J13.09, J17.69, P02.35-S,
P03.03-S, P03.07-S, P05.51-S,
P06.38-M, P06.42-M, P08.25-S,
P08.80-M, P09.118-M, P09.150-M,
P10.21-S, P10.42-M, P11.077-S,
P12.027-S, P14.42-M
mutational signatures: PL5.1
mutations of PAH gene: J08.20
mutations: J02.07, J02.17, J04.29,
J04.40, J10.07, J12.082, P02.25-S,
P05.67-S, P14.87-S, P17.30-M,
P17.61-S, S12.3
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528
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Pheochromocytoma: P12.110-M
phetal phenotype: J01.09
PHF6: P11.070-M
PHGDH gene: J12.092
Philadelphia Chromosome: J12.038
phosphatemic disorders: P06.18-M
Phosphatidylinositol-3-kinase/AKT/
mTOR: S07.3
Phospholipid metabolism: S18.3
Phospholipids metabolism: C16.2
photoperiod: P17.17-S
PHOX2B: P09.032-M, P09.033-S,
P12.111-S, P12.112-M
Physical Activity: J03.17
physical performance: J17.49
PI3K: S07.3
PI3K-AKT-mTOR signaling: P11.117-S
PIAI/PIA2 polymorphism: J05.18
Pierre Robin sequence: P11.116-M
Pierre-Robin Sequence: J04.28
PIEZO2: P11.041-S
pigmentation: P04.51-S
PIK3CA mutations: J12.093
PIK3CA: P04.52-M, P11.038-M,
P12.113-S
PIK3CA-related segmental overgrowth:
P11.117-S
PIK3R1 gene: P03.40-M
pitched voice: P08.41-S
Pitfalls: P16.61-S
Pitt-Hopkins Syndrome: J11.39,
P08.58-M, P11.118-M
Pituitary tumors: J12.006
pituitary: J03.19
PKAN: J09.47
PKD1: P03.34-M
PKD1gene: P03.07-S
PKD2: P03.34-M
PKHD1: P03.08-M, P11.100-M
PKP2: P05.11-S
PKU patients: J06.11
PKU: J06.20, P11.113-S
PLA2G6: P09.117-S
PLAN: P09.117-S
Planar Cell Polarity Pathway (PCP):
C18.4
plaques: P05.14-M
plasma cell differentiation: P07.25-S
plasma cell leukemia: J12.084
PLASMA CELL MYELOMA: J12.076
Plasma DNA: P12.114-M
plasma: J12.080
plasmid: P14.07-S
Plasminogen Activator Inhibitor:
J09.29
Plasminogen: J05.25
plastin 3: C10.2
Plateau iris: P02.32-M
Platelet concentrates: P17.79-S
Platelet endothelial cell adhesion
molecule-1: J05.10
platelet: P07.13-S
Platelets: ES1.2
platinum resistance: P12.104-M
PlGF: P01.079-S
PLOD1: J04.09
PLP1 gene: P11.119-S
PLS3: C10.1
PML/RARA fusion gene: J15.17
PML-RAR: J12.004
PMS2: J17.47, P12.081-S, P12.115-S
PNPLA1: J04.42
PNPLA2: P06.40-M, P06.41-S
PNPLA6: P06.04-M
PNPT1: P02.04-M
POAG: P02.33-S
podocyte genes: C02.2
POF: J01.47
Poikiloderma with Neutropenia:
P04.66-M
poikiloderma: J04.29
Poland Syndrome: P11.120-M
POLD1 gene: P11.094-M
POLD1: P12.116-M
POLE: P12.117-S
POLG: J06.21
POLG1: P06.49-S
Back to index
Q
Q104X: P07.09-S
Q56K: P07.09-S
QF_PCR: P01.061-S
QF-PCR: J01.53, J01.54, P01.092-M,
P13.39-S
qualitative method: P18.45-S
qualitative methods: EES2.1
qualitative research: P18.05-S
qualitative study: EPL8.2
Qualitative: EP38-M, EPL6.5
Quality Control: C11.4, P14.74-M
quality issues: EPL9.3
Quality Management: P14.30-S
quality of life: EP41-S, J03.06, J03.21,
J18.18
quality standards: P14.69-S
quality: P18.33-S, P18.38-M
Qualoty of clinical genetics services:
EP44-M
Quantification: J14.16
Quantitative genetics: P17.82-M
quantitative methods: EES2.1
quantitative trait prediction: P17.69-S
questionnaires: P18.19-S
R
R202Q: J17.22
RAD21: P03.12-M
RAD50: J12.098
Radiation: P01.093-S
Radiofrequency Waves: P01.060-M
Radiotherapy: P15.31-S
Random Aneuploidy: J13.13
random inbreeding: J17.31
randomised controlled trial: EPL6.2
randomized trial: C02.3
RANK gene: J17.08
rare BRAF variants: P12.121-S
Rare coding variants: C14.5
rare disease: PL2.3
Rare Diseases Day: P18.40-M
Rare diseases: P18.04-M, P18.39-S,
P18.42-M
rare disorders: J11.14
rare genomic deletion/duplication:
P11.123-S
rare microdeletion: J01.09
Rare Variant association tests:
P17.72-M
rare variant: P17.70-M, P17.71-S
rare variants: P05.49-S, P09.035-S,
P17.51-S, P17.59-S, P17.80-M
Rare-disease: P16.69-S
RAS signaling: PL1.1
RAS-MAPK: P18.32-M
RASopathies: P05.56-M, P09.107-S,
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