Human Genome and Diseases

MEETI NG ABSTRACTS Open Access
Proceedings of the International Conference on

Human Genetics and 39th Annual Meeting of
Indian Society of Human Genetics
Ahmadabad, India. 23-25 January 2013
Published: 21 January 2014
These abstracts are available online at http://www.molecularcytogenetics.org/supplement/7/S1
I NTRODUCTI ON
A1
Editorial
Jayesh Sheth
FRIGEs Institute of Human Genetics, FRIGE House, Satellite, Ahmedabad, India
Molecular Cytogenetics 2014, 7(Suppl 1):A1
The International Conference on Human Genetics and 39
th
Annual Meeting
of the Indian Society of Human Genetics organized from January 23-25,
2014 is an effort by the Foundation for Research in Genetics and
Endocrinology (FRIGE), Ahmedabad and the Institute of Life Sciences,
Ahmedabad University.
With the emergence of newer technologies and expansion of scientific
frontiers, the scope of genetics and allied sciences has crossed several
boundaries and has opened several applications in medical diagnostics and
treatment strategies. Human genetics and in particular molecular genetics has
demonstrated a significant contribution in prediction and detection of genetic
diseases (prenatal diagnostics) with advancement of techniques like Array-
CGH, FISH and many others. For instance, microarray-based comparative
genomic hybridization (array CGH) is a revolutionary platform that has been
developed to screen entire genome for copy number variations (CNV) and can
be used as the first line investigation modality in cases of non-syndromic
mental retardation, unexplained developmental delay (DD), intellectual
disability (ID), autism spectrum disorders (ASD) and multiple congenital
anomalies (MCA). It can also be used for molecular characterization, to size the
abnormality and study the gene content etc. In the field of cancer, several
genes have been identified as the target for the treatment and cure.
MicroRNAs (miRNAs) is yet another exciting advancement in the genetics. It
may play an important role in regulation of expression of genes involved in
cell competition at the post-transcriptional level. In silico screening of miRNAs
involved in a cell competition is an effort to identify potential miRNAs and to
reduce, economize and expedite experimental work. Under these
circumstances identification of few novel and functional genes in different
population can play an important role to define the future strategies for the
diagnosis and treatment of various diseases. Further, many studies are
emerging such that nutrition and certain micronutrients such as Vitamin B
12
,
Vitamin D, folic acid etc. may have influence gene expression and genetic
make-up. Therefore, the theme of the conference was chosen as Healthy
Genes - Healthy Life. The presentations planned and research papers received
are in conformity to this theme. There is a fair mix of papers with clinical and
basic topics to be presented and included in this issue of the journal. It
includes a vast area from basics of genetics to the complex of array CGH, SNP
arrays and Next generation sequencing; prenatal diagnosis, pre-implantation
genetics, epigenomics, pharmacogenomics, inborn errors of metabolic
disorders, storage disorders, latest from the human variome project, point of
care medicine and nanotechnology. We feel honoured and privileged to edit
this special issue of Molecular Cytogenetics that highlights the genomic
science presentations during this conference.
The Indian Society of Human Genetics (ISHG) imparts knowledge related
to Human genomics through annual meeting every year in January at
different places of the country to encourage young students in
biomedical science and invite learned scientists for the plenary talks.
FRIGE is a nationally recognised organization with national and
international alliances for research in human genetics. It is involved in
carrying out basic and translation research in Human Genetics and
Endocrinology with a motto of services to the society and imparting
knowledge to the young students and researchers.
Institute of Life Sciences is a part of the School of Science and Technology,
Ahmedabad University with a motto of Nurturing Science, Knowledge and
Innovation. The vision of the Institute is to undertake world class research
in nano biotechnology leading to affordable health care to enrich human
and environmental health. It has already forged alliances with national and
international academic organisations as well as industries.
We wish that the readers find these abstracts very interesting for their
future research work.
Jayesh Sheth
Alok Dhawan
Frenny Sheth
Ramesh K. Goyal
Sanjay Singh
Acknowledgements: The Organizers wish to put on record their sincere
appreciation for the generous financial support provided by the Department
of Science & Technology (Government of Gujarat), Gujarat State Biotech-
nology Mission, Department of Science & Technology (Government of India),
Department of Biotechnology (Government of India), Indian Council of
Medical Research, Council of Scientific and Industrial Research and other
industries like GenzymeSanofi India, Zydus Cadila and many others as an
exhibitors.
International Conference on Human Genetics and 39
th
Annual
Meeting of the Indian Society of Human Genetics
23
rd
25
th
January, 2014
Ahmedabad, India
Molecular Cytogenetics 2014, Volume 7 Suppl 1
http://www.molecularcytogenetics.org/supplement/7/S1
2014 various authors, licensee BioMed Central Ltd. All articles published in this supplement are distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
HUMAN GENOME / VARIOM PROJECT
I1
Human variome project current overview
Richard Cotton
1,2
1
Human Variome Project International Limited, Melbourne, Australia;
2
Department of Pathology, The University of Melbourne, Australia
E-mail: cotton@unimelb.edu.au
Molecular Cytogenetics 2014, 7(Suppl 1):I1
The Human Variome Project (HVP) was initiated in 2006 to foster discussion
around how patient outcomes could be improved by connecting and
promoting the disparate work of genetic variation database curators. In
the six years that have passed since that meeting, the concept of the
Human Variome Project has evolved and matured. The challenges and
complexities that face the field of human genetics, form a constantly
changing technology landscape. The influx of raw data that those changes
bring, has necessitated a shift away from the Projects initial passive stance
a forum for the sharing of ideas and collaborationto a more active
initiative that actively engages with partners and stakeholders to establish
and maintain the standards, systems and infrastructure that will enable the
global collection and sharing of genetic variation information to be
integrated into routine clinical practice. The Project is now well
established, with over 900 HVP consortium members from 72 countries, 16
countries officially developing HVP country nodes, 6 major disease groups
developing databases and over 140 databases having joined the Gene/
Disease Specific Advisory Council.
With the Project Roadmap 2010-2012 nearing completion, the establishment
of a new committee structure to govern the scientific aspects of the Projects
activities and the incorporation of a legal entity, Human Variome Project
International Limited, to act as the Projects International Coordinating Office
outlined in the Roadmap are now in place. Most importantly, the Project
Roadmap 2010-2012 proposed a strategy that would ultimately ensure the
collection of all genetic variation information worldwidethe One Project,
Two Channels, Multiple Locations Strategy. This strategy, the next Project
Roadmap 2012-2016 and progress will be outlined in the presentation.
I2
3D facial phenotyping
Peter Hammond
UCL Institute of Child Health, London, UK
E-mail: p.hammond@ucl.ac.uk
The recognition of facial dysmorphism remains an important skill for clinical
geneticists to acquire despite the expanding use of next generation
sequencing tools for the identification of gene mutations and related
anomalies. Phenotype-genotype correlations, for example, still have an
important role to play in diagnosis and prognosis. Facial morphology can
now be quickly captured in 3D and analysed with the support of
appropriate computer software. This talk will describe how: 3D images of
affected individuals can be combined to delineate characteristic facial
features of genetic and related conditions; static and dynamic visualisations
can help a clinician to identify what is atypical in an individual childs
craniofacial development; quantitative analysis of face shape can assist with
diagnosis and the study of phenotype-genotype correlations; links can be
established between facial dysmorphism and neuro-cognitive disability
arising from genetic anomaly and teratogen exposure.
SESSION II: COMPLEX DISEASE: DIABETES
& CARDIOVASCULAR DISORDERS
I3
Prakruti genomics and prameha-proclivity: relevance to metabolic
syndrome
Ashok DB Vaidya
ICMR-Advanced Centre of Reverse Pharmacology in Traditional Medicine,
Kasturba Health Society, Mumbai, India
E-mail: vaidya.rama@gmail.com
Ayurveda, an art-science of a healthy way of life and a system of
medicine, is being practiced in India for several millennia. Despite the
remarkable description of diabetes (Madhumeha) and its precondition
(Prameha), in Ayurveda, the current biomedical sciences stay generally
uninformed of its rich concepts.
The proclivity factors for Prameha are: Garbhakala Asatmyata (intrauterine
influence), Shaithilya (poor muscle and adipose firmness), Meda asarata
(dysfunctional adipocytes), Madhur Agraha (sweet tooth), Kapha chaya
(accumulation of sero-mucoids), Kapha prakopa (aggravation of biofilms),
Mansavahasrotadushti (hepatomuscular infiltration), Meda Vriddhi
(adiposity), Avyayama (distaste for exertion), Divasvapna (daytime sleep),
Medovahasrotadushti (adipose tissue inflammation), Kleda Dushti (excess
of extracellular fluid) etc. The relative significance of each of these factors
will depend on the individuals Prakruti. Genetic determinants of these
factors have to be investigated.
The profiling of Prakruti in patients with diabetes has been studied by
several groups. In a study from the Central Research Institute (Ayurveda),
Jaipur, the frequency of kapha/ with vata/pitta was much more (64 %) than
pure pitta/kapha/with vata (36 %). The response to the treatment with
Nisha amalaki was also better in patients of kapha prakruti. The other study
from the Banaras Hindu University showed a strong correlation of blood
glucose response to exercise with prakruti. In another study from BHU, an
association was found between the type 2 diabetes mellitus and 5,
10-methylene tetrafolate reductase MTHFR C677T. The CT genotype is
protective. The study did not show any association of genotypes with the
prakruti. MTHFR C677T gene polymorphism, common in the Chinese
population, presents a genetic risk factor for diabetic nephropathy in type
2 diabetic patients. So there is also a need to consider Prakruti genomics
for assessing the risk, onset and severity of complications in diabetes.
Reverse Pharmacolgy coupled with Ayurgenomics can be applied to detect
and measure the variation in drug response correlates vis--vis Prakruti.
In the development of obesity, variants of more than three dozen genes
are identified. For the type 2 diabetes, there are estimates of around
hundred genes being involved; most of them are not identified at present.
But it is being realized that it would be too simplistic to expect genomics
to provide all the answers. Besides proteomics and metabolomics, we
need a paradigm shift to consider the problem of proclivity to diabetes
from the perspective of human biology at the bedside. Prakruti, Pathya
and Ahar-Vihar of Ayurveda offer such bedside opportunities. These
interactions, at present, are formidable challenges to the dominant
reductionist paradigms in genomics and systems biology. However,
personalized prevention and management of the metabolic syndrome can
influence its resolution or progression to diabetes mellitus if one can
identify the sets and subsets of prakruti with objective clusters of genomic,
proteomic or metabolic markers; this would emerge by research at the
interface of Ayurveda and Modern Biomedicine.
I4
Next generation diagnostics on cardiomyopathy
Jean-Louis Blouin
1,2*
, Jeremy Bevillard
2
, Periklis Makrythanasis
2
,
Michel Guipponi
1,2
, Federico Santoni
2
, Stylianos E. Antonarakis
1,2
,
Siv Fokstuen
1
1
Genetic Medicine, University Hospitals of Geneva, Switzerland;
2
Genetic
Medicine and Development, University of Geneva School of Medicine,
Switzerland
E-mail: jean-louis.blouin@unige.ch
Cardiomyopathies are common, seemingly monogenic autosomal
dominant cardiac disorders known as the primary cause of sudden cardiac
death in young adults. These diseases are characterized by a remarkable
genetic heterogeneity, which makes it difficult to unravel the causative
mutation in a diagnostic laboratory that is very laborious and expensive by
Sanger sequencing.
To circumvent these limitations, we explored solutions of high throughput
sequencing of targeted exomes with the aim to implement this approach
in routine diagnostics. As a first test we designed a capture microarray
with the total genomic length of 1 Mbp that includes all exons/splicing
sites of 130 genes involved in cardiovascular mendelian disorders and
analyzed simultaneously four samples by multiplexing patients with
cardiomyopathies or Long-QT syndrome. Pathogenic mutations and
variants of unknown significance were found thus resolving the genetic
Page 2 of 59
causes of the cardiopathy in three. In the fourth patient the mutation
usually associated with hypertrophic cardiomyopathy was found with
Long-QT. Further developments to next generation diagnostics are now in
progress, and will be also discussed.
In conclusion, high throughput sequencing holds considerable promises
for molecular diagnosis of highly heterogeneous disorders in clinical
practice and allows a better understanding of the complexity of
mendelian disorders.
I5
Genetics of cardiovascular disorders: influence of maternal nutrition
Sukhinder K Cheema
Department of Biochemistry, Memorial University of Newfoundland, St.
Johns, Canada
E-mail: skaur@mun.ca
Cardiovascular disease (CVD) is the number one non-communicable disease
of the world. According to the World Health Organization, 30% of global
deaths in 2008 were caused by CVD, and it is estimated that by 2030, more
than 23 million people will die annually from CVD. Unfortunately, India will
be the leading country of the world to have the highest rate of CVD by
2030. Genetics, as well as diet and life style are the predominant factors
predisposing the population to an increased risk of CVD. Although genetic
makeup is generally considered as the culprit, recent phenomenon of
Developmental origins of health and diseases or in-utero programming
places significant importance on maternal diet in predisposing the next
generation to metabolic disorders. According to this phenomenon, the diet
of the mother during gestation and lactation affects the genetic makeup of
the growing foetus, thereby causing perturbations in the metabolic
regulations of the offspring, and later onset of diseases. Dietary fats
are known to be associated with an increased risk of CVD; however
the importance of maternal dietary fats in in-utero programming and the
onset of CVD in the offspring in later life is not clear. We have shown that a
maternal diet high in saturated fatty acids (SFA) increased the levels of
low-density lipoprotein (LDL) cholesterol of the offspring by inhibiting
hepatic LDL-receptor gene expression. Furthermore, maternal high fat diets
caused aortic endothelial dysfunction, which is an additional factor
associated with CVD. High fat diets during gestation and lactation also
induce fatty liver and myocardium hypertrophy, due to inhibition of beta-
oxidation through peroxisome proliferator activated receptors (PPARs). We
found that maternal high fat diets altered the gene expression of PPARs,
which likely involves epigenetic modifications. On the other hand, we found
that maternal diets high in omega (n)-3 PUFA reduced plasma lipid levels of
the offspring, thereby reducing the risk of CVD. Our findings have
established the importance of maternal dietary fat intake during gestation
and lactation to prevent the onset of CVD in the next generation. The Indian
population is predisposed to an increased risk of CVD due to their genetic
makeup. It is therefore recommended that the Indian population, especially
women in their child bearing years, should manage the intake of dietary
fat- both the quantity and the quality, to prevent the onset of CVD and
other metabolic disorders in the next generation.
SESSI ON I I I : CANCER GENETI CS
I6
Affordable diagnosis and prevention of genetic disease
John Burn
Institute of Genetic Medicine, Newcastle University, International Centre for
Life, Newcastle upon Tyne, UK
E-mail: john.burn@newcastle.ac.uk
Pathogenic variants in around 100 genes are responsible for a high risk of
early onset solid tumours in close to 1% of the worlds population. As
sequencing costs plummet and effective drugs are targeted at the
molecular structure of cancers, many more of these gene carriers will be
identified. They are a target population for low cost prevention strategies in
their own right. An added benefit is that similar molecular pathways are
disrupted in sporadic tumours, so effective chemoprevention strategies can
be extrapolated to the general population. The Cancer Prevention
Programme, CaPP, was developed 25 years ago to promote genetically
targeted cancer prevention trials. CAPP2, published in 2011 (Burn et al
Lancet) was the first trial with cancer as an endpoint to prove that regular
aspirin significantly reduced the burden of colorectal and other cancers in
people at risk of Lynch syndrome due to a defective mismatch repair gene.
600mg daily (2 tablets) for 2 years or more than halved the cancer rate after
a lag of five years. Long term follow up of the other trial which tested
cancer as a cancer preventive, the Womens Health Study, has now shown a
protective effect of very low dose aspirin taken on alternate days, but not
until 10 years from the trial start (Cook et al 2013). CaPP3 starts in 2014 and
will test the relative benefits of three different blinded doses of enteric
coated aspirin -600mg, 300mg and 100mg in 3000 gene carriers. Failure of
mismatch repair leads to the generation of frameshift peptides due to
unrepaired slippage in repetitive DNA stretches (microsatellites). Antibodies
to the resulting frame shift peptides will be tested as possible biomarkers.
A vaccine raised against the important frame shift peptides affecting genes
involved in carcinogenesis is also under investigation.
Our novel nanowire technology is being investigated as a means of
identifying microsatellite instability and BRAF mutations which help separate
sporadic from hereditary tumours. This will make case identification much
cheaper and easier in the context of countries with a developing economy.
I7
An empirical assay for assessing genomic sensitivity and for
improving cancer diagnostics
Diana Anderson
University of Bradford, Richmond Road, Bradford BD7 1DP, UK
E-mail: d.anderson1@bradford.ac.uk
Detection tests have been developed for many cancers, but there is no
single test to identify cancer in general. We have developed such an assay.
In this modified patented Comet assay, we investigated lymphocytes of
208 individuals: 20 melanoma, 34 colon cancer, 4 lung cancer patients,
18 suspect melanoma, 28 polyposis, 10 COPD patients and 94 healthy
volunteers. The natural logarithm of the Olive tail moment was plotted for
exposure to UVA through different agar depths for each of the above
groups and analyzed using a repeated measures regression model.
Response patterns for cancer patients formed a plateau after treating with
UVA where intensity varied with different agar depths. In comparison,
response patterns for healthy individuals returned towards control values
and for pre/suspected cancers, were intermediate with less of a plateau.
All cancers tested exhibited comparable responses. Analyses of Receiver
Operating Characteristic curves, of mean log Olive tail moments, for all
cancers plus pre/suspected-cancer versus controls gave a value for the
area under the curve of 0.87; for cancer versus pre/suspected-cancer
plus controls the value was 0.89; and for cancer alone versus controls
alone (excluding pre/suspected-cancer), the value was 0.93. By varying
the threshold for test positivity, its sensitivity or specificity can
approach 100% whilst maintaining acceptable complementary
measures. Evidence presented indicates that this modified assay shows
promise as both a stand-alone test and as a possible adjunct to other
investigative procedures, as part of detection programs for a range of
cancers.
I8
Mutational landscape of gingivo-buccal oral cancer: new cancer genes
and molecular subgroups identified
Partha P Majumder
National Institute of Biomedical Genomics, Kalyani, West-Bengal, India
E-mail: ppml@nibmg.ac.in
Gingivo-buccal oral cancer (GBOC), an anatomical and clinical sub-type of
head and neck squamous cell carcinoma (HNSCC), is prevalent in regions
where tobacco-chewing is common. Exome sequencing and other data
on 50 GBOC tumor/normal DNA pairs revealed (a) significantly and
recurrently mutated genes that are (i) specific (USP9X, MLL4, ARID2,
UNC13C and TRPM3), and (ii) shared with HNSCC (e.g., TP53, CDKN2A,
Page 3 of 59
PIK3CA, HRAS, NOTCH1); (b) new genes with recurrent amplifications
(e.g., DROSHA, YAP1) or homozygous deletions (e.g., DDX3X); (c) existence
of molecular sub-types, with distinctive mutational profiles; (d) high
proportion of C>G transversions, not noted earlier in HNSCC, among
tobacco users with high numbers of mutations; and, (e) enrichment of
alterations of pathways specific to GBOC, including Neurotrophin signaling,
Wnt signaling, dorso-ventral axis formation and axon guidance. Recurrently
mutated genes were validated on an independent set of 30 GBOC
patients. These findings open new vistas for biological characterization and
exploration of therapies.
I9
Functional genomics of lung cancer progression reveals mechanism of
metastasis suppressor function
Ram Krishna Thakur
1
, Vinod Kumar Yadav
2
, Akinchan Kumar
1
,
Richa Basundra
1
, Anirban Kar
1
, Rashi Halder
1
, Ankita Singh
1
, Pankaj Kumar
2
,
Aradhita Baral
1
, MJ Mahesh Kumar
3
, Krishnendu Pal
4
, Rajkumar Banerjee
4
,
Shantanu Chowdhury
1,2*
1
Proteomics and Structural Biology Unit;
2
G.N.R. Knowledge Centre for
Genome Informatics, CSIRInstitute of Genomics and Integrative Biology,
Delhi, India;
3
Animal House, CSIR-Centre for Cellular and Molecular Biology,
Uppal Road, Hyderabad, India;
4
Division of Lipid Science and Technology,
CSIR-Indian Institute of Chemical Technology, Hyderabad, India
E-mail: shantanuc@igib.res.in
The mechanism of action of NME2, a widely accepted metastasis-
suppressor gene, is poorly understood. Recently we found that NME2
directly regulates transcription of the c-MYC proto-oncogene. This
prompted a genome-wide study to ascertain whether NME2 exerts its
anti-metastatic action through transcriptional regulation. Chromatin-
immunoprecipitation followed by massively parallel sequencing (ChIPseq)
along with transcriptome profiling uncovered a network of genes involved
in intercellular contact, focal adhesion and actin assembly under direct
transcriptional control of NME2. In line with this, NME2-depleted cells
displayed increased focal adhesion points and altered actin stress fiber
organization. Our findings demonstrate that NME2 regulates transcription
of a key focal adhesion factor vinculin and its localization within adhesion
foci. NME2-depleted A549 lung cancer cells showed higher invasiveness in
vitro and seeded more metastases in vivo. Consistent with these findings,
expression of several NME2-transcriptional target genes related closely to
advanced tumor stages with metastatic proclivity, and NME2 levels
predicted patient survival.
I10
Non-coding rna based regulation of blood vessel development in
zebrafish and relevance to humans
Sridhar Sivasubbu
CSIR-Institute of Genomics and Integrative Biology, Delhi, India
E-mail: sridhar@igib.in
Non-protein coding RNAs (ncRNA) represent a variety of transcripts
having minimal or no protein coding capacity. The ncRNA have been
studied with interest for their function as regulators of gene expression.
Molecular studies on ncRNA have uncovered diverse interactions with
protein coding genes. It has been suggested that ncRNAs are an
additional layer of regulatory switches involved in gene regulation during
development and disease. Detailed studies deciphering the function of
ncRNAs have been limited to a few well-studied candidates. The function
of ncRNAs during vascular development has attracted the interest of our
laboratory. We designed reverse genetics screens to elucidate the role of
miRNAs conserved between human and zebrafish, and potentially
involved in regulating vascular development. In this screen we identified
several miRNAs involved in the development of vasculature. We also
undertook studies to understand transcription factor-miRNA interaction
during vascular development. We extended the study to identify long
non-coding RNAs (lncRNA) in diverse zebrafish tissues including vascular
tissues. We identified several novel lncRNAs from zebrafish tissues. The
functional importance of ncRNAs during zebrafish development will be
presented and their relevance to humans will be discussed.
SESSI ON I V: MOLECULAR
CYTOGENETI CS I N DI SEASE
DI AGNOSI S
I11
Small supernumerary marker chromosomes an update
Thomas Liehr
Jena University Hospital, Friedrich Schiller University, Institute of Human
Genetics, Kollegiengasse 10, D-07743 Jena, Germany
E-mail: i8lith@mti.uni-jena.de
Genotype-phenotype correlations in patients with small supernumerary
marker chromosomes (sSMC) are still difficult to asses.
The presently known influence of chromosomal imbalance induced by
sSMC size and origin, mosaicism of sSMC in different cells of the body
and uniparental disomy (UPD) of sSMCs sister chromosomes on the
clinical outcome is summarized according to data on ~5,000 sSMC cases
summarized on http://www.fish.uniklinikum-jena.de/sSMC.html.
Two third of sSMC carriers are clinically normal. In the remainder 1/3 of
sSMC patients, clinical symptoms may vary between slightly up to severely
affected, including intrauterine death. Besides the known sSMC related
syndromes Pallister-Killian-, isochromosome-15q12-, isochromosome-18p-,
cat-eye- and Emanuel-syndrome there are numerous other yet unnamed
and unidentified sSMC-syndromes. Recently, derivative-8- and derivative-
13/21 syndromes in complex sSMC were reported.
The influence of chromosomal imbalance induced by sSMC size and its
origin seems to have the largest impact on the phenotype of sSMC-
patients. Besides UPD of sSMCs sister chromosomes and mosaicism of
sSMC may be important for the clinical outcome. The latter is especially
important to be predicted in prenatal cases.
Acknowledgments: Supported in parts by Deutsche Forschungsgemeins-
chaft (DFG LI 820/22-1), Else Krner-Fresenius-Stiftung (2011_A42) and the
Deutscher Akademischer Austauschdienst (DAAD).
I12
Molecular cytogenetic characterization of chromosomal
rearrangements - utility in genetic counseling and research
Ashwin Dalal
Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics,
Hyderabad, Andhra Pradesh, India
E-mail: ashwindalal@gmail.com
Chromosomal abnormalities can lead to a number of human genetic
disorders. A large number of chromosomal rearrangements are known to be
associated with a disease phenotype as a result of interrupting or modifying
the expression of gene(s) localized in or close to the breakpoints. Structural
changes in chromosomes can be classified according to cytological types
and their effect on the phenotype. The main structural rearrangements are
translocations, inversions, deletions, insertions, isochromosomes, dicentric
chromosomes and ring chromosomes. Structural rearrangements alter
the genome architechture and may result in human disease phenotypes.
The identification of genes involved in human diseases resulting
from chromosomal rearrangements is important to understand the
pathophysiology of the disease, further it often provides new insights into
normal human development and biology. Chromosomal analysis with
routine methods gives a low resolution of about 5 Mb. Newer techniques
like Fluorescent In Situ Hybridization (FISH), array Comparative Genomic
Hybridization (CGH) have increased the resolution exponentially. These
recent advancements in molecular cytogenetic techniques have made it
possible to characterize the structural chromosomal rearrangements to very
high resolution. Some of these techniques and their application to
characterization of chromosomal rearrangements will be discussed using
different case scenarios.
Page 4 of 59
I13
Microdeletion syndromes
Chaitanya Datar
Sahyadri Genetics, Unit of Sahyadri Hospitals, Barve Memorial Complex, J.M.
Road, Pune, India
E-mail: dr.cdatar@gmail.com
Microdeletion syndromes are a group of disorders characterized by
the deletion of a small chromosomal segment (usually <5 Mb in size)
encompassing multiple disease genes, each potentially contributing to the
disease phenotype independently. The mechanism of disease causation is
usually due to haploinsufficiency of certain critical genes of that region.
The genetic changes of these microdeletion syndromes are often not
detected by the current band resolution using the routine or high
resolution karyotyping (2-5 Mb) but require application of molecular
cytogenetic techniques like Fluorescence in-situ Hybridization (FISH) or the
latest array CGH technique.
FISH is now the standard technique for the diagnosis of common
microdeletion syndromes like Prader Willi syndrome, Angelman syndrome,
Velocardiofacial (DiGeorge) syndrome, William syndrome etc. It is also
possible to diagnose rare syndromes like Wolf Hirschhorn syndrome,
Smith Magenis syndrome etc by FISH if the degree of clinical suspicion is
high. With the advent of chromosomal microarrays, detection of newer
microdeletion syndromes and better characterization of existing syndromes
has become possible.
SESSION V: CURRENT TRENDS IN
PRENATAL SCREENING OF GENETIC
DISORDERS
I14
Non-invasive prenatal diagnosis using massively parallel sequencing -
first experience in germany
Rolf-Dieter Wegner
1,2*
, Markus Stumm
1,2
, Wera Hofmann
3
1
Zentrum fr Prnataldiagniostik & Humangenetik Kudamm 199, Berlin,
Germany;
2
BG Berlin Genetics GmbH, Berlin, Germany;
3
LifeCodexx AG,
Konstanz, Germany
E-mail: wegner@kudamm-199.de
Non-invasive prenatal diagnosis (NIPD) of aneuploidies by cell free fetal DNA
(cff-DNA) from maternal plasma has reached the reliability to be applied in a
clinical setting. It started with an observation that fetal DNA fragments are
present in the blood of pregnant women. One main obstacle to be resolved
had been the low concentration of cff-DNA among maternal DNA fragments,
in general 2 10 % at 10
th
week of pregnancy. This problem was resolved by
the technical development of next generation sequencing applying massively
parallel sequencing of millions of DNA fragments out of a single blood sample
of 10 ml followed by bioinformatic processing of the data. Algorithms for
calculations of z-scores were validated to allow a highly accurate distinction of
pregnancies with an euploid fetus from those with a fetus carrying certain
aneuploidies. With focus on the reliability of results it should be in mind that
the cff-DNA is derived from the cytotrophoblast, e.g. from the placental tissue
which is analyzed in CVS short term culture. Hitherto collected clinical data
show sensitivity and specificity in agreement with a diagnostic test.
In Germany, aneuploidy testing by NIPD started in 2012. Initially, the
approach allowed the detection of trisomy 21. However, meanwhile probing
for trisomy 13, trisomy 18 and the sex chromosome constitution is feasible.
At the moment, the test is indicated for women with singleton pregnancies
at an increased risk for aneuploidies. Blood samples should not be taken
before 9+0 week of pregnancy. However, it is suggested to perform NIPD
only in conjunction with a first trimester ultrasonographic examination for a
profound judging of the fetal situation. For the time being, testing time is
reduced to 10 working days or even less.
In Germany, by now more than 4000 tests had been performed. In Berlin,
data exceeding 250 cases had been collected by BG Berlin Genetics. The
data of NIPD will be discussed in comparison to invasive prenatal
diagnosis.
I15
Basic principles of prenatal screening for aneuploidies
Prakash Gambhir
Birthright Genetic Clinic, Pune, India
E-mail: drprakashgambhir@yahoo.com
Prenatal screening processes are important public health interventions to
counter common genetic disorders. For India prenatal screening against
neural tube defects, thalassemia and Down syndrome is relevant to the
health needs. However, there are many misconceptions regarding the
principles of screening. Many clinicians wrongly consider it as substitute for
prenatal diagnosis. Basically, prenatal screening protocols can be applied on
a wide scale to identify women at risk of bearing a child with the common
genetic disorder in that population.
Prenatal screening for Down syndrome and other chromosomal
abnormalities is a rapidly evolving science. It initially started with offering
prenatal diagnosis to elderly mothers. The identification of low maternal
serum alpha-fetoprotein levels as a marker in Down syndrome led to quest
and recognition of many serum markers and maternal serum markers of
AFP uE3 and hCG were used in combination as triple test and with addition
of Inhibin as the quadruple test. The identification of Nuchal translucency as
a marker in 1
st
trimester led to much desired early risk prediction along with
PaPPA and free beta hCG. It was also found that this test led to early risk
determination and an early prenatal diagnosis with significantly better
detection rate of 85 to 90%. This detection rate is head and shoulders better
than the detection rate for the triple test rate of 65 to 70%.
It has also been noted that combination of these markers can also lead to
risk prediction of other chromosomal abnormalities. Presently risk prediction
is possible with some remarkable softwares for Trisomy 18, Trisomy 13,
Turner syndrome, triploidy, Cornela de Lange syndrome, Smith Lemlie Opitz
syndrome and others. New markers like ADAM 12 with PaPPA will lead to
early prediction of PIH and IUGR as well as prematurity. It is envisaged that
in future maternal serum markers in combination with fetal biometry will
give risk prediction for numerous chromosomal abnormalities many fetal
disorders as well as pregnancy disturbances. Thus prenatal screening has an
important place in antenatal care.
I16
Prenatal screening for mendelian disorders in antenatal care
Amar Verma
Department of Paediatrics & Neonatology,Rajendra institute of Medical
Sciences, Ranchi, Jharkhand, India
E-mail: draverma_2003in@yahoo.com
Antenatal screening for fetal abnormality should be offered to all
women, if available: In all cases of antenatal screening, the woman must
be fully informed and understand the implications of the test, be promptly
advised of their test result and be referred for further management and
definitive diagnosis if their screening test is positive or suggestive of high
risk.
A positive prenatal diagnosis poses many ethical issues and challenging
decisions for parents and clinicians. In those at increased risk of having a
baby with a genetic condition, the risk should be identified and discussed
fully before pregnancy and options for prenatal diagnosis discussed.
Genetic counseling should be provided.
At the present time around 5000 known disorders are inherited in a
monogenetic mendelian fashion. Foremost among them are autosomal
dominant, autosomal recessive and X-linked disorders, which carry a higher
risk of illness than that conveyed by age-related risk. An autosomal
dominant condition carries an a priori 50% inheritance risk where one
parent is affected. An autosomal recessive disease carries a 25% inheritance
risk for children of a healthy carrier couple. An X-linked recessive disorder
carries a 50% risk for the son of a carrier mother.
Specific, albeit non-screening genetic tests are currently available for
more than 1000 of these diseases. Unlike cytogenetic, prenatal diagnosis
based on maternal age, prenatal gene testing is not a screening test.
Given the individuality of each case, prior planning is essential. Two
differing strategies are possible: indirect and direct genetic testing.
Page 5 of 59
The following subsections cover the antenatal screening tests that are
routinely offered like screening for potential for neonatal infection,
Haemolytic disease of new born, Sickle cell disease and Thalassaemia,
Downs syndrome, Fetal anomaly, Measurement of fundal height etc.
The different types of tests like Biochemical, Cytogenetic and Molecular
genetic tests can be carried out by Chorionic villus sampling, Amniocentesis,
Cordocentesis / percutaneous umbilical blood sampling Fetoscopy, Fetal
radiology, Ultrasound-guided percutaneous skin and organ biopsy, Maternal
blood tests, Ultrasound-guided percutaneous skin and organ biopsy and
Preimplantation prenatal diagnosis.
At present, in most cases, accurate prenatal diagnosis requires invasive
testing. There is current research into noninvasive prenatal diagnosis
using PCR and molecular genetic techniques to examine fetal DNA
obtained from maternal blood.
I17
Non-invasive prenatal testing (nipt): a better option for patients
Ashish Fauzdar
Quest Diagnostics India Private Limited, A-17 Info City, Sector 34 , Gurgaon,
Haryana, India
E-mail: aashish.x.fauzdar@questdiagnostics.com
At present there are two methods to determine the chromosome health of
unborn fetus in pregnant women. One is through maternal serum screening
that is considered as simple but less sensitive method and the second
options involves invasive methods like amniocentesis or chorionic villus
sampling (CVS) for women who are found to be positive in maternal serum
screening. Recent studies have demonstrated that non-invasive prenatal
testing (NIPT) using cell-free fetal DNA (cfDNA) present in circulating
maternal blood is considered as one the most effective method of screening
for trisomy 21.
The single nucleotide polymorphism (SNP) based Non-invasive Pre-natal test,
being offered by Quest Diagnostics in technical collaboration with Natera Inc
to determine chromosomal copy number by looking for specific patterns of
(SNPs). This second generation technology analyzes cfDNA in a single
reaction targeting 19,500 single nucleotide polymorphisms which are the
most informative portions of an individuals DNA selected across all the
analyzed chromosomes.
Two published study demonstrated in the women undergone both invasive
chorionic villus samplings followed by karyotyping in conjunction with non-
invasive prenatal testing the efficacy of test. Data from Nicolaides et al., 2013
paper on total of 242 cases that include thirty two cases aneuploidy
included trisomy 21 (n=25) which were correctly identified with 100%
sensitivity and 100% specificity with no false positive and false negative.
Another study by Zimmerman et al., 2012 study on 166 samples from
pregnant women, including 11 trisomy 21, three trisomy 18, two trisomy 13,
two 45, X, and two 47,XXY samples including 146 low risk pregnancies. The
study also correctly reported the chromosome copy number of all five
chromosomes in 145 samples that passed a DNA quality test.
The studies had demonstrated that SNP based NIPT is considered as an
effective method of screening for common chromosomal abnormalities with
detection rate of more than 99% and false positive rate of less than 0.1%.
The initial finding shows NIPT approach has potential to avoid invasive
procedure and can be provided an option to high risk pregnancies i.e. those
with advanced maternal age, screens positive for biochemical screening and
history of pregnancies with previous aneuploidies.
SESSION VI: SINGLE GENE DISORDERS
I18
Molecular diagnosis of genodermatoses in india
Parag Tamhankar
ICMR-Genetic Research Centre, National Institute of Research in Reproductive
Health [NIRRH] Jehangir Merwanji Street, Parel, Mumbai, India
E-mail: tamhankarp@nirrh.res.in
Genodermatoses refer to inherited diseases of skin structure and function.
Several genodermatoses present with multisystem involvement lead to
increased morbidity and mortality. Genetic Research Centre focused on
identifying molecular basis of such dreadful skin diseases with recessive
inheritance. This would help us identify common mutations, founder effects
etc that would reduce the cost of screening patients and their carrier
parents. During the years 2011-13, 100 patients were referred to the centre
with genodermatoses. The commonest group was ichthyosis followed by
epidermolysis bullosa, ectodermal dysplasia, albinism, cutis laxa, progeroid
conditions, precancerous conditions xeroderma pigmentosum, Rothmund
Thomson syndrome, dyskeratosis congenita. Genetic heterogeneity is very
common and molecular diagnosis requires an extensive effort. Recurrent
mutations in unrelated families were seen in families with xeroderma,
Griscelli. Prenatal diagnosis could be provided for ichthyosis, infantile
hyalinosis and progeria. This is the largest cohort of mutation proven
patients with genodermatoses from India.
SESSION VII: GENETIC BASIS OF
REPRODUCTIVE DISORDERS
I19
Genetics of male infertility: Indian scenario
K Thangaraj
CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India
E-mail: thangs@ccmb.res.in
According to the recent epidemiological studies, nearly one out of every
10 couples face a problem in conceiving a child. Impaired fertility of male
partner is causative in approximately 50% of all couples unable to conceive
spontaneously. We have been studying the genetic factors associated with
male infertility among Indian population. We have earlier shown that about
8.5% infertility among Indian men is due to the Y chromosome microdeletions.
Further analysis of several autosomal (NR5A1, KLK3, CETN1, DEFB126, CAMK4,
UBE2B, TNP1 & 2 and PRM1, 2 & 3); Y chromosomal (DAZ deletions, TSPY1 copy
number) and mitochondrial genes accounted for additional 19.5% of the
genetic factors responsible for male infertility. However, etiology of a large
proportion (72%) of infertile men still remains unknown. Gene expression
studies from our lab using microarray approach, showed several genes are
many folds down regulated in the testicular tissue of infertile men, compared
to the fertile men. Targeted resequencing of these differentially expressed
genes and functional characterization of the observed mutations is in progress.
In addition, we have recently initiated exome sequencing of idiopathic male
infertile samples to identify additional genetic factors responsible for male
infertility. The results of the findings would be discussed at the time of
presentation.
I20
Fertility options and challenges for patients with cytogenetic infertility
& disorders of sex development
Rama Ashok Vaidya
*
, Jaya Gogte
Milan Polyclinic, 71-B Sarashwati Road, Santacruz (W), Mumbai, India
E-mail: vaidya.rama@gmail.com
Recent advances in assisted reproductive technology (ART), coupled with the
emerging understanding in molecular mechanisms of disorders of sex
development (DSD) and that of associated genetic infertility, have given
hopes for fertility in groups of patients who till recently were denied
biological parenthood.
Heterologous fertility potentiation, in the cytogenetic infertility by donor
oocytes, has been successfully used by us and others. However, homologous
fertility preservation for this group of patients is evolving. Cryopreservation
of either the mature oocytes following ovarian stimulation in post -pubertal
Turner Syndrome (TS) girls or that of ovarian tissue in prepubertal TS
patients are to be applied before actual ovarian failure occurs. Single
embryo-transfer and strict selection criteria and judicious use of this
advanced technology are advocated to minimize maternal morbidity and
mortality.
Microdissection testicular sperm extractions (Micro-TESE) in azoospermic
Klinfelter syndrome patients have resulted not only in successful retrieval
Page 6 of 59
of spermatozoa for intracytoplasmic sperm injection (ICSI) but also in
normal fertilization and clinical pregnancies. Swyers syndrome patients are
reared as females and have female external and internal genitalia.
However, with complete gonadal failure they need Oocyte donation with
ICSI/IVFET for fertility. Those with partial androgen resistance (PAIS) due to
genetic mutation of androgen receptor have a spectrum of abnormality
like hypospadias, micropenis, undescended testes and male infertility.
Though 46xy PAIS males have possibility of fatherhood complete AIS
patients are female phenotype and are raised as females. This category of
patients are encouraged and known to resort to fertilization of donated
oocyte using husbands sperms. Resultant embryos are transferred into
surrogate uterus.
Advances in reproductive medicine in general and those in assisted
reproductive technology in particular have revolutionized diagnosis of
disorders of sex development and the management of the associated
infertility. These advances have certainly provided fertility potentiation but
require judicious application coupled with expert genetic counseling.
SESSI ON VI I I : EPI GENOMI CS
I21
Uniparental disomy - clinical consequences due to imprinting and
activation of recessive genes
Thomas Liehr
Jena University Hospital, Friedrich Schiller University, Institute of Human
Genetics, Kollegiengasse 10, D-07743 Jena, Germany
E-mail: i8lith@mti.uni-jena.de
Uniparental disomy (UPD) is often considered as an event to be
characterized exclusively by molecular genetic or epigenetic approaches.
Still in at least one third of cases UPD emerge in connection with or due to
a chromosomal rearrangement.
Till date ~2,500 UPD cases detected in clinical, non-tumor cases are reported
in the literature (http://www.fish.uniklinikum-jena.de/UPD.html). Based on
this, the presently known imprinting syndromes, chromosomal contribution
to UPD phenomenon, and cytogenetic subgroups of UPD and segmental
UPD are reviewed.
UPD may arise in clinical cases, as well as it may be exclusively tumor
related. For clinical cases imprinting is quantitatively the most important
problem. Still isodisomy may also be a problem, due to activation of
recessive mutation-events, thus inducing rare autosomal recessive disorders.
Overall, as UPD is more but an interesting rarity, the genetic background
of each UPD-patient needs to be characterized besides by molecular
methods, also by molecular cytogenetics in detail.
I22
Epigenetic regulation of double c2 like domain beta (Doc2b) in cervical
cancer
K Satyamoorthy
*
, Samatha Bhat, Harish Rotti, K Shamaprasada
Manipal Life Sciences Center, Manipal University,Manipal, Karnataka, India
E-mail: ksatyamoorthy@manipal.edu
Associations of genetic changes and aneuploidy with tumor growth are
traditionally attributed to alterations in DNA sequence manifested as
mutations, deletions and amplifications. Inactive tumor suppressor genes
could serve as drivers of tumor progression due to not only altered or lack
of protein function but may also contribute to phenotypic changes that
may provide distinct growth advantage in a hostile environment in the
host. Human variation is also due to epigenetic alterations and heritable
change that leads to altered gene expression; the functional consequence
of which may contribute to definitive trait. A number of key regulatory
genes associated with epigenetic silencing due to DNA methylation in
cervical cancer have been reported. Elucidation of differentially methylated
genes may identify new targets and further strengthen our understanding
of molecular mechanism governing pathogenesis of cervical cancer. Thus,
to identify DNA methylation regulated genes in cervical cancer, we have
employed DMH based microarray experiments in pre-malignant and
malignant cervical sample. Microarray data analysis and validation using
bisulfite genomic sequencing lead to the identification of several CpG
island as altered during cervical carcinogenesis and showed the potential
for early screening of cervical cancer. One of the candidate gene identified
was Double C2 like Domain beta (DOC2B), a key calcium regulator protein
whose alteration has never been linked to cancer. We provide evidence
that DOC2B is depressed in cervical cancer due to promoter hyper-
methylation and act as a novel tumor suppressor gene by regulating
multiple pathways in cervical cancer.
I23
In search of epi-driver genes in head and neck cancer
Sumana Bhattacharjya
1
, Kumar Singha Roy
1
, Nitai P Bhattacharyya
2
,
Susanta Roychoudhury
1*
1
Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of
Chemical Biology, Kolkata, India;
2
Crystallography and Molecular Biology
Division, Saha Institute of Nuclear Physics, Kolkata, India
E-mail: susanta@iicb.res.in
Over the last decade, sequencing of a large number of tumour genomes
has identified thousands of mutations in many genes. Among these
mutations that confer selective growth advantage to the tumour cell
are called Mut-driver mutations. Furthermore, it has been proposed that
Epi-drivers are a class of driver genes that are not frequently mutated but
aberrantly-expressed in tumours through epigenetic means. Intriguingly, it
has been stated that the most obvious source of the proverbial dark
matter is in Epi-driver genes and human tumours contain large numbers
of epigenetic changes affecting DNA or chromatin proteins. However, it is
now clear that microRNAs (miRNAs) also have specific epigenetic functions
whereby they recognize and bind to specific mRNA targets to repress their
expressions. Using head and neck cancer tumour model we are trying to
identify such miRNAs and their target Epi-driver genes important in this
cancer. In this quest, we have carried out an in silico investigation of 53
miRNAs known to be deregulated in head and neck squamous cell
carcinoma (HNSCC) and the expression and mutation status of their
experimentally-validated target genes in the disease. Interestingly, our
results have put forward 224 target genes as potential Epi-drivers specific
to HNSCC. How miRNA regulation of mitotic genes could contribute to
the HNSCC development and thus might be considered as potential
Epi-drivers, will be discussed.
I24
Birth defects: etiology to prevention
Koumudi Godbole
Deenanath Mangeshkar Hospital and Research Center, Erandawane, Pune,
India
E-mail: koumudig@rediffmail.com
Structural birth defects together of are a prominent cause of mortality
and morbidity and are gaining importance with improving obstetric care
and reduction in infective causes. Advances in understanding genetic (G)
and environmental (E) factors and their interaction have added newer
dimensions to the etiology of birth defects. Epigenetic is one such
interesting field expected to provide mechanistic links between whether
and how the environment interacts with maternal and fetal genomes.
Some classical examples of environmental insults such as teratogens,
maternal medical disorders and nutritional deficiencies as well as G-E
interactions leading to birth defects will be discussed. It is important that
the relevant scientific information should reach professionals and public
for better awareness, appropriate policy decisions to prevent birth
defects.
I25
Genomic packaging and epigenetic regulation of genes
Rakesh Mishra
CSIR-Center for Cellular and Molecular Biology, Hyderabad, India
E-mail: mishra@ccmb.res.in
Page 7 of 59
Large proportion of the genome in higher eukaryotes does not code for
proteins. This non-coding DNA is emerging as key to the packaging of
the genome in form of chromatin, the functional form of genome, that is
critical for chromosomal organization and gene regulation. It is clear that
the packaging of the genome has regulatory consequences. We, however,
do not know what the packaging code of genome is, that allows as
many packaging options as the number of cell types in the organism.
What is clear is that packaging restricts enhancers/silencers that are
capable of functioning over long distances, to interact with only
appropriate promoters. Boundary elements that define topologically
independent chromatin domains are expected to flank each gene or
gene complex that is differentially regulated. Using comparative genomics,
large scale mapping of epigenetic modification and genetic approaches
we identified a large number of boundary elements by mapping
epigenetic chromatin features across the hox complexes of vertebrates
and use transgenic approaches to functionally analyze them. We find that
the regulatory elements that are involved in the regulation genes by
higher order chromatin structure are conserved across species - from flies
to mouse. Finally, we use powerful genetic approaches in Drosophila
melanogaster to explore the epigenetic mechanisms involved in the
genomic packaging and regulation of genes. Our findings highlight long-
range interactions involved in regulation of genes by means of genomic
packaging in cell type specific manner. We propose that accumulation of
non-coding DNA, including at least some of the repetitive elements, with
the evolution of complexity is then consequence of the regulatory function
embedded in this part of genome.
SESSI ON I X: NEXT GENERATI ON
SEQUENCI NG
I26
Exome sequencing in unspecific intellectual disability and rare
disorders
Anita Rauch
University of Zurich, Institute of Medical Genetics, Schlieren-Zurich,
Switzerland
E-mail: anita.rauch@medgen.uzh.ch
Identification of disease causing mutations in genetically heterogeneous
conditions such as intellectual disability by Sanger sequencing is time-
consuming, costly and often unsuccessful. The advent of NGS techniques is
paving the way for novel large scale approaches with an unforeseen
diagnostic power. However, the plethora of variants of unknown significance
detected by genome-wide approaches requires distinctive strategies to
identify actually disease-related mutations. We recently showed that exome
sequencing of patient-parent trios in sporadic cases of unspecific severe
intellectual disability may unravel disease causing mutation in more than
50% of previously unsolved cases. Thereby it became also evident, that the
current descriptions of phenotypes associated with mutations in a certain
gene, are heavily biased towards certain recognizable patterns. However,
while whole exome sequencing may currently provide theoretically the
highest cost-efficient diagnostic power, it may miss mutations due to
incomplete coverage of certain genes. Therefore in some phenotypes a
clinical exome limited to a set of genes with currently known monogenic
mutations may also be useful.
I27
New paradigms in whole genome sequencing: from lab bench to cell
phone
Jonathan OHalloran
QUANTUMDx GROUP LIMITED, Newcastle, UK
E-mail: jonathan.ohalloran@QuantuMDx.com
The DNA sequencing & MDx fields have seen dramatic advances since the
first draft of the human genome was published, with companies reporting
ever faster and cheaper methods. However, despite the race to attain the
$1000 genome producing a plethora of exciting technologies, capillary
electrophoresis (CE) is still being routinely used for targeted clinical
sequencing and expensive real time PCR devices are still the work-horse of
the MDx laboratory. QuantuMDx is developing a portable, handheld DNA
sequencing device for PGx and infectious disease applications, to provide
an alternative to slow and relatively expensive CE DNA sequencing &
expensive slow and lab based MDx & CDx.
I28
Personal genomes to precision medicine
Vinod Scaria
GN Ramachandran Knowledge Center for Genome Informatics, CSIR-Institute
of Genomics and Integrative Biology, Mathura Road, Delhi, India
E-mail: vinods@igib.in
The decade after the draft Human genome was published has seen
tremendous developments in the technologies that can sequence
genomes of individual Humans in a fast and affordable and accurate way.
Reading 3 billion odd bases which comprise of the Human genome is
today possible in realistic timelines and costs. This improvement was
primarily brought about by the significant advances in the throughput and
technology to sequence DNA and computational methods and resources
to assemble and interpret the data. In addition to the improvements in
technology, the miniaturization of technology that enables sequencing of
human genomes on bench top sequencers has been thought to be one of
the game-changers which could potentially accelerate the widespread
application of genome sequencing. The availability of the reference human
genome sequence has also in the last one decade accelerated the
discovery of human genetic variations and their associations with traits
which has provided a basis of annotating human genomes for predictive/
personalized medicine.
The availability of effective tools, resources and datasets has poised
genome sequencing in a unique position which could see its regular use in
clinical settings. The application of genomics in clinical settings would
primarily help in accurate and evidence based care, which encompass a
new field of medicine called Precision Medicine. Precision medicine
encompasses use of accurate clinical information and evidence to
appropriately manage a patient at an individual level or at a community
level. One of the major challenges which preclude the widespread
application of genome sequences in clinical settings is the lack of know-
how and expertise in analyzing and interpreting genome data in clinical
settings. This would require a new breed of clinicians who have good
clinical acumen, and are equally well versed with genomics and
computational tools and methodologies.
Towards accelerating the implementation of Precision medicine we have
established a pipeline for human genome/exome sequencing and analysis
in our laboratory and have developed a gamut of resources and tools for
discovery, modeling and annotation of clinically actionable variants in
genomes including Pharmacogenetic variations. In addition, we have
implemented a pipeline for validation of functional variations in zebrafish,
a popular vertebrate model system. Case studies and examples would be
detailed during the lecture.
SESSION X: DIAGNOSIS & THERAPEUTIC
APPROACH OF LYSOSOMAL STORAGE
DISORDERS
I29
Enzyme replacement therapy for lysosomal storage disorders in India
Mamta Muranjan
Genetic Division, Depart of Pediatrics, KEM Hospital, Parel, Mumbai-400012,
India
E-mail: muranjanmamta@rediffmail.com
Lysosomal storage disorders (LSD) are a heterogeneous group of inherited
metabolic disorders with a combined frequency of 1 in 5000 affected live
births. The commonest pathogenic mechanism is qualitative or quantitative
deficiency of one of the 50 known lysosomal enzymes (acid hydrolases)
Page 8 of 59
involved in the catabolism of a variety of molecules. The outcome of this
deficiency is progressive accumulation of partially degraded compounds
which stealthily leads to multiorgan dysfunction.
Unlike many inherited metabolic disorders, diet plays no role in the control
of LSD. However treatment in the form of Enzyme replacement therapy
(ERT) is available for a few disorders. The diseases for which ERT is currently
the standard of care are Gaucher disease (Imiglucerase, Velaglucerase,
Taliglucerase), MPS I (Laronidase), MPS II (Idursulfase), Pompe disease
(Alglucosidase alpha), Fabry disease (Agalsidase beta, Agalsidase alfa) and
MPS VI (Galsulfase). These drugs are human recombinant products
manufactured in in-vitro tissue culture systems. ERT alters the natural history
of these diseases and reverses many of the symptoms. However, as the
drugs do not penetrate the blood-brain barrier, there is no impact on CNS
disease.
Gaucher disease was the first LSD to be treated with ERT in India since
1999-2000. Six centres in India located in Mumbai, Delhi, Lucknow and
Chennai are treating patients with ERT.
The major factor limiting widespread access to ERT in the country is the
prohibitive cost, the need to import the drugs and lack of infrastructure for
comprehensive evaluation and supportive care. Access has been facilitated
for Indian patients through a charitable access program (INCAP). A board of
seven National experts and a panel of International experts constitute
the Indian Medical Advisory Board (IMAB) to screen patients for eligibility on
the basis of pre-determined objective criteria. The experts also provide
guidance for evaluation and management of LSD. To date approximately 60
individuals with Gaucher diseases are receiving ERT in India. Some of these
have been receiving ERT for more than 10 years and are young adults
pursuing graduate level academics. Additionally, 20 patients with MPS I, two
with Fabry and 20 with classic infantile Pompe are receiving ERT. ERT is
currently not available for MPS II and MPS VI in India.
Twelve individuals with Gaucher disease (10 with type I) have been
treated with Imiglucerase. Of these, one with severe disease resulting in
hepato-pulmonary syndrome expired and one was transitioned to oral
substrate reduction therapy.
I30
Molecular study of lysosomal storage disorders in India
Jayesh Sheth
FRIGEs Institute of Human Genetics, FRIGE House, Satellite, Ahmedabad,
India
E-mail: jshethad1@gmail.com
Lysosomal storage disorders (LSDs) are gaining greater attention of the
researcher and medical fraternity due to increasing awareness of
occurrence, diagnostic facility, prenatal diagnosis and options for
therapeutic intervention. Though the exact incidence is not known, they
are likely to occur with a ratio ranging from 1:6000-1:8000 with the highest
occurrence of Gaucher disease followed by GM2 gangliosidosis and
mucopolysachharide disorder. However, our knowledge about the
mutation spectrum for most of the LSDs and its phenotype consequences
is limited.
The present study was aimed to identify disease causing mutation for
HEXA and GBA gene and its phenotypic consequences.
Study was carried out in 37 biochemically confirmed subjects having
Tay-Sachs disease and 32 subjects with Gaucher disease. Molecular analysis
was carried out for all exons and exon-intron boundaries of HEXA and GBA
gene by bidirectional sequencing method. In silico analysis was carried out
using SIFT, Polyphen2 and Mutation T@ster softwares.
In HEXA gene, 21 mutations were identified in 34 unrelated families in
Tay-Sachs disease, 15 of which were novel, including 10 missense mutations
[E114K, D175A, T263I, G269R, D322N, D322Y, Q374P, R393P, E462V, G478R]
in 20 families, 4 nonsense mutations [Q237X, W474X, W485X, R510X] in
5 families, one 8 bp deletion [c.898_905TTCATGAG (p.F300HfsX21)] in one
family. Earlier known 6 mutations were also observed that include 2
missense mutations [R170W and R178C] in 2 patients, one 4bp insertion in 5
patients [c.1277_1278insTATC (p.Y427IfsX5)], 3 splice site mutations [c.805+1
G>C, c.459+5 G>A and c.672+30 T>G] in four families. Mutation was not
identified in 3 families. In GBA gene (Gaucher disease) we have identified 10
missense mutations in 32 unrelated families that include 9 known mutations
[E326K, V352M, G355D, S356F, R359Q, R368C, R395C, R463C and L444P] in
31 families and one novel mutation [G289A] in one family. In silico analysis
further confirmed the pathogenic effect of the novel mutations occurred at
highly evolutionarily conserved and functionally active domain residues in
the protein leading to conformational changes or mRNA producing
truncated protein resulting in the diminish or absent activity of the protein.
Present study demonstrated that E462V, D322Y and c.1277_1278insTATC
(p.Y427IfsX5) mutations are the most common mutations observed in
nearly 49% (18/37) of the children with TSD in India. Similarly, L444P
missense mutation is commonly observed in 21/32 (65.62%) children with
Gaucher disease. Additionally, the data also demonstrates that exon 5-12
in HEXA gene and exon 8-10 in GBA gene are the hot spot region where
~75% and 94 % mutations can be identified in Indian patients with the
aforementioned diseases respectively.
SESSI ON XI : ARRAY- CGH
I31
The advantages of SNP arrays over CGH arrays
Boris Keren
Genetic Department, La Piti-Salptrire Hospital, AP-HP, Paris, France
E-mail: boris.keren@psl.aphp.fr
In recent years, with the rapid development of Chromosomal Micro-array
Analysis (CMA), the resolution limit of 5Mb which was imposed by
conventional cytogenetics, has been significantly lowered. Currently, array
CGH is the most widely used CMA technology. With the inclusion of
thousands to millions of probes, it allows the detection of small copy
number variations (CNVs) of a few kb. SNP (Single Nuleotide Polymorphism)
arrays can also be used for CMA. They too enable the detection of CNVs, but
unlike array CGH, each probe is located at an SNP and can determine the
genotype of the corresponding SNP.
Here, we report on our 6 years experience of the use of SNP arrays in
cytogenetic diagnosis on more than 3000 patients and we will focus on
the main benefits of SNP arrays over array CGH. Because of their ability
to perform SNP genotyping, SNP arrays can detect long contiguous
stretches of heterozygosity (LCSH).
LCSH have 2 main interests:
1) they can detect uniparental isodisomies (UPD);
2) they can detect genetic identity by descent.
UPD can be responsible for imprinting disorders and both UPD and identity
by descent is associated withpromote the occurrence of autosomal recessive
disorders. Moreover LCSH analysis allows performing homozygosity
mapping and helping guide sequencing of candidate genes responsible for
recessive conditions. Because of an abnormal number of different alleles,
SNP arrays also enable the detection of polyplody and chimerism. Besides,
SNP arrays also are of interest in quality control: they can detect DNA
contamination and false paternity.
Thus, while array CGH is still a very efficient technique to detect CNVs,
the inclusion of SNP probes in arrays is desirable when possible.
I32
Cytogenetic microarray in prenatal and postnatal diagnosis
Shubha Phadke
Department of Medical Genetics, SGPGIMS, Lucknow, India
E-mail: shubharaophadke@gmail.com
Due to high resolution cytogenetic microarray (CMA) has replaced traditional
karyotyping for evaluation of individuals with intellectual disability, autism
and congenital malformations. The diagnostic yield of CMA is 10 to 12 %
and is more than any other investigation for evaluation of developmental
disabilities. Due to its high resolution CMA is also being used as a prenatal
test for chromosomal anomalies. The diagnostic yield is about 3% whatever
may be the indication. The yield is higher in cases with fetal anomalies. The
main concern with CMA is its ability detection of copy number variations of
unknown significance. This is a major problem in prenatal diagnosis and is a
challenge for the counselor and dilemma for the family in concern. With
accumulation of more and more data of pathological and polymorphic
variations in genome the variations of unknown significance will decrease.
Page 9 of 59
CMA is also useful in delineating abnormalities picked up by karyotyping.
Some cases with double segment rearrangement may point towards familial
chromosomal rearrangement and hence, CMA is indicated in familial cases
with developmental disabilities and birth defects.
Cost and availability of clinical cytogeneticists for appropriate interpretation
of CMA results are important concerns for wider application of the
technique.
I33
Genomic copy number variations in glaucomatous neurodegeneration
Arijit Mukhopadhyay
CSIR-Institute of Genomic and Integrative Biology, New Delhi, India
E-mail: arijit@igib.in
Copy number variation (CNV) is one of the major factors contributing
to genomic diversity and diseases. It has been shown especially for
neurodegenerative diseases that CNVs can play a very important role in
genetic predisposition of the disease. Glaucoma is a major neurodegenerative
disease causing irreversible vision loss across the globe. We wanted to
analyze the impact of CNVs in a genome-wide scale in patients of primary
open angle glaucoma (POAG) collected from the West Bengal state of India
and reproduce our results in another cohort of Caucasian origin. Genome-
wide data was generated on 347 POAG cases and 345 controls on Illumina
660W-Quad arrays and CNVs were called using PennCNV. The CNVs were
classified as small (<100 kb) and large (>100 kb) and analyzed seprately for
their involvement in the disease. A publicly available dataset of POAG cohort
of 624 cases and 404 controls from Caucasian origin (GLAUGEN study) was
used as a validation cohort and genome-wide CNV data of 208 HAPMAP
samples was used as global control. We analyzed genome-wide CNV from
1928 samples. For the large CNVs distribution was significantly skewed
toward larger size (>1 Mb) in cases compared to controls and this was
replicated in the GLAUGEN data. We found that CNVs >1 Mb are enriched for
gene rich regions in POAG patients with 125 genes while for controls a similar
percentage of large CNVs overlapped with only 5 genes. In 208 HAPMAP
samples CNV >1 Mb overlapped with 95 genes. Interestingly, genes found in
the patients were unique and did not overlap with controls or HAPMAP
samples. Within CNVs of >1 Mb gene-rich large deletions were ~2 fold
enriched in patients compared to duplications irrespective of their ethnic
background. Such a bias was not observed in the controls. In the smaller CNV
range we performed association analysis and identifed novel regions to be
under significantly higher CNV in patients comapred to controls. Particularly,
a CNV encompassing the transcription factor FOXE3 was significantly
enriched in patients of both Indian and Caucasian POAG patietns comapred
to their respective controls. A sequence analysis of the gene revealed novel
missense mutation in the patients. We have shown that genomic CNVs >1
Mb has significantly higher burden in POAG patients genome compared to
controls irrespective of the population background. We have also identified
candidate genes/regions which are uniquely present in POAG cases and
absent in controls from all over the world. Our data provide new insights into
role of CNV in pathogenesis of POAG.
I34
Clinical utility and dilemmas of SNP microarray testing
Virginia Kimonis
Division of Genetics and Genomics, Department of Pediatrics, University of
California, Irvine, CA 92868, USA
E-mail: vkimonis@uci.edu
The ability to diagnose patients with developmental delay, intellectual
disability, congenital anomalies, and dysmorphic features has significantly
improved with the introduction of SNP microarray technologies which
includes more than 1.8 million markers on a single array. This high-density
array will allow for sensitively detecting all known abnormalities with defined
loci of interest as well as the discovery of new syndromes as a cause of
mental retardation or autism. The most common micro deletion disorders
include15q11-q13 Prader-Willi/Angelman, 22q11.3 velo-cardiofacial, 17pl3.3
William, 1p36and 16p11.2 microdeletion syndromes. SNP arrays are able to
detect segmental uniparental disomy (UPD) as in Prader Willi syndrome and
UPD14. SNP arrays identify parental consanguinity which may otherwise go
undetected. Long stretches of homozygosity can be analyzed for recessive
genes in patients born to consanguineous parents. (http://www.ccs.miami.
edu/cgi-bin/ROH/ROH_analysis_tool.cgi)
Arrays cannot however identify balanced chromosomal rearrangements,
such as translocations or inversions, Marker chromosomes may also be
missed, depending on the size, marker composition, and array coverage of
the specific chromosomal region present on the marker. Dilemmas arise
when CNVs of uncertain clinical significance are identified, or if a parent
is not available for testing, thus making it difficult to identify if the
rearrangement is inherited or arose de-novo. Further dilemmas arise if a
parent is identified with the same rearrangement and is apparently
asymptomatic, should one then consider the presence of a mutation in a
recessive gene in the other allele to explain autism, mental retardation or
other disorders.
Overall SNP array technology has become the test of choice, permitting a
5.9% detection rate in patients with negative microarray-based CGH and
an overall detection rate approaching 29%.
I35
Detection and Inheritance Pattern of Copy Number Variations (CNVs)
in Children with Multiple Congenital Anomalies
Frenny Sheth
FRIGEs Institute of Human Genetics, FRIGE House, Satellite, Ahmedabad,
India
E-mail: fshethad1@googlemail.com
Microscopically visible chromosomal alterations or segmental aneusomy are
one of the major causes for congenital anomalies and require cytogenetic
investigations. Conventional G-banding analysis is limited to the detection
of imbalances greater than 5-10 Mb. Expressivity of a phenotype generally
correlates with the degree of genetic imbalance, therefore, patients with a
greater genetic imbalance are more likely to be investigated, which creates
a diagnostic bias. This does not reflect the full range of phenotypic
presentation that may be associated with an imbalance of a particular
chromosomal segment. Systematic clinical diagnosis of a rare syndrome can
be carried out using FISH to characterize known deletion/duplication
breakpoints or by screening for known micro-deletion syndromes and sub-
telomeric imbalances where routine banding technique suggests normal
karyotype. In the past decade, array- Comparative Genomic Hybridization
(a-CGH) has made it feasible to detect cryptic imbalances in more number
of cases with apparently normal chromosomal makeup in addition to
characterize structurally rearranged chromosome. The study of inheritance
pattern by a combination of conventional cytogenetic and a-CGH
techniques has helped to determine the imbalance as either de novo or
inherited. a-CGH could also provide information on whether an imbalance is
pathogenic or polymorphic, which could aid in calculating the recurrent risk
for future pregnancies. It provides several advantages over conventional
cytogenetic techniques such as whole genome coverage in a single
experiment with resolution of 20-150 kb, faster turnaround time, higher
sensitivity and specificity and non-requirement of dividing cells. Having an
extensive coverage of the genome and high resolution, this technique has
been pinned as the first line genetic test to study cryptic genetic
imbalances in cases with developmental delay and congenital anomalies,
where G-banded karyotype is normal.
SESSI ON XI I : GENETI CS OF
NEUROBI OLOGY
I36
Neuroferritinopathy: iron in the brain
John Burn
Life, Newcastle upon Tyne, UK
E-mail: john.burn@newcastle.ac.uk
Page 10 of 59
Careful attention to clinical phenotypes can identify new diseases which are
now amenable to molecular genetic elucidation. In the late 1980s I met a
family labelled as having Huntingtons disease but with absence of
dementia. Scanning revealed unusual cavitation of the basal ganglia. The
pedigree was extended my connection to a second family using birth
records. We mapped the gene to chromosome 19 and went on to identify
an unusual mutation in the E helix of light chain ferritin. Staining for iron
revealed huge accumulation of iron/ferritin complexes in the brain leading
to neurodegeneration. We named the condition neuroferritinopathy and
tested desferrioxamine without success. We have shown that accumulation
of iron commences in childhood and are now preparing to test deferiprone
as an iron chelator which crosses the blood brain barrier.
I37
Clinical aspects of neuroregression: our experience on batten disease
Mahesh Kamate
KLES, Belgaum, Karnataka, India
E-mail: drmaheshkamate@gmail.com
Neuroregression in a child is an important clinical problem faced by a
pediatric neurologist. Depending on the initial clinical features they can be
broadly divided into grey matter disorders, white matter disorders or
combined. The age of onset and the progression of symptoms also help us
in further characterization. Involvement of other systems and neuroimaging
findings helps us in formulating the differential diagnosis and guides us in
the laboratory evaluation and selection of appropriate confirmatory tests.
After brief discussion on the clinical approach to neuroregression, here I
would like to present our experience with one of the important
poliodystrophies in children- Neuronal ceroid lipofuscinosis.
Neuronal ceroid lipofuscinosis is a group of progressive neurodegenerative
disorders characterized by accumulation of ceroid lipopigment in lysosomes
in neurons and other cell types. Over a period of four years we have
diagnosed 20 children with neuronal ceroid lipofucinosis. Of the 20 patients,
5 had infantile type and 15 had late-infantile neuronal ceroid lipofuscinosis.
Diagnosis was confirmed by appropriate enzyme assay. Clinical presentation
was quite varied. Common presenting features included refractory seizures,
developmental delay/regression, and abnormal movements. Visual failure
was not common in the present case series, and novel neuroimaging
finding in the form of isolated dentate nucleus hyperintensities in PPT
related neuronal ceroid lipofuscnioses was noted. During follow-up, all
patients had a progressive downhill course and one patient died. Prenatal
diagnosis could be offered to one family. Our experience suggests that
infantile and late-infantile neuronal ceroid lipofuscinosis is not uncommon in
this region of the country and the phenotype is different.
I38
Early stress evokes age-dependent biphasic changes in hippocampal
neurogenesis, epigenetic regulation of the bdnf gene, and cognitive
behavior
Vidita Vaidya
Department of Biological Sciences, Tata Institute of Fundamental Research,
Mumbai, India
E-mail: vvaidya@tifr.res.in
An experience of stress in early life is predominantly associated with
negative consequences including increased anxiety and depressive
behavior, as well as a failure to mount appropriate stress responses. It has
remained a source of debate whether early stress also evokes potentially
adaptive consequences that equip animals to cope better with their
environment. We have shown that early stress exposure facilitates
transient, adaptive changes in hippocampal neurogenesis, enhanced
trophic factor expression and improved cognitive performance, thus
providing possible competitive advantages in stressful environments.
However, middle-aged animals with a history of early stress exhibit
aladaptive effects on hippocampal neurogenesis, reduced trophic factor
expression and impairments in cognitive performance. Our study provides
novel insights into the short and long-term consequences of early stress,
demonstrating biphasic, as well as unique, age-dependent changes at the
molecular, epigenetic, neurogenic and behavioral level. These results
compel a reappraisal of the traditional notion that early stress is
deterministic for future negative outcomes. Our studies suggest that when
observed across a life-span, early stress experience evokes both adaptive
as well as maladaptive changes that emerge in a temporally regulated
manner, with early adaptive outcomes that may eventually exert a high
cost, evoking maladaptive consequences.
SESSI ON XI I I : I NBORN ERRORS OF
METABOLI C DI SORDERS
I39
Dysmorphology of inborn errors of metabolism
Virginia Kimonis
Division of Genetics and Genomics, Department of Pediatrics, University of
California, Irvine, CA 92868, USA
E-mail: vkimonis@uci.edu
As we discover the molecular mechanism of disorders, eventually all
dysmorphic syndromes will ultimately be considered biochemical defects.
An overview on the recognition and classification of dysmorphic features
will be provided. Categories of inborn errors of metabolism associated with
dysmorphic manifestations will be discussed. For e.g. abnormal eye
findings are an important clue to the diagnosisin galactosemia, cystinosis,
Lowe syndrome, and homocystinuria. Unusual kinky hair is seen in Menkes
disease. Skin findings typically lead to the diagnosis in Fabry, Hunter and
steroid sulphatase deficiency. Infants who have peroxisomal disorders
(Zellweger) pyruvate dehydrogenase deficiency, cholesterol biosynthetic
disorders (Smith-Lemli-Opitz), multipleacyl-CoA dehydrogenase deficiency
(glutaric aciduria type II), infants of mothers with phenylketonuria have
striking facial dysmorphism and structural anomalies at birth. In other
disorders dysmorphic features may not be present at birth but may
develop with age at varying rates such as in lysosomal storage disorders
(mucopolysaccharidoses, oligosaccharidoses), congenital disorders of
glycosylation and mitochondrial disorders. Salient clinical features,
biochemical defects, molecular basis, and diagnostic strategies will be
discussed to permit early treatment of these disorders.
I40
New born screening program in India: ICMR multicentric experience
Roli Mathur
Indian Council of Medical Research, New Delhi 110029, India
E-mail: rolimath@icmr.org.in
Inborn Metabolic Disorders (IMD) are common genetic disorders imposing
huge burden on health care infrastructure though many of these are
treatable conditions. Indian Council of Medical Research (ICMR) recognized
the need of initiating a multicentre Task Force (NTF-IMD) study to
systematically collect data for congenital hypothyroidism and congenital
adrenal hyperplasia to help in early diagnosis and management to prevent
disability.
This study was done to evaluate the feasibility of a newborn screening for
different geo-ethnic regions of the Indian Population and attempt to
define the incidence of the selected inborn metabolic errors i.e., CH and
CAH in the population and also to develop the capability of treating and
diagnosing inborn errors of metabolism.
A series of brainstorming sessions were conducted between 2008-2013 at
5 regional centers in the country before the start of the study. The NTF-
IMD Group developed a common protocol for implementation at all study
sites for comprehensive screening, management, treatment of affected
newborns, counseling, high risk screening in sick newborns, enrolment in
quality assurance program, development of tools for advocacy, setting up
of a dedicated website etc. A sample size of 100,000 newborns was set as
a target and 1000 sick children admitted in ICUs at study sites were also
included.
Page 11 of 59
Coordinated effort of a dedicated team of investigators at 5 newborn
screening centers, 3 high risk screening centers, data coordination centre,
quality assurance centre and central coordination centers could lead to
successful completion of newborn in more than 1,00,000 newborns from
both urban and rural areas. The study involved the use of common
protocols and involved data compilation and analysis to prepare
comprehensive results from all centers and set a data representative for
the country following thorough quality assurance procedures. The study
helped to establish the incidence of CH and CAH in Indian population and
prevented disability in affected newborns following early diagnosis and
treatment. The study has far reaching implications as it helped to build
regional capacity and in setting up an invaluable model for future
newborn screening programs in India as well as other developing
countries.
I41
Newborn screening- the roadmap for India
Seema Kapoor
Department of Pediatrics, Maulana Azad Medical College, New Delhi, India
E-mail: drseemakapoor@gmail.com
India witnessed a major transition in 2013. It marked the completion of
50 years of the activities of the Indian Academy of Pediatrics, the
completion of a taskforce study conducted by ICMR, organization of the
Asia Pacific meeting on newborn screening and inclusion of congenital
hypothyroidism as an important activity of a landmark program by the
Government, the Rashtriya Bal Suraksha Kayakram. It has also created a
platform for inclusion of many Delhi Hospitals in newborn screening.
These activities have created 3 major thrust points in the country which
include awareness about the need for screening, the feasibility of its
execution in the metropolitan cities of our country and the commitment to
make the special diets available. Both public and private players have
developed deep commitments which are likely to change the face of
newborn screening in the country. Screening for 5 nearly completely
reversible and treatable disorders is possible. These include Congenital
Hypothyroidism, Congenital Adrenal Hyperplasia, Glucose-6-phosphate
dehydrogenase deficiency, Biotinidase deficiency and Galactosemia. These
have been included in different pilot programs across India.
Challenges which invite discussion are the feasibility of coverage in less well
developed areas, hilly terrains and the home deliveries. MCTS (Mother child
tracking system) an initiative launched by the Government of India once in
full implementation may play a pivotal role in this program. Availability of
good counselors and a well integrated follow up system needs to be
developed so that all screen positive babies can be followed up. Generation
of epidemiologic data for the currently untreatable conditions being the
non availability of diets needs to be simultaneously addressed so that we
can gear up for the expanded phase later.
Generating ethnic cutoffs, ensuring quality compliance, improving
availability of confirmatory tests needs to be addressed. The volumes are
formidable but also suggest that with high rates of consanguinity and
inbreeding one is likely to encounter a significant proportion of these in the
country. The most positive aspect is the commitment to this noble cause
which will help us cross and reach the horizon.
I42
Treatment of inborn errors of metabolism
Anil B Jalan
Navi Mumbai Institute of Research in Mental and Neurological Handicap, C-
116, Om Rachna Society, Sector 17, Vashi, Navi Mumbai, India
E-mail: jalananil@yahoo.com
Inborn errors of metabolism (IEM), though individually rare are collectively
common. Average incidence of 50+ common IEMs is considered to be
approx 1 in 1,000 live births. With annual birth rate of approximately 25
million babies in India, we can expect at least 25,000 babies being born with
IEM in India and hence it is a significant burden to the families and societies.
Over the last 3 decades many new and effective therapies have emerged for
the management and treatment of IEMs. As of today approximately 70
different forms of therapeutic agents are available to help people suffering
from IEM. Besides these we have various forms of transplantsliver cell
transplants, liver transplant (both ALT and OLT), bone marrow or HSCT and
kidney transplant now easily available at various parts of India. Amongst
hundreds of IEM, certain disorders are common and treatable with simple
forms of therapeutic agents. Urea Cycle Disorders and Organic Acidemias
are top on the list. Every pediatrician and neonatologist must be aware of
emergency management of these two group of disorders as they may
present at any age especially in the 1
st
decade and more so in infancy. Both
of these groups of disorders may present with hyperammonemia as their
first manifestations and needs to be treated with easily available
medications as oral form of Sodium Benzoate (250500 mg/kg/day in 2-3
divided doses), low protein diet or temporary stoppage of protein intake till
acute crisis is under control. For UCDs one can use arginine (granules or
powder) hydrochloride or base250 mg/kg/day in 2-3 divided doses except
for Arginase deficiency. For disorders like CPS and NAGS deficiency Carbaglu
of Carbamylglutamate or N Acetyl Glutamate can also be used in the dose
of 100300 mg/kg/day. Now a days it is also recommended for treatment of
certain organic acidemias e.g. propionic acidemia. Other products like
Sodium Phenyl butyrate or injectable forms of Arginine (10%) or Sodium
Benzoate+Sodium Phenylbutyrate) are not available in India and are very
costly for an average Indian patient. Once acute crisis is managed, special
diets with low protein are successful in managing most of the UCDs.
For Organic acidemias like Propionic acidemia, Methyl malonic Acidemia
and Isovaleric acidemia, L-Carnitine in the dose of 100300 mg/kg/day
must be used. Injectable form of L-Carnitine is also available for emergency
management. Correction of acidosis is very important along with supple-
mentation of adequate amount of dextrose. One can use Dextrose-Insulin
drip in emergency. Besides these, other medications like Betaine, NTBC,
Dextromethrophane, Diazoxide, certain vitamins e.g. Biotin, Vit-B12,
Thiamine, Riboflavin, Folic acid, Folinic acid, Pyridoxine, Pyridoxal5-
Phosphate, Vit C, certain aminoacids like Glycine, Ornithine, Citrulline etc are
also available for the management of various types of IEMs. Of late many
enzymes are available for enzyme replacement therapies of LSDs e.g.
Gaucher, Pompe, Fabrys Disease, MPS I, II and VI.
SESSION XIV: INHERITED BLOOD
DISORDERS: HEMOGLOBINOPATHIES
I43
Thalassemias: can we reduce the national burden?
Roshan Colah
ICMR-National Institute of Immunohaematology, Parel, Mumbai, India
E-mail: colahrb@gmail.com
The burden of inherited disorders of hemoglobin, the commonest group of
single gene disorders in India is huge. With a population of 1.21 billion and
an average prevalence of b-thalassemia carriers being around 3.5-4%, there
would be 35-45 million carriers and the estimated number of births of
affected babies annually would be 10,000-12,000. The carrier rates vary from
1-17% in different ethnic groups. Apart from b-thalassemia, Hb E is common
in the north eastern region and in West Bengal (4 to > 50%) and Hb S is
prevalent in parts of central, western and eastern India (5-40%). Thus
interaction of the b-thalassemias with these Hb variants is not uncommon
and can lead to a severe disorder.
One way to combat the burden is by prenatal diagnosis but the only
approach to reduce the national burden is by a comprehensive community
control programme. Awareness is very limited in different states (<20%
among pregnant women) and the entire public health infrastructure from
medical colleges to district hospitals and down to the community health
centres must be mobilized for education and generating awareness on
these disorders. Experience shows that screening for carriers in India will
have to be done at multiple levels schools, colleges, antenatal clinics as
well as cascade screening where extended family members of an affected
child are screened. However, antenatal screening with subsequent testing
of the husbands of carrier women would be the most cost effective way to
identify couples at-risk and give them the option of prenatal diagnosis. For
this, obstetricians must recognize the implications of hypochronic and
microcytic red cell indices (MCV <80 fl, MCH < 27 pg and a high RBC
count) and ask for a b-thalassemia screen by estimation of HbA
2
levels.
Several laboratories in the country use automated HPLC for reliable HbA
2
estimation and identification of heterozygotes is not a problem. Late
registration at antenatal clinics (only 15-20% in the first trimester in public
Page 12 of 59
hospitals) is an impediment resulting in many couples at-risk being
identified late and requiring second trimester fetal diagnosis. Social
stigmatization is an issue to be dealt with during premarital screening of
marriage partners of carrier individuals. Only education can reduce this
barrier. Many State Governments in India are now undertaking population
screening and counselling programmes and Gujarat and West Bengal have
taken the lead.
There are 10-12 centres offering prenatal diagnosis by CVS and DNA
analysis and recently the Indian Council of Medical Research has established
6 more centres in different regions. However, many more centres would be
required once there is an increasing demand. The spectrum of mutations
and their distribution are now known which would facilitate prenatal
diagnosis.
Thus, there are many challenges a large and diverse population, limited
awareness, late registration in antenatal clinics and inequality of available
services (urban v/s rural areas) with around 70% of the population residing
in rural areas. There is a need for the Central and State Governments to
join hands and involve NGO groups to form networks in different regions
which when backed by political will could gradually reduce the national
burden of hemoglobinopathies in this vast country.
I44
Haemophilia - diagnosis and management challenges
Shrimati Shetty
ICMR-National Institute of Immunohaematology Parel, Mumbai, India
E-mail: shrimatishetty2@gmail.com
Haemophilia care in India is slowly progressing but the diagnostic and
management challenges continue until optimum care for haemophilia
patients become a reality in this country. Against an anticipated severe
haemophilia population of more than 0.1 million, only 16000 haemophilia
patients are registered in the 74 Haemophilia Chapters across the country.
As per the recent WFH Survey, 43% of the World haemophilia population
live in India, Bangladesh, Indonesia and China, out of which only 12% is
diagnosed in these countries. Additionally these countries represent only
2% of the Worlds total factor usage. The per capita use of FVIII in India has
been shown to be 0.0075 (1IU per capita is 20000 IU FVIII per haemophilia
patient) which is approximately equal to mean consumption of 1654 IUs
per PWH against 112508 IUs per PWH in United States. Haemophilia in
India is thus a grossly under diagnosed disorder, mainly due to lack of
awareness and diagnostic facilities in this country.
Except a few Corporate Hospitals and a few Medical Colleges in India
majority of the 630 District hospitals and Medical Colleges do not have even
screening coagulation facilities. Haemophilia Federation (India) has taken
wide initiatives in providing diagnostic facilities to the PWH in this country,
through various measures such as supporting the cost of diagnosis of PWH,
networking private laboratory facilities, setting up coagulation laboratories
in close proximity to Haemophilia Chapters, involving faculty members of
Medical Colleges in the activities of Haemophilia Chapters and so on. Indian
Council of Medical Research has also initiated through its Translation
Research Programme several workshops in the Laboratory diagnosis of
bleeding disorders at Mumbai and several places in East and North Eastern
parts of India. As quality control is an important exercise in a coagulation
laboratory, many of the laboratories in India fail to establish factor and
inhibitor screening assays in their respective laboratories.
The prevalence of inhibitors in India is estimated to be 8.213%. Post
operative inhibitors are another important problem which our PWH is
facing in India. About 30% of PWH undergoing surgeries for various
indications develop inhibitors postoperatively. Diagnosing inhibitors,
demands specialized laboratories and expertise. The management of PWH
and inhibitors comprises several approaches involving prompt treatment
of bleeding episodes, managing its complications, preventing bleeds, and
conserving and restoring joint function. The ultimate goal of treating PWH
and inhibitors is to permanently eradicate inhibitors by immune tolerance
therapy (ITT), or by use of alternative products like rFVIIa or FEIBA.
However, because of prohibitive cost and logistics constraints many of our
PWH are not able to utilize these products. Availability of free factors in
some states of our country is a refreshing and welcome change for PWH.
Significant progress has been made in awakening and alerting the State
Governments for providing free factors to all our PWH. WFH has
contributed to haemophilia care in India through a series of international
twinning programme by initially twinning upcoming chapters in India or
institutions of repute in India with some of the best centres in Europe and
USA leading to the development of expertise in the area of genetic
diagnosis and physical therapy.
Despite this, there are several newer challenges that the hemophilia
community will face in the coming years. Normalization of patients lives as
a result of improved treatment has led to new problem areas. Malignancies
specifically hepatocellular carcinoma (HCC) in HCV infected patients and
non Hodgkins lymphoma in HIV infected patients are on the rise. About
25% of our patients is HCV infected. A less common albeit potentially
severely morbid event is ICH. Osteoporosis in general has been considered
to be an important cause of morbidity in patients with haemophilia and
other bleeding disorders.
I45
Current status of sickle cell disease in India: how can you attenuate?
Dipika Mohanty
Apollo Hospitals, Bhubaneswar- 751 005, Odisha, India
E-mail: mohantydipika09@gmail.com
Sickle cell disease is the most prevalent monogenic disorder worldwide
resulting from single DNA mutation in beta globin gene. The mapping on
the pattern of its distribution in India has been studied to a great extent.
However paucity of adequate data throughout the country regarding the
clinical manifestations, natural history of the disorder and correlation with
genotype and phenotype of the SCD cases is observed. This communication
will focus on following aspects: (i) prevention and control of the SCD in
India. (ii) the good clinical management with particular reference to pain
during the vaso-occlussive crisis. (iii) neonatal screening and genetic
counseling in SCD in tribals of India.
Between August 2009 and July 2010, 1668 newborns were enrolled and
screened for SCD in Kalahandi district of Odisha. An average incidence of
17.62% of sickle cell trait (HbAS) was recorded for the area with the
highest incidence observed in the tribal dominated part (19.03%). The data
till date reveals a striking fact that more than 20 per thousand live births in
the district are born with the disease. All the 34 cases of SCA detected
were confirmed by parents testing, of which confirmation of 4 cases of
compound heterozygosity for HbS and b-thalassaemia is an interesting
finding.
For pain management with an informed approved consent in 15 patients
treated at Apollo Hospital, Bhubaneswar we have given nitric oxide
inhalation at 80ppm for 6-12hrs and 90% of patients responded well, pain
score being relieved significantly >60%. In 2 patients there was recurrence
of pain on 2
nd
day and they were given again the same therapy. Another
1 patient did not respond very well to pain. On investigation she was
found to have superficial vein thrombosis of left hand.
This NBS programme design provides scope for catering the benefit in rural
areas and follow up for newborns. It also stresses on the acute need of such
kind of extensive reach-out programme for early and confirmed detection of
SCD in other places of the country. This will ultimately reduce the child
mortality and morbidity in SCD. Nitric Oxide inhalation for pain relief in VOC
of SCA is a very effective and less expensive method.
I46
Community genetics approaches in the prevention of beta-thalassemia:
towards achieving Zero beta-thalassemia status in India
V R Rao
Department of Anthropology, University of Delhi, Delhi, India
E-mail: drraovr@yahoo.com
The estimates of prevalence of b-thalassemia in India are grossly
underestimated, as it is based on 0-5% carrier frequency, while population
surveys indicated communities with as high frequency as 24%. The overall
carrier frequency distribution of b-thalassemia, is highly heterogeneous with
wide geographical foci of high risk communities with 8% and above carrier
frequency all over the country. As of now, there is no mechanism to
evaluate whether the frequencies are increasing or decreasing in the
populations. Prenatal diagnosis and prevention of births of b-thalassemia
homozygotes is the most preferred approach adopted in India. However,
the extent of distribution and the occurrence across various stratification in
the society with large component of rural masses, it is difficult to assume
Page 13 of 59
that prenatal diagnosis alone can bring in Zero b-thalassemia status in
India. Countries like Cyprus could achieve such status where large scale
population carrier screening programs involving education and premarital
counselling were also effectively employed besides prenatal diagnosis. In
India, there is urgent need to initiate large scale population carrier screening
of high risk communities along with education and awareness.
The extent of b-thalassemia distribution, identifying high risk communities
and geographical regions presenting a population design for carrier
screening program in India, will be presented.
SESSI ON XV: HUMAN
CHROMOSOMAL DI SORDERS
I47
Chromosomal instability and molecular mutations in multi spectrum
disease of Fanconi anemia
Babu Rao Vundinti
ICMR- National Institute of Immunohaematology, Parel, Mumbai, India
E-mail: vbaburao@hotmail.com
Fanconi anemia (FA; MIM no. 227650), the most commonly inherited bone
marrow disorder, has an overall prevalence of 15 per million and an
estimated carrier frequency of 1:200-300 in most populations. Demonstrating
either an autosomal or X-linked recessive mode of inheritance, FA is
characterized by childhood progressive bone marrow failure and
predisposition to acute myelogenous leukemia, older patients are at
increased risk for squamous cell carcinomas of the head, neck, and
genitourinary tract. Congenital abnormalities are present in approximately
70% of FA patients and may include radial ray defects, cafe au lait spots or
hypopigmentation, short stature, microphthalmia, malformations of kidneys,
gastrointestinal tract, and heart, mental retardation, and hearing defects.
Because of the high degree of phenotypic variability exhibited by FA patients,
diagnosis may be difficult on the basis of clinical manifestations alone.
Because FA patient-derived lymphocytes and fibroblasts exhibit
hypersensitivity to DNA crosslinking agents such as diepoxybutane (DEB) and
mitomycin C (MMC), resulting in a high rate of chromosomal breakage and
radial formation, analysis on the basis of this hypersensitivity has been
routinely used to confirm clinical diagnosis.
Molecular diagnosis of FA has been challenging because of the genetic
heterogeneity associated with the disease. To date, 15 FA gene products
(FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O and P) have been identified and
they constitute the FANC pathway, which is thought to function in
preventing genome instability. The FA core complex comprises FAAP24,
FAAP100, and 8 FA proteins (FANCA, B, C, E, F, G, L, and M) and mediates
DNA-damage-induced or replication-stress induced monoubiquitylation of
FANCD2 and FANCI. Monoubiquitinated FANCD2 and FANCI translocate to
chromatin and function in DNA repair at least partially by recruitment of
FAN1 nuclease. Cloning of the FANC genes has provided a means, via
complementation group analysis, to routinely pinpoint the patients specific
FA gene harboring the mutations, thus greatly simplifying subsequent
molecular analysis. Although retroviral-mediated complementation analysis
of FA patient-derived cells is commonly used to identify the patients genetic
subtype as a prerequisite for mutation screening, such analysis has not been
formally validated for clinical use. Previous studies have considered Fanconi
complementation Group A (FA-A) assignments, but none have performed
comprehensive sequencing to verify complementation assignment and/or
evaluated complementation assignments performed using retroviral-
mediated rescue as evidenced by correction of MMC and DEB-induced
chromosome breakage and radial formation. Among all complementation
groups, FAA accounts for the majority of FA patients (6065%) followed by
FAC (10%) and FAG (9%). Mutations in the gene for complementation group
FA-A (FANCA; MIM no. 607139) confer autosomal recessive inheritance and
account for approximately 66% of all FA cases. The FANCA gene is mapped
to chromosome 16q24.3,11 spans approximately 80 kb of genomic DNA
(gDNA), and consists of 43 exons. With more than 200 different mutations
described thus far, the FANCA mutation spectrum is very heterogeneous.
Because of the presence of a large number of Alu repeat sequences within
the FANCA gene, large intragenic deletions involving multiple exons have
been reported to account for more than 40% of all FANCA mutations.
First time we have carried out a molecular study in the Indian population.
We have studied FA by chromosomal breakage study using mitomycin C
(MMC) and diepoxybutane (DEB) induction and FANCD2 monoubiquitination
by western blotting to understand gene defects in FA pathway. The
complementation analysis was done by retroviral transfection. The molecular
study was carried out by Multiplex Ligation-dependent Probe Amplification
(MLPA) and direct sequencing of FANCA, C, G, E, F, L, and M genes. The
complementation analysis results showed different spectrum as compared to
world literature. Our study revealed FA-A and E gene defects in 69% cases
and followed by FANCE gene defect. The molecular analysis showed the
large deletions in 11 patients and FANCA gene mutations were in 12 FA
patients. Out of 12 mutations 5 mutations (c.3678 C>G, p.Ser 1226 X,
c.3993G>A p.Leu 1331 Pro, c.1274 C>G p.Glu 425 His, c.2630 C>G p.Ser 877
X) found to be novel mutations. In our series the single nucleotide
polymorphisms (SNP); Exon9,c.796A>G(p.Thr266Ala), Exon16,c.1501G>A(p.
Gly501Ser), Exon26, c.2426G>A(p.Gly809Asp), of FANCA gene also observed
in 23 patients, and these polymorphisms in disease association was reported
in FA database, however, it needs to be established in Indian population.
Interesting finding of our study is the existence of FANCE mutation in Indian
population, and the same was reported rarely in other places of the world.
The study also highlighted the uncharacterized FA patients, which may
associated with new genes in Indian population and these patients should
be studied molecularly and genotype-phenotype correlation need to be
established, which helps in better understanding and management of the
disease.
SESSION XVI: NANOTECHNOLOGY IN
BIOMEDICAL RESEARCH
I48
Unlocking the chemistry of bile acids for cancer therapeutics
Avinash Bajaj
Laboratory of Nanotechnology and Chemical Biology, Regional Centre for
Biotechnology, Gurgaon, India
E-mail: bajaj@rcb.res.in
Breast cancer is the second leading cause of cancer deaths today after lung
cancer and is the most common cancer among women. The primary drug
tamoxifen is used treat breast cancer has several problems including poor
oral bioavailability. Bile acids/salts are known to be components of
endogenous molecular pool that solubilizes, absorbs dietary fat/lipid
molecules in the form of micelles. Bile acids are interesting chemical scaffold
for drug conjugation due to presence of different number of free hydroxyl
groups and a free carboxylic acid. We have been exploring bile acids as
drug carriers for cancer therapy. We used three bile acids: lithocholic acid
(LCA), deoxycholic acid (DCA) and cholic acid (CA) to engineer bile acid
tamoxifen conjugates with free amine and acid functionalities. In this talk,
I would present the interactions of these new drug carriers and their
therapeutic potential for breast cancer therapy.
SESSION XVII: PRE-IMPLANTATION AND
PRENATAL DIAGNOSIS OF GENETIC
DISORDERS: INDIAN SCENARIO
I49
Pre-implantation and polar body diagnosis in cases of parental
chromosomal translocations applying array-CGH
Rolf-Dieter Wegner
1,2*
, Markus Stumm
1,2
, Heide Mundt
2,3
, Matthias Bloechle
4
1
Zentrum fr Prnataldiagnostik und Humangenetik Kudamm 199, Berlin,
Germany;
2
BG Berlin Genetics GmbH, Berlin, Germany;
3
Freie Universitt
Berlin, Germany;
4
Kinderwunschzentrum an der Gedchtniskirche, Berlin,
Germany
E-mail: wegner@kudamm-199.de
Parents with a balanced chromosomal translocation show an increased risk
of reduced fertility including spontaneous abortions and chromosomally
Page 14 of 59
unbalanced offspring. In the course of genetic counseling the parents
frequently decide to seek artificial reproductive techniques. In vitro
fertilization (IVF) offers the chance to perform Polar Body Diagnosis (PBD) or
Preimplantation Diagnosis (PID). Female translocation carriers can opt for
analysis of polar body, blastomeres or trophectoderm cells while male
translocation carriers have only a choice between blastomere and
trophectoderm diagnostic.
In Germany, up to the year 2010 a restrictive Embryo Protection Law did
allow only PBD excluding male translocation carriers from diagnostics. Thus,
experience with PBD applying FISH or array-CGH was collected in cases of
maternal translocations. For such cases, advantages and limits of array
analysis as compared to the FISH approach will be presented. Furthermore,
the resolution power of the BlueGnome 24sure and 24sure+ arrays will be
shown. In particular we report here on the segregation pattern of maternal
translocation chromosomes in 16 cases including 24 cycles and analyzing 97
first polar bodies. In addition to the distribution of the translocation
chromosomes the number of segregation errors leading to aneuploidy in
the 1
st
. polar body will be presented.
Since a German high court ruled that PID of trophectoderm cells should
be legal under very stringent conditions, the Embryo Protection Law had
been slightly modified in 2011. In future this will allow the application of
array analysis also in cases of paternal translocation.
I50
Challenges in prenatal and pre-implantation genetic diagnosis studies
Prochi Madon
Department of Assisted Reproduction and Genetics, Jaslok Hospital and
Research Centre, Mumbai, India
E-mail: prochi_madon@yahoo.com
Prenatal diagnosis (PND) of chromosomal anomalies by karyotyping and
fluorescence in situ hybridization (FISH) is a routine procedure in many
cytogenetic laboratories. However, PND of single gene disorders is
available in only a few centres specializing in molecular genetic testing in
India. Preimplantation genetic diagnosis (PGD) is a state-of the art
technique involving biopsy of a single cell from a cleavage stage embryo
obtained after intra-cytoplasmic sperm injection (ICSI). This is an additional
step during in-vitro fertilization (IVF). The biopsied cell is then fixed on a
slide in a critical step where the cytoplasm is removed to expose the
nucleus. Single cells from many embryos of the couple are fixed and then
screened for common aneuploidies by FISH using multicolour DNA probes.
A maximum of 5 chromosomes can be checked in 1 hybridization. Hence
the probes are stripped and slides are rehybridized in 2-4 rounds to study
more chromosomes within a limited time frame. Only the chromosomally
normal embryos are transferred or cryopreserved for a subsequent transfer.
Karyotyping of couples with recurrent early miscarriages helps to identify the
cause if the husband or wife is a carrier of a balanced translocation. PGD can
help such couples to have a healthy baby earlier than by normal chance.
Specific FISH probes have to be ordered especially for reciprocal
translocations and a pre-PGD work up is important. This helped to identify
an additional cryptic translocation between chromosomes 9 and 12 in a
case from a neighbouring country, where the husband had inversion 12.
Hence PGD included testing of chromosome 9 in this case. The wife has an
ongoing pregnancy in the first attempt. Currently, ours is the only centre in
India which offers PGD even for reciprocal/ Robertsonian translocations and
microdeletions. We have had success with the birth of healthy babies.
PGD for single gene disorders is yet in its infancy in our country. It involves
genetic testing from DNA extracted from a single cell. Pre-PGD work up is
more demanding, especially in cases where a HLA matched normal savior
sibling is desired to cure a child affected with a hematological disorder such
as Thalassemia.
I51
Consanguinity and perinatal medicine - the berlin perspective
Rolf Becker
Centre for Prenatal Diagnosis and Human Genetics, Berlin, Germany
E-mail: bedaktari@t-online.de
The present study was conducted to assess the impact of consanguinity
on the prevalence of major anomalies in a prenatal study group of Berlin/
Germany.
Over a time-span of 19 years (1993-2011), 34,900 fetuses were examined by
prenatal sonography. In 659 cases (1.9%) the parents were consanguineous,
with 45.2% related as first cousins (F = 0.0625) and 54.8% beyond first
cousins (F< 0.0625). Detailed information on the pregnancy outcome of
31,145 fetuses was retrieved either through direct report by families or by
active inquiry of patients or their physicians, 555 of these fetuses (1.8%) had
consanguineous parentage.
The prevalence of major anomalies among fetuses with non-
consanguineous parents was 2.8% (863/30,590). By comparison, in the
sub-group of fetuses with consanguineous parentage the prevalence
was 11.0% (61/555 fetuses). Within the consanguineous sub-group a
causal association between fetal anomaly and consanguinity was assessed
as probable in 6.5% (36/555) of cases, as possible in a further 3.4%
(19/555) of cases, and as improbable in 1.1% (6/555) of the diagnosed
anomalies.
The data indicate that the prevalence of major fetal anomalies associated
with consanguinity was approximately eight percentage points higher
than in non-consanguineous offspring. As a proportion of these
anomalies result either in intrauterine death or medical termination of
pregnancy, the prevalence of consanguinity-associated defects diagnosed
post-birth is equivalently lower, thus under-estimating the overall adverse
effect of intra-familial marriage on fetal and neonatal well-being.
SESSI ON XVI I I : SI GNI FI CANCE OF
PHARMACOGENOMI CS I N
CLI NI CAL RESEARCH
I52
Pharmacogenetics: polymorphism and genotype-phenotype correlation
of drug response in indian population
Harish Padh
Sardar Patel University, Vallabh Vidyanagar, Gujarat, India
E-mail: hpadh@yahoo.com
Inter-individual genomic variations have recently become evident with
advances in sequencing technologies. Polymorphism and human
variants have been linked to disease susceptibility as well as drug
efficacy and toxicity. Although polymorphism can occur at any loci and
can affect any protein structure and function, it is largely studied in
enzymes involved in drug metabolism where much correlation has
been established with drug efficacy and toxicity. Among genetic
variations SNPs are widely studied and better defined because of
availability of large scale detection platforms. Besides SNP, insertion-
deletions (INDELs), inversions, copy number variations (CNVs) and larger
structural variations (> 3 Mb) have come to light in recent years,
however, their link to health and disease remain ill-defined. CNVs are
defined as the segment of DNA larger than 1 Kb in size, and compared
to reference genome vary in copy number.
All types of genomic variations are bound to play vital role in disease
susceptibility and drug response. In this presentation, genetic variation in
many DMEs like CYP2D6, CYP2C9, CYP2C19 will be discussed, and its
effect on pharmacokinetic (PK) parameters like AUC, Cmax, Tmax and
T1/2 will be presented. PK variation as phenotype will be compared and
correlated with genotype variation in Indian population. Examples of CNV
data in Indian population will be presented and compared with other
populations. Available literature will pose significant policy issues about
drug approval procedure. The issue of incorporation of local
pharmacogenetic consideration in drug approval will be analysed.
I53
Pharmacogenomics of cardiovascular drugs
C Adithan
Department of Clinical Pharmacology, JIPMER, Pondicherry, India
E-mail: adithan@yahoo.com
Page 15 of 59
Cardiovascular diseases account for the second largest number of non-
communicable disease after mental illnesses. Coronary heart diseases,
hypertension, atherosclerosis, congestive heart failure, arrhythmias are
important cardiovascular diseases. Beta blockers, statins, antiplatelet drugs,
anticoagulants, drugs modifying renin angiotensin systems and many others
are being used for treating these ailments. Still, satisfactory treatment of these
diseases is still elusive. Drug response in influenced by many environmental
factors besides host factor. Besides the above, genetic factor is also important
in modifying response to cardiovascular drugs. Now there is reasonable
amount of evidence exists that Indian population is genetically distinct from
other major ethnic groups. The allele and genotype frequencies of genes
encoding important drug metabolizing enzymes, drug transporters and
receptors of Indian population are different from Caucasians and Orientals.
Studies done in our laboratory and other Indian laboratories suggested that
pharmacogenomics of clopidogrel, warfarin, acenocoumarol, beta blockers
etc. may have clinical significance in treating cardiovascular diseases of Indian
population. There is a need for multi-institutional and multi-disciplinary
approach for large scale implementation of pharmacogenomics and
personalized medicines in cardiovascular diseases.
I54
Point of care testing for improving risk- benefit ratio of aspirin and
warfarin
Harsh Sheth
1*
, Emma Northwood
2
, Faye Elliott
2
, Michael Jackson
1
,
Mauro Santibanez Koref
1
, John Tyson
3
, Ann Daly
4
, Jonathan OHalloran
3
,
Jayesh Sheth
5
, Frenny Sheth
5
, Keyur Parikh
6
, D Timothy Bishop
2
, John Burn
1
1
Life, Newcastle upon Tyne, UK;
2
Leeds Institute of Molecular Medicine, St.
James Hospital, Beckett Street, Leeds, UK;
3
QuantuMDx Ltd., International
Centre for Life, Newcastle upon Tyne, UK;
4
Institute of Cellular Medicine,
Medical School, Newcastle University, Newcastle upon Tyne, UK;
5
FRIGEs
Institute of Human Genetics, FRIGE House, Satellite, Ahmedabad, India;
6
CIMS
Hospital, Nr. Shakun Mall, Sola, Ahmedabad, India
The increase in identification of putative biomarkers and opportunities to
develop tailored treatments are due to emergence of omics technologies.
Application of pharmacogenetic knowledge with the help of quick and
cheap companion diagnostics in the primary care setting is expected to
deliver improved treatment and reduced heathcare costs. Warfarin and
aspirin are the two most widely prescribed drugs for preventing
cardiovascular diseases. Long term aspirin use has also been shown to
reduce risk, recurrence and mortality from colorectal cancer. However, they
both have narrow therapeutic windows and several genetic polymorphisms
have been noted to influence their dose and efficacy. We therefore have
launched two collaborative projects: first, to study the genetics of warfarin
safety in the Gujarati Indian population and second, to identify further
polymorphisms that modulates aspirins colorectal cancer chemopreventive
efficacy. Understanding the impact of polymorphisms on dose and efficacy
for these drugs would lead to development of a combined panel of
markers that would predict accurate therapeutic dose with minimal risk for
adverse reactions. These markers will be deployed at the point of care
settings using a novel handheld genotyping device which will use
disposable microfluidic cassettes and silicon nanowires currently developed
by QuantuMDx. Results, future work, opportunities and barriers will be
examined.
SESSI ON XI X: GENE DI SCOVERY -
TRANSLATI ONAL RESEARCH
FROM BENCH TO BEDSI DE
I55
Digital health, microfluidics, and bedside genetic testing
Syed Hashsham
Department of Civil and Environmental Engineering, Michigan State
University, USA
E-mail: hashsham@egr.msu.edu
Decentralized, bedside, or point of care analysis of nucleic acids (DNA/RNA/
mRNA/microRNA)-based markers is expected to be a key component of
digital healthcare. Nucleic acids-based approaches allow for screening of
disease and pathogens, disease surveillance, selection of treatment,
treatment effectiveness, differential diagnosis, risk assessment, staging,
and prognosis. Hand-held genetic analysis systems have the potential to
provide all these capabilities in a simple, affordable, field deployable, rapid,
multiplexed, and robust manner without the need for electrical power or
refrigerated reagents. Even the need for specific language can be
eliminated by translatable or visual graphical user interface truly
integrating the analytical system with the communication network.
The presentation will introduce Gene-Z and iDx, two networkable
platforms that are developed to provide simplified analysis of genetic
markers. Gene-Z has Android/iPod based operation using Bluetooth
TM
and capable of carrying out quantitative isothermal amplification for 64
reactions in a disposable microfluidic chip. The device is battery operated
and can be charged by solar panels integrated at the top of the device.
A smaller version (iDx) working as an attachment to cell phones, also with
real time amplification and quantification capabilities, allows 8 reactions in
parallel. Both these devices are networkable and currently being validated
for a number of key applications important to human and animal health,
plant safety, industrial microbiology, and environmental protection.
Simplification, reliability, and cost-effectiveness are key factors ensuring
successful implementation of such approaches. Business model for
adoption of such low cost approaches, however, is still evolving and major
challenges need to be addressed about how to sustain a business that is
built on small margins usually by small companies engaged in biomedical
research.
I56
G protein signaling in tumor cell growth and metastasis
Danny Dhanasekaran
Peggy and Stephenson Cancer Center, University of Oklahoma Health
Sciences Center, 975 NE 10
th
Street, BRC 1417, Oklahoma City, OK, 73012,
USA
E-mail: danny-dhanasekaran@ouhsc.edu
G protein signaling has been implicated in different aspects cancer growth
and progression. Our studies have identified that the G12-family of
G proteins that defines the gep family of oncogenes are critically involved
in tumor cell proliferation and metastasis. Defining these pathways has
shown that the gep protooncogene GNA12 is specifically involved in the
proliferation of ovarian cancer cells whereas GNA13 is involved in cancer cell
metastasis. Consequently, the silencing of the gep proto-oncogenes potently
inhibited tumor growth of ovarian cancer cells in a mouse xenograft model,
thus suggesting the dominant role for the gep oncogenes in ovarian cancer
growth and progression. In addition we demonstrate a similar role
for GNA13 in the invasive migration of pancreatic cancer cells. Furthermore,
we demonstrate that an eleven amino acid peptide derived from the gep
oncogenes Ga
12/13
can effectively disrupt LPA-stimulated oncogenic
pathways. Thus, in addition to unraveling the molecular mechanism
underlying cancer progression and metastasis, our results provide evidence
that the G protein signaling nodes can be targeted for cancer
chemotherapy.
This work was supported by grants from the National Institutes of Health
(CA123233, CA 125752, CA 116984).
I57
Vitiligo: a complex disease and a complex approach
Rasheedunnisa Begum
1*
, Yogesh S. Marfatia
2
, Naresh C Laddha
1
,
Mitesh Dwivedi
1
, Mohmmad Shoab Mansuri
1
, Mala Singh
1
1
Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao
University of Baroda, Vadodara, India;
2
Department of Skin & V.D., Faculty of
Medicine, The Maharaja Sayajirao University of Baroda, Vadodara, India
E-mail: rasheedunnisab@yahoo.co.in
Vitiligo is an acquired, circumscribed hypomelanotic skin disorder,
characterized by milky white patches due to loss of functional melanocytes
Page 16 of 59
from the epidermis. Prevalence of vitiligo is found to be very high in
Gujarat i.e., ~8.8%. Vitiligo is a multifactorial polygenic disorder with a
complex pathogenesis, linked with both genetic and non-genetic factors.
Several theories have been proposed to explain the etiopathogenesis of
vitiligo, but none of the hypotheses explains the entire spectrum of this
disorder. We are addressing this complex disease in our Gujarat population
with various approaches. Our study mainly deals with the evaluation of
oxidative stress, autoimmune, genetic and neurochemical hypotheses in
Gujarat vitiligo patients. We have shown that our vitiligo patients exhibit
significant oxidative stress and thus, systemic oxidative stress could play a
pathophysiological role in precipitation of vitiligo in Gujarat population.
Our studies revealed that presence of increased antimelanocyte antibodies
and the imbalance of T-cell (CD4
+
/CD8
+
and Tregs) subsets along with
their functional defects might result in melanocyte destruction in vitiligo
patients. Our results on selected candidate genes in conferring oxidative
stress and autoimmunity suggest that HLA-A*33:01, HLA-A*02:01,HLA-
B*44:03, HLA-DRB1*07:01 and a few studied polymorphisms in IL4, CTLA4,
SOD2, SOD3, GPX1, NALP1, MYG1, TNFA, TNFB, IFNG and IL10 genes are
strongly associated with vitiligo susceptibility, whereas a few studied
polymorphisms in PTPN22, MBL2, ACE, CAT, G6PD and SOD1 genes are not
found to be significantly associated with Gujarat vitiligo patients. Gene
expression studies of the IL4, CTLA4, SOD2, SOD3, NALP1, MYG1, TNFA,
TNFB, IFNG, IL10 and ICAM1 genes suggest that these genes are strongly
associated with vitiligo susceptibility. We are also addressing the role of
immune-regulatory genes with respect to their expression in skin along
with the effect of selected cytokines on in vitro cultured melanocytes
derived from healthy and vitiliginous human skin to have an insight
towards vitiligo pathogenesis. We are also exploring the potential
microRNAs involved in pathogenesis of vitiligo. This integrated study will
provide a better understanding of the role played by oxidative stress and
autoimmunity in the pathogenesis of vitiligo in Gujarat population and
also to develop selective therapy and the genetic marker/s for vitiligo.
Various factors such as the antioxidant status, LPO (oxidative stress) levels
and antimelanocyte antibody titer decide the selective therapy for our
vitiligo patients. The pathogenesis of vitiligo though partially understood still
remains complex and enigmatic to a greater extent. Though the condition
may be precipitated by multiple etiologies, the interaction of oxidative stress
and immune system clearly appears to be the key convergent pathway that
initiates and/or amplifies the enigmatic loss of melanocytes in vitiligo.
I58
STR Markers in clinics: a rapid prenatal diagnosis by quantitative
fluorescent-pcr for aneuploidies
Sarita Agarwal
*
, M Srinivasan, Shubha Phadke
Department of Medical Genetics, SGPGIMS, Lucknow, India
E-mail: saritasgpgi@gmail.com
In the past few decades, prenatal diagnosis of fetal chromosomal
abnormalities has relied on conventional cytogenetic analysis of cultured
amniocytes, chorionic villi, or fetal blood. In recent years, the clinical
validity of a newer technique, QF-PCR, to detect the common aneuploidies
has been reported by a number of investigators. This technique has the
advantage of providing rapid results for the diagnosis or exclusion of
aneuploidy in chromosomes 13, 18, 21, X or Y. It is now possible to choose
standard chromosome analysis or QF-PCR for the prenatal diagnosis of
chromosomal abnormalities, or to perform both tests, depending on the
clinical indication for testing.
QF-PCR exploits the distribution of STR markers, 2-6bp tandem repeats,
across the genome. This DNA marker is easily amplified by polymerase
chain reaction (PCR) without the problem of differential amplification; that
is, the PCR products for STRs are generally similar in amount, making
analysis easier. An individual inherits one copy of an STR from each parent,
which may or may not have similar repeat sizes. The number of repeats in
STR markers can be highly variable among individuals, which make these
STRs effective for diagnosis and human identification purposes. It has an
added advantage of identifying maternal or paternal origin of
nondysjunction.
The establishment of QF-PCR has been initiated with the aim of providing
rapid prenatal diagnostic service and to reduce anxiety of patients about
their at risk pregnancies.
For validation, we performed analysis on 100 confirmed cases of Down
syndrome with the markers D21S1435, D21S11 and D21S1411, whose
heterozygosity has been studied in North Indian population, with
heterozygosity of 70.1%, 83.1% and 93.6% respectively. We observed
100% concordance with the clinical diagnosis as well as cytogenetic
analysis. With these results we extended the methodology in prenatal
services were chromosomal studies are very common for suspected
Down syndrome pregnancies. However, in addition we included 2 more
markers D21S1411 and D21S1413 for 21 chromosome, D13S238 and
D13S631 for 13 chromosome, D18S391 for 18 chromosome and AMEL,
XHPRT, X22 and SRY for sex chromosomes. Addition of 13, 18 and sex
chromosomes marker were helpful at drawing conclusive reports for
maternal contaminated samples and simultaneously to screen for rare
aneuploides like 13, 18 trisomy and Klinefelter syndrome.
The test was offered to the pregnancies with triple test positive as well as
abnormal findings in ultrasonography. The source of DNA was 3 5 ml of
amniotic fluid while in few cases chorionic villi sample. 190 pregnancies
were studied to date, 3 pregnancies (1.6%) were turned to be Down
syndrome positive while rest was negative for Down syndrome while 1 case
was turned out to be homozygous for all 4 markers of 21chromosome in
which we required additional markers to do reporting. However, karyotying
showed this sample to be negative for Down syndrome. Rest all the finding
was consistent with that of karyotyping results.
The technique has its own advantage that can utilize the maternal blood
contaminated samples, to add more, low volume of sample requirement
and possibility to do reporting within 6 12 hours. Still we are working to
record heterozygosity of rest of the 8 markers and to tune up the marker
panel accordingly.
SESSI ON XX: STEM CELL AND ERT
THERAPEUTI CS
I59
Mitochondrial donation by stem cells: potential for novel therapeutics
Anurag Agrawal
CSIR-Institute of Genomics & Integrative Biology, New Delhi, India
E-mail: a.agrawal@igib.res.in
There is increasing interest in whether mesenchymal stem cells (MSC) act
as mitochondrial donors for rescuing injured cells. Islam et al (Nature
Medicine, 2012) showed conclusively, using a mouse model of acute lung
injury, that MSC mediated mitochondrial donation can lead to survival
benefits (3). Contemporaneous work from Dr. Anurag Agrawals lab reveals
the molecular regulation of mitochondrial movement during donation and
shows how this can be engineered to increase therapeutic efficacy of MSC.
They find that Miro1, a calcium sensitive mitochondrial Rho-GTPase that
attaches mitochondria to KIF5 motor proteins on microtubules and
regulates neuronal mitochondrial trafficking, is necessary for mitochondrial
transfer by MSC. Overexpressing Miro1 in MSC (MSCmiro
Hi
) led to
increased mitochondrial transfer and therapeutic efficacy in mouse models
of lung injury as well as asthma. This is a significant addition to the field of
mitochondrial biology and stem cell therapeutics. It is also of general
interest to physicians and members of public with interest in regenerative
medicine, because this is highly translatable into more effective therapies.
I60
Haematopoetic stem cell transplantation at apollo group hospitals
Chirag Shah
Apollo Hospitals International Limited, Gandhinagar, Gujarat, India
E-mail: drchiragashah@gmail.com
Hematopoietic stem cell transplantation (HSCT) is the transplantation of
multipotent hematopoietic stem cells, usually derived from bone marrow,
peripheral blood, or umbilical cord blood. It is a medical procedure in the
fields of hematology and oncology, most often performed for patients with
certain cancers of the blood or bone marrow, such as multiple myeloma or
leukemia. HSCT can be divided in two types, autologous and allogenic
Page 17 of 59
transplant based on the source of stem cells. Autologus is when stem cells
are collected from patients own body. Allogeneic is when stem cells are
collected from someone else.
Apollo group hospitals perform largest number of HSCT in private sector.
HSCT requires multidisciplinary effort with team of many experts including
haematologist/oncologist, intensive care specialist, trained nursing staff,
experts for venous access, infectious disease specialist, transfusion
medicine expert, counsellors and many other experts.
Over 700 patients have been treated by HSCT. The majority of these patients
have haematological malignancies, consisting of acute and chronic
leukaemias, lymphomas, plasma cell myeloma and other haematological
neoplasms. Patients with other benign blood diseases including thalassemia,
sickle cell disease, aplastic anemia, Fanconi anemia, and others have also
been treated. The major facility for these patients is a specially designed
haematology ward with HEPA-filtered air.
The hemato-oncology department manages both adult and paediatric
haematopoietic stem cell transplantation cases, where more than a hundred
allogeneic and autologous transplantations are now performed every year.
The full range of allogeneic transplantation is performed, with
haematopoietic stem cells coming from HLA-identical siblings, matched
unrelated donors, umbilical cord blood and haploidentical donors.
I61
Perspective of stem cell research & therapy in diabetes
Sarita Gupta
Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao
University of Baroda, Vadodara, India
E-mail: saritagupta9@gmail.com
In the area of regenerative medicine, researchers have been exploring the
potential of Stem Cells in preclinical and clinical studies to improve
therapies that can resolve injuries by enhancing endogenous repair,
opening a new paradigm in cell therapy.Stem cells may it be embryonic
stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells
have been used as potential sources for beta cell replacement therapy
across the globe. Numerous studies have shown the differentiation
potential of ESCs and iPSCs towards producing pancreatic progenitors but
after terminal differentiation into insulin producing cells these lacked
various important pancreatic markers and have low index of glucose
stimulated insulin secretion (GSIS). This along with other ethical
conundrums makes it difficult to use ESCs as a source for cell therapy.
Adult stem cells provide clinically and ethically accepted option over
embryonic or induced pluripotent stem cells, as they can be used as
autologous source for cell transplant in treatment of diabetes. Widely used
option of cadaveric islet therapy has its own problems due to insufficient
amount of islets available for transplantation. Hence, our research is
focused on increasing islet mass from various adult stem cells using
neutraceutic bioactive compounds isolated from a plant demonstrating
efficient anti-diabetic activity and further explored them as potent
differentiating agent under in-vitro and in-vivo condition. In-vitro
differentiated islets from various adult stem cells sources were assessed at
morphological, molecular, immunological and functional level and further
evaluated for glucose lowering effect after transplantation in
Streptozotocin (STZ) induced diabetic balb/c mice.
ORAL PRESENTATI ON
O1
A Poisson regression model for association mapping of count
phenotypes
Saurabh Ghosh
1*
, Abhishek Chakrabortty
2
1
Human Genetics Unit, Indian Statistical Institute, Kolkata, India;
2
Department
of Biostatistics, Harvard University, Cambridge, USA
E-mail: saughosh@gmail.com
Molecular Cytogenetics 2014, 7(Suppl 1):O1
Background: Clinical end-point traits are often characterized in terms of
quantitative precursors. For psychiatric disorders, traits such as symptom
counts often serve as endophenotypes of interest for understanding the
genetic basis of the clinical end-point trait. Since such traits are discrete
in nature, it may not be optimal to use standard approaches such as
Analysis of Variance (ANOVA) to detect association.
Methodology: For population level data, we propose a Poisson regression
approach that computes the likelihood of the count phenotype conditional
on an additive allele count at a SNP. The test statistic is asymptotically
distributed as chi-squares with one degree of freedom under no association
between the SNP and the phenotype. For family-based data involving trios
with at least one heterozygous parent at a SNP, we use a similar Poisson
regression model conditional on two indicator variables: the marker allele
transmitted by the heterozygous parent and the marker allele transmitted
by the other parent. A one degree of freedom test based only on the
coefficient of the first indicator is protected against population stratification
as it tests for association in the presence of linkage. Two degrees of freedom
test based on both the indicators is also a valid test for association, but is
susceptible to population stratification.
Results and conclusions: Based on extensive simulations under different
genetic models, we find that for population level data, while the asymptotic
tests for ANOVA yield an inflated rate of false positives, especially when
there is heteroskedasticity in the distribution of the trait across the QTL
genotypes, our proposed method maintains the correct size. Moreover, our
method yields uniformly more power compared to ANOVA for the different
genetic parameters in our simulations. For the trio design, we find that the
two degrees of freedom test is more powerful than the one degree of
freedom test. We applied our method to analyze externalizing symptoms, an
endophenotype correlated with alcoholism using data generated in the
Collaborative Study On the Genetics of Alcoholism (COGA) project. We
found significant evidence of association in the class 1 alcohol
dehydrogenase subunit ADH1C in the 4q22.3 region.
O2
Therapeutic potential of histone deacetylase inhibitor for treatment of
Niemann-Pick type C1 disease
Nina H Pipalia
*
, Frederick R Maxfield
Department of Biochemistry, Weill Cornell medical College, 1300 York Ave.
New York, NY 10065, USA
E-mail: nhp2001@med.cornell.edu
Background: Niemann-Pick type C (NPC) is a rare, autosomal recessive
neurodegenerative lysosomal storage disorder (LSD) caused due to
mutation in either npc1 or npc2 genes. However, 95% of the reported
cases are caused due to mutation in npc1 gene and only 5% due to the
mutation in npc2 gene. Mutations in either of these two genes results in
similar phenotype such that there is an abnormal accumulation of
unesterified cholesterol and glycosphingolipids in late endosomes/
lysosomes (LE/Ly) of many cell types. NPC1 is a multi-spanning
transmembrane protein localized in limiting membrane of LE/Ly whereas
NPC2 is soluble cholesterol binding protein localized in the lumen of LE/Ly.
There are no therapeutic options to treat the disease and the children
born with this defect die before the age of 20 years. Miglustat, a drug used
to treat Gaucher disease has been reported to stabilize NPC patients and
has also been approved for treating NPC patients in Europe. Infusion of
very high dose of chemical chaperone like 2-hydroxypropyl-b-cyclodextrin
(HPBCD) several times a week has also been shown to reduce the
cholesterol load in peripheral tissues. Unfortunately, the blood brain barrier
(BBB) cross over capability of HPCD is very poor and hence requires
intrathecal CNS injections.
Results: We have found that the treatment of several histone deacetylase
inhibitors (HDACi), corrects the NPC defect specifically in human patient
derived NPC1, but not in NPC2 fibroblast. Amongst the HDACi tested
Vorinostat is a FDA approved drug that has been shown to cross BBB and
is currently used in clinic for treatment of cutaneous T-cell lymphoma and
many other type of cancer. Our initial study was conducted on most
common mutation I1061T found in human patients but since then we
have tested the effect of Vorinostat and other HDACis on several
different patient derived mutant fibroblasts.
Conclusions: Our data indicate that Vorinostat is effective in rescuing the
phenotype in most cases but to a varying degree. This is a promising
breakthrough example of repurposing existing FDA approved drug for
the treatment of NPC1 and other LSDs.
Page 18 of 59
O3
Genetics of autism spectrum disorder & BDNF gene
Usha P Dave
Medical Geneticist & Neuroscientist, Mumbai, India
E-mail: ushadave26@gmail.com
Autism is a group of complex neurodevelopmental disorders which
manifests problems with social interaction, language, communication and
behavior deficits like stereotype and repetitive activities. Autism
prevalence rate in last one decade has shown astonishing level of increase
from 1 per 10,000 to 1 in 110 children in USA (CDC, 2007). Various
environmental & genetic factors or combination are suggested as
contributing factors in this clinically diagnosed neurobehaviour syndrome.
However, similar data on prevalence are scarce in India. The etiology of
autism still remains unknown, with many factors implicated in the
development of autism phenotype. Autism Spectrum Disorder (ASD) is
evaluated by a clinical psychologist using DSMR- IV criteria & may manifest
mild to severe autistic features, clinical symptoms & low to high
intellectual functioning. There are few well characterized genetic/
metabolic conditions (e.g. Rett Syndrome, Tuberous sclerosis, PKU) and
chromosomal syndromes (e.g. Fragile-X, Angelman and Prader willi ) where
autism has frequently associated features. Multiple genes are thought
to be involved in the pathogenesis, but no evidence involving any
one particular gene. The recent research focus is also on epigenetic
mechanisms operating in complex autism.
Brain Derived Neurotrophic Factor (BDNF) is a neurotophin in the mammalian
Central Nervous System & important in neuronal survival, neurogenesis, and
synaptic plasticity. BDNF gene is located on chromosome 11. In humans,
Val66Met is probably the most investigated SNP of the BDNF gene. Since
BDNF readily crosses the Blood-Brain-Barrier, the serum concentrations
correlate directly to brain concentration, therefore plasma studies of BDNF
are thought to accurately reflect CNS concentration. The significance of
serum BDNF in ASD to explore its precise role in pathogenesis of ASD and
therapeutic relevance is increased with the evidence of BDNF linked with
autism. A genetically heterogeneous population of India where consanguinity
and endogamous marriages are prevalent genetic risk factors, ASD is a
challenge. An attempt is made to study BDNF level & mutations in
correlation with the severity of neurobehavioral deficits in Indian patients
with ASD & mental retardation. This will be discussed in the light of current
scenario.
O4
Identification and clinical evaluation of segments of homozygosity,
uniparental disomy and complex chromosomal abnormalities revealed
by copy-number SNP arrays
Jia-Chi Wang, Leslie Ross, Loretta W. Mahon, Renius Owen, Morteza Hemmat,
Boris T Wang, Mohammed El Naggar, Kimberly A Kopita, Mary Haddadin,
Fatih Z Boyar, Arturo Anguiano, Charles M Strom, Trilochan Sahoo
*
Cytogenetics Laboratory, Quest Diagnostics Nichols Institute, 33608 Ortega
Highway, San Juan Capistrano, CA, USA
E-mail: tsahoo001@gmail.com
Background: Presence of such segments of homozygosity (SOH) may be
due to parental relatedness, chromosomal recombination or rearrangements
and provides important clues regarding ancestral homozygosity, parental
consanguinity or uniparental disomy. We have determined the frequency
and nature of copy neutral segments with allelic homozygosity identified in
cases interrogated by oligonucleotide-SNP microarrays.
Materials and methods: We collected cases from consecutive specimens
sent to our clinical laboratory over the past two years. The cases were
reported based on the presence of a contiguous SOH >10 Mb in a single
region or >5 Mb in at least two regions. The percentage of the genome
encompassed by SOH regions was calculated based on the total coverage of
about 2,700 Mb.
Results: Of 14,574 cases analyzed by SNP arrays, 872 (6%) cases harbored
SOH, with 659 (76%) cases harboring multiple SOH and interpreted as
arising due to identity by descent (IBD), 213 (24%) cases with SOH
involving a single chromosomal segment and suspected or confirmed as
resulting from UPD. For the cases with IBD, the coefficient of inbreeding
was calculated: 5% cases due to first degree or closer parental relatedness,
9% second, 19% third, 16% fourth, and 51% fifth. Cases with UPD cases
involved every single chromosome. In eight cases, identification of SOH
was crucial to diagnosing autosomal recessive disorders.
Conclusions: This study demonstrates that the identification of SOH, in
addition to CNVs, is much more frequent than previously recognized,
reflecting close parental relatedness, and often ascertains autosomal
recessive diseases or unravels UPD in many cases.
O5
Utility of CD-138 negative fraction for chromosome analysis in Plasma
Cell Dyscrasias (PCD): a novel approach
Gopalrao Velagaleti
*
, Christina Mendiola, Gihan Mohamed, William Ehman Jr.,
Vinaya Noronha, Veronica Ortega
Department of Pathology, University of Texas Health Science Center, 7703
Floyd Curl Drive, San Antonio, TX, USA
E-mail: velagaleti@uthscsa.edu
Background: FISH analysis is superior to chromosome analysis in
detecting important prognostic genetic abnormalities in PCD. However, its
sensitivity is hampered due to paucity of plasma cells in whole bone
marrow and often shows false-negative results when frequency of
abnormal cells is below the cut-off values. Studies have shown that the
abnormality detection rate in enriched plasma cells (EPC) is greater than
unselected plasma cells, but purification techniques are limiting to only
FISH when bone marrow volumes are inadequate. The inability to perform
chromosome analysis may compromise patient care since chromosome
analysis is equally important for detecting non-plasma cell related
abnormalities, such as secondary myelodysplastic syndrome. To resolve
this critical issue and optimize limited quantity received, we designed a
study where an immuno-magnetic CD138 enriched positive selection of
plasma cells was used for FISH while the negative selection was used to
retrieve the remaining cellular components (RCC) for chromosome analysis.
After validating this approach in a pilot study, we implemented this
strategy in 2012 for routine clinical diagnosis in patients with PCD. When
there was adequate sample volume available, both whole bone marrow
and RCC were used for chromosome analysis.
Results: We found 100% success rate for chromosome analysis using RCC.
Identical PCD related genetic abnormalities were observed by both
chromosome analysis using whole bone marrow and EPC FISH in 4.2%
(4/96) cases while 8.3% (8/96) cases showed discordant results. Population
variants such as loss of Y chromosome were observed in 6.3% (6/96) cases.
Karyotypic abnormalities of myeloid origin or population variants were
found both in whole bone marrow and RCC cultures. Karyotypes with PCD
related aberrations were seen only in whole bone marrow cultures in 37.5%
of the cases (3/8) while the remaining 62.5% (5/8) of cases showed them in
both whole bone marrow and RCC cultures. All PCD related aberrations
showed concordant EPC FISH results.
Conclusions: Our results confirm the feasibility of retrieving RCC from the
CD138 negative fraction, and prove to be an innovative strategy for
performing chromosome analysis on PCD patients with insufficient
sample volumes.
O6
Prenatal screening above aneuploidies that is Pre-Eclampsia
Gurjit Kaur
Department of Physiology, Consultant Incharge, Genetic Centre, GMCH-32,
Chandigarh, India
E-mail: gurjitkaur123@rediffmail.com
Background: Pre-Eclampsia is defined as high blood pressure and excess
protein in the urine after 20 weeks of pregnancy in a normotensive
woman. Even a slight increase in blood pressure may be a sign of
preeclampsia. Despite extensive clinical trials no therapeutic approaches
are available for either treatment or prevention of preeclampsia. Removal
of placenta remains the only solution for resolution of preeclampsia which
necessitates premature deliveries and can have adverse outcomes of low
Page 19 of 59
birth-weight babies, at the same time increasing the risk of maternal and
neonatal mortality and morbidity worldwide. The concentrations of several
placental proteins, inflammatory cytokines and growth factors are altered
in the maternal circulation of women with preeclampsia. Nonetheless,
early pregnancy screening for preeclampsia remains insufficient, and
randomized controlled trials that used biomarkers to identify high risk
women have been disappointing, perhaps because the sensitivity of most
of these markers is high in the second or third trimester, long after the
placental dysfunction is already established. At Genetic Centre, GMCH-32,
Chandigarh we have tried to correlate first trimester biochemical and
ultrasonographic markers (viz. Pregnancy Associated Plasma Protein-A,
Placental Growth Factor, Doppler Pulsatility Index) with establishment of
preeclampsia and other obstetrical complications (fetal growth restriction,
pre-mature delivery, risk of miscarriage, still-birth) in pregnant women. This
study will help in determining the possible diagnostic utility of PAPPA,
PlGF, and Doppler Pulsatility Index as sensitive and specific biomarkers for
screening early onset of pre-eclampsia.
Materials and methods: All pregnant women visiting Government
Medical College and Hospital, Chandigarh in first trimester of pregnancy are
tested for PAPPA, PlGF, and Doppler Pulsatility Index after thorough
counselling and written informed consent. PAPPA, PlGF and Doppler
Pulsatility Index are quantitatively analyzed and multiple of medians are
obtained of approximately 1000 pregnant ladies.
Result and conclusion: In order to relate mode of delivery (NVD/LSCS)
with PAPPA, PlGF, and Doppler Pulsatility Index an initial sample size of 222
was taken for analysis. The association of mode of delivery was carried out
using chi square test and it has been observed that PAPPA risk has a
significant association with mode of delivery however PlGF and Doppler
Pulsatility Index do not have a significant association. However this is an
ongoing study and statistical analysis of more samples is needed to get a
significant association.
O7
Suspected microdeletion syndromes and molecular cytogenetic
techniques: an experience with 330 cases
Ashutosh Halder
*
, Manish Jain, Isha Chaudhary
Department of Reproductive Biology, AIIMS, New Delhi 110029, India
E-mail: ashutoshhalder@gmail.com
Background: Microdeletion syndromes are characterized by small (< 5Mb)
chromosomal deletion in which one or more genes are involved. They are
frequently associated with multiple congenital anomalies. The phenotype is
the result of haploin sufficiency of genes in the critical interval. Fluorescent In-
Situ Hybridization (FISH), Multiplex Ligation-dependent Probe Amplification
(MLPA), Quantitative Fluorescent Polymerase Chain Reaction (QFPCR) and
Array (microarray) Comparative Genomic Hybridization (aCGH) techniques are
commonly used for precise genetic diagnosis of microdeletion syndromes.
Methods: This study comprised of 330 cases of suspected microdeletion
syndromes. There were 184 cases of 22q11.2 microdeletion, 52 cases of
William, 47 cases of Prader Willi/Angelman, 18 cases of Miller Dieker, 14
cases of Retinoblastoma (bilateral infantile), 5 cases of Trichorhinophalangeal
(TRP) and 10 cases of other microdeletion syndromes. FISH was carried out
on all 330 clinically suspected microdeletion syndrome cases using non-
commercial FISH probes. Subsequently, we have performed aCGH in 100
cases (77 cases of 22q11.2; 9 cases of William Syndrome, 8 cases of Prader
Willi Syndrome, 4 cases of Miller Dieker Syndrome and 2 cases of other
microdeletion syndromes) including one monozygotic twin pairs with
discordant phenotype. Another 50 cases, including 22q11.2 microdeletion,
aCGH experiment and analysis are in progress.
Results: FISH was confirmatory in 28 cases only (8.48%; 19 cases of
22q11.2 microdeletion, 5 cases of Prader Willi, 3 cases of William and 1
case of TRP syndrome). There were 8 cases with mosaicism and 20 cases
with pure deletion. Microarray was picked up copy number variation (CNV)
with or without copy neutral loss of heterozygosity (LOH) in approximately
70% of cases, mostly involving several chromosome loci. However, aCGH
was failed to pick up mosaic cases (with even 45% deleted cell lines).
Clinically suspected specific locus CNV was detectable in approximately
24% cases only by aCGH. Variation in deletion sizes and or break point
differences (with genes involvement variations) as well as other CNVs with
or without LOH was evident.
Conclusions: We conclude that FISH in this format should not be the
method of choice for clinically suspected microdeletion syndromes as cost,
labor & time versus benefit is unjust. Microarray seems better technique, in
clinically doubtful cases. However, microarray is likely going to miss mosaic
cases, if deleted cell lines concentration is less than 50%. It seems time
has come to follow strict clinical criteria for FISH testing or preferably to
follow better methods viz., DNA microarray (array comparative genomic
hybridization). We think that whole genome screening should be adopted as
first line of investigation and FISH may be used for detecting mosaicism,
screening family members and prenatal diagnosis. Furthermore, microdeletion
syndrome best fitted with genomic disorder as several chromosomal loci are
involved in CNV with or without LOH and alteration in deletion size or
breakpoint. Our study has not found identical deletion profile in any cases,
thus explaining reason for phenotypic variability between deletion positive
cases.
O8
Molecular characterization of mutations in galactosemia genes:
structural and functional implications
R Singh
1
, BR Thapa
2
, G Kaur
3
, R Prasad
1*
1
Department of Biochemistry, Postgraduate Institute of Medical Education
and Research, Chandigarh, India;
2
Department of Gastroenterology,
Postgraduate Institute of Medical Education and Research, Chandigarh, India;
3
Department of Physiology, GMCH, Chandigarh, India
E-mail: fateh1977@yahoo.com
Background: Galactosemia is an autosomal recessive metabolic disorder
caused by the deficiency of enzymes involved in galactose metabolism
resulting in complications like cataracts, hepatocellular damage and
developmental delay. Nonetheless, no report is available on mutations in
galactosemia genes from our population. The objective of the present
study was to determine blood GALT and GALK activity in infants with
cholestasis and congenital cataracts and to establish a spectrum of
mutations in GALT and GALK genes.
Material and methods: 430 infants (2 days-11 months) with cholestasis
admitted in Pediatric Gastroenterology over 3.5 years were evaluated for
galactosemia. Basic investigations included hemogram, liver function tests,
blood culture, urine culture, urine for non-glucose reducing substances and
eye evaluation. Screening for GALT deficiency was done using PerkinElmer
neonatal GALT kit. The levels of galactose-1-phosphate were also measured.
Also, 115 patients with congenital cataracts were screened for the galacto-
kinase (GALK) deficiency. Mutation analysis for most common Q188R and
N314D mutations in GALT gene was performed by Restriction Fragment
Length Polymorphism (RFLP). Single Stranded Conformational Polymorphism
(SSCP) analysis and subsequently DNA sequencing were done for
identification and characterization of unknown and novel mutations in GALT
and GALK genes.
Results: 55 (12.7%) infants were found to have reduced GALT activity with
male: female: 37:18, jaundice in 55 (100%), hepatomegaly in 54 (98%),
splenomegaly in 32 (58%), coagulopathy in 23 (42%), encephalopathy in 9
(16%), septicemia in 10 (18%) and cataracts in 12 (22%) were observed.
Increased galactose-1 phosphate levels were fraternized with reduced
activity of GALT. A total of 16 mutations and 4 polymorphisms were
detected. 10 were novel mutations. Reduced blood galactokinase activity
was found in 13 (10.2%) patients with congenital cataracts. 4 novel
mutations were found in GALK gene.
Conclusions: N314D mutation was found to be the most common
mutation in our population. 10 and 4 novel mutations were also detected
in GALT and GALK genes respectively.
ARRAY- CGH
P1
Use of array CGH for molecular characterization of genetic disorders
Ashish Bahal
*
, Rajitha P , Ashwin Dalal
Center for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad
500001, India
E-mail: ashishbahal@cdfd.org.in
Molecular Cytogenetics 2014, 7(Suppl 1):P1
Page 20 of 59
Background: Microarray-based comparative genomic hybridization (array
CGH) is a revolutionary platform that has been developed to screen
entire genome for copy number variations (CNV) with resolution beyond
the capacity of light microscope. ACMG has recommended that array
CGH can be used as the first line investigation modality in cases of non-
syndromic mental retardation. We present here three cases in which use
of array CGH has provided an insight of genetic abnormality.
Cases studies: Case 1 was a 2 month old child who presented with
multiple dysmorphic features of face and extremities. The karyotype of the
child showed presence of additional chromosomal material of unknown
origin on chromosome 18. Array CGH was carried out which showed
duplication of region 2q31.1-q37.3. Whole chromosome paint probe for
chromosome 2 showed presence of chromosome 2 material on 18q.
Case 2 was a 10 year old girl having intellectual disability and facial
dysmorphism. The initial cytogenetic evaluation did not reveal any
abnormality. Further, array CGH showed presence of gain in chromosome 1
from bases 1959715 to 2787707. The segment was analysed using
computational software (Decipher) which showed it to be a pathogenic CNV
for Intellectual disability.
Case 3 was from two affected children in a consanguineous family with
hypotonia and delayed motor milestones. Molecular analysis for Spinal
Muscular Atrophy (SMA) was negative. Array CGH was done to look for
regions of loss of heterozygosity which were detected in multiple regions.
Further analysis of these regions was done to look for the genes associated
with SMA and we found PLEKHG5 gene within homozygous regions.
Conclusions: Our study demonstrates the usefulness of array-CGH in
detailed characterization of chromosomal rearrangements, detection of
submicroscopic copy number variations and detection of regions of
homozygosity in single gene disorders in consanguineous population.
P2
Diagnostic utility of array-based Comparative Genomic Hybridization in
a clinical setting
Frenny Sheth
1
, Chaitanya Datar
2
, Koumudi Godbole
3
, Joris Andrieux
4
,
Manisha Desai
1*
, Bhumi Patel
1
, Jayesh Sheth
1
1
FRIGEs Institute of Human Genetics, FRIGE House, Jodhpur Gam Road,
Satellite, Ahmedabad, India;
2
Sahyadri Genetics, Unit of Sahyadri Hospitals,
Barve Memorial Complex, J.M. Road, Pune, India;
3
Deenanath Mangeshkar
Hospital and Research Center, Erandawane, Pune, India;
4
Laboratory of
Medical Genetics, Jeanne de Flandre Hospital CHRU de Lille, Lille Cedex,
France
Background: Submicroscopic genomic imbalances are a major cause of
congenital and developmental abnormalities including unexplained
Developmental Delay (DD), Intellectual Disability (ID), Autism Spectrum
Disorders (ASD) and Multiple Congenital Anomalies (MCA). Submicroscopic
imbalances are not visible at the resolution level offered by conventional
cytogenetics techniques but could potentially be analysed with array based
comparative genomic hybridization (aCGH) which offers high resolution
scan of the genome. We present the motion backed by clinical evidence for
the diagnostic utility of aCGH in a clinical settings for aforementioned cases.
Material and methods: aCGH analysis was carried out in 37 non-syndromic
individuals that clinically presented with either one or a combination of
aforementioned phenotypes. Of which, 13 cases had congenital anomalies
with or without mental retardation [Group-1]. Remaining 24 cases presented
developmental and learning disabilities with seizure [Group-2]. Conventional
cytogenetic [GTG-] banding analysis showed apparently normal chromosomal
pattern at 550-band resolution. Hence, all 37 cases were further analyzed
using Agilent 60k oligonucleotide array-Comparative Genomic Hybridization
(aCGH). Sex matched genomic DNA (Promega Corporation, Madison, WI, USA)
was used as reference. Relative fluorescence intensity data was analyzed with
the aCGH analysis software v3.4 (Agilent Technologies Inc., Santa Clara, CA,
USA) by applying Z-score segmentation algorithm with a window size of 10
points to identify chromosome aberrations. Parents in 12 out 16 families were
analysed for genomic imbalances using q-PCR to confirm the mode of
inheritance.
Results: Cryptic quantitative genomic alteration was detected in a total of
16 cases that include 31% [4/13] from group-1 and 50% [12/24] from
group-2. The chromosomes involved in submicroscopic alterations were
1p, 2q, 5q, 6q, 7q, 8q, 10q, 11q, 12q, 15q, 18q, 19q, 22q, Xp and Xq.
Single copy number variation was detected in 14 cases whereas in the
remaining two, multiple CNVs were detected. De novo alteration and
parental inheritance was confirmed in 4 and 7 cases respectively. One
case is currently under investigation and four cases did not provide
consent for further investigation.
Conclusions: aCGH is currently practiced as a front line diagnostic
technique for patients with developmental delay and intellectual disability in
various countries. This technique is also being applied to understand why
patients get developmental disorders as a part of Deciphering Development
Delay (DDD) study. Accurate information of etiology aids in genetic
counselling and calculating risk for future pregnancy. Our clinical data
indicate the application of aCGH in cases with complex phenotypes and
apparently normal karyotype.
P3
Combined classical cytogenetics and array Comparative Genomic
Hybridisation for genomic copy number analysis
Meena Lall
*
, Pushpa Saviour, Ratna Puri, Preeti Paliwal, Surbhi Mahajan,
Ishwar Verma
Center of Medical Genetics, Sir Gangaram Hospital, New Delhi 110060, India
E-mail: lallmeena@gmail.com
Background: There is a great need for reporting and cataloging of
genotype phenotype correlation of clusters of individuals sharing similar
genomic rearrangements and phenotypes. This will facilitate diagnosis and
personalized genetic counseling and will also improve our understanding of
gene function and disease. Karyotyping and FISH are microscopic classical
cytogenetic methods of detecting gain, loss or rearrangement of genetic
material. Array Comparative Genomic Hybridisation (CGH) can used for
molecular characterization, to size the abnormality and study the gene
content.
Materials and methods: A pilot study was designed: Combined classical
cytogenetic, Array CGH and phenotypic findings were correlated in
20 patients with autism, mental retardation or congenital malformations.
Results: Ten out of 20 patients with autism, mental retardation or
congenital malformations had a normal karyotype and ten had abnormal
karyotype. Ten normal subjects were included as normal controls, of which
two had balanced translocations, which were not recognized by ACGH.
Conclusions: Copy number variations detected by ACGH can be novel or
extremely rare such that uncertainty may remain as to whether the
aberration is pathogenic or simply a benign variant. Copy number variant
(CNV) loci have been listed in the database of genomic variants (DGV).
Using this and the UCSC browser the analysis of the size of the
aberration and gene content was done in all individual cases, with
normal or abnormal karyotypes. These findings were correlated with the
phenotype to recognize individual syndromes.
Conclusions: This pilot study is just the beginning. Identification of more
patients, who share a region of genomic duplication or deletion and have
phenotypic features in common, will allow greater certainty to be given to
the pathogenic nature of the rearrangement and delineation of new
syndromes. Most data available is western. Therefore there is a need to
document and register Indian data accumulated to recognize the more
common syndromes affecting our population and the common benign
CNVs which can be discounted in our population, during the CNV analysis.
CANCER GENETI CS
P4
A study of human Kallikrein-2 gene polymorphism with special
reference to prostate cancer patients in India
Rajesh Biswas
1*
, Shiv Deep Bansal
2
, Kakoli Biswas
3
, Shrawan K. Singh
4
1
Department of Zoology, Government Home Science College, Sector-10,
Chandigarh, India;
2
National Centre for Human Genome Studies and
Research, Panjab University, Chandigarh, India;
3
Department of
Biotechnology, DAV College, Sector-10, Chandigarh, India;
4
Department of
Urology, Postgraduate Institute of Medical Education and Research,
Chandigarh, India
E-mail: rajeshbiswas63@yahoo.co.in
Page 21 of 59
Background: Prostate cancer is the third most common cancer among
men in the world. The human kallikrein-2 gene (KLK2 gene) contains a
mutant (T) for wild-type (C) allele substitution, resulting in a Trp to Arg
change on the 250
th
amino acid at the 784
th
nucleotide of KLK2 cDNA.
The reports on polymorphism in the KLK2 gene in relation to prostate
cancer are contradictory among American and Korean population.
Reports are not available regarding polymorphism in KLK2 gene in
relation to prostate cancer in Indian population. The relationship between
mutants (T) for wild-type (C) allele substitution of the KLK2, circulating
human kallikrein-2 (hK2) levels and prostate cancer risk in Indian
population has been examined.
Materials and methods: The study was limited to 15 subjects that include
seven patients with prostate cancer and six controls who were persons with
no cancer. Peripheral blood samples were collected from subject and
control. DNA was extracted from these samples using standard protocol.
DNA was dissolved in 1X TE buffer and used for PCR reactions for the
amplification of 322bp KLK2 gene. Polymerase chain reaction- Restriction
fragment length polymorphism (PCR-RFLP) procedure was employed using
MspI to detect this polymorphism. The enzyme recognizes the 4bp
sequence 5CCGG3 at codon 226 of KLK2 gene. PCR-RFLP products ware
separated in 2% agarose gel and viewed under Gel-doc system.
Results: The median age of the eight subjects diagnosed for prostate
cancer were 60, out of which 6 are found to be homozygous for CC allele of
KLK2 gene, 1 was heterozygous having CT allele and 1 was homozygous for
TT allele. From 7 subjects diagnosed negative for prostate cancer 2 were
having the CT genotype and 5 were found to have the TT genotype.
Conclusions: The study indicates a possible correlation among the C/T
polymorphism of the KLK2 gene and circulating levels of hK2 and, in
combination, may be predictive for prostate cancer. Though significant
association has been found with small subjects, uses of allele marker for
prostate cancer need further extensive study with large number of
subject.
P5
Role of betel quid in changing oral pathology
Aniket Adhikari
1*
, Subhrajyoti Mukherjee
2
, Kaustav Roy
2
,
Ranjan Roychowdhury
3
, Madhusnata De
1
1
Department of Genetics, Vivekananda Institute of Medical Sciences,
Ramakrishna Mission Seva Pratisthan, 99- Sarat Bose Road, Kolkata 700 026,
West Bengal, India;
2
Department of Oral and Maxillofacial surgery of
Ramakrishna Mission Seva Pratisthan, 99- Sarat Bose Road, Kolkata 700 026,
West Bengal, India;
3
Department of ENT of Ramakrishna Mission Seva
Pratisthan, 99- Sarat Bose Road, Kolkata 700 026, West Bengal, India
E-mail: aniket_adhikari@rediffmail.com
Background: Chewing of betel quid (BQ) and areca nut is an ancient
custom in South East Asia. More than 600,000,000 people chew areca nut
worldwide. In India there are 75,000 to 80,000 cases of oral cancer each year
and incidence rates of cancers of the oral cavity in both male and female in
all urban cancers are among the highest in the world. In India, 13 -50% of
the student chew BQ and pan masala frequently and prevalence rate is 14
-15% among 11-15 year of children. Areca nuts, Catechu, Slaked lime are
major components of Betel quid. Nitrosamines formed from alkaloids in
betel nut and reactive oxygen species (ROS) are generated due to slaked
lime during betel quid chewing may be implicated in the etiology of oral
cancer. Micronuclei (MN) have been proposed as a good biomarker to
assess cytogenetic damage. Among the xenobiotics metabolizing enzymes,
the CYP2A family is characteristics of its catalytic properties to nitrosamines.
Micronuclei (MN) and CYP2A6 genetic polymorphism were studied among
the Eastern and North eastern population.
Materials and methods: In this present study subjects were screened from
different camps and Department of E.N.T. & Oral and Maxillofacial surgery of
RKMSP hospital, Kolkata. Exfoliated cell from the buccal mucosa were
examined for micronuclei (MN). Polymorphism of CYP2A6 gene was studied
from EDTA blood.
Results: Micronuclei percentage was higher in subjects who had betel
quid chewing habit. Early metabolizers are susceptible to oral cancer
whereas in case of poor metabolizers chances are less.
Conclusions: Oral pathology seems to be altered due to chewing betel
quid and its ingredients with increased susceptibility to cancer.
P6
In silico screening of alleged miRNAs associated with cell competition:
an emerging cellular event in cancer
Manish S Patel
1*
, Bhavesh V Antala
1
, Neeta Shrivastava
1,2
1
Department of Biotechnology, National Institute of Pharmaceutical
Education and Research (NIPER)-Ahmedabad, Ahmedabad-380054, Gujarat,
India;
2
Department of Pharmacognosy and Phytochemistry, B. V. Patel
Pharmaceutical Education and Research Development (PERD) Centre,
Ahmedabad-380054, Gujarat, India
E-mail: patel_manish22002@yahoo.com
Background: Cell competition is identified as a crucial phenomenon for
various conditions like cancer, aging and organ development. MicroRNAs
(miRNAs) may play an important role in regulation of expression of genes
involved in cell competition at the post-transcriptional level. In silico
screening of miRNAs involved in a cell competition is an effort to identify
potential miRNAs and to reduce, economize and expedite experimental
work. In the present study we have identified miRNAs of Drosophila
genome involved in a cell competition using in silico screening strategy.
Material and methods: We used four steps of in silico (i) Selection of cell
competition related genes of Drosophila genome through literature survey;
(ii) Identification of miRNAs that target selected cell competition-related
genes; (iii) Sequence conservation analysis of identified miRNAs with Human
genome; (iv) Identification of genomic location of that Drosophila miRNAs
and exploration of their expression profiles in tissues by use of host gene
expression profile.
Results and conclusions: In this study we have identified seven potential
Drosophila miRNAs that are most probably involved in cell competition.
Human homologs of these seven potential miRNAs may be very important
in cell competition related diseases like cancer. Proof of this concept will be
established with wet lab experimentation to reassert the role of miRNAs in
cell competition.
P7
Analyzing the expression of candidate microRNAs in primary tumors
of oral squamous cell carcinoma
Mayakannan Manikandan
*
, Deva Magendhra Rao A K,
Arasambattu Kannan Munirajan
Department of Genetics, Dr. ALM PG Institute of Basic Medical Sciences,
University of Madras, Taramani campus, Chennai 600113, Tamil Nadu, India
Background: Accumulating evidences suggest that aberrant expression of
microRNAs (miRNAs) contribute to the initiation, development, invasion
and metastasis of human cancers including Oral Squamous Cell Carcinoma
(OSCC).
Materials and methods: In this study, we investigated the differential
expression of six candidate miRNAs namely hsa-miR-21, hsa-miR-125b-2,
hsa-miR-138, hsa-miR-155, hsa-miR-184 and hsa-miR-205 in thirty two
OSCC primary tumors in comparison to five normal control tissue samples
by employing TaqMan MicroRNA Assays.
Results: Amongst the studied candidates, miR-125b-2 and miR-205 were
significantly down regulated while miR-21 and miR-155 were significantly
upregulated in the primary tumors compared to controls. The other two
miRNAs, miR-184 and miR-138, in general were down regulated in the
cancer samples although not statistically significant. These observations
suggest that microRNA dysfunction could be one among the major
factors for initiation and progression of oral cancer with miR-125b-2 and
miR-205 functioning as tumor suppressor miRs, and miR-155 and mir-21
acting as oncomiRs. Functional annotation of the experimentally validated
targets of these miRNAs highlighted that miR-21, miR-125b-2 and
miR-155 were predominantly targeting genes of the apoptotic pathway,
while miR-205 was targeting genes involved in transcriptional and
metabolic processes.
Conclusions: It is concluded that microRNA dysfunction could be one
among the major factors for initiation and progression of oral cancer.
However, further experimental studies in more OSCC primary tumors are
required to facilitate the use of these miRNAs as molecular biomarkers or
as diagnostic/prognostic indicators.
Page 22 of 59
P8
The role of direct DNA repair gene O6-methylguanine-DNA
methyltransferase (MGMT) in high grade malignant glioma
M Jeru Manoj
1*
, G K Chetan
1
, KVL Narasinga Rao
2
, HN Venkatesh
1
, MK Sibin
1
,
Ch Lavanya
1
1
Department of Human Genetics NIMHANS, Bangalore-29, India;
2
Department of Neurosurgery, NIMHANS, Bangalore-29, India
E-mail: jeru.manoj@gmail.com
Background: High-grade [WHO, 2007- grade III-IV] gliomas are undifferen-
tiated or anaplastic, are highly infiltrating and termed as malignant. The
overall prognosis and survival rate is very low. Only few highly sensitive and
specific biomarkers for Glioma have been identified, but are yet to be put
for routine use due to challenges in their clinical validation for early disease
detection, diagnosis and monitoring to improve long-term survival of
patients. The inefficacy of currently available chemotherapeutic and radio-
therapeutic agents largely depends on a number of resistance mechanisms
among which DNA repair plays an important role. Hence bio-molecules
involved in DNA repair mechanisms will be promising biomarkers. Current
status in the validation of DNA repair genes include, studying the promoter
methylation of the only direct DNA repair gene O6-methylguanine-DNA
methyltransferase (MGMT). MGMT is epigenetically inactivated via
hypermethylation of the 5-CpG islands located in promoter region. It is
associated with prediction of successful alkylating agent therapy with
temozolomide in combination with radiotherapy and also longer overall
survival in patients. The silenced MGMT gene possibly enhances radiation
effects by inducing radiation-mediated double stranded DNA (dsDNA)
breaks and suppression of dsDNA repair pathways after radiation exposure.
Materials & methods: Post surgery excised tumor samples from 20
patients diagnosed histopathologically with high grade glioma were tested
for MGMT promoter region methylation status. DNA extraction and
bisulphate conversion using suitable commercially available kits (Qiagen)
followed by Methylation specific PCR using specific methylated and
unmethylated primers was carried out. Correlations are underway with
clinical profile and treatment regimen.
Results: Hypermethylation of the promoter region was observed in about
60%, (12) samples. The other samples only demonstrated unmethylated
regions.
Conclusions: Assessment of promoter methylation of the MGMT gene
helps in the therapeutic choice of the patients. It has gained importance
not only as a prognostic but also as a predictive biomarker in molecular
profiling of high grade gliomas.
P9
Correlation o P16 and Bmi1 gene expression in human high grade
glioma
M K Sibin
1*
, GK Chetan
1
, Ch Lavanya
1
, Manoj M Jeru
1
, Dhananjaya I Bhat
2
1
Department of Human genetics, National Institute of Mental Health and
neurosciences, Hosur road, Bangalore-29, India;
2
Department of
Neurosurgery, National Institute of Mental Health and neurosciences, Hosur
road, Bangalore-29, India
E-mail: siboottan@gmail.com
Background: Gliomas are neoplasm of the central nervous system and
make up approximately 80% of all malignant brain tumors. Even with the
advancement in the field of surgery, chemotherapy and radiotherapy, the
prognosis of glioma patients is dismal. Many molecular alterations of
various oncogenes and tumor suppressor genes are seen in glioma and
can be used for molecular characterization of tumor. The Bmi1 is a
Polycomb group of protein which is associated with various cancers. The
main alterations in Bmi1 gene in glioma includes copy number variation,
over expression of mRNA and protein levels when compared to the non
glioma brain samples. The CDKN2A (p16) is a tumor suppressor gene,
mapped to 9p21. Alterations of the 9p21 locus have been implicated in
many types of cancer, indicating the role of the tumor suppressor genes
CDKN2A which encodes for p16
INK4a
/p14
ARF
. In this study we wanted to
check the gene expression pattern of p16 and Bmi1 genes in glioma and
their clinical correlations.
Material and methods: 50 glioma tissues were collected from
Neurosurgery department and histologically confirmed as glioma. Total
RNA was isolated and 1g was converted to cDNA using RT kit. Real time
RT PCR was performed using Taqman probes specific for p16 and Bmi1
and GAPDH as internal control.
Results: We showed that the p16 mRNA levels in glioma found to be
decreased when compared to the non glioma tissues. Bmi1 gene was
found to be over expressed in glioma. There is a correlation in the
expression of both genes. Bmi1 is the main upstream regulator of p16
and we found that gene expressions of both are correlated in glioma.
Conclusions: It is concluded that p16 and Bmi1 genes are important in
glioma and can be used as independent biomarker in glioma characterisation.
P10
Promoter methylation of PTEN gene in high grade gliomas
C Lavanya
1*
, GK Chetan
1
, MK Sibin
1
, Manoj M Jeru
1
, Bharath MM Srinivas
2
1
Department of Human genetics National Institute of Mental Health and
Neurosciences, Bangalore, India;
2
Department of Neurochemistry, National
Institute of Mental Health and Neurosciences, Bangalore, India
E-mail: lavanya.nimhans@gmail.com
Background: Gliomas are the most common primary malignant central
nervous system tumors arising from glial cells accounting for 80% of all
malignant brain tumors. Many mutations occur frequently in genes that
control cell cycle and proliferation leading to tumor progression. Major genes
that are associated with the malignant tumors are MGMT, hTERT, TP53, PTEN,
P16. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN)
is a tumor suppressor gene deleted or mutated in many cancers. It plays a
significant role in cellular processes like cell cycle progression, apoptosis,
translation, growth, proliferation and migration by negatively controlling PI3/
AKt pathway. Deregulation of PI3K signaling pathways proceed from genetic
alterations in the PTEN gene on 10q23 at the level of mutation, LOH and
methylation identified in 60% of glioblastoma. Recent reports show that
absence of PTEN was associated with increased tumor malignancy in
gliomas. This seems to have prognostic significance and accepted as
independent prognostic factor. In this study 15 high grade glioma tissues
were collected from neurosurgery department and confirmed as glioma
histologically.
Material and methods: DNA was isolated using Qiagen kit and
bisulphite DNA conversion was done by Qiagen Epitect. PCR was
performed to check the methylation status in the promoter region of
PTEN gene.
Results: We noticed that around 50% of patients DNA show methylation in
promoter region of PTEN gene when compared with healthy control DNA.
We are looking at the response of these patients to chemotherapy and
radiotherapy. We are also looking in to changes in the PI3/AKt signaling
pathway.
Conclusions: There is a possibility of correlation between the methylation
status and patients survival.
P11
Role of p16 deletion and Bmi1 copy number variation in glioma
GK Chetan
1*
, M K Sibin
1
, Dhananjaya I Bhat
2
, Ch Lavanya
1
, Manoj M Jeru
1
,
N Geethashree
1
1
Department of Human genetic, National Institute of Mental Health and
neurosciences, Hosur road, Bangalore-29, India;
2
Department of
Neurosurgery, National Institute of Mental Health and neurosciences, Hosur
road, Bangalore-29, India
E-mail: drchetangk@gmail.com
Background: Malignant gliomas are the most common and lethal
intracranial tumors. Glioma exhibit a relentless malignant progression
characterized by widespread invasion throughout the brain, resistance to
traditional and newer targeted therapeutic approaches. The classical genetic
alterations in glioma target pathways governing cellular proliferation, cellular
survival (apoptosis and necrosis), invasion, and angiogenesis. The p16 is the
second most altered tumor suppressor gene and frequent mutations and
Page 23 of 59
deletions of p16 in human cancer cell lines first suggested an important role
for p16 in carcinogenesis. The Bmi-1 is an important oncogene and its
expression is found to be elevated in many types of cancers. Role of Bmi1
copy number variation in glioma is still under debate. In this study we
analyzed the alterations in p16 and Bmi1 genes in glioma.
Material and methods: 50 glioma samples from patients were collected
from the neurosurgery OT. Tissues having >95% tumor cells were
processed and DNA was isolated. For the analysis of p16 deletion multiplex
PCR was done using primers specific for all 3 exons of p16. Copy number
variation in Bmi1 gene was analyzed using real time PCR with Bmi1
specific primers.
Results: Our results showed that there is 20% of p16 deletion in our
samples and the deletion pattern vary with exons. There was no copy
number variation found in Bmi1 gene in glioma.
Conclusions: We concluded that the p16 deletion is a common alteration
found in glioma and Bmi1 gene amplification is not the common mechanism
to increase the expression of this protein in glioma.
P12
Analysis of DNA damage in cells excreted in urine of cervical cancer
patients using alkaline comet assay
Avani Patel
1*
, Mihir Shah
1
, Pinaki Patel
2
, Trupti Patel
1
1
School of Biosciences and Technology, Vellore Institute of Technology,
Vellore 632014, Tamil Nadu, India;
2
Sardar Patel University, Vallabh
Vidyanagar, Anand, India
E-mail: avani9patel@gmail.com
Background: Cancer is one of the most unwanted menaces in the
human body. Cancers of lower abdomen are not only life threatening but
also painful and survival rate is low. Cervical cancer is second most
common worldwide and fifth deadliest in women. It affects about 16 per
100,000 women per year and kills about 9 per 100,000 in a year. In
developing countries occurrence rate is 80%. It is possible that there may
be no symptom until an advance stage of the cancer is progressed. The
single cell gel electrophoresis assay also called comet assay, which is
versatile, reliable, powerful, uncomplicated and sensitive technique for
detection of DNA damage at the level of individual eukaryotic cell.
Understanding the extent of DNA damage in neoplastic cells discarded in
the urine of cancer patients through comet assay. Usually normal urine
sample have very rare cells. In case of cancer patient the number of cells
increases drastically. The type of cells passed in the urine of cancer
patient contains mainly leukocytes, some neoplastic cells and some
infected tissue cells.
Materials & methods: The analysis was carried out on 10 subjects
having cervical cancer and different levels of DNA damages were seen in
cells which were separated from urine. The choice of sample is so, as it is
non-invasive.
Results: The cells have damaged DNA as a result of cancer, which do not
have proper binding with histone proteins giving a tail in comet assay
which can be easily seen by ethidium bromide staining.
Conclusions: Comet assay can be used to diagnose the cancer in the
suspected patient as an alternative approach to detect the stages of the
cancer instead of biopsy and cytology which are painful methods. Early
stages of cancer will be detected by non-invasive technique.
P13
Vitamin D receptor gene polymorphisms modulate the clinico-
radiological response to vitamin D supplementation in knee
osteoarthritis
Divya Sanghi
1*
, RN Srivastava
1
, Sarita Agarwal
2
1
King Georges Medical University, Near Daligunj Chauraha, Lucknow, India;
2
Post Graduate Institute of Medical sciences, Raibareily Road, Lucknow, India
Background: Vitamin D receptor (VDR) gene plays an important role in
bone mass regulation and is demonstrated in human articular chondrocytes
of cartilage. We have previously shown a beneficial effect of vitamin D on
symptomatic improvement in Osteoarthritis knee (KOA) patients. This study
investigated whether the clinic-radiological response to vitamin D was
modulated by VDR gene.
Materials and methods: This randomized placebo-controlled trial
recruited 103 KOA cases as per American College of Rheumatology (ACR)
guideline having vitamin D insufficiency (25(OH)D50 nmol/L). Enrolled
cases were randomly allocated in two groups to receive placebo (51) and
vitamin D (52). Primary outcome measures: pain and functional disability
which were recorded by knee specific WOMAC index and secondary
outcome measure were radiological features (joint space width and
osteophytes). The serum levels of vitamin D were assessed by ELISA
method using IDS, UK kit. Detection of VDR polymorphisms (Taq1 & Apa I)
were done by PCR-RFLP technique. 25(OH)D levels, clinical and radiological
features were recorded at baseline and at one year follow up.
Results: At one year, in vitamin D group, TT genotype of TaqI polymorphism
showed increment in 25(OH)D levels in comparison to Tt and tt genotype
whereas in placebo group it remained same. No such association was
observed for ApaI polymorphism. In clinical features, pain significantly
increased in Tt and tt genotype of placebo group whereas it decreased,
although not significant in each genotype of vitamin D group. Total WOMAC
scores significantly decreased in both Tt and TT(p<0.05) genotypes in vitamin
D group, whereas functional disability only in Tt(p<0.05). In radiological
features, decreased Medial-JSW was observed in tt genotype and increased
Osteophyte was observed in TT and Tt genotype of placebo group whereas
no significant changes were noted in vitamin D group. No such effect was
observed with ApaI polymorphism.
Conclusions: VDR gene polymorphisms influence the clinico-radiological
response to vitamin D supplementation in KOA Bottom of Fo with low
25-OHD levels.
P14
Anti cancer activity of colon specific osmotic drug delivery of acetone
extract of Quercus Infectoria Olivier, Fagaceae in 1,2-
dimethylhydrazine-induced colon cancer in rats
Roshni Solanki
1*
, Dhaval Madat
2
, Ramesh K. Goyal
3
1
Faculty of Pharmacy, Dharmsinh Desai University, Nadiad, India;
2
TBC;
3
Institute of Life Science, Ahmedabad University, Ahmedabad, India
Background: Adenoma of colon and rectum is known as colorectal
cancer. It is the fourth most common form of cancer in the United States.
Various colon drug targeting systems are currently under development to
minimize drug degradation, prevention of side-effects and increase drug
bioavailability in the required zone. Galls of Q.infectoria contains about 50%-
70% tannin mainly gallotannic acid which is reported to be antimutagenic.
In present investigation colon targeted AEQI formulation was prepared and
evaluated by in vitro drug release study and in vivo study using 1,2-
dimethylhydrazine(DMH) induced colon cancer in rats with or without
diabetes.
Materials and methods: Pellets of AEQI were prepared by extrusion-
spheronization method using Chitosan as osmotic agent. Optimized
formulation was tested further for in vitro study, stability study and release
kinetic model fitting study. In vivo study was carried out using 1,2-
dimethylhydrazine (DMH) 20 mg/kg s.c. in rats. Diabetes was induced by
streptozotocin 40 mg/kg i.v. Colon targeted formulation of AEQI of 100mg/
kg and 200 mg/kg was administered for 16 weeks. At the end of study
blood samples were collected for estimation of antioxidant and oxidant
parameters and measurement of TNF a and TGF b levels. Colon was
isolated at the end of study and development of aberrant crypt foci (ACF)
was observed in histopathology and tissue VEGF levels were measured.
Statistical analysis was carried out using analysis of variance followed by
multiple comparison Tukey (p<0.05).
Results: In vitro study showed that optimization gave sustained release of
drug for 24 hr 98.37% release and stability with respect to release pattern.
The release of drug follows Weibull model with minimum F value (22.40).
In vivo treatment showed improvement in oxidant and antioxidant
parameters significantly with improved histopathological characters in
colon as compared to non treated model control group. Moreover
significant reduction in TNF a, TGF b and VEGF levels was observed with
treatment. Diabetes associated with colon cancer increases severity of
colon cancer and it was prevented by treatment.
Page 24 of 59
Conclusions: Our data suggest that microbially triggered colon specific
osmotic pump delivery have a potential to be used as targeted therapy
for cancer.
P15
Genetic variation in PSCA gene and bladder cancer susceptibility in
North Indian population
Praveen K Jaiswal
*
, Vibha Singh, Rakesh Kapoor, Rama D Mittal
Department of Urology and Renal Transplantation, Sanjay Gandhi Post
Graduate Institute of Medical Science, Raebareli Road, Lucknow 226014, Uttar
Pradesh, India
Background: Bladder cancer (BC) is a significant health problem worldwide.
Prostate stem cell antigen (PSCA) gene has been reported earlier in Genome
Wide Association Study (GWAS) for BC risk. It is highly expressed in bladder
cancer and considered to be involved in the cell proliferation inhibition and/
or cell-death induction activity. It has been assumed as a useful marker for
diagnosis and progression of bladder cancer.
Materials and methods: We genotyped PSCA rs2978974G/A and PSCA
rs2294008C/T gene by Real Time Taqman probes to evaluate the risk of
bladder cancer (BC) in histologically confirmed 225 BC patients and 240
healthy controls (age and gender matched) from North Indians in a hospital
based case control study. Further statistical analysis for association studies
was done by SPSSver16.0.
Results: Significant increased BC risk was observed to be associated with
heterozygous CT genotype of PSCA rs2294008C/T having 1.86 folds risk
(p=0.004;OR=1.86). The variant allele T was also significantly associated
with BC risk (p=0.027;OR=1.38) for PSCA rs2294008C/T. In case of PSCA
rs2978974G/A, no significant association was observed with BC at
genotypic level. Smoking significantly modulated the BC risk in patients
with heterozygous CT genotype (p=0.025) for PSCA rs2294008C/T gene
polymorphism. A significant BC risk was observed when risk was evaluated
with tumor-grade-stage level for PSCA rs2294008C/T with heterozygous CT
genotype (p=0.045;OR=1.02). Furthermore, BC patients receiving BCG
treatment showed no significant association with any genotype of PSCA.
Bioinformatics analysis (in-silico analysis) showed no significant association
with BC risk.
Conclusions: Our study has unveiled a complex intervention of PSCA
rs2294008C/T conferring a higher risk of BC risk among North Indian
population. Further studies in large sample size and different ethnic
group are needed for validation.
P16
Replicative study of GWAS reported TP63C/T rs710521, TERTC/T
rs2736098 and SLC14A1C/T rs17674580 with susceptibility to bladder
cancer in North Indians
Vibha Singh
*
, Praveen Kumar Jaiswal, Rakesh Kapoor, Rama Devi Mittal
Department of Urology, Sanjay Gandhi Post Graduate Institute of Medical
Science, Raebareli Road, Lucknow 226014, India
Background: Genome Wide Association Studies (GWAS) have confirmed
association of rs710521, rs2736098 and rs17674580 gene variants with
susceptibility to Bladder Cancer (BC) in European and Caucasian. However,
the risk conferred for BC in north Indians is unknown. In present study, we
replicate GWAS findings of TP63C/T(rs710521), TERTC/T(rs2736098) and
SLC14A1C/T(rs17674580) gene polymorphisms for their association with BC
susceptibility in North Indian population.
Material and methods: Three SNPs were genotyped by real-time
polymerase chain reaction in histologically confirmed 225 BC cases and 240
healthy controls (age and gender matched) from North Indians in a hospital
based study. To evaluate SNP effects on BC susceptibility, odds ratio and
confidence interval were calculated by SPSSver.16.0. Bioinformatics analysis
was done by F-SNP free online web server.
Results: In case of TP63C/T(rs710521), variant genotype (TT) showed
significant reduced risk for BC (p=0.045;OR=0.53). On Combining
heterozygous and variant genotype, it showed reduced risk for BC (p<0.001;
OR=0.54). In case of TERTC/T(rs2736098) heterozygous genotype (CT) as well
as variant genotype (TT) showed significant risk for BC susceptibility
(p=0.031;OR=1.77,p=0.004;OR=2.78 respectively) along with T allelic level
(p<0.001;OR=4.19). Further SLC14A1C/T(rs17674580), variant genotype (TT)
also showed significant high risk for BC susceptibility (p=0.006;OR=3.01)
along with variant T allele level (p=0.003;OR=1.52). Interestingly smoking
was also found to modulate risks for BC in case of TERT and SLC14A1 variant
genotype (TT). Further tumor-grade-stage level of cases supports the
genotypic data with TERT and SLC14A1 for BC risk. Bioinformatics analysis
supported our result at genotypic level for the BC risk for TERTC/T and
SLC14A1C/T.
Conclusions: Our results indicated that TERTC/T(rs2736098) and SLC14A1C/
T(rs17674580) showed high risk for BC in North Indian population. However,
TP63C/T(rs710521) showed reduced risk of BC susceptibility. More study
with large sample size and diverse ethnicity are required to validate our
observations.
P17
Association of miR-27a, miR-181a and miR-570 genetic variants with
gallbladder cancer susceptibility on North indian population
Annapurna Gupta
1*
, Kiran L Sharma
1
, Annu Yadav
1
, Vijay Kumar
2
,
Sanjeev Misra
2
, Ashok Kumar
3
, Balraj Mittal
1
1
Departments of Genetics, Sanjay Gandhi Post Graduate Institute of Medical
Sciences (SGPGIMS), Lucknow, India;
2
Department of Surgical Oncology,
KGMU, Lucknow, India;
3
Surgical Gastroenterology, SGPGIMS, Lucknow, India
Background: MicroRNAs are small endogenously expressed short non-
coding RNAs. They appear to be critical regulators of tumor biology as
their aberrant expression is well characterized in cancer progression. The
role of microRNA is not fully understood in gallbladder carcinoma, so in
present study we investigated the role of miR-27a, miR-181a and miR-570
genetic variants with gallbladder cancer (GBC) susceptibility.
Material and methods: In this case-control study, we evaluated the role
of miR-27a, miR-181a and miR-570 genetic polymorphisms with GBC
susceptibility in North Indian population. The present study included 515
GBC patients and 200 healthy controls from North India. Genotypes were
determined by TaqMan probes. Statistical analysis was done by SPSS ver.
16. In silico analysis was performed using Bioinformatics tools (F-SNP,
FAST-SNP).
Results: Logistic regression analysis showed no significant association
of miR-27a, miR-181a and miR-570 genetic polymorphism with GBC
susceptibility (P> 0.05). On stratifying our data on the basis of gall stone
status, the [AG+GG] genotypes of miRNA rs895819 (A>G) were significantly
associated with increased risk of GBC in patients without stone (p=0.003
OR=1.83 [(95%CI) 1.23-2.72]. The genetic risk by miR-27a, rs895819 (A>G)
was also modulated by tobacco consumption as the heterozygotes (AG)
were at higher risk p=0.005 OR=1.94 [(95%CI) 1.22-3.08]. However, there was
no association of miR-181a and miR-570 polymorphisms with disease risk in
subgroup analysis. In-silico analysis showed change in transcriptional
regulation of miR-27a and miR-570 variations.
Conclusions: We found significant association of miRNA rs895819 A>G
with gallbladder cancer risk through gallstone independent pathway and
tobacco usage.
P18
Selection of suitable housekeeping genes for gene expression analysis
in glioma using quantitative real-time PCR
Madhuri GS Aithal, Narayanappa Rajeswari
*
Department of Biotechnology, Dayananda Sagar College of Engineering,
Bangalore, Karnataka, India- 560078
E-mail: nraja7@gmail.com
Background: Quantitative real-time PCR (qPCR) is the most reliable tool
for gene expression measurements from tissue samples. Selection of
housekeeping genes (HKGs) that are most stably expressed among tissues
is vital to carry out accurate gene expression profiling of target genes.
Expression of HKGs varies among tissues and experimental conditions.
There is no universal housekeeping gene having stable expression in all
tissues under all experimental conditions. So, it is extremely important to
Page 25 of 59
identify most appropriate internal control genes for a particular tissue and
experimental conditions. The aim of the present study is to identify most
suitable HKGs for gene expression analysis in glioma tissue samples.
Material and methods: Based on literature survey six most commonly
used HKGs reported to be invariant in human gliomas were chosen for gene
expression analysis. We performed qPCR using RNA from formalin fixed
paraffin embedded glioma patient samples and normal brain samples to
investigate the expression pattern of six HKGs [Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT),
b2 microglobulin (B2M), TATA-binding protein (TBP), 18S ribosomal RNA
(RN18S1) and ribosomal protein L13a (RPL13A)] with different abundance.
A simple Ct approach was employed to calculate the fold change.
Results and conclusions: Our data showed that out of the six genes
studied, expression of GAPDH, B2M and RPL13A were found to have most
constant expression across all the tumor samples in our experimental
setup. Thus, these three genes proved to be the most suitable to be used
as internal controls for gene expression analysis in human glioma. Except
for GAPDH, none of the other conventionally used HKGs in glioma studies
e.g., HPRT and TBP were found to be suitable as they showed large
variation in RNA expression. Validation of housekeeping genes is therefore
highly specific for a particular experimental setup and is important in
assessing any new setup.
P19
Development of allele specific hybridization assay and computer aided
tool for detection of genetic variations in folate pathway genes
Ravishankara
1*
, Shreeshakala
1
, S Padmalatha Rai
1
, Kerthana Prasad
2
,
C Deepthi
2
, PM Gopinath
1
, K Satyamoorthy
1
1
Division of Biotechnology, School of Life Sciences, Manipal University
Manipal, India 576104;
2
School of Information Sciences, Manipal University
Manipal, India 576104
Background: Most current single nucleotide polymorphism (SNP)
genotyping methods are still too slow and expensive for routine use
in large association studies that requires hundreds or more SNPs in a
large number of DNA samples and for diagnostic purpose. Clinical
implementation and ultimate use of nucleic acid biomarkers necessitate
development of pragmatic detection assays that deliver adequate
sensitivities, mutation selectivity and high throughput capabilities in a
rapid, robust and cost effective manner. In the present study we have
developed a sensitive and cost effective genotyping technique with
computer aided tool for allele discrimination and implemented it to
genetic epidemiology study.
Material and methods: Modified allele specific oligonucleotide
hybridization assay with elimination of DNA isolation step for PCR and novel
allele discrimination method was used to screen folate metabolism gene
polymorphisms in acute lymphoblastic leukemia patients and normal
individuals. The plasma homocysteine and global DNA methylation was
performed by HPLC method.
Results: The developed novel allele discrimination method along with the
newly developed image analysis software increases the sensitivity and
elimination of DNA isolation step for PCR reduces the cost of the
genotyping technique. The average kappa value of all designed probes
was > 0.81. The technique was successfully designed for both diagnosis
and research purpose. The evaluation of effect of gene polymorphism on
plasma folate, homocysteine level and global DNA methylation explored
that the only MTHFR C677T gene polymorphism is associated with
elevated homocysteine level in healthy young adults. The genotyping of
22 folate metabolism genes explored that only RFC1 80GA and RFC1 80AA
genotypes were significantly associated with increased risk of Acute
Lymphoblastic Leukemia (n=203) when compare to healthy individuals
(n=245) in south Indian population.
Conclusions: The developed visual based allele specific oligonucleotide
hybridization assay will be useful in clinical diagnostic system where large
scale screening is often necessary without any sophisticated detection
systems. RFC1 80GA and RFC1 80AA genotypes were significantly associated
with increased risk of acute lymphoblastic leukemia. In young adults,
only MTHFR C677T polymorphism is associated with elevated homocysteine
level.
P20
Cytogenetic and interphase Fluorescence in Situ Hybridization studies
in patients with multiple myeloma
G Perumal
1*
, RS Chandra
2
, P Prabu
1
, N Indhumathi
1
, Anil Tarigopula
1
,
Rama Mani
1
1
Department of Molecular Biology and Cytogenetics, Apollo Hospitals, No.
21, Greams Lane, Off. Greams Road, Chennai-600 006, Tamilnadu, India;
2
Department of Genetics, Dr. ALM PG. Institute of Basic Medical Sciences,
University of Madras, Taramani, Chennai - 600113, Tamilnadu, India
E-mail: perumalyogi@gmail.com
Background: Multiple myeloma (MM) is characterized by the clonal
proliferation and accumulation of malignant plasma cells in the bone
marrow, monoclonal protein in the blood or urine and associated organ
dysfunction. Some patients may show a slow progressive evolution from
monoclonal gammopathy of undetermined significance while others may
be associated with features of high clonal aggressiveness such as plasma
cell leukemia or extramedullary plasmacytomas. Chromosomal abnormalities
(mainly IgH translocations and trisomies) have been shown to be of
prognostic significance in MM. Interphase fluorescence in situ hybridization
(i-FISH), in particular, has been much more effective in identifying these
trisomies/ monosomies and specific translocations.
Materials and methods: The bone marrow aspirates were processed for
conventional cytogenetic and interphase FISH analyses using three probes
to detect del(13)(q14.3), t(4;14)(p16.3:q32) and del(17)(p13.1). A total of 30
newly diagnosed patients were studied between May 2012 and March
2013. The affected were mostly elderly people with median age of 55
years (range: 32 to 80 years) at the time of diagnosis. There were 21 males
and 9 females.
Results and conclusions: Chromosomal abnormalities were detected in
only 7 patients because of the low proliferation rate of plasma cells and the
non-availability of analyzable metaphases. On the other hand, i-FISH
revealed an abnormality in 14 patients and a normal pattern for the selected
probes in the remaining 16 patients. The most frequent abnormality was
found to be 13 monosomy (complete/ partial) in 13 cases followed by t
(4;14) seen in 4 patients. However these abnormalities were not recognized
in the karyograms. None of the cases showed a p53 deletion. Further studies
employing the complete FISH panel are required for better diagnosis and
prognosis.
P21
APC is epigenetically down regulated in advance cases of gallbladder
cancer
Dinesh Tekcham Singh
1
, Satish Poojary
1
, Shushruta Bhunia
1
,
Mustafa Ahmed Barbhuiya
1
, Manisha Kakkar
2
, Vishwajit Jalaj
2
, Sanjeev Gupta
2
,
Braj Raj Shrivastav
2,3
, Pramod Kumar Tiwari
1*
1
Centre for Genomics, Molecular and Human Genetics, School of Studies in
Zoology, Jiwaji University, Gwalior-474011, India;
2
Cancer Hospital and
Research Institute, Gwalior-474007, India;
3
Gajra Raja Medical College,
Gwalior-474007, India
E-mail: pk_tiwari@hotmail.com
Background: The mortality rate of gallbladder cancer (GBC) is considerably
high in India and world over. The Adenomatous Polyposis Coli (APC) gene is
widely reported for its role in cancer. APC is known to have two promoter
regions, 1A and 1B, that show differential role in various cancers. However,
role of these promoters and their exons in the molecular pathogenesis of
GBC is obscure. Our aim was to study the epigenetic control of APC
promoters in GBC and to evaluate their utility as prognostic/diagnostic
biomarker of GBC.
Methods: We carried out methylation specific PCR of the modified
genomic DNA from GBC and GSD tissues, followed by semi-quantitative
and quantitative PCRs (qPCR). We compared the transcript levels of the
two exons of APC in GBC and GSD as compared to their adjacent control
tissues. We also performed Immunohistochemistry (IHC) on tissue micro
array (TMA) of GBC and GSD. The scoring of different tissue cores was
done by the expert pathologist and students t-test was performed to
check the significance.
Page 26 of 59
Results: The APC 1A promoter was found significantly methylated in both
GBC (96%; p=0.0155) and GSD (80%; p=0.015), but 1B was not. The down-
regulation of APC exon 1 was observed in both GBC and GSD, whereas exon
2 appeared normally expressed. Immunohistochemistry of APC protein on
Tissue Microarray (TMA) revealed down regulation with negative score of
34.48% in GBC (p=0.057), 1+ in 24.14% GBC (p=0.005), 2+ in 25.85% in early
stage or GSD (p=0.091).
Conclusion: We infer that APC is epigenetically down regulated in
advance cases of GBC and might be a key step in the tumorigenesis of
gallbladder. In future, APC may be considered for diagnostic, prognostic
and therapeutic purposes in GBC.
P22
Bladder-Exstrophy-Epispadias-Complex risk and C677T polymorphism
in MTHFR gene: case-control study among Indian children
Abid Ali
*
, Rajeev Kumar Pandey, Sukanya Gayan, Minu Bajpai
Department of Pediatric Surgery, All India Institute of Medical Sciences, New
Delhi-110029, India
Background: Bladder exstrophy is a congenital anomaly in which part of
the urinary bladder is present outside the body. It is rare in occurrence
but the frequency is increasing very rapidly. 5, 10-methyltetrahydrofolate
reductase (MTHFR) enzyme, which catalyzes the synthesis of 5-methylene-
tetrahydrofolate and C677T polymorphism in MTHFR, shows a significant
role with a thermolabile enzyme and decreased specific MTHFR activity.
The objective of the present investigation was to study the case-control
association between C677T polymorphism in relation to Bladder-Exstrophy-
Epispadias-Complex.
Materials and methods: For the present study, a total of 50 patients
classified as Bladder-Exstrophy-Epispadias-Complex & cloacal exstrophy
patient and 50 healthy school going children (as control) were recruited
to participate in this study. Genomic DNA was extracted from peripheral
blood lymphocytes by using commercially available kits. Genotypes of the
MTHFR C677T polymorphisms were detected by polymerase chain
reactionrestriction fragment length polymorphism (PCR-RFLP).
Results: Frequency distributions of genotypes and combined genotypes
were obtained. The overall distribution of the C677T genotype was found to
be significantly associated with cloacal exstrophy but not with epispadias as
compared to the controls.
Conclusions: Genotyping of 50 patient cases and 50 controls revealed a
significant association between C677T polymorphism and Bladder-
Exstrophy.
P23
MLPA analysis of transcriptional gene(s) variations in patients with
congenital heart septation defects
Rajasekhar Moka
*
, Neetha John, Yashvanthi Borkar, K Ranjan Shetty
School of Life Sciences, Manipal University, Manipal-576104, India
E-mail: rsmoka@gmail.com
Background: Congenital heart diseases (CHD) occur in ~1% live births and
are among the most common birth defects. It accounts nearly about 46% of
all deaths from congenital malformations. Adults with repaired congenital
heart disease represent a complex and heterogeneous group of patients
that are increasingly surviving beyond childhood. Advances in our molecular
understanding of normal heart development have led to the identification
of numerous genes necessary for cardiac morphogenesis. The etiological
factors of many genetic syndromes and familial CHD have been identified,
but the genetic basis of majority of them remains unknown. Objective of
this study was to screen the transcriptional genes for novel mutations since
majority of these involved in the early stages of cardiogenesis.
Materials and method: Multiplex Ligation-dependent Probe Amplification
(MLPA) was employed in to detect copy number changes in 50 patients
DNA using the P311-A1 CHD SALSA MLPA Kit for the genes NKX2-5,
GATA4, TBX5, BMP4 and CRELD1 in 25 genomic regions.
Results: Copy number changes were identified in four (8%) two
heterozygous deletions in BMP4 and TBX5 were observed in common in
two patients atrial septum defects.
Conclusions: MLPA assay can be used for detection of copy number
changes in patients with non-syndromic CHDs as a first line of screening
test.
P24
COL6A1 loss of function mutation underlie atrioventricular septal
defects in down syndrome patients
Priyanka Ghosh
*
, Pranami Bhaumik, Subrata K Dey
West Bengal University of Technology, Department of Biotechnology &
Biological Sciences, Kolkata, West Bengal, Pin-700064, India
E-mail: priyankaghosh.in@gmail.com
Background: The present investigation was undertaken to explore the role
of COL6A1 variants on cardiovascular septal defects in Down syndrome.
Materials and methods: We sequenced the entire reading frame of
COL6A1 in the samples from Kolkata and adjoining areas. Four hundred
participants were included in the genetic association study and they were
stratified as Down syndrome (DS) with atrioventricular septal defect (AVSD),
DS without AVSD, euploid with AVSD and euploid without AVSD. DNA of
Down syndrome individual with atrioventricular septal defect and Down
syndrome individuals without AVSD were sequenced for the SNP analysis of
COL6A1 gene.
Results: A missense mutation p.E887K in exon 35 of COL6A1 gene was
identified in three DS patients with complete AVSD and was absent in
control population. The causative potential of a sequence variation was
evaluated by bioinformatics software like Mutationtaster, MutPred and
SOPMA which predicted. The mutation was predicted to be deleterious and
is predicted to affect normal protein function. Genetic analysis of the
mutation carriers available family members showed that the substitution
co-segregated with AVSD transmitted in an autosomal dominant pattern.
The p.E887K variation was automatically predicted to be disease causing.
Conclusions: Our data suggest that missense mutation p.E887K in exon
35 of COL6A1 mutation may serve as a potential biomarker for diagnosis
of AVSD in individuals thus suggesting potential implication for early
prophylaxis and personalised treatment of AVSD.
P25
An intronic rare mutation in Presenilin-1 (PSEN-1) gene may be
involved in the developement of Alzheimers disease
Pranami Bhaumik
*
, Priyanka Ghosh, Subrata K. Dey
Human Genetics Research Laboratory, Department of Biotechnology and
Biological Sciences, West Bengal University of Technology, Kolkata 700064,
India
E-mail: pranami.bhaumik@gmail.com
Background: The Presenilin-1 gene (PSEN-1) encodes a protein component
of gamma-secretase complex which is involved in processing of amyloid
precursor protein (APP). The PSEN-1 is involved in many cardinal mechanisms
in several molecular pathway which when impaired leads to the
manifestation of Alzheimers disease (AD). The aim of the study was to
investigate the role of PSEN-1 gene in the developement of AD in Indian
Bengali population.
Materials and methods: Blood samples were collected from 96 AD
patients and 173 age matched control individuals. DNA was isolated from
each sample and then sequencing was performed for the exon 8 and its
flanking introns of PSEN-1 gene.
Results: A rare mutation rs201992645 was identified within intron 8 and
several in. silico analyses (Bioinformatic tools like Human Splicing Finder,
SpliceAid and mutation t@sting) revealed the mutation as potentially
damaging at the transcript splicing level. The genotypic frequencies of
mutant heterozygotes were 0.031 AD, but it was not found in the control
population.
Conclusions: We hypothesize that this rare mutation may be involved in
the malfunctioning of Presenilin-1 protein and thus may play a role in
the manifestation of Alzheimers disease. Further study with large
population size may establish this mutation as a potential biomarker for
diagnosis of AD.
Page 27 of 59
P26
Identification and characterization of disease causing genetic variant
by conventional genotyping and whole genome sequencing in
familial tooth agenesis
Tanmoy Sarkar
1*
, Rajesh Bansal
2
, Parimal Das
1
1
Centre for Genetic Disorders, Faculty of Science, Banaras Hindu University,
Varanasi- 221 005, India;
2
Faculty of Dental Sciences, Institute of Medical
Sciences, Banaras Hindu University, Varanasi- 221 005, India
E-mail: tanmoy258819@yahoo.co.in
Background: Congenital tooth agenesis (CTA), a type of craniofacial
disorders affects approximately 20% (including 3
rd
molar or Wisdom
teeth) and 2-10% (excluding 3
rd
molar) of the world population. Five
major candidate genes known to be associated with syndromic and non-
syndromic CTA, these are PAX9, MSX1, AXIN2, EDA and WNT10A. The
present investigation was undertaken to identify and characterize disease
causing genetic variant by conventional genotyping and whole genome
sequencing in familial tooth agenesis
Material and methods: We have identified two Indian families (DEN11 and
DEN12) segregating two distinct types of non-syndromic tooth agenesis with
autosomal dominant (DEN11) and apparently indistinguishable either
autosomal or X-linked dominant pattern of inheritance (DEN12) family. Eight
affected and four unaffected members from DEN11 and four affected and
two unaffected members from DEN12 family were enrolled for the present
study to identify causative genetic defect associated with the disease. While
direct DNA sequencing and microsatellite based genotyping were carried
out to screen all but EDA genes in affected (II-5,-7, III-1,-7,-9, IV-2,-3,-4) and
unaffected (III-2,-3,-5, IV-1) family members of DEN11, whole genome
sequencing was carried out for two affected (III-2 and IV-4) family members
from DEN12 along with four unaffected unrelated controls using Illumina
2500 Next Generation Sequencing platform for identification and
subsequent characterization of causative variant/s.
Results and conclusions: In the microsatellite and SNP based haplotype
analysis we identified a haplotype block of a ~9.64 Mb region containing
PAX9 gene located between D14S70 and D14S288 markers associated with
CTA in DEN11. However, absence of any DNA sequence variant within the
exons and exon-intron boundaries of the linked PAX9 was found and this
indicates the involvement of other pathogenic mechanism. In the whole
exome sequencing we identified 86 nonsynonymous novel nucleotide
variations distributed among 84 different genes across the nuclear
genome of two affected members of DEN12 subjected for investigation.
Using bioinformatics, among those variations a specific variation in
Ectodysplasin-A (EDA), c.956G>T transversion leading to p.Ser319Ile, at the
Tnf homology domain, was considered as a potential pathogenic variation.
This variation was not observed in any unaffected family member and
in 100 unrelated control chromosomes as assayed through Sanger
sequencing and/or RFLP. In silico analysis using SwisSPdb Viewer V4.1.0
reveals that this change destroys an H-bond between p.319 Ser and p.332
Cys establishing this as a plausible pathogenic variation for tooth agenesis
in DEN12 family.
P27
Identification of NKX2-5 and GATA4 sequence variations in patients
with cardiac septation defects
Yashvanthi Borkar
1*
, K Ranjan Shetty
2
, K Krishnanand
2
, Rajasekhar Moka
1
1
Department of Biotechnology, School of Life Sciences, Manipal University,
Manipal, India;
2
Department of Cardiology, KMC, Manipal University, Manipal,
India
E-mail: borkaryashvanthi@gmail.com
Background: Cardiac septation defects (CHD) are the problems with the
structure of the heart during the development and is one of the most
common among congenital heart defect (CHD) which occurs ~1% in
general population. The etiology for the majority of these defects may be
multifactorial. However, advancement in understanding the molecular
basis of normal heart development has revealed the necessity of
numerous genes which are predominantly expressed during the heart
morphogenesis. Many cohort studies have shown that cardiac transcription
factors NKX2-5, GATA4 and TBX5 are the master regulators during heart
development. The mutations in any of these genes results in the failure of
normal development of heart leading to septal defects. The study was
carried out to identify the nucleotide sequence variation in the patients
with Atrial Septal Defects (ASD), Ventricular Septal Defects (VSD) and
Atrioventricular Septal Defect (AVSD).
Materials and methods: Fifty seven patients with ASD (n=51) and
VSD (n=6) were included in the study after a thorough evaluation of
Echocardiography and clinical phenotypes. All the coding regions and
flanking introns of NKX2-5 and GATA4 which are predominantly expressed
in the heart were amplified by polymerase chain reaction and sequenced
using ABI PRISM 3130 automated sequencer.
Results: No patient was found to have NKX2-5 and GATA4 mutations,
however single nucleotide polymorphism (c.63A>G; rs2277923) in NKX2-5
was observed in 20 patients with Ostium secundum ASD.
Conclusions: Since the mutation frequency is very low (NKX2-5 ~1-4%;
GATA4 ~1%) in these cases, we need to study more number of patients
to get expected frequency.
P28
Factor V Leiden and MTHFR mutations as a combined risk factor for
hypercoagulability in referred Patients population from Western India
Priya Bansal
Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Four
Bungalows, Andheri West, Mumbai, India
E-mail: priya.b9@gmail.com
Background: Factor V Leiden mutation is a recognized most prevalent
genetic risk factor for venous thromboembolic disease. Factor V mutations,
are known to potentiate the effect of MTHFR on deep vein thrombosis. The
thermo labile variant of the MTHFR gene (C677T) increases the plasma
homocysteine levels and hyperhomocysteneimia is a known risk factor of
deep vein thrombosis. Results of studies concerning interaction of
Hyperhomocysteneimia and thromobophilic risk factors like Factor V are
contradictory. Some studies have shown an increased risk (10-50 times) of
deep vein thrombosis because of MTHFR and FVL mutations combined, yet
other studies fail to conclude similarly. We attempt to address this paradox
in our study referred for DVT, Hyperhomocysteneimia and pulmonary
embolism and assess the importance of the synergistic effects of FVL and
MTHFR mutations.
Material and methods: In this study we analyzed 190 (79 females and 111
males) cases referred to Kokilaben Dhirubhai Ambani Hospital and Medical
Research Institute (Jan 2009 to Aug 2013) for DVT, Hyperhomocysteneimia
and pulmonary embolism. MTHFR mutation was studied in 27 of the above
subset. The detection of FVL mutation by PCR was studied by Restriction
Fragment length polymorphism and for MTHFR (C677T & A1298C)
mutations detection was done using commercially available kit.
Results: FVL mutation was found to be present in 10% (19/190) in our
study population. Of these, 18 patients were heterozygous and 1 was
homozygous for this mutation. Of which 25% (19/76) patients with deep
vein thrombosis were positive for variants of FVL. 74% (20/27) of the
patients screened for MTHFR were found to be positive (5 for C677T, 4
were compound heterozygous & 11 for A1298C). 2 out of 4 patients who
were positive for both FVL and C677T MTHFR mutations had poor
prognosis and died.
Conclusions: Our study reconfirms the Synergistic role of factor V Leiden
and MTHFR (14.8%) in hypercoagulability disorders like DVT, throm-
boembolism and others leading to poorer prognosis.
P29
RAGE gene polymorphism and expression: risk factor for vascular
complications in type 2 diabetes mellitus
Diwesh Chawla, Savita Bansal, Pawan K Kare, Basu Basu Dev,
Sri Venkata Madhu, Ashok K Tripathi
*
Department of Biochemistry and Department of Medicine, University College
of Medical Sciences and G.T.B. Hospital, Delhi-110095, India
E-mail: aktripathiucms@gmail.com
Page 28 of 59
Background: Advanced glycation end products (AGEs) are formed due to
hyperglycemia in T2DM. Interaction of AGEs with its receptor RAGE induces
signal transduction that culminates in vascular complications, the major
cause of morbidity and mortality in diabetic subjects. Some functional
polymorphism of this gene show differential activity of this receptor
and therefore may be associated with the development of diabetic
complications. In the present study we investigated the association of
expression of RAGE gene and its polymorphism namely -374T/A and -429T/
C in the promoter region and Gly82Ser polymorphism in the exon 3 region
with vascular complications in T2DM patients.
Materials and methods: We screened 820 subjects which includes
200 healthy controls, 200 type 2 diabetes mellitus (T2DM) subjects without
any vascular complications (DM), 220 T2DM subjects with microvascular
complications (DM-Micro) and 200 T2DM subjects with macrovascular
complications (DM-Macro) for -374 T/A, -429 T/C and Gly82Ser
polymorphisms of RAGE gene. DNA isolated from the enrolled subjects was
genotyped by PCR-RFLP. RAGE expression was determined by quantitative
real-time PCR.
Results: Mutant variant of -429T/C and Gly82Ser RAGE polymorphism was
about three times more prone to develop macrovascular and microvascular
complications respectively in T2DM subjects while -374A allele showed
reduced risk towards the development of macrovascular complications (OR
= 0.57, p = 0.006). Further, haplotype analysis revealed that CTG haplotype
was significantly associated with the development of macrovascular
complications while haplotype TAG was observed to be significantly
protective towards development of macrovascular complications in T2DM
subjects. The expression of RAGE correlated significantly with the genotypic
variation of the RAGE gene.
Conclusions: Mutant genotypes of RAGE gene and its enhanced
expression may be considered as risk factor for vascular complications in
North Indian T2DM patients.
P30
Association of Matrix Metalloproteinase 1 and 3 (MMP1 and MMP3)
gene polymorphisms with susceptibility to ESRD risk in North
Indian population
Archana Verma
*
, Aneesh Shrivastava, Rama D Mittal
Department of Urology and Renal Transplantation, SGPGIMS, Lucknow, India
Background: End Stage Renal Disease (ESRD) is influenced by genetic and
epigenetic factors. Few studies have been reported earlier depicting the role
of Matrix Metalloproteinase in different populations for ESRD risk, but we
have reported few novel and functional genes in North Indian population.
The prime objective of the present study was to find out the association of
four variants viz. MMP1-519(A/G) and MMP1-1607(1G/2G) and MMP3+5356
(A/G) and MMP3 -1161(A/G) with ESRD risk.
Materials and methods: 4 SNPs were genotyped by Polymerase Chain
Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) in 200
ESRD patients and 200 healthy controls, that were age and gender
matched. To evaluate the SNP effects on ESRD, odds ratio (OR) and
confidence interval (CI) 95% were calculated by SPSSver16.0. Haplotype
analysis was done for MMP1 by SNPanalyzer1.2 ver.
Results: In case of MMP1-519(A/G) the variant genotype GG showed
significant reduced risk of ESRD (p=0.005, OR=0.254). On combining the
heterozygous and variant genotypes marginal reduced risk was observed
(p=0.055, OR=0.677), although we found significance reduced risk of ESRD
(p=0.007, OR=0.639) in G allele. In case of MMP1-1607(1G/2G) the variant
genotype 2G2G showed significant reduced risk of ESRD (p=0.008,
OR=0.476) whereas the combination of heterozygous and variant
genotypes reduced risk was obtained (p=0.027, OR=0.614). At allelic level,
2G allele showed reduced risk (p=0.006, OR=0.675) of ESRD. Gene-gene
interaction was also performed for above gene variants. We found a
significant reduced risk in combination GG+AA (p=0.034, OR=0.185) on
interacting MMP1-519(A/G) and MMP3-1161(A/G). We also found reduced
risk for ESRD for MMP1-519(A/G) and MMP1-1607(1G/2G) while looking risk
for ESRD at haplotype level. Bioinformatics analysis (in-silico) showed no
association with ESRD.
Conclusions: Our results indicated that polymorphism in MMP1-519(A/G)
and MMP1-1607(1G/2G) showed reduced risk for ESRD in North-Indian
population. More relevant studies with large sample size and diverse
ethnicity are required to validate these observations.
P31
Association of PKD1 sequence variants with pathophysiology of
ADPKD in Indian patients
Sonam Raj
1*
, RG Singh
2
, Parimal Das
1
1
Centre for Genetic Disorders, Faculty of Science, Banaras Hindu University,
Varanasi, India;
2
Dept. of Nephrology, Institute of Medical Sciences, Banaras
Hindu University, Varanasi, India
E-mail: sonamraj01@gmail.com
Background: Autosomal dominant polycystic kidney disease (ADPKD) is a
systemic conditions with cardinal symptom of bilateral multiple renal cysts
of variable sizes. ADPKD is genetically heterogeneous, Mendelian disorder,
shows late onset and contributes 8-10% cases of end stage renal disease
(ESRD). Linkage analysis in ADPKD familial cases revealed approximately
85% cases of PKD1, rest cases of PKD2 and an unidentified locus.
Materials & methods: In the present study occurrence of mutation,
amino acid change and three frequently observed SNPs from 3single copy
region (exon 44-46) and in 5 duplicated region (exon 2-11) of were
screened in 47 patients (24 sporadic and 23 familial) and in control
individuals to search the responsible and/or susceptible spots for ADPKD
in Indian patients using direct DNA sequencing of specified exons and
RFLP for SNP assay. PKD1 exons 44-46 encoding intracellular and exons
2-11 encoding ligand binding domains, Ig-like PKD domains in extracellular
compartment with their suggested role in cell signaling and cell adhesion
were chosen for this study.
Results: Screening of exons 44-46 in 47 patients identified five exonic
(I4045V, 4059V, A4092A, L4137L, P4210P) and two intronic (IVS44+15G>A,
IVS+22delG) variants. Assessment of the three SNPs viz. I4045V, A4059V, and
A4092A screened in a multiplex family with four affected (II-1, II-7, II-9, III-12)
and 30 unaffected members along with 100 ages and sex matched controls
showed higher occurrence (2X) in patients compared to controls indicating
their role for disease susceptibility. Screening of exons 2-7 in 19 patients
uncovered six exonic (A92A, c.445delC, T191N, G340G, N416N, A443T) and
two intronic (IVS4+21delC, IVS6+12delC) variants while screening of exons
8-11 in 15 patients detected twelve exonic (Q554X, T558M, Q562R, A637V,
P676L, c.1987delC+2016_2017insG, D910D, L811L, L845S, N854S, V873A,
N890N) and one intronic (IVS9+14_27del14) variants. Two exonic changes
(c.445delC, c.1987delC+2016_2017insG) generate 288 amino acids long
truncated protein and a stretch of nine altered amino acids sequence in
LDL-A domain respectively.
Conclusions: The spectrum of changes detected in various affected
individuals indicates that the clinical as well as allelic heterogeneity
compounding the pathophysiology of ADPKD in both sporadic and familial
cases. Screening of remaining region of PKD1 and other candidate genes is
therefore planned for future for better understanding.
P32
Identification of mutations in bone morphogenetic protein 2, 4 and 7
in congenital heart disease patients from Indian population
Ritu Bhardwaj
1*
, Damyanti Agrawal
2
, Ashok Kumar
3
, Bhagyalaxmi Mohapatra
1
1
Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University,
Varanasi, India;
2
Department of Cardio Thoracic & Vascular Surgery, Banaras
Hindu University, Varanasi, India;
3
Department of Pediatric Medicine, Institute
of Medical Sciences, Banaras Hindu University, Varanasi, India
E-mail: g_ritu89@yahoo.com
Background: Congenital heart disease (CHD) is the most common
congenital defect, affecting 1% of all livebirths. Initial cardiac development
and the subsequent maturation of the heart are temporally and spatially
regulated by several Bone Morphogenetic Proteins (BMPs). BMP signaling
plays an important role in the specification and patterning of the early
embryo. Animal model studies have established a strong link between
BMPs and cardiac development. At least 6 BMPs (BMP2, BMP4, BMP5,
BMP6, BMP7 & BMP10) are expressed in the heart where they have both
independent and redundant functions. BMP2, 4 & 7 are expressed in the
Page 29 of 59
AtrioVentricular cushion and Out Flow Tract (OFT) and helps in their
formation. However due to functional redundancy of BMP ligands,
relatively little is known about the role of individual BMPs. The association
of BMPs with CHD has not been studied so far in human subjects. Here we
report the genetic variants in BMP2 (NM_001200.2), BMP4 (NM_001202.3)
and BMP7 (NM_001719.2) in individuals with CHD.
Materials & methods: We screened 190 unrelated probands with CHD
and 100 healthy controls by PCR and DNA sequencing for 3 genes.
Results & conclusion: We have identified 6 novel sequence variants in
BMP2. Out of which 3 (H321L, E328K & S351C) are missense variants. One
nonsense variants (K241X) has also been identified in one case having
Patent Ductus Arteriosus and is not found in 100 controls. In BMP7, 10 novel
sequence variants have been identified, out of which 3 (D85V, R175W &
N372Y) are missense, 3 (c.366G>A, c.381G>A & c.384C>T) are in 5UTR, 1
synonymous change (c.682G>T) and 3 (c.947-20G>C, c.1140+14G>A &
c.1289+68G>A) are intronic variants. In BMP4, 3 sequence variants are
found, out of which only one novel (c.1695A>C) in 3UTR region has been
found in one individual. Other than these some already reported single
nucleotide polymorphisms have also been identified in these 3 genes and
some of them are more frequently found in CHD cases. In-silico analyses
based on evolutionary, biochemical and structural aspect of the gene
revealed the disease causing effect of these variants. Further biochemical
analysis would reveal the regulatory circuits involving these cardiac
transcription factors and their possible role in pathogenesis of the disease.
P33
Vitamin D Receptor (VDR) gene polymorphism and risk of ischemic stroke
Puttachandra Prabhakar
1*
, Rita Christopher
1
, Dindagur Nagaraja
2
1
Department of Neurochemistry, National Institute of Mental Health and
Neuro Sciences, Bangalore-29, India;
2
Department of Neurology, National
Institute of Mental Health and Neuro Sciences, Bangalore-29, India
E-mail: prab.mys@gmail.com
Background: Vitamin D deficiency is associated with various chronic
disease conditions. The expression and nuclear activation of vitamin D
receptor (VDR) are essential for the effect of Vitamin D, because vitamin D
is involved in various signaling cascades. Few polymorphisms in VDR gene
have been reported to be associated with cardiovascular disease and
hypertension. We sought to examine the association between VDR gene
variants and risk of ischemic stroke in Indian population.
Methods: We recruited 250 patients diagnosed with ischemic stroke and
300 age and gender-matched healthy control subjects, after informed
consent. 4 SNPs in VDR gene (ApaI, BsmI, FokI & TaqI), were genotyped by
using PCR-RFLP method. Serum Vitamin D levels were determined by ELISA.
The association of each SNP with the risk of stroke was analyzed by multiple
logistic regressions by adjusting with age, gender, smoking, alcohol,
hypertension and diabetes.
Results: The VDR BsmI bb and FokI Ff genotypes were associated with
increased risk for ischemic stroke (OR: 1.76; 95% CI; 1.01 3.08; P = 0.04) &
(OR: 1.52; 95% CI; 0.99 2.31; P = 0.05) respectively. No association was
observed between TaqI and ApaI polymorphisms and ischemic stroke.
Vitamin D deficient (<20ng/ml) subjects with Bsm I Bb & bb genotypes had
significantly higher risk for ischemic stroke (OR: 2.24; 95% CI; 1.11 4.56; P =
0.03) & (OR: 2.27; 95% CI; 1.05 4.99; P = 0.034), respectively. Similarly,
increased risk for stroke was observed in Vitamin D deficient subjects with
TaqI Tt genotype (OR: 2.77; 95% CI; 1.45 5.31; P = 0.002), and ApaI Aa
genotype (OR: 2.456; 95% CI; 1.25 4.81; P = 0.009).
Conclusions: In this first study to elucidate the role of VDR gene
polymorphism in ischemic stroke patients, we found that genetic variants in
VDR gene was associated with an increased risk for stroke especially
in Vitamin D deficient subjects. Our findings could contribute to the
development of strategies for the prevention of ischemic stroke.
P34
Clinical Characterization of Idiopathic Restrictive Cardiomyopathy
having rare variant (E949K) in b-cardiac myosin heavy chain gene
Mitali Kapoor
1*
, Amitabh Biswas
1
, Soumi Das
1
, Sandeep Seth
2
,
Balram Bhargava
2
, Vadlamudi Rao
1
1
Department of Anthropology, University of Delhi, Delhi, India;
2
Department
of Cardiology, AIIMS, India
Background: Idiopathic Restrictive cardiomyopathy (IRCM) and
hypertrophic cardiomyopathy (HCM) reflects the same or very similar
disorders showing restrictive physiology with different names due to
discretionary crosscuts in the LV wall thickness rather than two separate
distinct diseases. The perspective of this study is to clinically evaluate the
IRCM proband and comparison between the two distinct disease phenotype
IRCM and HCM as an outcome of same genotype i.e.E949K in gene MYH7.
Methods: Diagnosis was based on ECG, 2D-echocardiography, Cardiac
Catheterisation and Endomyocardial biopsy. 5ml of Blood sample was
collected and DNA was isolated using phenol-chloroform method.
Sequencing of the hotspot region of MYH7 gene i.e. exon 23 by Sanger
method (ABI 3730) was done. Screening of family members available was
also done clinically as well as genetically. The study was ethically approved
by Institutional committee and informed written consent was taken from all
participants.
Results: The proband, 11 year old male reported with chest discomfort,
syncope, palpitations and arrthymias since last six months. Echocardiography
showed marked biatrial enlargement (LA=46mm, RA=54mm), normal sized
ventricles (IVS=8mm, LV=9mm) with mild MR and severe TR. Cardiac
catheterization revealed grade III MR, severe TR severe PAH, mild RV systolic
dysfunction. With these evidences a diagnosis of Idiopathic RCM was made.
Sequencing of exon 23 of MYH7 gene led to the identification of rare variant
E949K (c.2845G>A) in MYH7 gene. This variant was not found in our
screening of 80 cardiomyopathies patients and 78 unaffected family
members. This mutation was previously reported by Watkins et al.1992 in a
case of familial HCM and suggested late onset of disease with severe LV
hypertrophy and Rayment I et al.1995 also reported that this mutation
shows loss of tensile strength of stiffness which represents the main
phenotype of IRCM. We found this rare mutation in IRCM patient with early
onset and no ventricular hypertrophy.
Conclusion: This study represents the phenotypic heterogeneity of same
rare variant in two ethnically different probands indicating environmental
role or mutations in some other genes. This study reiterates the importance
of whole genome sequencing approaches in clinical practice.
P35
A novel donor site mutation in LMNA gene leading to severe form of
Dilated Cardiomyopathy in a proband of a family from Bihar, India
Soumi Das
1*
, Amitabh Biswas
1
, Mitali Kapoor
1
, Sandeep Seth
2
,
Balram Bhargava
2
, Vadlamudi Rao
1
1
Department of Anthropology, University of Delhi, Delhi, India;
2
Department
of Cardiology, AIIMS, India
Background: Dilated Cardiomyopathy (DCM) is poorly understood in
terms of their mechanistic pathways. It may lead to sudden cardiac death
with a prevalence rate of 0.04-0.2%. The cause of DCM is still unknown and
referred to as Idiopathic Dilated Cardiomyopathy. There are many
candidate genes associated with the DCM but most of the mutations are
found in the LMNA and MYH7 genes.
Methods: The proband underwent Echocardiography and ECG to confirm
the diagnosis. 5ml blood was collected and DNA was extracted using
Phenol-chloroform method. The hot spot regions exon 23 of MYH7 gene,
exon3 and exon 4 along with the intron3 of LMNA gene were sequenced
using Sanger sequencing (ABI 3730xl). ACE 287bpI/D and TNNT25bpI/D
polymorphisms were also genotyped. In silico analysis of this novel mutation
by using softwares, Human splicing finder (HSF) and MaxENT to understand
the effect of mutation on splicing. The study was ethically approved by
Institutional committee and informed written consent was taken from all
participants.
Results: The proband aged 50yrs diagnosed with DCM, age of onset 45yrs,
showing severe symptoms such as dyspnea, palpitation, fatigue and pedal
edema under NYHA III classification showing dilated LV with EF 30%.
Probands mother died due to heart problem but was not clinically
confirmed for DCM and his sister had a suspicious death. The son of the
proband aged 24 years has the same LMNA mutation but is asymptomatic
presently.
A Novel donor splice site mutation G>C transversion in intron3 of LMNA
gene and a synonymous mutation (C>T at codon 923) was found in MYH7
gene in proband. ACE and TNNT2 polymorphisms showed a heterozygous
(ID) and homozygous (II) genotypes respectively. In silico analysis by HSF
and MaxENT shows that the elimination of natural donor splice site leading
Page 30 of 59
to the use of a cryptic donor site which is located 7 nucleotide upstream of
native splice site.
Conclusion: As reported in previous studies, LMNA gene mutations are
associated to the severe form of DCM. From the above results, it may be
concluded that the severe form of the disease is due to the splice site
mutation.
COMPLEX TRAI T & POLYGENI C
DI SORDERS
P36
Association of Vitamin D
3
levels with Glycemic Control in Type 2
Diabetes Subjects from Gujarati population-India
Avisek Majumder
1*
, Bhavik Doshi
1
, Frenny Sheth
1
, Manan Patel
1
,
Navneet Shah
2
, Thakor Premal
3
, Rama Vaidya
4
, Jayesh Sheth
1
1
Satellite, Ahmedabad-380015, India;
2
Department of Diabetes and
Endocrinology; Sterling Hospital, Ahmedabad- 380052, India;
3
Gujarat
Diabetic Association, Ahmedabad-380007, India;
4
Kasturba Health Society,
Medical Research Centre, Mumbai-400056, India
E-mail: avi.biocv@gmail.com
Background: Considering the active role of Vitamin D
3
in the functional
regulation of pancreatic b-cells, present study was carried out to know the
occurrence of Vitamin D
3
deficiency in Type 2 diabetic [T2D] and non-
diabetic subjects and demonstrate its influence on glycemic control.
Materials and Methods: This prospective study comprises of 508
individuals (including 210 T2D & 298 controls). All subjects were categorized
into 3 groups according to Vitamin D
3
levels. Group-I: included 171
individuals (61 T2D out of 171) with normal Vitamin D
3
concentration (25
nmol/l), group-II: included 264 subjects (118 T2D out of 264)with mild to
moderately Vitamin D
3
deficiency (15-24.9 nmol/l) and group-III included 73
subjects (31 T2D out of 73) having severe Vitamin D
3
deficiency (14.9
nmol/l). Vitamin D
3
and glycosylated hemoglobin [HbA1C] level was
analyzed for all the subjects.
Results: Overall 66.34% subjects (both T2D & Controls) were found to have
Vitamin D
3
deficiency. This was more common in T2D patients (71%)(mean
SD Vitamin D
3
level was 17.777.19nmol/L) as compared to controls (63%)
(meanSD Vitamin D3 level was 24.38 9.30nmol/L)(p<0.05).Female
subjects were more prone for Vitamin D
3
deficiency as compared to male
(71.09% vs 61.09%, p<0.03) subjects. Moreover, a gradual increase in mean
HbA1C level was observed as Vitamin D
3
level when reduced from its
normal level in T2D subjects (7.95- 9.08% in group-I through group-III)
[bHbA1C, Vit. D3= -0.07, r = -0.28, p<2.7310
-5
]. No such changes in mean
HbA1C level were observed in controls.
Conclusion: Present study demonstrates the high prevalence (66.34%) of
Vitamin D
3
deficiency in Gujarati population from India, more so for subjects
with T2D. It is likely that Vitamin D
3
has a role in regulating insulin
sensitization; resulting in poor glycemic control in subjects with low Vitamin
D
3
levels. This study also indicates that females are likely to be at a higher
risk for Vitamin D
3
deficiency compared to male in T2D and control subjects.
P37
Effect of PPAR-g2 Gene Pro12Ala Polymorphism (Rs1801282) and
Vitamin D
3
on Glucose Homeostasis in Type 2 diabetic Subjects from
Gujarat-India
Avisek Majumder
1*
, Frenny Sheth
1
, Manan Patel
1
, Bhavik Doshi
1
,
Navneet Shah
2
, Premal Thankor
3
, Rama Vaidya
4
, Jayesh Sheth
1
1
2
3
Gujarat
Diabetic Association, Ahmedabad-380007, India;
4
Medical Research Centre, Mumbai-400056, India
E-mail: avi.biocv@gmail.com
Background: Pro12Ala polymorphism in PPAR-g2 gene is known to be
involved in insulin sensitization and metabolic deregulation in Type 2
Diabetes (T2D) subject. Considering beneficial effects of Vitamin D
3
in
functional regulation of pancreatic-b cells, present population based
study was designed to determine the association of Pro12Ala
polymorphism with serum Vitamin D
3
level and its effects on glucose
homeostasis in T2D and non-diabetic subjects.
Materials & methods: Total 508 subjects (including 210 T2D & 298
controls) were divided into two groups according to serum Vitamin D
3
level.
GrouPI: included 338 subjects (150 T2D out of 338) having Vitamin D
3
deficiency (25.0 nmol/l) and group-II: included 170 subjects (60 T2D out of
170) with normal vitamin D
3
level (>25.0 nmol/l). All cases were investigated
for Vitamin D
3
, glycosylated hemoglobin (HbA1C) level and Pro12Ala variant
of PPAR-g2 gene.
Results: It was observed there is 12Ala allele frequency of PPARg2 gene in
10.19% of T2D and 9.46% in control subjects (p>0.36). The mean HbA1C
was better controlled in group-II T2D subjects with 12Ala allele compared
to group-I T2D subjects having same allele (7.260.44% vs. 8.350.43%,
p>0.014). In contrary to the above, patients who were homozygous for
12Pro allele, the mean HbA1c remains high irrespective of the normal
Vitamin D
3
levels (8.590.18% vs. 8.290.28% in group-I and group-II
respectively).
Conclusions: Significant decrease in mean HbA1C level was observed in
T2D patients with 12Ala allele and normal Vitamin D
3
level compared to the
patients having same allele with Vitamin D3 deficiency. No such effect was
observed in T2D patients with homozygous status for 12Pro allele. This
indicate that biologically active form of Vitamin D
3
(125(OH)D
3
) together
with 12Ala allele may affect glucose homeostasis by some gene-nutrition
interactions.
P38
Transferrin (rs3811647) gene polymorphism in iron deficiency anemia
K Sri Manjari
1*
, KSPS Teja
1
, M Sujatha
1
, A Jyothy
1
, Pratibha Nallari
2
,
A Venkateshwari
1
1
Institute of Genetics and Hospital for Genetic Diseases, Osmania University,
Hyderabad;
2
Department of Genetics, Osmania University, Hyderabad
E-mail: srimanjari18@gmail.com
Background: Iron-deficiency anemia (IDA) is the most common type of
anemia, caused by inadequate iron availability for hemoglobin production
due to the lack of dietary iron or insufficient uptake of iron. Transferrin (TF)
exerts a crucial function in the maintenance of systematic iron homeostasis.
The expression of the TF gene is controlled by transcriptional mechanism,
although little is known about its influence on IDA. Hence, the aim of the
current investigation was to determine the functional polymorphism
(rs3811647) of TF gene in iron deficiency anemia.
Material and Methods: A total of 207 school children of age from 12-16
years were selected from Government High School Seetaphalmandi,
Secunderabad, Andhra Pradesh and screened for iron deficiency. Out of
which 70 school children had iron deficiency anemia with hemoglobin
levels less than 11.5 g/dl and 137 were normal. Demographic details and
blood samples were obtained from all the subjects. Genotyping was
carried out by tetra-primer ARMS PCR followed by agarose gel
electrophoresis, and appropriate statistical analysis.
Results: The genotype distribution of TF (rs3811647) region were 4.3%
(AA), 81.4% (AG) and 14.3% (GG) in iron deficient children compared to
8% (AA), 72.3% (AG), and 19.7% (GG) in normal children. No significant
variation was observed with respect to the allelic distribution [OD = 1.035
(0.67 1.59), p = 0.917] in the IDA group when compared to normal
group.
Conclusions: There was no significant association of the TF (rs3811647)
gene polymorphism with iron deficiency anemia. Thus, our study
highlights the importance of other genetic variants influencing the
outcome of iron deficiency anemia. However, larger samples have to be
analyzed to confirm the same.
P39
CD36 gene variants and their potential in determining T2DM
susceptibility
Monisha Banerjee
1*
, Sunaina Gautam
1
, CG Agrawal
2
1
Molecular & Human Genetics Laboratory, Department of Zoology, University
of Lucknow, India;
2
Department of Medicine, King Georges Medical
University, Lucknow, India
E-mail: banerjee_monisha30@rediffmail.com
Page 31 of 59
Background: Type 2 diabetes (T2DM) is a non-communicable disease
affecting huge populations in India. Several groups all over the world
are in search of prognostic genetic markers for the early detection of
T2DM. Single nucleotide polymorphisms (SNPs) in CD36, a macrophage
scavenger receptor were implicated in the pathogenesis of T2DM and its
complications. This molecule is responsible for the uptake of free fatty
acids specially oxidized low density lipoprotein (Ox-LDL) during diseased
conditions.
Aim of the present study was to find out the allelic and genotypic
frequencies of 11 SNPs spanning the entire CD36 gene in north Indian
population and find their association with T2DM. In addition, haplotypic
analysis was undertaken to find out the risk haplotype in individuals of
families with a history of T2DM.
Material and Methods: All the 11 SNPs were genotyped in at least
100 each of controls and T2DM subjects using polymerase chain reaction-
restriction fragment length polymorphism (PCR-RFLP) and single-strand
conformation polymorphism (SSCP) methods. Haplotype analysis was
done by SHEsis software. Ten families with diabetic history were
identified and blood samples were collected from available family
members. DNA was extracted by salting out method and three SNPs viz.
rs1761667 (G>A) in exon 1A, rs3211938 (T>G) in exon 10 and rs3212018
(16 bp del) in exon 14 were genotyped in all individuals. Anthropometric
characteristics viz. systolic/diastolic blood pressures, body mass index
(BMI), waist hip ratio (WHR) and biochemical parameters were measured
in all individuals.
Results: Out of 11 SNPs, six were polymorphic (rs1984112, A>G; rs1761667,
G>A; rs1527479, C>T; rs3211938, T>G; rs1527483, C>T; rs3212018, 16bp del)
in the study population. Individuals having a haplotypic combination
AACGC1 showed highest significant association with T2DM (P<0.001).
Family studies showed that individuals with risk genotype GA of rs1761667,
G allele of rs3211938 (T>G) or G allele of rs1984112 (T>G) had an
increased BMI and relatively higher glucose levels.
Conclusions: Therefore, we conclude that haplotypes/genotypes/alleles of
the CD36 gene variants can be potential markers in determining diabetes
risk and such analyses may be useful for early identification of individuals
susceptible to T2DM and its complications in the north Indian population.
P40
Mitochondrial-nuclear epistasis contributes to phenotypic variation in
wild yeasts
Swati Paliwal
*
, Anthony C. Fiumera, Heather L. Fiumera
Department of Biological Sciences, State University of New York at
Binghamton, 4400 Vestal Pkwy E, Binghamton, NY 13902, USA
E-mail: swatzpaliwal@gmail.com
Background: Mitochondria are ubiquitous organelles and are the
main source of cellular energy. Mitochondria affect nearly every cellular
process, including energy production, metabolite biosynthesis, ion
homoeostasis, growth, cellular differentiation and apoptosis. In human and
mice models, mitochondrial DNA variation is associated with various
metabolic diseases. Mitochondrial functions require intricate interactions
between mitochondrial (mt) and nuclear (n) genomes. The extent to which
mitochondrial-nuclear (mt-n) interactions contribute to phenotypic variation
in a population is unknown. We have made an attempt to find out if
different combinations between the different mt and n genomes are found
in natural populations would may lead to the expression of complex traits
through epistatic interactions.
Materials and methods: We created a novel population of 100 novel
yeast strains with all possible combination of 10 naturally occurring and
polymorphic, nuclear and mitochondrial genomes. These strains were
phenotyped for metabolic growth under a variety of conditions, including
high temperature, carbon source, and oxidative stress using high-
throughput micro-cultivation plate readers. The results were analysed
statistically for the growth rates using ANOVA.
Results: It was found that mt-n epistasis significantly contributes to
phenotypic differences among these strains, explaining up to 40% of
phenotypic variation. A large number of strains were found to contribute,
indicating the mt-n epistasis is a wide-spread phenomenon. The patterns of
genome interactions vary across environments, indicating that multiple
interactions affect fitness. Interestingly, we found that certain strains
harboring their native mitochondrial genomes are more fit than
when harboring non-native mtDNA, suggesting nuclear-mitochondrial
co-evolution.
Conclusions: It is suggested that mt-n epistasis may provide a fitness
landscape upon which selection can operate. This work highlights the need
to consider mt-n epistasis when characterizing the genetic basis behind
complex traits and missing heritability and in developing treatments for
mitochondrial disorders in humans.
P41
Association of Pro-inflammatory Cytokine Gene Polymorphisms with
Schizophrenia in South Indian Population
Lekshmy Srinivas
1*
, NV Neetha
1
, Chandrasekharan Nair
2
, Priya M. Allencherry
3
,
Moinak Banerjee
1
1
Human Molecular Genetics Laboratory, Rajiv Gandhi Centre for
Biotechnology, Trivandrum, Kerala, India;
2
Nairs Hospital, Ernakulam, Kerala,
India;
3
Mental Health Centre, Trivandrum, Kerala, India
E-mail: lekshmys@gmail.com
Background: Schizophrenia is a severe and debilitating mental illness.
Around 0.26% of people in South India suffer from schizophrenia. It is a
complex disorder which may involve multiple genes with mild to moderate
effect and non-genetic risk factors like environmental and psychological
assaults. Cytokines, regulators of immune/inflammatory reactions and brain
development, emerge as part of a common pathway of genetic and
environmental components of schizophrenia. Our study explored the
association of polymorphisms in cytokine genes with schizophrenia, the
interaction of these genes in the causation of the disease and the
population genetics of cytokine gene polymorphisms.
Materials and methods: We performed a case-control association study
using 248 patients from Kerala, South India and 244 ethnically matched
normal healthy controls. We screened polymorphisms in 10 cytokine genes
(IL1A, IL1B, IL1RN, IL3, IL4, IL6, IL10, IFNG, TNFA and TGFB1). Genomic DNA
was isolated from blood and genotyping was carried out by PCR-RFLP,
TaqMan allelic discrimination and KASPar assays. Allelic, genotypic,
haplotypic and diplotypic frequencies were calculated and compared. Gene-
gene interactions among cytokine genes were also assessed.
Results: We found significant association of SNPs in pro-inflammatory
cytokine genes IL1A, IL6, TNFA and IFNG with schizophrenia in the Kerala
population. No significant association was observed with any of the anti-
inflammatory cytokine genes IL4, IL10 and TGFB1. Our study provides
significant evidence for strong gene-gene interactions among pro-
inflammatory cytokine genes in the development of schizophrenia. Our
findings support the immune hypothesis in the predisposition to
schizophrenia.
Conclusions: The phylogenetic analysis of Kerala population with HapMap
populations show proximity to HapMap Gujarati Indian population (GIH),
Mexican population (MEX) and the African populations ASW, MKK and YRI.
This similarity can be attributed similar selection pressures that lie in the
same latitudinal belt indicating similar environmental conditions with
respect to temperature, rainfall, pathogens, sunlight etc. This information
might facilitate the identification of clinical subgroups of patients with
strong immunological basis for the outcome of the disease.
P42
Glutamate Transporter Genes Are Associated With Schizophrenia in
South Indian Population
Ajay Jajodia
1*
, Harpreet Kaur
1
, Ruchi Baghel
1
, Gurpreet Kaur
1
, Sanjeev Jain
2
,
Ritushree Kukreti
1
1
CSIR-Institute of Genomics and Integrative Biology, Mall Road Delhi, India;
2
NIMHANS, Honsur Road, Bengaluru, India
Background: Schizophrenia is a neurodevelopmental disorder and is
manifested by disruption in cognitive ability along with positive and
negative symptoms. Neurodevelopment abruptions involve pathologic
processes caused due to genetic and environmental factors. Study aims to
evaluate the association between the genetic polymorphisms of the
neurodevelopmental gene and the risk of schizophrenia.
Page 32 of 59
Method: The study includes 482 schizophrenia cases and 401 age, sex
matched controls. Genotyping was performed for single nucleotide
polymorphisms (SNPs) of genes by primer extension reaction followed by
the MALDI-TOF mass spectrometry (Sequenom
TM
). Polymorphisms involved
in neurodevelopment processes like synaptic plasticity, synaptogenesis,
signal transduction and activity of receptors/transporters. Genotypic tests
were applied to examine the association of SNPs with disease. SNPs with
p value < 0.1 were investigated by haplotypes analyses. Further, p values
were adjusted with clinical variables using multivariate logistic regression. To
evaluate interaction between the loci multifactor-dimensionality reduction
(MDR) test was performed. Test for multiple corrections was applied using
10000 MaxT permutations.
Results: Single locus association analysis showed association of rs2033267
(SLC1A3) OR=3.08, 95%-CI=1.98-4.77, rs10430590(PIP4K2A) OR= 1.71, 95%-
CI=1.29-2.26. We identified a significant three marker haplotype of SLC1A3
significantly associated with disease with OR=3.81, 95%-CI=2.03-7.16. MDR
analysis reveal interaction between rs2494750 and rs3803300 of v-AKT
murine thymona viral oncogene homolog 1(AKT1) and rs16917204,
rs56164415 of Brain Derived Neurotrophic Factor (BDNF) with cross
validation 8/10 and Pvalue 0.0001,OR=13.07,95%-CI=8.97-19.03.
Conclusions: We report a association of SLC1A3(5p13) with risk of
schizophrenia development, which codes for glutamate transporter found
in glial cells that functions to regulate neurotransmitter concentration at
excitatory synapse. In addition interaction study revealed AKT1 and BDNF
genes both having neuroprotective role.
P43
Association of HLA-G 14bp INS/DEL Polymorphism with brain
morphology in Schizophrenia
Ashwini Rajasekaran
1,2*
, Venkataram Shivakumar
2
, Deepthi Venugopal
1,2
,
Sunil V. Kalmady
2
, Anekal C. Amaresha
2
, Mahavir Agarwal
2
,
Janardhanan C. Narayanaswamy
2
, Ganesan Venkatasubramanian
2
,
Monojit Debnath
1
1
Department of Human Genetics, NIMHANS, Hosur Road, Bangalore-560029,
India;
2
Translational Psychiatry Laboratory, Department of Psychiatry,
Neurobiology Research Centre, NIMHANS, Hosur Road, Bangalore-560029,
India
Background: Multiple lines of evidence have implicated dysregulated
immune processes in the pathogenesis of schizophrenia. Being an
important immuno-modulatory molecule, Human Leukocyte Antigen
(HLA)-G plays a pivotal role in successful pregnancy. Altered expression of
HLA-G due to environmental and genetic variations not only lead to
pregnancy complications but also a range of immunopathologies, some of
which are being considered to confer risk for schizophrenia. One of the
polymorphic marker, 14bp insertion/deletion (INDEL) located within the 3
UTR region of the HLA-G locus in Chr.6p21.3 is associated with HLA-G
expression and function. The current study is aimed at analysing the role
of 14bp polymorphism and the impact of feto-maternal compatibility/
incompatibility at this locus on the risk of schizophrenia. In addition, the
effect of 14bp INDEL on brain structure alterations in schizophrenia
patients was also investigated.
Methods: A total of 151 (male-75 and female-76) schizophrenia patients
and 113 (male-68 and female-45) ethnicity matched healthy controls (HC)
were considered. In addition, mothers of 64 schizophrenia patients
were also recruited. Genotyping of 14bp INDEL was determined by PCR.
Structural brain images were acquired using a 3-Tesla MRI in 108 HC and
76 schizophrenia patients. Voxel-Based Morphometry toolbox in SPM8 was
utilized for brain imaging analysis using bilateral masks of the following
brain regions: dorsolateral prefrontal cortex (DLPFC), hippocampus,
parahippocampal gyrus (PHG) and posterior cingulate gyrus (PCG)
[uncorrected p<0.01; small-volume-correction for the respective mask
(family-wise-error) p<0.05; 10-voxel threshold].
Results: There were no significant allele and genotype differences between
patients and controls. However, a significant increase of heterozygous
(+14bp/-14bp) genotype was observed in the female patients (p0.05).
Interestingly, mother and the female patients also shared increased +14bp/-
14bp compatibility. Imaging analysis indicated that patients exhibiting
+14bp/-14bp genotypes had significantly deficient volume in the right
hippocampus [30, -13, -12] and right PHG [41, -38, -11].
Conclusion: Our results demonstrate a possible role of HLA-G
polymorphism and feto-maternal matching of HLA-G in conferring the
risk of schizophrenia. Importantly, this genetic variant also influences
brain morphometric measures. Taken together, these findings suggest
HLA-G could be an important biomarker for schizophrenia.
P44
Stress and 5-HTT (SLC6A4) as an indicator of suicidal behavior: a
population study among the Dubla tribe of Daman
Sweta Saha
*
, Masan Kambo Newmei, Shivani Pasi, Piyoosh Kumar Singh,
Vadlamudi Rao
Department of Anthropology, University of Delhi, Delhi, India
E-mail: sweta_saha007@yahoo.co.in
Background: Suicide is considered to be characteristic of modern
societies and thought to be an alien feature in tribal societies. Few earlier
studies have reported cases of suicide in tribal communities but there is
a dearth of evidences from India and around the globe investigating
suicide among the tribal communities. The stress diathesis model is the
best framework to understand the complex mechanisms interacting
throughout the relationship between stress and suicide. The present
study aims to understand the genetically determined vulnerability of
suicide under stressful life events among the Dubla tribe of Daman.
Method: 84 unrelated individuals were recruited for the study. Face to
face interview was conducted to collect information. Columbia Suicide
severity Rating Scale (C-SSRS) and Presumptive Stressful Life Event Scale
(PSLES) was administered to measure suicidal behavior and stressful life
events respectively. Alcohol abuse was assessed using DSM-IV based
questionnaire. Sociodemographic variables were also recorded. Buccal cell
samples were collected for genetic analysis. Genotyping of 5HTT-VNTR
(Stin2) was done with 55 samples.
Result: Bivariant analyses showed significant difference in stressful life
events among suicide ideators and non-ideators (OR=8.625, p= 0.00007).
Also, stressful life events were higher in suicide attempters both in males
and females [OR=8.438, p= 0.02 (males); OR=9.454, p= 0.04 (females)]. The
frequency of the 5HTT VNTR 10 repeat allele and12repeat allele was 27%
and 73% respectively in the studied population. Significant difference was
observed in 10 repeat allele (Stin 2.10) for suicide attempters and non
attempters among females (odds ratio, OR = 4.263; p = 0.000003).
Conclusion: Stress was found to be a major predictor of suicide among the
Dubla tribe. 5HTT VNTR 10 repeat allele was associated with suicide attempt
among females. This finding of 5-HTT genes associated with female suicide
is justified by the dimorphic nature of the serotonergic system.
CYTOGENETI CS
P45
Significance of Gain and Loss of Chromosomal Abnormalities in AML:
An Indian Experience
Pina J Trivedi
1*
, Manisha M Brahmbhatt
1
, Dharmesh M Patel
1
, Esha N Dalal
1
,
Geeta Joshi
2
, Prabhudas S Patel
1
1
Cell Biology Division, Gujarat Cancer Research Institute, Ahmedabad, India;
2
Department of Cancer Biology, Deputy Director, The Gujarat Cancer &
Research Institute, Ahmedabad, Gujarat, India
E-mail: pjt1410@gmail.com
Background: Acute myeloid leukemia (AML) is a heterogeneous group of
disorder. Recurrent translocations are generally recognized to be a major
parameter for prognostication in AML. Recurrent chromosomal translocation
t(15;17),t(8;21) and inv(16) have good prognosis, whereas, loss and gain of
different chromosomes and/or chromosome segments play a vital role by
different mechanism in leukemogenesis. Cytogenetics is one of the most
powerful independent prognostic indicators in AML. It serves to identify
biologically distinct subsets of disease and has been widely adopted to
provide the framework for risk-adapted treatment approaches. The aim of
the present study was to appraise the clinical significance of numerical and
structural chromosomal abnormalities in AML patients in terms of loss and
gain of chromosomal material.
Page 33 of 59
Materials & Methods: Bone marrow and peripheral blood lymphocytes of
321 AML patients were carried out by cytogenetics and FISH studies. Short
term cultures and GTG banding were done for karyotyping. FISH and
Multicolour FISH; were carried out as and when required as per standard
protocols.
Results: Out of all 321 patients, trisomy 8 showed the highest prevalence
(n=14) and found as sole, complex and secondary change. Apart from most
commonly observed recurrent chromosomal abnormalities, there were loss
and gain of different chromosomes also observed. The loss of sex
chromosome was observed in the highest frequency (n=20). Gain of whole
chromosomes were; 8 (X23), 10 (X5), 19 (X7), 21 (X10), 22 (X6) and loss of X
(X6), Y (X17). Mainly involved breakpoints in structural abnormalities were
gain or loss of different chromosomes i.e. 1q (X8), 5q (X5), 8q (X4), 9q (X5),
11p (X5), 11q (X8), 17q (X9), and 22q (X 6).
Conclusions: Study revealed that the, loss of chromosomal material was
observed much more often than gain in AML with aberrant karyotypes.
Hence, loss of tumor-suppressor genes may occur which may involved in
mechanism of leukemogenesis. Numerical abnormalities in karyotype may
affect gene-dosage and may play a significant role in the pathogenesis of
AML. This study also highlights the importance of diagnostic cytogenetics as
an independent prognostic factor in AML, providing the allocation for a
stratified treatment approach of the disease.
P46
Chromosomal Abnormalities and Hormonal Imbalance in Patients with
Amenorrhea in Tamilnadu
G Bhavani
1*
, RS Chandra
1
, D Anuradha
2
, ST Santhiya
1
1
Dr ALM Post Graduate Institute of Basic Medical Sciences, University of
Madras, Taramani, Chennai-600113, India;
2
Department of Medical Genetics,
Institute of Obstetrics and Gynecology, Madras Medical College, Government
Hospital for Women and Children, Egmore, Chennai-600008, India
E-mail: Bhavani.sekaran@gmail.com
Background: Amenorrhea is the absence of menstrual bleeding in women
of reproductive age. Multifarious causes such as pregnancy, absence of
uterus and vagina, hormonal imbalance, excess of male testosterone,
endometritis and improper functioning of ovaries could be attributed. It is
the sixth major cause of female infertility. The present study was undertaken
to determine the prevalence of chromosomal anomalies in patients with
amenorrhea and to correlate the karyotype with the clinical condition.
Materials & Methods: The study comprised of cases with provisional
diagnosis of Primary Amenorrhea [n=74], Secondary Amenorrhea [n=13],
Turner Syndrome [n=8], Gonadal dysgenesis [n=4] and a case of
Androgen insensitivity syndrome.
Results: In the present study, Eighty-one cases revealed normal karyotype
(46,XX) while, the remaining 19 cases showed abnormal chromosomal
pattern. The most frequent abnormal karyotype in these patients was of
46,XY females [n=6] followed by 45,X [n=5]. Two individuals showed 45,X/
46,X,i(X)(q10) and a single case each of 45,X/46,XX; 45,X/46,XY; 45,X/46,X,r
(X); 46,X,i(Xq); 46,X,del(X)(q21.2) and 46,X,del(X)(p11) respectively were also
observed. The percentage of chromosomal abnormalities in patients with
PA and SA were 18% and 7.6% respectively. Hormonal profiling wherever
possible revealed an elevated level of FSH in 47 individuals [52.8%] and
that of LH in 34 [43.2%] of them. Altered levels of other hormones such as
PRL [3.09%], T3 [11%], T4 [9%] and TSH [12%] were seen. Most of the
patients had hormone values appropriate to the post-menopausal range.
Detailed molecular analysis of possible candidate genes in cases of 46,XY
females has been proposed to resolve genetic etiology.
Conclusion: This study has shown the spectrum of chromosomal
abnormalities underlying amenorrhea.
DYSMORPHOLOGY
P47
Partial Deletion of Distal Long Arm Encompassing Jacobsen Syndrome
Manisha Desai
1*
, Bhumi Patel
1
, Chaitanya Datar
2,3
, Anand Pandit
3
,
Prakash Ghambhir
4
, Darshana Nayak
5
, Jayesh Sheth
1
, Frenny Sheth
1
1
Satellite, Ahmedaba, India;
2
Sahyadri Genetics, Unit of Sahyadri Hospitals,
Barve Memorial Complex, J.M. Road, Pune, India;
3
KEM Hospital, Department
of Paediatrics, Rasta Peth, Pune, India;
4
Birth Right Genetic Clinic,
Erandawane, Pune, India;
5
Asian Child Neuro Clinics, Park Avenue, Ellisbridge,
Ahmedabad, India
E-mail: manisharushik@yahoo.co.in
Background: The terminal 11q deletion syndrome also known as
Jacobsen syndrome (JS) is a rare genetic disorder associated with
multiple dysmorphic features and occurs in 1 in 100,000 live births. The
etiology behind the disorder is loss of contiguous set of genes due to
7-20Mb deletion that has proximal breakpoint at 11q23.
Materials and Methods: Segmental aneuploidy or alteration involving
#11q arm was detected in 2 cases during conventional cytogenetic analysis
carried out in children having multiple congenital anomalies (MCA).
Chromosome preparations were obtained from PHA stimulated lymphocyte
cultures according to the standard procedure at 500-band level in both
patient and their parents. Evaluation of the break point region was
performed by 60K oligonucleotide array-Comparative Genomic Hybridization
(aCGH) using Agilent platform. Female genomic DNA (Promega Corporation,
Madison, WI, USA) was used as a sex-matched reference, which was
analyzed with the aCGH analysis software v3.4 (Agilent Technologies Inc.,
Santa Clara, CA, USA) by applying Z-score segmentation algorithm with a
window size of 10 points to identify chromosome aberrations.
Results: Chromosomal study demonstrated structural rearrangements on
#11q arm in both the cases i.e. 46,XX,der(11)del(11)(q24). Oligonucelotide
aCGH analysis was performed using 3-points filter and 0.2 variation which
lead to the confirmation of partial deletion of 11.8-11.9Mb at 11q24.1q25
[arr 11q24.1q25(123,045,174-134,868,407)x1] in case-1 where presence
of deletion was suggested by banding technique. Whereas, a 13.9-
14Mb deletion at 11q23.3q25 together with 7.3-7.6Mb duplication at
12q24.32q24.33 was detected in the second case [arr 11q23.3q25
(121,000,318-134,868,407)x1 and 12q24.32q24.33(126,482,698-133,767,986)
x3]. The complex structural rearrangement was missed by the conventional
cytogenetic analysis in case 2. Paternal inheritance was confirmed in the
latter case.
Conclusion: Conventional cytogenetic analysis provided an overview of
the deleted segment whereas aCGH analysis added detailed information
about the breakpoint region. The complex structural rearrangement
detected only by aCGH indicates its utility in diagnosis of rare genetic
diseases especially in cases with multiple congenital anomalies.
GENETI C TOXI COLOGY
P48
In Vitro Fluoride Induced Genotoxic Effect on Human Blood
Lymphocyte Cells and its Amelioration by Emblica Officinalis Extract
Swati B Thakur
*
, Mandava V Rao
Human Genetic Unit, Department of Zoology, School of Sciences, Gujarat
University, Ahmedabad, Gujarat, India
E-mail: zooldeptgu@satyam.net.in
Background: Fluoride is a widespread industrial pollutant. Although, acute
and chronic exposure of fluoride results in adverse health effects, in vitro
studies demands for further evidences to conclude on the role of F as
genotoxic agent. We have investigated the genotoxic properties of fluoride
on peripheral blood lymphocyte cells and evaluated the protective effect
of Emblica officinalis (Amla) against fluoride toxicity.
Materials and Methods: Peripheral blood lymphocytes were cultured and
treated with different concentrations of fluoride (17 M, 34 M, and 51M)
and supplement with amla extract(20 g) for the study of various
genotoxic parameters such as sister chromatid exchanges (SCEs) and
cytokinesis block micronucleus (CBMN) assay. To rule out the antioxidant
properties of amla, indices like 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay
and High performance thin layer chromatography (HPTLC) were done.
Results: Fluoride exhibited a significant increase in SCEs per metaphase
plate (p<0.001) and SCEs per chromosome (p<0.05). Similarly, cell cycle
proliferative index significantly decrease (p<0.001) in a dose-dependent
manner in the three fluoride dose groups. Genotoxic indices such as
nuclear deformities and frequency of micronucleus significantly (p<0.001)
Page 34 of 59
elevated with increased fluoride concentration. Furthermore, nuclear
division index (NDI) and cell viability also noticed to be declined in fluoride
treated cultures. Cultures with high dose of fluoride co-supplement with
amla extract indicated a remarkable recovery in these genotoxic indices as
comparable to control cultures. Antioxidant analysis of amla extract
showed high free radical scavenging activity with EC
50
value of 55.44
0.12g/ml.
Conclusion: Amla has a strong antioxidant system to scavenge the free
radicals generated through toxic effect. Amla showed an antigenotoxic
effect against fluoride and thus has a great potential for the application in
medicinal products.
P49
Genetic Damage Biomarkers in Buccal Epithelial Cells of Healthy
Individuals Staying Near Three Mobile Phone Base Stations
Naresh Mahajan
*
, Akbar Bhat, Gursatej Gandhi
Department of Human Genetics, Guru Nanak Dev University, Amritsar, India
E-mail: nareshmahajan1431@yahoo.co.uk
Background: Massive surge in mobile phone usage throughout the world
has increased the installation of mobile phone base stations to meet the
subscribers demand. However, as mobile phone towers and handset use
microwave radiation for signal transmission, their arise health concerns
from exposures to these radiations especially for people living in areas
where the mobile phone base stations have been installed.
Materials and Methods: In cross sectional case control study, effort was
made to assess genetic damage in some individuals (n=50; exposed
group) residing in an area with three base stations close by (150 meters)
with power density ranging from 172.6 to 550.3w/m
2
at the threshold of
residences from where sampling was done. Healthy controls (n=50) with
no exposure were contacted from areas with no base stations (power
density 0.0023w/m
2
).The standard buccal cytome assay was performed to
assess DNA (genetic) damage, all proliferation and cell death biomarkers.
Results and Conclusion: Genetic damage (micronuclei and nuclear buds)
was significantly (p<0.05) higher in the exposed group while cell
proliferation markers (basal cells and binucleated cells) were not
significantly raised. However, among the cell death markers (condensed
chromatin, karyorrhectic cells, pyknotic cells and karyolitic cells), the
condensed chromatin (p<0.05) and karyolitic (P<0.05) cells also showed
significant increase in the exposed group. The observed significant
increase in genetic damage assessed in the buccal epithelial cells in
individuals residing near mobile phone base stations is of concern as all
neoplasia initiate from genetic damaging events.
P50
Role of -Aminolevulinic Acid Dehydratase (ALAD) Gene Polymorphism
in Lead Induced Nephrotoxicity
Mugdha Tiwari
1*
, LJ Bhagia
2
, I Shaikh
2
, D Rohila
1
1
Environmental Carcinogen Unit, National Institute of Occupational Health,
Ahmedabad, India;
2
Hygiene Department, National Institute of Occupational
Health, Meghani Nagar, Ahmedabad, India
E-mail: mugdhatiwari1@gmail.com
Background: Lead is an important environmental and occupational
pollutant and it can cause nephrotoxicity even at low doses. Early
detection of renal disease from occupational and environmental
exposure to nephrotoxic chemicals is currently limited by the lack of
sensitive or chemical specific tests. There are growing body of evidence
that supports Kidney injury molecule-1 (KIM-1) as a specific marker for
nephrotoxicity, specifically- ischemic renal injury. - aminolevulinic acid
dehydratase (ALAD) gene polymorphism is known as an important
factor affecting workers susceptibility to lead toxicity, further role of
ALAD-1-1 and ALAD2-2 genotype is not clear yet. Correlation among the
blood lead level, ALAD genotype present, urinary KIM-1 level of workers
can provide better understanding about lead intoxication pattern and
the role of genetic factor in susceptibility towards lead intoxication
among workers.
Methods: A total 200 biological samples (blood and urine) collected from
workers working in lead-acid battery manufacturing unit, were analysed for
blood lead levels, urinary KIM-1 and presence of ALAD genotype.
Results: In the present study significant correlation between blood lead
levels and kidney toxicity were found. In individuals having ALAD 1-1
genotype, blood lead levels were observed significantly higher.
Conclusion: Higher KIM-1 level in urine in lead exposed workers denotes
that KIM-1 can be considered as early kidney injury for lead induced
nephrotoxicity.
GWAS STUDY
P51
Genome Wide Association Study to Identify SNPs Associated with
Homocysteine, Vitamin B
12
and Holotranscobalamin in Indian
Population
Vinay Singh Tanwar
1*
, Gaurav Garg
1
, Ankur Mukherjee
2
, Ganesan Karthikeyan
3
,
Sandeep Seth
3
, Analabha Basu
2
, Shantanu Sengupta
1
1
CSIR-Institute of Genomics and Integrative Biology, Delhi, India;
2
National
Institute of Biomedical Genomics, Kalyani, West Bengal, India;
3
Department
of Cardiology, All India Institute of Medical Sciences, New Delhi, India
Background: Vitamin B
12
, a cofactor for the enzyme methionine synthase,
catalyzes the remethylation of homocysteine to methionine. About 50-60%
of the Indian population are deficient in vitamin B
12
, a micronutrient that is
only synthesized by microorganisms while mammals have evolved ways
for its absorption from diet. Of the various factors involved in Vitamin B
12
absorption, Transcobalamin II (TC II) is most important as vitamin B
12
bound to TCII is bioavailable. Thus, the objective of our study was to
identify the genetic variants that are associated with homocysteine,
vitamin B
12
and holotranscobalamin levels.
Methods: A total of 3024 healthy individuals of Indo-European ethnicity
were included in the study. Biochemical parameters like homocysteine,
vitamin B
12
, holotranscobalamin etc were determined for each individual.
In the first phase (discovery phase) Genome wide association studies were
performed using Illumina Omni express chip and 524 individuals were
genotyped for 731,442 single nucleotide polymorphisms (SNPs). Statistical
analysis was performed using PLINK software (v 1.07) after stringent
quality control. In the second phase SNPs that were found to be
significantly associated were genotyped in 2500 healthy individuals.
Results: Several genetic variants, some of which are novel, were found to
be significantly associated with homocysteine, vitamin B
12
and holotransco-
balamin. This is the first GWAS for holoTC which we found is a better predic-
tor of vitamin B12 status.
Conclusion: Although we found several SNPs earlier reported to be
significantly associated with the biochemical traits measured in our
population also, many of the SNPs were previously not reported to be
associated with the biochemical traits measured.
METABOLI C DI SORDERS
P52
Identification of Novel Mutations in Glucocerebrosidase (GBA) Gene in
Indian Patients with Gaucher Disease (GD)
Chitra Ankleshwaria
1*
, Jayesh Sheth
1
, Mehul Mistri
1
, Ashish Bavdekar
2
,
Sheela Nampoothiri
3
, Sarita Gupta
4
, Frenny Sheth
1
1
Institute of Human Genetics, FRIGE House, Jodhpur Gam Road, Satellite,
Ahmedabad, India;
2
KEM Hospital, 489, Rasta peth, Sardar Mudliar Road,
Pune, India;
3
Amrita Institute of Medical Sciences and Research Centre, AIMS
Ponekkara, Kochi, India;
4
Department of Biochemistry, The MS University,
Baroda, India
E-mail: chitraankleshwaria@yahoo.co.in
Background: Gaucher disease (GD) is the most common glycolipid
storage disorder due to inherited deficiency of lysosomal enzyme acid
b-glucosidase (glucocerebrosidase, E.C.3.2.1.45) occurring due to mutations
in the GBA gene. More than 300 mutations have been reported with
higher frequency of most common mutant allele N370S (c.1226A>G),
Page 35 of 59
Leu29AlafsX18 (c.84dupG), L444P (c.1448T>C), IVS2+1G > A (c.115+1G>A)
in Jewish population. The objective of the investigation was to identify
mutations in Indian patients with GD and to understand genotype/
phenotype correlation.
Materials and Methods: Our study comprises of forty five patients with
GD and we have reported two novel mutations observed in two of the
patients. Genomic DNA was extracted from whole blood by salting-out
method and was screened for the common N370S (c.1226A>G), L444P
(c.1448T>C), R463C (c.1504C>T), and IVS2 (+1) G>A (c.115+1G>A)
mutations by RFLP PCR. Bidirectional sequencing was carried out using
automated sequencer in absence of above common mutations. In silico
analysis was carried out using Polyphen2, SIFT and Mutation t@sting
softwares.
Results: Our study has identified two novel missense mutations G289A
(c.866G>A) in homozygous state in Exon-7 and I466S (c.1397T>G) in
heterozygous state in Exon-10 in two patients respectively. Experimental
program Polyphen 2 showed G289A (c.866G>A) and I466S (c.1397T>G) as
probably damaging with a score of 0.963 (sensitivity: 0.78, specificity:
0.95) and 1.000 (sensitivity: 0.00, specificity: 1.00) respectively. SIFT/
PROVEAN Human program showed G289A (c.866G>A) and I466S
(c.1397T>G) mutation as deleterious with score of -3.233 and -5.045
respectively and Mutation testing showed G289A (c.866G>A) as disease
causing mutation and I466S (c.1397T>G) mutation as polymorphism.
Conclusion: In this study, we have observed two novel GD mutations
G289A (c.866G>A) and I466S (c.1397T>G) and effect of this genotype was
studied using protein modeling. Identification of the genotype helps in
predicting phenotypic expression, therapeutic response, and carrier
screening for genetic counselling.
P53
Identification of Novel Mutations in HEXA Gene in Children Affected
with Tay-Sachs Disease from India
Mehul Mistri
1*
, Chaitanya Datar
2
, Frenny Sheth
1
, Sarita Gupta
3
, Jayesh Sheth
1
1
FRIGEs Institute of Human genetics, Ahmedabad, Gujarat, India;
2
Sahyadri
Medical Genetics and tissue engineering facility (SMGTEF), Pune,
Maharashtra, India;
3
The M. S. University, Vadodara, Gujarat, India
Background: Tay-Sachs disease (TSD) is an autosomal recessive
storage disorder due to impaired activity of the lysosomal enzyme
b-Hexosaminidase-A (EC 3.2.1.52) due to the mutation in HEXA gene. As per
HGMD database, 134 mutations have been reported from different ethnic
groups, while in India only few mutations have been reported till date. Here
we reported three new novel mutations in HEXA gene causing TSD in
children from Maharashtra. The objective of the investigation was to
determine the disease causing mutations in HEXA gene in children affected
with Tay-Sachs disease confirmed by deficient enzyme activity of
b-Hexosaminidase-A.
Materials and Methods: Seven children in the age range of 1 month to
1.5 years were enrolled in this study. Enzyme study was carried out using
4-MU substrate (MUGS) specific for b-Hexosaminidase-A. The exons and
exon-intron boundaries of HEXA gene were bidirectionally sequenced
using automated sequencer. In silico analysis was carried out using SIFT,
Polyphen2 and Mutation T@ster softwares. Written consent was obtained
from guardian of the study subjects.
Results: Overall, we have identified 8 mutations in seven unrelated
families, three of which are novel, including combined heterozygous
missense mutations c.524A>C (p.D175A) and c.805G>C (p.G269R)
was observed in one case and one homozygous nonsense mutations
c.1528C>T (p.R510X) in one case. A previously known missence mutations
c.532C>T (R178C), c.964G>T (p.D322Y), c.1385A>T (p.E462V), 4bp insertion
c.1277_1278insTATC (p.Y427Ifs5) and splice site mutations c.459+5 G>A
were observed in 5 children. In silico analysis further confirmed the
pathogenic effect of the novel mutations occurred at highly evolutionarily
conserved and functionally active domain residues in the protein leading
to conformational changes or mRNA producing truncated protein resulting
in the diminish or absent activity of the protein.
Conclusion: Mutations responsible for TSD in Indian population are
unique. We have found 3/8 (37.5%) novel mutations [D175A, G269R and
R510X] in present study along with 5/8 (62.5 %) previously reported
mutations [E462V, D322Y, R178C, c.1277_1278insTATC (p.Y427IfsX5) and
c.459+5 G>A]. This study further confirms that nearly 37.5 % of TSD
children harbor novel mutations in India and 62.5 % have common or
earlier reported mutations. Overall study provides the new insights into
the molecular basis of the disease that can be utilized for the molecular
screening of TSD.
P54
Live-Cell Imaging of Compartment-Specific Redox Changes in Menkes
Disease Fibroblasts
Ashima Bhattacharjee
1*
, Martina Ralle
2
, Svetlana Lutsenko
1
1
Johns Hopkins School of Medicine, Baltimore, Maryland, USA;
2
Oregon
Health and Science University, Portland, USA
E-mail: abhatta6@jhmi.edu
Background: Copper is an essential micronutrient and its misbalance in
the body is associated with severe neurodegenerative disorders. While
the importance of copper in the body is evident, mechanisms by which
copper misbalance induces pathologic changes and disease symptoms
are poorly understood. In this study, using fibroblasts from Menkes
disease patient, as a cellular model of copper accumulation, we examined
whether excess copper triggers specific and distinct changes in the redox
environment of different cellular compartments.
Subjects and Methods: Skin fibroblasts from Menkes disease patient (YS
cells, ATP7A
-/-
) and his heterozygous mother (ATP7A
+/-
) were used as an
experimental system. Glutathione mediated redox environment and levels
of H
2
O
2
were investigated in nuclei, cytosol, and mitochondria of live
cells by tagging the respective ratiometric sensors (GRX-roGFP and HyPer)
with a compartment-specific localization signal.
Results: Under basal conditions, the YS and XS cells show similar glutathione
mediated redox environment in the nucleus and cytosol. However, the
mitochondria are oxidizing in YS cells. YS cells were observed to accumulate
higher level of peroxide in the cytosol and mitochondria. We also found that
copper accumulation in cytosol and nuclei of YS cells sensitize cells to
glutathione depletion suggesting importance of glutathione in protection of
cells against copper overload
Conclusion: Our experiments revealed differential response of cellular
compartments to excess copper in cells. Nuclei, in spite of being a site of
copper accumulation, do not show marked redox changes, suggesting
presence of robust protective mechanisms operating in this compartment.
H
2
O
2
accumulation in cytosol in YS cells does not change glutathione
balance, whereas mitochondria appear most affected, since both H
2
O
2
levels
and glutathione balance are altered. We propose that mitochondria could
be a primary site of copper toxicity in Menkes fibroblasts. Molecular
mechanisms underlying differential redox responses are presently being
investigated.
MOLECULAR CYTOGENETI CS
P55
Characterization of prenatally detected small Supernumerary Marker
Chromosomes (sSMC) by molecular cytogenetic technique: FISH
Bhumi Patel
1*
, Thomas Liehr
2
, Manisha Desai
1
, Bindu Parikh
3
, Jayesh Sheth
1
,
Frenny Sheth
1
1
FRIGEs Institute of Human Genetics, FRIGE House, Satellite, Ahmedabad-380
015, India;
2
Jena University Hospital, Institute of Human Genetics,
Kollegiengasse 10, D-07743 Jena, Germany;
3
Satyam Hospital, 6/65 Nilam
Park, Bapunagar, Ahmedabad India
Background: Microscopically recognized chromosomal trisomies and
monosomies are clinically well described. However, clinical effects in the
unborn baby having imbalances of small chromosomal regions resulting
from karyotypes containing small supernumerary marker chromosomes
(SMCs) in addition to normal chromosomal count are less predictable.
Moreover, due to extreme heterogeneity of sSMCs in size, structure and
chromosomal origin, full characterization of sSMCs by molecular techniques
FISH has become imperative.
Materials and methods: Two out of 1600 cases investigated showed sSMC
during amniotic fluid (AF) analysis. In case-1, single sSMC (47,XN,+mar1)
Page 36 of 59
[100%] was detected during third gravida in a young couple having previous
child with Down syndrome and second pregnancy ending in first trimester
miscarriage. In case-2, two sSMCs were detected in the foetus of an elderly
couple during primi gravida (i.e. 48,XN,+mar1,+mar2)[100%]. In both cases,
fetal anomaly scan and triple marker study were normal. Parental
chromosomal analysis at 500 band resolution was apparently normal at the
time of prenatal study confirming de novo origin of the SMCs. Various
FISH probes were applied such as (acro-)cenM-FISH, SRY, and subtel
X/Ypter; besides immunohistochemistry using antiboies CENPB (all
centromeres apart from Y) and CENPC (all active centromere) was done.
Microdissection and reverse painting FISH was also performed for complete
characterization.
Results: Microdissection and reverse FISH was carried out in case-1 and
showed signal only on the SMC. cenM-FISH did not yield any further
information. Since the fetus had two X chromosomes, reverse FISH was
carried out on a normal male control which gave signal on the #Yp.
Additional probes specific to the SRY and subtel X/Y pter gave two
signals confirming a neocentric inv dup(Y) i.e. 47,XN,+mar.ish inv dup(Y)
(pter/Yp11.2::Yp11.2/pter)(SRY++)(subtelX/Y++)[100%]. The sSMC thus
consisted of euchromatin exclusively. In case-2, sSMC were characterized
as inv dup(13 or 21)(q10) and del(13 or 21)(q10) i.e. 48,XN,+inv dup(13 or
21)(q10)(CENPC++)(D13/21Z1+),+del(13 or 21)(q10)(CENPC+)(D13/21Z1+)
[100%]. Marker chromosomes in case 2 solely consisted of heterochromatic
material according to FISH.
Conclusions: This shows that molecular technique FISH is one of the
most powerful tools for precise identification and detail characterizations
of SMCs and thereby providing valuable information to the families
regarding genotype-phenotype correlation during prenatal diagnosis.
P56
Assessment of 1p/19q deletion by Flourescence Insitu Hybridization
(FISH) in Glioma Patients from Andhrapradesh
Eppa Kavitha
1*
, Iravathy Goud
1
, Swarna Latha
2
, Meenakshi Swain
2
,
Michelle De Paude
2
, Tejal Modi
2
, Anuradha
2
, Ravi V
1
, Sakina Aneeb
1
,
Adi Maha Lakshmi M
1
, Vijayanand Reddy P
3
1
Molecular Biology and Cytogenetics Department, Apollo Health City
Building, Apollo Hospitals, Jubilee Hills, Hyderabad, India;
2
Department of
Histopathology, Apollo Hospitals, Jubilee Hills, Hyderabad, India;
3
Department of Oncology, Apollo Hospitals, Jubilee Hills, Hyderabad, India
E-mail: matamkavitha@gmail.com
Background: Oligodendroglial tumors represent approximately 4-7% of
all gliomas, however, in some series the incidence has been reported to
be as high as 10-20% due to improved histological appreciation and
recently recognized molecular signatures. The discovery of 1p and 19q
chromosomal arms deletion in glial tumors influences both more
objective diagnosis and more accurate prediction of chemotherapy
response. As a result an attempt has been made to detect deletion using
fluorescence in-situ hybridization (FISH) and to determine its prognostic
value in a cohort of glial tumor patients from Andhra pradesh.
Materials and Methods: FISH was performed on 66 FFPE tissue sections
by using Vyis LSI 1p36/LSI 1q25 and LSI 19p13/LSI 19q13 dual coloured
FISH probe sets. Signals were scored from at least 150-250 non-
overlapping, intact nuclei.
Results: Simultaneous occurrence of both 1p and 19q deletions was
observed in (21/35) 60% of oligodendroglyomas which included (8/21) 38%
of grade II and (13/21) 61.9% of grade III. Isolated 19q deletion was seen in
(1/21) 4.76% & lone 1p loss was not observed in oligodendroglyomas. In
Mixed Oligoastrocytomas combined 1p/19q loss was observed in (7/16)
43.75% cases, including one grade II and 6 grade III tumors and 1/16
(6.25%) showed isolated 1p loss & 19q deletion. This disorder was not
observed in astrocytomas. The oligodendroglial phenotype was found to be
significantly associated with a loss of 1p (P<0.05), a loss of 19q (P<0.05) and
a combined loss of 1p and 19q (P< 0.05). Frontal location of a tumor
occurred to be a statistically significant factor unfavourable for prognosis,
p<0.05.
Conclusion: In the work presented the FISH was successfully applied to
identify deletion 1p/19q. Its incidence depends on the type of diagnosed
glioma. Deletions also have prognostic significance in the test group
what constitutes the basis for inclusion of determining deletion 1p/19q
into diagnostic and treatment algorithm in gliomas.
P57
Split Hand/Foot Malformation Type 1 Associated with 7q21.3 Deletion -
A Case Report
S Aswini
1*
, S Ambika
2
, KS Pooja
2
, D Anuradha
3
, JS Kadandale
1
, CR Samuel
1
1
University of Madras, Taramani, Chennai, Tamil Nadu, India;
2
Centre for
Human Genetics, Bangalore, Karnataka, India;
3
Department of Medical
Genetics, Institute of Obstetrics and Gynecology, Madras Medical College,
Government Hospital for Women and Children, Egmore, Chennai , India
E-mail: aswini.sivasankaran@gmail.com
Split hand/split foot malformation (SHFM) is a rare congenital deformity
involving limb development. SHFM, also known as ectrodactyly, is
characterized by absence of digits, fusion of remaining digits, and a deep
median cleft in the hands and feet. It has been observed to occur at a
prevalence of approximately 1:18,000 newborns. This malformation is
genetically heterogeneous involving several loci including 7q21-q22.1,
Xq26, 10q24-q25, 2q31 and 3q27. New loci requiring further validation
have also been suggested as valuable candidate regions.
Chromosomal rearrangements involving7q21-q22 is most commonly
associated with isolated or syndromic ectrodactyly, referred to as SHFM
type 1. We report a case of SHFM1 in an eight-month-old female baby
with developmental delay. Follow up after two years revealed bilateral
hearing loss also. Chromosome analysis using high resolution banding
technique showed an interstitial deletion of the sub-band 7q21.3. FISH
using BAC clones resolved the break to have occurred within the band
7q21.3. Cytogenetic evaluation of her parents showed the deletion to be
of a de novo origin.
SFHM1 is expressed as an autosomal dominant trait with reduced
penetrance and variable expression and is accompanied by deafness in
35% of the patients as observed in the proposita. Several studies have
pointed out the probable role of three genes present in this region
DLX5, DLX6 and DSS1 - in limb development.
This report reiterates the importance of high resolution banding and
molecular cytogenetic techniques such as FISH in the detection and
delineation of subtle abnormalities. Array CGH will help in further refining
the deleted region and thus in the discovery of candidate genes for the
phenotypic characteristics.
MOLECULAR STUDY
P58
Autosomal Dominant Mutation in COL7A1 Gene causing Epidermolysis
Bullosa Dystrophica
Jayesh Sheth, Mehul Mistri, Harsh Patel, Chitra Ankleshwaria, Aradhana Parikh
*
FRIGEs Institute of Human Genetics, FRIGE House, Ahmedabad, Gujarat, India
E-mail: aradhanaparikh@yahoo.com
Background: Epidermolysis bullosa dystrophica is a rare genetic disease
involving skin fragility, resulting in blistering of the skin either spontaneously
or following minor skin contact, or trauma. Mutations in COL7A1 gene are
associated with all forms of dystrophic epidermolysis bullosa. The gene
which is present on the short arm of chromosome 3, codes for a protein
called Collagen alpha-1(VII) chain which functions as an anchoring fibril
between the external epithelia and the underlying stroma. The aim was to
examine a de novo Mutation in Gene causing Epidermolysis bullosa
dystrophica in a thirty year old female patient.
Materials and Methods: The case of a thirty-year-old female patient was
analyzed. The patient had vesiculobullous lesions, peeling of skin on
pressure, milia formation and dorsa of both hands. Lichenified lesions
with few fresh bullae were present over the knees and elbows. The
histopathological examination was carried out for skin biopsy samples.
For the molecular analysis, a sequence study of exons 73 to 75 of the
COL7A1 gene was conducted.
Page 37 of 59
Results: The histopathological examination of the skin biopsy revealed
separation of epidermis at dermoepidermal junction along with
acanthosis, hyperkeratosis and focal parakeratosis of the overlying
epidermal layer. Dermis revealed moderate inflammatory cell infiltration.
The immunofluorescence study was negative for IgG, IgM, IgA and C3.
These findings were consistent with clinical diagnosis of EB Dystrophica.
Molecular Analysis showed that the patient was heterozygous for c.6712
G>A (p.G2043R) mutation in exon 73 of the COL7A1 gene. Interestingly,
the patient had no family history of the disease indicating that the
mutation was de novo.
Conclusion: It is one of the rare examples of a patient who was
heterozygous for the mutation with dominant type of EB dystrophy.
P59
A Balanced Reciprocal Translocation T (X;20) in A Girl with Seizures and
Intellectual Disability Disrupting ARHGEF9
Usha R Dutta
1*
, Vijaya Kumar Pidugu
1
, Vera M Kalscheuer
2
, Ashwin B Dalal
1
1
Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India;
2
Max
Planck Institute for Molecular Genetics, Berlin, Germany
E-mail: ushadutta@hotmail.com
Background: Chromosomal aberrations are a significant cause of human
disorders. The purpose of the present study is to characterize a balanced
reciprocal translocation identified in a girl who presented with seizures,
disturbed sleep, intellectual disability and focal hypopigmentation on the
skin and identify the gene(s) involved.
Materials and Methods: Several methods like GTG banding, array CGH, X-
inactivation studies by methylation specific PCR for the human androgen-
receptor gene (HUMARA), FISH (Fluorescence-in situ-hybridization) with
whole chromosome paint probes (WCP) and with Bacterial Artificial
Chromosome (BAC) clones from the regions of interest, RT-PCR expression
analysis for ARHGEF9 gene were used.
Results: The chromosomal analyses revealed a translocation between the
long arm of chromosome X and the short arm of chromosome 20 [46,X,t
(X;20)(q12;p13)]. This result was confirmed by WCP FISH. Additionally, array
CGH ruled out any gains or losses at the breakpoints or elsewhere in the
genome. Also, X-inactivation studies by methylation specific PCR for
HUMARA indicated skewed X-inactivation of the normal X chromosome.
Breakpoint mapping of both derivative chromosomes was performed by
serial FISH using BAC clones and RP11-943J20 from chromosome X
showed split signals on patient derivative translocation chromosomes,
indicating that this clone spanned the breakpoint. The breakpoint on
20p13 was mapped to a region of about 28 kb. Subsequent in silico
analysis of the fine mapped breakpoint regions showed that on
chromosome X, ARHGEF9 was likely disrupted by the chromosome
rearrangement, whereas on chromosome 20 the breakpoint region does
not seem to harbor a known gene. RT-PCR expression analysis of ARHGEF9
using RNA isolated from the patients lymphoblastoid cell line and a
control suggested that in the patient the breakpoint maps between exons
1 and 2 of this gene. Further, the rearrangement has potentially resulted in
fusion genes, suggested by the low expression of ARHGEF9 exons 2 to 10
in the patient.
Conclusion: We have previously reported another chromosome
rearrangement that truncated ARHGEF9 in a patient with epilepsy,
anxiety, aggression, insomnia and learning and memory loss. Given the
similar clinical phenotypes of both patients we propose that in the
patient reported here ARHGEF9 loss of-function is likely to be the cause
of disease.
P60
Molecular analysis of mucopolysaccharidoses: identification and
characterization of pathogenic mutations in Indian population
Anusha Uttarilli
*
, S Jamal Md Nurul Jain, Ashwin B Dalal, Prajnya Ranganath,
Shubha R Phadke, Girisha Kumar, Sankar, SJ Patil, Madhulika Kabra,
Sumita Danda
Diagnostics Division, CDFD, Nampally, Hyderabad, India
E-mail: uttarillianusha@gmail.com
Background: Mucopolysaccharidosis (MPS) are a group of rare inherited
metabolic disorders which are caused due to the deficiency of a specific
lysosomal enzyme involved in the catabolism of glycosaminoglycans. These
disorders show a wide clinical spectrum ranging from severe, intermediate
and mild phenotypes. Most of them show overlapping clinical features such
as corneal clouding, coarse facies, hepatosplenomegaly, skeletal dysplasia,
short stature, dysostosis multiplex, joint stiffness, joint contractures,
cardiovascular and respiratory difficulties. Certain MPS disorders also show
impaired neurological functions leading to mental retardation. They are
inherited as an autosomal recessive with exception of MPS II, which is X-
linked recessive disorder. They exhibit clinical, genetic as well as molecular
heterogeneity. To date more than 200 mutations in IDUA, ~ 500 in IDS,
~ 200 in GALNS and ~ 200 in ARSB have been reported worldwide. The
mutation spectrum of MPS disorders in Indian population is not
characterized and established yet. This study was done to establish
mutation spectrum in the Indian population that can be useful for the
design of cost-effective strategies towards the molecular diagnosis of MPS
disorders.
Materials and Methods: We have carried out mutation analysis of a total
of 90 different MPS disorder patients (MPS I, II, IV and VI) and identified a
total of 64 different mutations comprising majorly missense, nonsense,
small deletions and splice site mutations. The mutations were further
characterized using mutation prediction software and protein structure
analysis for pathogenicity prediction.
Conclusion: The characterization of mutational spectrum of Indian
patients is likely to be useful in provision of better carrier diagnosis and
prenatal diagnosis for patients with MPS disorders and also aids in
designing of newer therapeutics for efficient treatment. It also helps in
establishment of genotype-phenotype correlation and provision of better
genetic counselling to the families with MPS affected members.
P61
Association of ESR and FOXP3 Gene Polymorphisms with Outcome of
Ovarian Stimulation in Infertile Females Undergoing IVF
Arun Kiran Patnam
1*
, R Vinu
2
, J Vijayalakshmi
2,1
, P Venkatachalam
1
,
G Usha Rani
3
1
Department of Human Genetics, Sri Ramachandra University, Porur,
Chennai, India;
2
Department of Biomedical Sciences, Sri Ramachandra
University, Porur, Chennai, India;
3
Department of Obsteritics and Gynecology,
Sri Ramachandra University, Porur, Chennai, India
E-mail: arunkiran2222@gmail.com
Background: Estrogen receptor (ER) gene plays a major role in
folliculogenesis, maturation of oocytes and fertilization of embryo. FOXP3
gene is a crucial regulatory factor for the development and function of Treg
cells; it takes part as a central role in the induction and maintenance of fetal-
maternal immunologic tolerance. Therefore, the present study was aimed to
investigate the association of ESR and FOXP3 gene polymorphisms in
infertile women who are undergoing in vitro fertilization (IVF) with poor
ovarian reserve upon controlled ovarian hyperstimulation (COH) induced by
follicle-stimulating hormone (FSH).
Materials and Methods: Genomic DNA was extracted from EDTA-
peripheral blood by using high salting-out method, polymerase chain
reaction-restriction fragment length polymorphism (PCR-RFLP) analysis
was done followed by DNA sequencing to confirm the results for ESR1
gene on intron 1, PvuII T/C (rs2234693) and XbaI A/G (rs9340799); ESR2:
RsaI G/A (rs1256049) on exon 5; and FOXP3 gene: HaeIII T/C (rs2294021)
single-nucleotide polymorphisms (SNPs). Genotyping was carried out in
infertile female (n=25) undergoing in-vitro fertilization (IVF) with poor
ovarian reserve and healthy fertile controls (n=25).
Results: Statistical analysis of ESR 1 (rs2234693) gene polymorphism
showed a significant association (p<0.05) between cases and controls.
Whereas, ESR 1 (rs9340799), ESR2 (rs1256049) and FOXP3 (rs2294021)
genotypes showed no significant association (P>0.05) in cases and
controls.
Conslusion: As polymorphism rs2234693 (PvuII) showed a significant
association in dominant model (Odds ratio: 3.19 (95%CI: 1.00-10.17);
P: 0.04), genetic variability of the Estrogen receptor gene may exert an
indirect effect on the pregnancy outcome of IVF patients by affecting the
Page 38 of 59
development of the follicles, oocytes and embryos. Still, further studies
are necessary to confirm our findings with larger sample size that might
improve our understanding of ESR1 gene polymorphisms and its
importance for advancing infertility diagnoses, treatment and genetic
counselling.
P62
Protein structure prediction for novel mutations in Arylsulfatase-A gene
M Divya
*
, S Jamal Md Nurul Jain, SR Phadke, Ratna Kishore, Mahesh Kamate,
Neerja Gupta, Ashwin Dalal
Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics,
Hyderabad, Andhra Pradesh, India
E-mail: divya.bioinfo4@gmail.com
Background: Protein structure prediction is the prediction of three-
dimensional structure of a protein from its amino acid sequence. It is
useful in determining the effect of a mutation on protein structure and
associated function in detail. Along with the use of mutation prediction
servers (Mutation taster, Polyphen etc.) protein structure prediction is an
additional approach to functionally annotate genetic variants detected
from different individuals.
Methods: The X-ray structure of the P15289 protein was used for protein
structure prediction of effect of mutations (PDB code 1AUK). TRANSEQ pro-
gram of EMBOSS was used to obtain translated products of the deletion
and insertion mutants. PyMOL and Swiss PdbViewer were used for
performing structural analysis. Potential changes in overall hydrophobi-
city due to non-synonymous mutations were calculated using CLC
workbench.
Results: We performed protein structure prediction for six novel non-
synonymous missense mutations (P180Q, Y33S, Q139K, R299P, G34E and
R311P) and four frameshift mutations (c.188-189insA, c.752-753insT,
c.576delC and c.445-446insT) obtained from sequence analysis of ARSA
gene. The residue Q139 is in 3
10
helix and R311 is in a b-sheet. Missense
mutations at these positions affect the secondary structure of the protein.
R299P mutation predicted to destabilize surrounding helical structure by
hydrogen bond disruption. G34E, P180Q, R299P, and R311P showed a
wide range of alterations in the overall hydrophobicity at the sites of
mutation. Y33S and R311P were involved in active site modification and
P180Q affecting the catalytic ability. All frameshift mutations were
predicted to be leading to nonsense mediated decay.
Conclusion: Protein structure prediction helps to provide a means of
generating a plausible protein structure resulting after the mutation and
understanding the effect of mutations on a protein whose effects have
not been experimentally determined.
P63
A Family Based Study on T-C Transition Polymorphism in Cyp17a1
Gene in Indian Children
Sukanya Gayan
*
, Abid Ali, Rajeev Kumar Pandey, Minu Bajpai
Department of Pediatric Surgery, All India Institute of Medical Sciences, New
Delhi, India
Background: Congenital adrenal hyperplasia (CAH) or 46, XX DSD is a
result of a defect in the P450 adrenal enzymes responsible for the
conversion of progesterones to glucocorticoids and mineralocorticoids.
This syndrome affects both males and females but causes ambiguous
genitalia only in females. Mutations in the CYP21 cause 90% of cases of
CAH. The remainder of CAH cases is distributed among deficiencies of
P450c11, CYP17 or steroid acute regulatory protein, depending on the
ethnic origin of patients. Rarely, 46, XX, DSD can result from exposure to
exogenous androgens such as those given in the past to prevent loss of
pregnancy.
The present study was conducted to replicate a family based correlation of
C to T transition polymorphism in Congenital adrenal hyperplasia (CAH).
Materials and Methods: A total of 60 samples (20 families) within a
period of one year (October 2012 2013) associated with CAH, were
collected from patient visiting the Out Patient Door (OPD) facility of the
department of Paediatric Surgery, All India Institute of Medical Sciences
(AIIMS). Genomic DNA was isolated from peripheral blood leukocytes of
twenty patients and their family members using the phenol - chloroform
method. Polymerase chain reactionrestriction fragment length
polymorphism (PCR-RFLP) was used to detect the polymorphism in
CYP17A1 using restriction enzyme, MSPA1.
Results: This study revealed no association between CAH risk and
CYP17A1 gene in Indian children.
Conclusion: Our results do not suggest a role of CYP17A1 as a high
susceptibility gene for Congenital adrenal hyperplasia in Indian family.
P64
Frequency analysis of Spinocerebellar ataxia types 1, 2, 3 & 6 in
patients with ataxia from Gujarat
Harsh Patel
*
, Mehul Mistri, Chitra Ankleshwaria, Frenny Sheth, Jayesh Sheth
Institute of Human Genetics, FRIGE House, Ahmedabad, Gujarat, India
Background: The autosomal dominant spinocerebellar ataxias (ADCA) are
a clinically and genetically heterogeneous group of neurodegenerative
disorder characterized by progressive deterioration in balance and
coordination as well as cerebellar ocular disturbance. There is a lack of
information about the frequency of SCAs in Gujarat (western part of
India), which can be used as a common screening test in our population.
The study was conducted to analyze the frequencies of SCA1, SCA2, SCA3
and SCA6 in patients with ataxia from Gujarat.
Materials and Methods: Prospective analyses of 64 unrelated patients
were included after an informed written consent. They were presented
with progressive cerebellar ataxia with other features like gait and speech
disturbance, saccadic eye movements and tremors. The study included 35
males and 29 females in the age range of 20 years to 85 years. All
patients were investigated for CAG repeat expansion in SCA1, SCA2, SCA3
and SCA6 gene by PCR. Genomic DNA was used for the molecular study.
Results: CAG repeat expansion was detected in 27 patients (42.1%) of 64
unrelated individuals. Among these, 7.41% (female-2) subjects were found
to have SCA1 with CAG repeat expansion copy in the range of 49-56 in
ATXN1 gene, 59.3% (female-9, male-7) were found to have SCA2 with
CAG repeat expansion copy in the range of 37-47 in ATXN2 gene, 33.33%
(female-3, male-9) were found to have SCA3 with CAG repeat expansion
copy in the range of 63-82 in ATXN3 gene. While none of the subject
was found to have SCA6 as has been shown by normal CAG repeats in
the range of 10-16 in CACNA1A gene. Age range of onset of the affected
subjects was 3
rd
- 5
th
decade in SCA1, 3
rd
- 6
th
decade in SCA2 and 3
rd
-
8
th
decade in SCA3.
Conclusion: Our study demonstrates that SCA2 is the commonest
dominant spinocerebellar ataxia in Gujarati population followed by SCA3
affecting in the 3
rd
- 6
th
decade of life.
P65
Genetic susceptibility of Henoch-Schnlein purpura in children
Ritu Aggarwal
*
, Anju Gupta, Jasmine Naru, Neha Nanda, Manila Salaria,
Deepti Suri, Surjit Singh
Department of Immunopathology and Allergy and Immunology Unit,
Deptartment of Pediatrics, Post Graduate Institute of Medical Education and
Research, Chandigarh, India
E-mail: ritu_immunopath@yahoo.co.in
Background: Henoch-Schnlein purpura (HSP) is a small vessel vasculitis
typically observed in children, 3-10 years old. The aetiology is unclear.
Interaction of several environmental factors, including infections and
multiple genes has been proposed to play a role in pathogenesis. An
increased familial occurrence is an indicator of genetic predisposition;
association with a major histocompatibility complex is plausible. The aim
of the study was to investigate the association of HLA-DRB1 (HLA class II
antigen) with HSP.
Subjects and methods: The study was prospective and hospital based.
Patients up to the age of 14 years, who fulfilled the diagnostic criteria of
HSP, laid by the European League Against Rheumatism were enrolled.
Age matched healthy controls were included as well. One ml blood in
EDTA was collected from patients as well as controls. DNA extraction was
Page 39 of 59
performed using commercially available kit. The quantity and quality of
DNA was estimated by spectrophotometer and PCR for housekeeping
gene beta actin, respectively. PCR with 24 sequence specific primers for
HLA-DRB1 antigen was performed. Commercially available HLA-DR tissue
typing kit (Inno-train, Kronberg im Taunus, Hesse, Germany) was utilized.
Frequency of HLA-DRB1 was correlated with gastrointestinal and renal
involvement.
Results: The study included 43 patients and 53 controls. The mean age
of the patients and controls was 8.5 years (range: 3-14) and 7.4 years
(range: 1-14), respectively. Frequency of HLA-DRB1*11 was significantly
increased in patients (p=0.006). A greater gastrointestinal (p=0.03) as well
as renal (p=0.004) involvement was observed in patients with HLA-
DRB1*11.
Conclusion: This is the first study from India to report the HLA
susceptibility genes in children with HSP. Presence of HLA-DRB1*11 was
observed to predispose to HSP in children, as well as for greater
likelihood of gastrointestinal and renal involvement.
P66
Evaluation of galectin-3 genetic variants and lipid profile in RA patients
in North Indian population
Tarnjeet Kaur
1*
, Manpreet Kaur
1
, Jatinder Singh
2
, Sukhdev Singh
2
,
Sumeet Arora
3
1
Department of Human Genetics, Guru Nanak Dev University, Amritsar, India;
2
Department of Molecular Biology and Biochemistry, Guru Nanak Dev
University, Amritsar, India;
3
Rheumatology Clinic, Amritsar, India
Background: Rheumatoid arthritis (RA) is a chronic, inflammatory,
systemic disease characterized by inflammation and destruction of
peripheral joints which leading to deformity and disability. Galectin- 3 is
emerging as one of key molecules in pathogenesis of RA. The aim of the
present study was to evaluate association of two genetic variants rs4644
and rs4652 of galectin-3 with susceptibility towards RA in North Indian
population. The study further involved evaluation of lipid profile variables
in cases and controls.
Methods: The present case-control study involved 200 RA patients
diagnosed according to 1987 revised criteria of American college of
Rheumatology and 200 unrelated age, sex and ethnicity matched
controls. Genomic DNA was isolated from blood samples and genotyping
was done with PCR-RFLP. Sample size for genetic association was
calculated by CaTS Power calculator (http://www.sph.umich.edu). Serum
was analyzed for lipid profile biomarkers using standard reagents and
kits. Genotypic distribution of control and RA was compared by odds
ratio statistics using medcal software. Differences in lipid profile were
analyzed by independent `t test using SPSS version 18.0 (IL, USA, and
Chicago)
Results: The genotypic distribution of +191(A/C) showed significant
differences between patients and controls (odds ratio = 1.9552, 95% CI =
1.0461-3.6542, p<0.05). AA genotype was found to be more prevalent in
patients in comparison to controls. However, genotypic distribution for
+292 (C/A) showed no significant difference between controls and cases
(odds ratio = 0.2768, 95% CI = 0.0541-1.4149, p>0.05 ). RA patients were
found to be dyslipidemic as indicated by the significantly higher
atherogenic index as compared to controls (p<0.01).
Conclusion: Galectin-3 may play an important role in pathogenesis of RA.
P67
Possible involvement of altered expression of BDNF exon II gene and
specific dopamine receptors in simvastatin induced beneficial effects in
depression
Digvijay G Rana
1*
, Amrut Patel
2
, CG Joshi
2
, Ramesh K Goyal
3
1
Department of Pharmacology, Sigma Institute of Pharmacy, Vadodara, India;
2
Department of Biotechnology, Anand Veterinary College, Anand, India;
3
Institute of Life Sciences, Ahmedabad University, Ahmedabad, Gujarat, India
E-mail: dgrana3755@yahoo.com
Background: The up regulation of central BDNF gene expression has
been suggested in the treatment of major depression. Chronic
administration of dopaminergic agents activates the function of CREB
which results into the up regulation of the BDNF gene expression. Statin
therapy is associated with a reduced risk of depression and could be of
therapeutic potential for major depression.
Materials and methods: We have examined a possible link amongst
simvastatin, bromocriptine, haloperidol and levodopa in accordance with
BDNF exon II gene expression using RT-PCR method in mice treated with
standard paradigm of chronic mild stress procedure for 14 days. We
specifically determined if the oral administration of simvastatin would
affect the efficacy of bromocriptine, haloperidol or levodopa in mediating
the regulation of the BDNF exon II gene expression.
Results: The results of RT-PCR method revealed the differential
expression patterns for the expression of BDNF exon II gene in brain of
mouse by indicating the three different bands, as evidenced previously to
be the three different exon II transcript variants in mouse namely BDNF
IIA, BDNF IIB and BDNF IIC. Mice treated with bromocriptine or levodopa
in combination with simvastatin for 14 days could synergize the up
regulation of for the expression of specific BDNF exon II transcript as
compared to simvastatin alone whereas the mice treated with haloperidol
in combination with simvastatin for 14 days could abolish the for the
expression of up regulation of specific BDNF exon II transcript compared
to simvastatin alone.
Conclusion: The results of the above study suggests linkage between the
function of dopamine or dopamine D
2
like receptor and differential
expression patterns for the expression of BDNF exon II gene in brain of
mouse which further strengthen the emerging hypothesis, suggesting the
ability of neuronal systems to exhibit the appropriate adaptive plasticity
could contribute to the treatment of depression. Further, the
dopaminergic agents in accordance with the cholesterol lowering drug as
adjuncts may reduce the depressive like behavior more significantly and
facilitation of antidepressant action of dopaminergic agents may be
correlated with HMGR or cholesterol or mevalonate pathway.
P68
Genetic, metabolic and cellular factors influencing intracellular
localization of the Wilson disease protein, ATP7B
Arnab Gupta
1*
, Ashima Bhattacharjee
2
, Nesrin Hasan
2
, Lita Braiterman
1
,
Svetlana Lutsenko
2
, Ann L Hubbard
1
1
Department of Cell Biology, Johns Hopkins University, Baltimore, USA;
2
Department of Physiology, Johns Hopkins University, Baltimore, USA
Background: Wilson disease (WD) is a disorder of copper accumulation
in liver and brain caused by mutations in the copper-transporting ATPase
ATP7B that affects 1 in 5000 live births. Under basal conditions, ATP7B
protein localizes to the trans-Golgi network (TGN) but traffics to vesicles
in response to high copper. The purpose of the study is to identify
genetic, metabolic and regulatory factors that regulate ATP7B function
and localization to maintain normal copper homeostasis in cells. The
study is divided into three parts, (a) Role of copper in normal protein
folding and its ER exit, (b) Role of regulatory phosphorylation of ATP7B in
its trafficking from TGN to vesicles (c) Interaction of ATP7B with
regulatory proteins in its trafficking route.
Material and methods: Recombinant ATP7B (wt and variants), Confocal
microscopy, NMR, Mass Spectroscopy, targeted knockdowns and other
basic molecular biology techniques were utilized in this study.
Results: We demonstrate that the polymorphism (producing the
Gly
875
>Arg substitution) of ATP7B drastically alters the intracellular
properties of ATP7B, while copper reverses the effects. Unlike the
wtATP7B, the Arg
875
variant is located in the endoplasmic reticulum (ER)
and does not deliver copper to the TGN. Elevated copper rectifies the
ATP7B-Arg
875
phenotype. Analysis by NMR suggests that the ER retention
of ATP7B-Arg
875
is due to increased unfolding of the Arg
875
-containing.
conserved domain (b) I characterized the sites of copper dependent
regulatory phosphorylation of ATP7B critical in normal ATP7B trafficking. I
discovered that a stretch of Ser (S
340-343
) at the N-terminal of ATP7B are
the sites of phosphorylation and that phosphorylation alters the
interaction between two conserved domains of ATP7B, causing the
protein to attain a TGN exit conformation (c) Finally, while identifying
the cellular machinery that is involved in trafficking of ATP7B vesicles, I
found that MyosinVb is a major regulator of ATP7B trafficking. Presently,
the molecular mechanism of the role of MyoVb in ATP7B trafficking is
being investigated.
Page 40 of 59
Conclusion: We characterized an ATP7B genetic variant (c.2623A/G) that
is causal to the disease in Indians and is a non-disease causing SNP in
Chinese population.
P69
Association of microRNA-146a and their target gene IRAK-1
polymorphism with enthesitis related arthritis category of juvenile
idiopathic arthritis
Sushma Singh
1*
, Geeta Rai
2
, Amita Aggarwal
1
1
Department of Clinical Immunology, Sanjay Gandhi Post Graduate Institute
of Medical Sciences, Lucknow, India;
2
Department of Molecular and Human
Genetics, Faculty of Science, Banaras Hindu University, Varanasi, India
E-mail: sushma2502@gmail.com
Background: MicroRNAs (miRNAs) are non-coding RNA molecules that
play pivotal role in modulating the expression of multiple target genes at
the post-transcriptional level. Single Nucleotide Polymorphisms (SNP) in
pre-miRNAs can alter miRNA expression, and polymorphism in target
molecules can affect binding to target mRNA. Studies have shown an
association between miR-146a polymorphisms and autoimmune disease.
Taking into account that interleukin-1 receptor-associated kinase-1 (IRAK-1)
is a target of miR-146a, we studied the association between SNPs of
miRNA-146 and its target IRAK-1 with susceptibility to Juvenile Idiopathic
Arthritis-Enthesitis Related Arthritis (JIA-ERA).
Methods: One hundred and fifty patients with JIA-ERA (ILAR criteria) were
included in the study. 216 blood donors (201 male) with a mean age of
30.5 years served as controls. miR-146a (C/G) (rs2910164) and its target
IRAK-1(C/T) (rs1059703) at Exon 12 region and IRAK-1 (A/C) (rs3027898) at
3UTR polymorphisms were analyzed using PCR-RFLP method.
Results: Among 150 patients, 134 were males and the mean age at onset of
disease was 11 (4-16) years, mean disease duration was 4.5 (0.3-12) years. 22
had uveitis and 21 had positive family history. 73 had enthesitis and 75 had
inflammatory back pain and all had arthritis. 116 were HLA B27 positive.
Genotype frequency of miR-146a gene was in Hardy Weinberg equilibrium
in healthy controls whereas in IRAK-1 genotype the frequency was contrary
owing to its presence on chromosome X.
The genotype frequency for miR-146a were different in controls and
patients [GG (51.85% vs 50.0%), GC (42.13% vs 37.29%) and CC (6.02% vs
12.71%), OR = 2.18; 95% CI 1.02 4.68; p value = 0.0418]
IRAK-1 (rs1059703) allelic frequencies in controls and patients were similar
[CC (33.8% vs 32.9%), and TT (66.2% vs 67.0%)]. IRAK-1 (rs3027898) allelic
frequencies were also similar among control and patients [CC (70.1% vs
76.1%) and AA (29.8% vs 23.9%)].
Conclusion: The CC genotype of the miR-146a rs2910164 polymorphism
was significantly associated with the susceptibility to JIA-ERA.
P70
TMC1 may be a common gene for nonsyndromic hereditary hearing
loss in Indian population
Pawan Kumar Singh
*
, Shipra Sharma, Manju Ghosh, Shivaram S Shastri,
Neerja Gupta, Madhumita Roy Chowdhury, Madhulika Kabra
Division of Genetics, Department of Pediatrics, All India Institute of Medical
Sciences, New Delhi, India
Background: Hearing impairment is very heterogeneous and most common
sensory disorder. The prevalence of prelingual hearing loss is 1:500, with both
environmental and genetic factors being equally responsible. To date, more
than 128 loci and 74 genes responsible for nonsyndromic hereditary hearing
loss have been identified, of which GJB2 gene is the most common across
populations. Transmembrane channel like 1 or transmembrane cochlear-
expressed gene 1 (TMC1) at DFNB7/11 locus at 9q13q21 is another gene
that is responsible for prelingual, severe to profound hearing impairment. It
contains 24 exons and encodes 760 amino acids long 87.8 kDa multipass
transmembrane protein, which is required in maintaining electrochemical
homeostasis, structure and function of neurosensory hair cells in the inner
ear. More than 29 different mutations have been reported in 48 families.
Materials and methods: DNA was extracted from 47 multiplex families.
Sanger sequencing identified connexin 26 mutations in six families and in
the rest of the 41 families, homozygosity mapping was done using 35
fluorescent markers for eight common loci. DNA sequencing of TMC1
gene was done in all individuals of two families, in which linkage to
DFNB7/11 locus was seen.
Results and conclusion: Linkage to DFNB7/11 by four markers was found
in two families. DNA sequencing of TMC1 gene in this locus identified a
reported homozygous mutation, c.100C>T (p.R34X) in one family. In the
other family, a novel homozygous change, c.1283C>A (p.Ala428Asp) was
found in all the affected children. The mutation segregated with the hearing
loss in both the families. Online protein prediction software SIFT and
PolyPhen 2, predicted this novel change as damaging and probably
damaging respectively. As TMC1 gene mutations have also been reported
earlier in Indian and Pakistani families, it appears that TMC1 may be a
common gene after GJB2 in the Indian subcontinent. c.100C>T is a common
mutation in this gene. TMC1 gene should be considered in routine diagnosis
if GJB2 is negative.
P71
Significance of nucleophosmin1 (NPM1) gene mutation status on acute
myeloid leukaemia patients with normal karyotype in South India
R Sureshkumar
1*
, S Santhi
1
, V Sangeetha
1
, N Geetha
2
, S Hariharan
1
1
Division of Cancer Research, Regional Cancer Centre, Thiruvananthapuram,
Kerala, India;
2
Division of Medical Oncology, Regional Cancer Centre,
Thiruvananthapuram, Kerala, India
Background: Acute myeloid leukaemia (AML) is a heterogeneous group
of haematological malignancy. In spite of recurrent chromosomal
abnormalities present in a significant proportion of AML patients, more
than 50% of the patients have a normal karyotype (NK-AML) and lack
reliable molecular markers. NPM1 has been recently characterized as the
most frequently mutated gene in AML Patients. The objective of the
present study was to profile the karyotype and to assess the types of
NPM1 gene mutations in AML patients in south India.
Subjects and methods: A total of 200 de novo AML patients were
investigated in this study. Cytogenetic G- banding analysis, Fluorescence
in situ hybridization was performed with standard methods. Mutation
screening of NPM1 mutation hotspot exon12 performed in DNA by PCR-
SSCP and suspected samples were sequenced.
Results: Among 200 de novo AML patients 119 showed normal karyotype.
Fourty out of two hundred patients showed mutations in NPM1 exon12.
Most of the NPM1 mutation were detected in NK-AML (37/40) (92.5%).Three
of them showed abnormal karyotype. The highest incidence of mutation
was detected in AML-M1 with 47.5% followed by M5, M4, M2 and M3.
Predominant type of mutation was a type A mutation (c.860-863dupTCTG)
(75%). Other type of mutations includes tetranucleotide insertion of TGCA
(10%), CTGC (10%), GTCA (2.5%) and GTAG (2.5%). All the mutated cases
were heterozygous in nature retaining wild type allele and have a distinct
sequence in the protein c-terminal.
Conclusion: Presence of 31% mutation in the NK-AML in the present
study indicates that it is the most prevalent mutation in NK-AML patients
in south India. NPM1 mutations are associated with good prognosis in
AML. So predominance of NPM1 mutation in the AML especially in NK-
AML patients suggests that it could be used as a reliable molecular
marker for those patients.
P72
Role of TNF-A, IL-6 and IL-4 with the susceptibility to chronic
periodontitis in North Indian population: a multi-analytic approach
Garima Prakash
1*
, Sadhna Ajay
2
, KK Gupta
2
, Balraj Mittal
1
1
Dept. of Genetics, SGPGIMS, Lucknow, India;
2
Dept. of Periodontics,
SPPIDMS, Lucknow, India
E-mail: garimaprakash1987@gmail.com
Background: Periodontitis is a chronic inflammatory disease causing
destruction of tooth supporting tissue and loss of teeth. Inter-individual
variation in genetic factors and one or more variations in genes could play
critical role in susceptibility to Chronic Periodontitis and its pathogenesis.
Therefore, in present study, we aimed to perform combined risk genotype
analysis and Multifactor dimensionality reduction (MDR) to identify
combination of alleles in modifying the risk of chronic periodontitis.
Page 41 of 59
Materials and methods: A total of clinically diagnosed 200 periodontitis
cases and 200 age and gender matched controls were genotyped for TNF-a,
IL-6 and IL-4 gene polymorphisms using ARMS PCR and PCR-RFLP methods.
Multi-analytic approach (combine risk genotype analysis and MDR) were
used to find out the combination of allele contributing to risk of chronic
periodontitis. All statistical analyses were performed using SPSS ver.15.0 and
MDR analysis by MDR 2.0 Beta 8.4.
Results: Single locus analysis showed association of TNFa-308GA (rs1800629)
with risk of CP whereas IL-6-174GC (rs1800795) and IL-4-590CT (rs224325)
polymorphisms did not show any significant differences. However, combined
risk genotypes of all three polymorphism depict the increased risk of CP
(P
trend =
0.001) where MDR analysis revealed TNF-a-308GA and IL-6-174GC as
a best polymorphic model for the risk of CP. When subjects were stratified on
the basis of gender, only TNFa-308GA polymorphism showed association
with risk of CP in North Indian population.
Conclusion: TNF- a -308G>A polymorphism is associated with chronic
periodontitis. However, multi-analytical approach suggests TNF-a-308GA
and IL-6-174GC genetic variants interact to confer significant risk for
chronic periodontitis.
Acknowledgment: ICMR, New Delhi, India.
P73
Bio-chemical and molecular analysis in cardiomyopathy patients
Rutvik J Raval
1*
, Tapan A Patel
1
, PL Sachapara
2
, Mandava V Rao
1
1
Gujarat Genetic Diagnostic Center (GenDiCe), Department of Zoology,
University School of Sciences, Gujarat University, Ahmedabad, Gujarat, India;
2
Samarpan Hospital, Madhavjyot, Kalubha road, Bhavanagar, Gujarat, India
Introduction: Cardiomyopathy refers to diseases of the heart muscle.
Symptoms of cardiomyopathy includes: Shortness of breath, Chest pain,
Dizziness, Lightheadedness or fainting, Palpitations. Cardiomyopathies are
divided in two types that are Primary (intrinsic) and Secondary (extrinsic)
cardiomyopathies. Primary cardiomyopathies are divided in three types i.
e. genetic, mixed and acquired.
Present study was planned to evaluate the biochemical alterations in
patients with cardiomyopathy or myocardial Infarction using bio-chemical
and molecular indices.
Methods: Blood samples were collected from patients (n=12) with
cardiomyopathy/myocardial infarction and same age and sex matched
healthy controls (n=12). Serum myoglobin and serum creatine kinase-MB
(CK-MB) levels were evaluated. Genomic DNA was isolated from peripheral
blood and its quality and quantity were checked. PCR amplification of the
various exons of genes MYH7 (exon 8 and 9) and TNNT2 (exon 8) was
carried out. PCR products were checked by 2% agarose gel electrophoresis.
Further, mutations were screened by Polymerase Chain Reaction-Single
Strand Conformation Polymorphism (PCR-SSCP) technique.
Results: In cardiomyopathy patients the ratio of Male: Female was 1:1.
Out of twelve patients four patients showed high levels of CK-MB and
two patients with high Myoglobin levels and only one patient reported
high levels of both myoglobin & CK-MB. None of the patient showed
mutation in exon8 and 9 of MYH7 gene & exon 8 of TNNT2 gene.
Conclusion: In Gujarat population, cardiomyopathy patients exhibited
alterations in biochemical parameters, but did not reveal any mutation in
exon 8 and 9 of MYH7 gene &exon 8 of TNNT2.
P74
Cytogenetic and Yq microdeletion screening studies in infertile males
J Suganya
1*
, Kamala Selvaraj
2
, SS Muthaiah
3
, RS Chandra
1
1
University of Madras, Taramani, Chennai, TamilNadu, India;
2
G.G. Hospital, 6-e,
Thirumoorthy Nagar, Nungambakkam High Road, Nungambakkam, Chennai-,
TamilNadu, India;
3
Kanmani Fertility Centre, 43, South Usman Road, T Nagar,
Chennai, TamilNadu, India
E-mail: mydecipheringcode@gmail.com
Infertility is defined as the inability of a sexually active couple to achieve
pregnancy despite unprotected intercourse for a period of greater than
12 months. Infertility affects 15% of the infertile couples and in about
half of them, male factor is responsible. It is due to low number,
abnormal or immobile sperm production, or blockages that prevent the
delivery of sperm. Illnesses, injuries, chronic health problems, lifestyle
choices and other factors can also play a role. A total of 130 infertile
males (43 azoospermic, 31 asthenospermic, 7 cryptozoospermic, 20
oligoasthenozoospermic, 29 normozoospermic) were analyzed for the
presence of chromosomal abnormalities and Y chromosome classical
microdeletions in the azoospermia factor (AZF) regions. Multiplex PCR
amplification of genomic DNA was performed using two STS markers for
each of the three non-overlapping regions, AZFa (sY84, sY86), AZFb
(sY127, sY134) and AZFc (sY254, sY255) following standard guidelines.
The markers ZFX/ZFY and sY14 (SRY) were included as internal controls.
Two individuals showed Klinefelter syndrome (47,XXY) and three cases
exhibited reciprocal translocations involving autosomes - t(3;17)(q26.2;
p12), t(1;11)(p22.3;p13) and t(1;10)(q25;q24). Polymorphisms involving the
heterochromatic regions of Y and the acrocentric chromosomes were
seen in seven patients. The rest of the individuals exhibited a normal
chromosomal pattern. The occurrence of chromosomal abnormalities was
confined to only the azoospermia category which could reflect the small
sample size. A deletion involving all the three AZF regions was detected
in one individual while another showed a deletion in AZFb (partial) and
AZFc regions. These results indicate a distinctly lower frequency of Y
chromosomal microdeletions in the selected group of men with
idiopathic infertility. The study needs to be extended using more primer
sets defining these loci which play a major role in spermatogenesis.
P75
Molecular studies on ARX gene in syndromic and non-syndromic
mental retardation
Gayatri Kulkarni
*
, Suvidya Ranade
Department of Chemistry, University of Pune, Pune, Maharashtra, India
Background: Mental retardation (MR) is frequently the result of genetic
mutations. Syndromic mental retardation is intellectual deficits associated
with other medical and behavioral signs and symptoms. Non-syndromic
mental retardation refers to intellectual deficits that appear without other
abnormalities. The newly identified ARX (Aristaless related homeobox) gene
consists of five exons and encodes 562 amino acid proteins and is thought
to regulate brain development. Mutations in the ARX gene are associated
with a diverse spectrum of phenotypes ranging from severe developmental
abnormalities of the brain to syndromic and nonsyndromic forms of
X-linked mental retardation (XLMR) syndromes that can be associated with
normal or abnormal brain morphology. Mutations in the human ARX gene
are the major cause of developmental and neurological disorders.
Material and methods: The present study was focused on screening of
exon 2 of ARX gene in Indian families with mental retardation in order to
obtain the relative prevalence of ARX mutations. Method adapted in present
study was amplification of target exon by using polymerase chain reaction,
qualitative conformation of amplicons by agarose gel electrophoresis and
their use for conformation sensitive gel electrophoresis to find heteroduplex
formation which is followed by sequencing.
Conclusion: In present study we have found insertion mutation at
genomic position 25031668, this change leads to loss of homeobox, OAR
domains of ARX protein. The other mutation obtained was substitution of
C to A/G is observed and does not affect protein functioning.
P76
Squared nasal root, nasal voice -indicators of 22 q11.2 deletion in
patients with psychiatric illness
Jyothilakshmi Annavarapu
*
, Prabhavathi Halappa, Niby J Elackatt,
Mitesh Shetty, Sridevi Hegde
Dept. of Medical Genetics, Manipal Hospital, Bangalore, India
E-mail: genetics@manipalhospitals.com
Background: 22q11.2 Deletion Syndrome DGS (22q11) is a micro deletion
syndrome caused by the deletion on chromosome 22. It is a multi system
disorder which affects Cardiovascular system, immune system, facial
Page 42 of 59
features covered by acronym CATCH22 (Cardiac defects aortic arch
anomalies, conotruncal anomalies, ventricular septal defect, patent ductus
arteriosis and tetra logy of fallot; Abnormal facies ; Thymic hypoplasia;
Hypocalcemia). Few children will not present with all of the above clinical
features but only delayed motor mile stones, learning disability and mild
behavioral issues which may progress onto psychiatric illness in adulthood.
In this study, we aim to study the prevalence of DGS in patients with
psychiatric illness.
Materials and methods: To further explore physical, behavioral and
psychiatric findings associated with 22q11 deletion in adults with
psychiatric illness, we assessed 12 patients. All were confirmed psychiatric
cases referred from a well-known Institute for mental health studies and
also had mild facial dysmorphism. All patients were screened using the
clinical checklist for DGS and Fluorescence In Situ Hybridization(FISH)
studies was conducted using the probe specific for TUPLE1 gene located
on chromosome 22 q11.2 in cultured blood samples.
Results: Out of 12 cases, six cases (50 %) tested positive for 22q11 deletion
indicating a strong association between 22q11.2 deletion syndrome and
psychiatric illness in adult population. All six patients presented with
squared nasal root, and nasal voice in addition to psychosis and one also
had cardiac abnormality (VSD). Recent studies report that Digeorge critical
region (DGCR) spanning up to 2 Mb on chromosome 22q11 region
contains several genes like TBX1, GNB1L, PRODH and ZDHHC8 which are
strong candidate genes for schizophrenia susceptibility. In conclusion, all
patients with psychiatric illness, squared nasal root and nasal voice should
be investigated for DGS.
NANOTECHNOLOGY
P77
TiO2 nanoparticles induce cytotoxicity and genotoxicity in human
alveolar cells
Krupa Kansara
*
, Pal Patel, Darshini Shah, NV Srikanth Vallabani, Ritesh K Shukla,
Sanjay Singh, Ashutosh Kumar, Alok Dhawan
Institute of Life Sciences, School of Science and Technology, Ahmedabad
University, Ahmedabad Gujarat, India
E-mail: krupa.kansara@ils.ahduni.edu.in
Background: Engineered nanoparticles (ENPs) such as TiO
2
are widely
used in products such as cosmetics, clothing, food packaging, drug
delivery systems, etc. due to their unique physicochemical properties. This
has increased the liklihood of ENP exposure in humans. As the ENPs are
having small size and high diffusion coefficient, they can migrate rapidly in
the air. Therefore, inhalation is considered to be the primary route of
exposure to such ENPs. Hence, in the present study an attempt was made
to assess the potential toxicological effects of TiO
2
NPs in human alveolar
cell line (A549).
Materials and methods: The average hydrodynamic size, size distribution,
zeta potential and stability of TiO
2
NPs in DMEM-F12 media were
determined by dynamic light scattering (DLS). Internalisaiton of ENPs in
cells was detected using flow cytometry. Cytotoxicity was assessed using
the MTT and neutral red uptake (NRU) assays. The genotoxic potential of
TiO
2
NPs was assessed by cytokinesis block micronucleus (CBMN) assay
and flow cytometry based assays.
Results: The mean hydrodynamic diameter of TiO
2
NPs in DMEM-F12
media, as measured by DLS was 23.27 2.1nm and the zeta potential was
-10.1 1 mV. The particles were also found to be stable in the media for
upto 72 hr. A significant (p<0.05) concentration dependent uptake of TiO
2
NPs was obseverd as evident by an increase in the side scatter (SSC)
intensity in flow cytomtery after 6 hr of exposure. A reduction in cell viability
was observed as evident by the results of MTT and NRU both as a function
of NP concentration as well as time of exposure. Moreover, significant
(p<0.05) induction in the micronucleus formation was observed by
conventional and flow cytometry based methods at non cytotoxic
concentrations.
Conclusion: Our data demonstrate that TiO2 ENPs are internalised in the
human alveolar cells and induce cyto- and genotoxicity. This warrant
minimizing the unwanted exposure to the nanotechnology based
products and suggests ensuring its safe use both by consumers and
industry.
Acknowledgements: The financial assistance for the Centre for
Nanotechnology Research and Applications (CENTRA) by The Gujarat
Institute for Chemical Technology (Grant no. ILS/GICT/2013/003) is
acknowledged.
P78
PEGylated nanoceria protect human epidermal cells from reactive
oxygen species
Ragini Singh
*
, Ritesh K. Shukla, Ashutosh Kumar, Alok Dhawan, Sanjay Singh
Institute of Life Sciences, Ahmedabad University, Ahmedabad, Gujarat, India
E-mail: sanjay.singh@ahduni.edu.in
Background: Cerium oxide nanoparticles (CeNPs) have shown promise as
catalytic antioxidants in cell culture and animal models due to its
superoxide dismutase and catalase mimetic activities. CeNPs can exist in
+3 and +4 oxidation states, which have been suggested as the
mechanism behind the free radical scavenging activity. It has also been
shown that unique crystal structure of CeNPs with surface oxygen defects
promote the shuffling between the +3 and +4 oxidation states that help
in eliminating the free radicals. This activity of CeNPs has been suggested
as the mechanism, at least in part, behind increase in cellular longevity
and decrease in the toxic insults in mammalian cells/tissues. Further, the
uniform distribution of CeNPs, upon cellular uptake, within the cells could
prevent the accumulation of reactive oxygen species in the cells.
Material and method: PEG coated CeNPs were synthesized by methods
described by Karakoti et al. CeNPs were characterized by UV-visible spectra
and dynamic light scattering. Cytotoxicity of CeNPs was done by MTT and
Neutral Red Uptake (NRU) assay in a time and dose dependent manner.
The level of intracellular ROS generation was estimated by using 2,7-
dichlorofluorescein diacetate (DCFDA) dye by time and dose dependent
approach. Superoxide dismutase (SOD) mimetic activity of ceria
nanoparticles was determined by a ferricytochrome C based assay.
Results: The mean hydrodynamic diameter of PEG CeNPs in MillQ and
culture medium were 250.6 nm and 153.3 nm respectively whereas Zeta
potential were found to be -7.86 mV and -10.4 mV respectively. CeNPs did
not exhibit cytotoixicity at the tested doses and time period in human
epidermal cells (A431) as evident by MTT- and NR uptake- assays. The co-
incubation of CeNPs with A431 cells showed significant (p<0.05) decrease
in ROS generation as evident by a decrease in the DCFDA fluorescence.
Conclusion: The present study demonstrates that CeNPs are stable in cell
culture media conditions, non cytotoxic to A431 cells up to a relatively
high concentration. CeNPs show free radical scavenging activity when
co-incubated with A431 cells.
acknowledged.
P79
TiO2 nanoparticles induced micronucleus formation in human liver
(HepG2) cells: comparison of conventional and flow cytometry based
methods
NV Srikanth Vallabani
*
, Ritesh K Shukla, Dinesh Konka, Ashutosh Kumar,
Sanjay Singh, Alok Dhawan
University, Ahmedabad Gujarat, India
Background: TiO
2
nanoparticles (TiO
2
NPs) are extensively used metal
oxide NPs in cosmetics, as an additive in pharmaceuticals, food colorant etc.
Being widely used NPs, their toxicity assessment studies help in
understanding the adverse effects to the humans. It is likely that NPs
exposure to the humans can be through different routes but will finally
reach the liver. Therefore, an attempt was made to explore the genotoxicity
of TiO
2
NPs on human liver cells (HepG2).
Materials and methods: TiO
2
NPs were characterized by transmission
electron microscopy (TEM) for their primary size, shape and dynamic light
scattering for their size, size distribution and zeta potential in culture
medium. Cellular uptake of TiO
2
NPs in HepG2 cells was detected using
Page 43 of 59
flow cytometry method. Moreover, ultrathin sections of cells were
analysed using TEM to visualise the internalisation of TiO
2
NPs. The
genotoxic potential of TiO
2
NPs was assessed by micronucleus assay
using the conventional and flow cytometry methods.
Results: TEM analysis of TiO
2
NPs revealed a mean diameter size of 30-70
nm and DLS measurements showed a mean hydrodynamic diameter and
zeta potential of 192.5 10 nm and -11.4 1.2 mV, respectively. The
electron microscopy and flow cytometry studies for particle internalisation
showed a significant cellular uptake of TiO
2
NPs in the human liver cells
(HepG2). A significant (p < 0.05) induction in micronucleus formation was
observed at 20 g/ml of TiO
2
NPs exposure when compared to control cells.
However further treatment to HepG2 with higher concentrations (40 and
80 g/ml) showed a decrease in micronucleus formation than 20 g/ml.
In contrary, the flow cytometric results exhibited a significant concentration
dependent induction of micronucleus in HepG2 cells exposed to all
concentrations (20, 40 and 80 g/ml) of TiO
2
NPs than control.
Conclusion: The difference in the micronucleus frequency obtained from
conventional and flow cytometry methods in HepG2 may be due to the
accumulation of TiO
2
NPs on prepared slides, which hinders the counting of
micronucleus (in conventional method). However, in the flow cytometry
analysis, these nanoparticles do not interfere with the optics. Hence, it is
proposed that in the case of NPs treatment, flow cytometry based
micronucleus assay should be used instead of the conventional method.
acknowledged.
P80
Host-guest mediated sensing of biologically relevant small molecules
using supramolecular nanoassembly
Alok Pandya
1*
, Pinkesh G Sutaria
2
, Anand Lodha
2
, Heena Goswami
2
,
Shobhana K Menon
2
1
Institute of life Sciences, School of Science and Technology, Ahmedabad
University, Ahmedabad, -380 009, Gujarat, India;
2
Department of Chemistry,
School of Sciences, Gujarat University, Ahmedabad, 380 009, Gujarat, India
E-mail: alokpandya20@gmail.com
Background: A high concern for human health and safety has motivated
dynamic research on the potential impact of transition metal ions or
molecule and their toxic effects. Thus, selective detection of biologically
relevant molecule have enormously gained its attention due to involvement
in a variety of fundamental environmental and biological process in
organism because its deficiency and excess can induce a variety of diseases.
Therefore, biomolecule detection have received a great deal of study. Here,
we have designed an efficient strategy using supramolecular nanoassembly
to detect biologically relevant small molecules with high specificity and
selectivity and applicable to the biological milieus.
Method: In our method, we designed a microwave assisted new
promising approach using Silver (Ag) and Gold (Au) nanoparticles (NPs)
based colorimetric sensing system (ANCSS) which form calix[4]arene-
functionalized Ag and Au nanoprobe complex (CX-AgNPs/AuNPs) for the
detection of biologically relevant small molecules such as ferric ion and
glucose in water.
Result: Driven by the need to detect trace amounts of Fe
3+
and Glucose
from blood samples, a molecular receptor based on calix[4]arene
functionalized AuNPs/AgNPs was designed which proficiently and selectively
recognizes glucose and Fe
3+
in nanomolar level from aqueous solution with
excellent discrimination against other heavy metals and biomolecule. The
assembly was characterized by, DLS, UV-Vis, FT-IR, ESI-MS and
1
H NMR
spectrometry which demonstrates the higher binding affinity for Fe
3+
and
Glucose via weak forces. It is easy to operate and, most importantly, it
exhibits fast response time (<80 s) and has long shelf-life (>4 weeks).
Conclusion: The calix[4]arene functionalized Ag or AuNPs are able to
selectively detect Fe
+3
and glucose from aqueous medium and even from
human blood. The key to the successful formation of this strategy is multi
binding site of functionalized calix[4]arene which sensitively and
selectively target to small molecules. The developed functionalized
calixarene-Ag or AuNPs based human heme and gluocse biosensor has
been proved to be a simple, reliable and accurate which can also indirectly
assist to medeco-legal system for the routine investigation process.
P81
Cytotoxicity assessment of ZnO nanoparticles on human
epidermal cells
Pal Patel
*
, Krupa Kansara, Darshini Shah, NV Srikanth Vallabani, Ritesh K Shukla,
Sanjay Singh, Alok Dhawan, Ashutosh Kumar
University, Ahmedabad, Gujarat, India
Background: Nanotechnology is growing rapidly worldwide and
engineered nanoparticles have found tremendous applications in
consumer and industrial products. Metal oxide nanoparticles (NPs),
especially zinc oxide (ZnO), are widely used in cosmetics, catalysis,
electronics, biosensors, medicine, paints, food packaging and imaging. As,
the ZnO NPs have major application in cosmetics, their exposure will
mainly be through the skin, which is the largest organ of the body and
could serve as an important portal route for entry. Therefore, the present
study was carried out to assess the cytotoxicity of ZnO NPs on human
epidermal cells (A431).
Materials and methods: The average hydrodynamic size, size distribution,
zeta potential and stability of ZnO NPs were determined by dynamic light
scattering (DLS). The detection for the internalization of ZnO NPs was carried
out using flow cytometry. Cytotoxicity response was assessed by neutral red
uptake (NRU) and MTT [3-(4, 5-dimethylthiazoyl-2-yl)-2, 5-diphenyltetrazolium
bromide] assays. Further, to analyze the ability of ZnO NPs to induce the
reactive oxygen species (ROS) generation and oxidative damage, 2, 7-
dichlorofluorescein diacetate (DCFDA) dye was used. The effect of ZnO NPs
in progression of cell cycle was also assessed using propidium iodide dye by
flow cytometer.
Results: The mean hydrodynamic diameter and zeta potential of ZnO NPs
in DMEM with 10% FBS was 30.95 3.72 nm and -12.8 0.6 mV
respectively. In flow cytometry, a concentration dependent significant
(p<0.05) increase in the side scatter (SSC) intensity of treated cells was
observed after 6 hr exposure. A significant (p<0.05) dose and time
dependent cytotoxicity as evident from MTT and NRU assays was also
observed. Additionally, a significant (p<0.05) induction in ROS generation
was also observed in a dose dependent manner.
Conclusion: Our study demonstrates that ZnO NPs induce cytotoxicity in
human epidermal cells. Hence, proper precautions need to be taken while
handling such NPs.
acknowledged.
P82
TiO2 NPs induced hepatic injury in mammals: a mechanistic approach
Ritesh K Shukla
*
, Ashutosh Kumar, NV Srikanth Vallabani, Sanjay Singh,
Alok Dhawan
Institute of Life Sciences, Ahmedabad University, Opposite University Bus
Stand, University Road, Navrangpura, Ahmedabad, Gujarat, India
Background: The rapid advancement in nanotechnology has increased the
production of metal oxide nanoparticles (NPs) especially TiO
2
for consumer
and industrial products. This has also increased the likelihood for their
exposure to human. TiO
2
NPs exposure to humans can occur through
different routes, but will finally reach to liver through the circulatory system.
Hence, the present study was planned to assess the effects of TiO
2
NPs in
mammalian liver and their possible mechanism.
Materials and methods: TiO
2
NPs were characterized by transmission
electron microscopy (TEM) and dynamic light scattering (DLS). Genotoxicity
assessment of TiO
2
NPs was carried out by fpg-modified Comet assay both
in in vitro (HepG2 cells) and in vivo (mice liver). Additionally, to understand
Page 44 of 59
the mechanism of hepatotoxicity, biochemical parameters, oxidative stress
markers, reactive oxygen species (ROS), and expression profile of different
stress proteins, tumour suppressor apoptotic/antiapoptotic proteins were
investigated.
Results: TEM measurements and DLS analysis showed that TiO
2
NPs were in
nano size regime, stable and mono-dispersed in different exposure vehicles,
making them suitable for in vitro and in vivo toxicity studies. Our data from
in vitro and in vivo study exhibited that TiO
2
NPs induced significant
(p<0.05) oxidative DNA damage assessed by the fpg-Comet assay. This
could be attributed to a concentration-dependent significant (p<0.05)
increase of ROS generation as evident from the enhanced fluorescence
intensity of DCFDA dye. A significant alteration in the level of different
hepatic enzymes in TiO
2
NPs treated mice was also observed.
Furthermore, immunoblot analysis revealed a significant increase in the
expression profile of Hsp60, Hsp70, p53, BAX, Cyto-c, Apaf-1, caspase-9 and
caspase-3 protein and a concomitant decrease in the level of antiapoptotic
protein Bcl-2. Our data demonstrate the role of mitochondrial intrinsic
pathway for TiO
2
NP induced apoptosis in liver cells.
Conclusion: The present study using fpg-modified Comet assay, blood
biochemical parameters, oxidative stress markers and immunoblot analysis
confirmed that oxidative stress induced by TiO
2
NPs trigger the DNA
damage, which consequently initiates the expression of apoptotic proteins
resulting in hepatic injury. Hence the use of such nanoparticles should be
carefully monitored.
acknowledged.
P83
BSA coated gold nanoparticles exhibit size dependent interaction with
lung cancer (A549) cells
Rahul Purohit
*
, NV Srikanth Vallabani, Ritesh K Shukla, Ashutosh Kumar,
Alok Dhawan, Sanjay Singh
University, Ahmedabad 380009, Gujarat, India
Background: Due to high surface to volume ratio and other unique
properties, nanomaterials interact within living system (cells, tissues,
organisms) significantly different. Therefore, developing a rational basis for
investigation and understanding about how these nanomaterials interact
with living system is still a fundamental challenge. Further, it has been well
documented that nanomaterials get coated by the proteins present in their
surrounding thus constructing a protein corona. This event also plays a
major role in determining the interaction of nanomaterials with cells/tissues.
Therefore, to probe this we have chosen BSA coated gold nanoparticles
(AuNPs) as a model system to study the interaction with a mammalian lung
cell culture model system.
Materials and methods: AuNPs of different sizes (15, 30, 50 and 70 nm)
were synthesized using sodium citrate as a reducing agent. Size distribution,
zeta potential and stability of coated (AuNPSBSA) as well as uncoated AuNPs
were determined by dynamic light scattering (DLS) and UV-vis spectroscopy.
Cytotoxicity response was assessed by neutral red uptake (NRU) and MTT
[3-(4, 5-dimethylthiazoyl-2-yl)-2, 5-diphenyltetrazolium bromide] assays.
Results: Zeta potential values for AuNPs of different sizes were found
negative, which was decreased significantly after BSA coating. Further, the
stability of AuNPSBSA in saline solution (1M and 500 mM NaCl) and cell
culture media (DMEM-F12), which showed that BSA coated AuNPs were
stable in both saline solution and serum containing culture media, as
compared to uncoated AuNPs. Cytotoxicity studies revealed that BSA coated
and bare AuNPs were non-toxic up to 50 M, however, at higher
concentration (above 50 M) bare AuNPs were found to be more toxic as
compared to AuNPSBSA. Further, the cytotoxicity of AuNPs on A549 cells
was also found to be size dependent.
Conclusion: This study demonstrates the size and BSA coating on AuNPs
significantly control the cytotoxicity on lung cancer cells. BSA coating
imparts the biocompatibility thus non toxic nature of these particles even at
higher concentration. However, the relation between cytotoxicity and
internalization of AuNPs (bare and BSA coated) in A549 cells remains to be
seen.
acknowledged.
P84
A data mining approach for identifying novel target specific small
molecules
Varun Khanna
*
, Alok Dhawan
Institute of Life Sciences, Ahmedabad University, Opp. University Bus Stand,
Navrangpura, Ahmedabad - 380009, Gujarat, India
E-mail: varun.khanna@ahduni.edu.in
Background: There has been a paradigm shift in drug discovery from
being a single-target approach to a multi-target comparative analysis. This
has shifted the emphasis from designing lead candidates with desirable
pharmacokinetic properties against individual targets to synthesizing small
molecules active at family and subfamily levels. Therefore, in the present
study, protein targets and small molecule ligands available in PubChem
BioAssay and DrugBank databases were comparatively analyzed to identify
novel target specific small molecules.
Materials and methods: The data obtained from public databases was
mined using shell scripts and R-statistical computing software (2.15.3)
installed under Linux environment.
Results: Our data from small molecule analysis showed that 210 FDA
approved drugs bind to a single protein target whereas 157 bind to two
targets, 82 bind to three targets and rest bind to four or more targets. This
shows that out of 1541 FDA approved drugs only 14% are specific binders
whereas majority of the drugs are promiscuous binders. Similarly in
PubChem BioAssay dataset 20% of the compounds bind to a single target
whereas 11% compounds bind to two targets while the rest of the
compounds bind to three or more targets. Further, our results from the
comparison of target datasets showed that there are 2097 and 1271
unique domains in DrugBank and PubChem BioAssay target datasets
respectively. There are 1190 domains common to protein targets in
DrugBank and PubChem BioAssay datasets. Further, we note that of the
top 10 domains 8 domains are shared between both the datasets.
Conclusions: The above observations have implications in drug design
approaches where the goal is to find target specific small molecules. Our
analysis shows that promiscuity plays a major role in drug discovery
therefore, should not be overlooked while designing novel drugs. From
domain analysis of target proteins we conclude that protein target space is
fairly narrow and the majority of the drug design efforts are concentrated
only on few target classes containing limited domains.
Nanotechnology Research and Applications (CENTRA) by The Gujarat Institute
for Chemical Technology (Grant no. ILS/GICT/2013/003) is acknowledged.
NEXT GENERATION SEQUENCING
P85
Next generation sequence analysis of the transcriptional response to
neonatal hyperoxia
Soumyaroop Bhattacharya
*
, Zhongyang Zhou, Min Yee, Ashley Lopez,
Valarie Lunger, Bradley Buczynski, Gloria Pryhuber, Thomas Mariani,
Michael OReilly
Division of Neonatology, Department of Pediatrics, University of Rochester
Medical Center, Rochester NY, USA
E-mail: soumyaroop_bhattacharya@urmc.rochester.edu
Background: Bronchopulmonary Dysplasia (BPD) is a major complication
of preterm birth associated with significant morbidity. BPD is a debilitating
condition characterized by inflammation, enlarged airspaces, vascular
dysmorphia and aberrant extracellular matrix accumulation that is typically
described as arrested lung development. Rodent models involving
Page 45 of 59
neonatal exposure to excessive oxygen concentrations (hyperoxia) have
been used to study the mechanisms contributing to BPD pathology.
Transcriptomic assessment of the effects of hyperoxia in neonatal mouse
lungs using RNASeq will help to identify genes and pathways associated
with BPD.
Materials and methods: Whole lung tissue from newborn C57BL/6 mice
exposed to 100% oxygen for 10 days (n=8) and room air-exposed age
matched controls (n=6) were compared. Total RNA was isolated from
individual whole lung tissues (n=14) and pooled in duplicates to perform
transcriptome Sequencing (RNA-seq). Alignments were generated using
multiple algorithms (CASAVA; TopHat; and SHRiMP). Raw counts obtained
from each alignment algorithm (using HT-Seq) were further and filtered to
remove undetected genes. Differentially expressed genes were detected
using Significance Analysis of Microarrays (SAM) and CuffDiff2, on each
version of mapped and normalized data. Ingenuity Pathway Analysis (IPA)
was used for pathway and network analyses. Expression patterns for
selected genes were examined by quantitative polymerase chain reaction
(qPCR).
Results: 248 genes were identified as differentially expressed between
hyperoxia and control samples by both SAM (median FDR = 0) and CuffDiff2
(p<0.05) and had a fold-change 2. We successfully validated 17 of 24 genes
by qPCR. Canonical pathways significantly dysregulated in hyperoxia lungs
included Nrf2-mediated oxidative stress signaling, p53 signaling, hepatic
fibrosis and sildenafil pathways. Interestingly most genes significantly
affected following hyperoxia exposure (~70%) showed a pattern of
expression consistent with an arrest in lung development. A subset of
the genes dysregulated in hyperoxic neonatal mouse lungs, were also
differentially expressed in human BPD lung tissue.
Conclusions: We have identified genes dysregulated in mouse of BPD-like
pathology. Further analysis of these data will enhance our current
knowledge of BPD, and may be useful for developing novel therapeutic
strategies.
P86
Comparative analysis of human mitochondrial methylome show distinct
patterns of epigenetic regulation in mitochondria
Sourav Ghosh
1*
, Shantanu Sengupta
1
, Vinod Scaria
2
1
Genomics and Molecular Medicine, CSIR Institute of Genomics and
Integrative Biology, Mall Road, Delhi, India;
2
GN Ramachandran Knowledge
Center for Genome Informatics, CSIR Institute of Genomics and Integrative
Biology, Mall Road, Delhi, India
Background: Understanding the epigenetic regulation of genes and their
functional implications in physiological and pathophysiological states has
been one of the emerging areas of genomics. DNA methylation and histone
modifications across the nuclear genome have been recently extensively
analyzed in this perspective. However, the mitochondrial epigenome,
though has been discussed widely has not been analyzed at genome-scale
resolutions. The recent availability of genome-scale epigenome datasets
allowed us to analyse profiles of methyl cytosine across mitochondrial
genomes.
Method: We analyzed methyl-cytosine profiles as evident from Methylated
DNA immunoprecipitation across 39 tissues and cell lines that were available
as part of the NIH RoadMap Epigenomics project. The mitochondrial
reference genome used in the current study was derived from the UCSC
human genome build hg19. We used custom scripts to retrieve reads
mapping to the mitochondrial genome.
Results: Our analysis suggests that the general profile of methylated
cytosines across the samples show a distinct pattern. This pattern was
generally conserved across the datasets considered, with exceptions of a
few regions which showed variability in methylation amongst datasets
analyzed. We show that certain regions of the mitochondria could be
differentially methylated in datasets which show distinct temporal and
functional characteristics, like Brain and Blood. One such region harbors the
loci associated with mitochondrial encoded NADH dehydrogenase
(MT-ND6), variations in which are associated with neurological disorders. To
date this is the first and comprehensive analyses of genome-scale
methylation data for Human mitochondria.
Conclusion: This study describes the first comprehensive map of methyl-
cytosines across the Human mitochondrial genome.
P87
Defining the effects of prematurity on the lymphocyte transcriptome
Soumyaroop Bhattacharya
1*
, Ravi Misra
1
, Heidi Hyuck
1
,
Christopher Slaunwhite
1
, Shannon Castiglione
2
, Deanna Maffett
1
,
Anne Marie Reynolds
2
, Gloria Pryhuber
1
, Thomas Mariani
1
1
Division of Neonatology, Department of Pediatrics, University of Rochester
Medical Center, Rochester NY, USA;
2
Division of Neonatology, Department of
Pediatrics, Womens and Childrens Hospital, University at Buffalo, Buffalo NY, USA
E-mail: soumyaroop_bhattacharya@urmc.rochester.edu
Background: We hypothesize that intrinsic and extrinsic factors associated
with oxidative stress drive lymphocyte dysfunction contributing to lung
disease in premature infants. Comprehensive transcriptomic assessment of
CD8
+
lymphocytes may identify biomarkers, and define disease-related
mechanisms of chronic lung disease in premature infants.
Methods: Peripheral blood samples were collected from premature infants
at the time of hospital discharge at multiple sites and shipped to a central
laboratory. Freshly purified PBMCs were isolated by Ficoll gradient
centrifugation, sorted into individual lymphocyte cell types, and processed
for total RNA. RNA isolated from CD8
+
lymphocytes (n=79) was used for
RNA-Seq analysis using the Illumina HiSeq2500. Sequences were aligned
using the SHRiMP algorithm and expression values were summarized using
HTSeq. Normalized gene expression data were analyzed for significant
changes in expression using various statistical approaches. Ingenuity
Pathway Analysis software was used for gene set interpretation.
Results: Sufficient blood was collected to complete sorting from 137 of the
183 subjects recruited and discharged. The total lymphocyte frequency from
the PBMC fraction was highly variable, with an average of 29.69.3%.
Lymphocyte sub-type frequencies were also highly variable across subjects,
with CD8
+
cell averaging 52%. Total RNA yield from sorted cells varied
according to cell type with CD8
+
RNA averaging 177140ng. RNA-Seq
analysis using 1ng total RNA was completed for CD8
+
cells from 79 subjects,
generating an average of 115 million reads/sample with detection of 67
4% of the transcriptome. One-way ANOVA identified 147 genes that were
differentially expressed among the 79 samples. A total of 47 genes were
identified as differentially expressed between subjects born before 29-weeks
and those born after 29-weeks of gestation. Pathways analysis of these
genes identified mechanisms related to B and T cell signaling.
Conclusion: These data demonstrate both the fidelity of our methodology
and purity of the samples. Although we assessed a homogeneous cell type,
our data includes substantial gene expression variability across subjects, and
with respect to gestational age at birth. Our results support the feasibility of
using these data and methods to identify biomarkers of, and mechanisms
for, chronic respiratory morbidity following premature birth.
OTHERS
P88
Oxidantantioxidant imbalance in the serum of Myotonic Dystrophy
type 1 (DM1) patients correlates with the progression of disease
Ashok Kumar
1*
, Sarita Agarwal
1
, Sunil Pradhan
2
, Shubha Phadke
1
1
Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of
Medical Sciences, Lucknow, 226014, India;
2
Department of Neurology, Sanjay
Gandhi Post Graduate Institute of Medical Sciences, Lucknow, 226014, India
E-mail: chemistry.ashok83@gmail.com
Background: Myotonic dystrophy type 1 (DM1) is the most common form
of muscular dystrophy affecting adults and is due to trinucleotide sequence
(CTG) in the 3 UTR region of DMPK gene located at 19q13.3 chromosome.
The increased levels of ROS/free radicals and lipid peroxides and decreased
antioxidant levels play an important role in the pathogenesis of DM1.
Aim: The intention of the present study is to assess the lipid peroxidation
and antioxidant status and its association with clinical phenotypes in DM1
patients of Indian origin.
Material and methods: Clinically diagnosed 20 DM1 patients (16 men
and 4 women; median age 32.8 years9.3, range 1752) and 40 age and
sex matched controls (32 men and 8 women; median age 31.0 years8.6,
range 1654) were included in the study. The collected blood samples
were processed for serum separation used in measurement of MDA
Page 46 of 59
(Cat. No. 10009055-96), SOD (Cat. No. 706002-96), GPX (Cat. No. 703102-
96), GST (Cat. No. 703302-96), GSH (Tietze et al. 1969 method) and total
antioxidant status or TAS (Koracevic et al. 2001 method) levels and its
association with clinical phenotype were evaluated.
Results: Analysis revealed significantly higher levels of MDA (p=0.002), SOD
(p=0.006) and TAS p= 0.004) and lower level of GPX (p=0.003), GST (P<0.001)
and GSH (P=0.016) in DM1 patients. A significant negative correlation of
MDA level with dyspepsia and CK-MB and GST level with serum SCK, CK-MB
and diabetes were observed. However, a significant positive correlation of
SOD level with serum CK-MB, CK-MM and diabetes and negative correlation
with facial weakness were noted. Though, GSH level had significant positive
correlation with learning and writing disability, speech and languages
disability yet found negative correlation with duration of the disease. The
GPX and TAS showed no correlation with any clinical findings.
Conclusion: Our data supports the pathogenic role of oxidative stress in
DM1 of Indian origin and supports the opportunity to undertake clinical
trials with antioxidants in this disorder.
P89
Mental retardation in younger children
Grishma Shukla
1*
, Swati B Thakur
1
, GT Swamy
2
, MV Rao
1
1
Gujarat Genetic Diagnostic Center (GenDiCe), Department of Zoology,
University School of Sciences, Gujarat University, Ahmedabad, Gujarat, India;
2
B M Institute of mental health, Ahmedabad-09, Gujarat, India
Background: It is a generalized disorder appearing before adulthood,
characterized by significantly impaired cognitive functioning and deficits in
two or more adaptive behaviours. The present study was aimed to
investigate the delayed development and psychological aspects in
symdromic and non syndromic mental retarded individuals.
Material and methods: This study was conducted at B.M. Institute,
Ahmedabad. The aetiology was ascertained after proforma studying and
number of patients observed. The spectrum of causative conditions and
contribution of genetic disorders was established. A total of 40 patients
were observed in which 30 were male & 10 were female between the ages
of 3 -11 years. The various aetiological categories were: chromosomal
disorders in 17, non chromosomal syndromes in 23. In which chromosomal
disorders include Down syndrome, non-chromosomal disorders include
delayed development and cerebral palsy.
Results: Among non syndromic mental retardation (NSMR) children; the
walking is delayed by 1 yr in 22% of children, 2 yrs in 57 % of children, 3 yrs
in 4% and 13 % children were not able to walk. In syndromic mental
retardation (SMR) walking is delayed by 1 yr in 41 % of children, 2 yrs in 23 %
of children, 3 yrs in 24 %, 6 % were not able to walk. Other developmental
milestone such as sitting and speech in both syndromic and non syndromic
mental retardation also showed the same pattern of delayed development as
seen in the walking. In IQ, level the highest is SMR which is 44% severe & 45%
moderate while in NSMR 31% severe & 38% moderate level were observed.
Conclusions: The present investigation concluded that there was a
delayed milestone in mental retarded children which was supported by
IQ levels in this study.
P91
Role of miRNA binding site SNPs in candidate genes in a North Indian
schizophrenia cohort
Jibin John
1*
, Smita N Deshpande
2
, Vishwajit L Nimgaonkar
3
, BK Thelma
1
1
Department of Genetics, University of Delhi South campus, Benito Juarez
Road, New Delhi, India;
2
Department of Psychiatry, Dr. RML Hospital, New
Delhi, India;
3
Department of Psychiatry, Western Psychiatric Institute and
Clinic, University of Pittsburgh School of Medicine, PA 15213, USA
Schizophrenia (SZ) is a debilitating neuropsychiatric disorder with ~80%
heritability. Despite several genetic studies including linkage and candidate
gene association and more recently GWAS, which have identified several risk
variants, the total heritability of SZ remains elusive. In addition, a number of
gene expression studies have reported dysregulation of candidate genes
both in brain and blood of SZ cases compared to controls. Although, the role
of coding, promoter, intergenic and UTR SNPs, have been demonstrated,
very little is known about the role of miRNA binding site SNPs. In this study,
we undertook to investigate the association, if any, of this important class of
regulatory variants with SZ. Using in silico prediction tools, 27 functionally
relevant SNPs from around 150 candidate genes were prioritized and
genotyped in a north Indian SZ cohort (n=507 cases; n=522 controls).
Test of association of these SNPs showed only one variant rs7430 in PPP3CC
to be associated (p=0.01) with SZ. Analysis of genotype data in a subset of
patients (TD positive n=89; TD negative n=160) with Tardive dyskinesia (TD),
an iatrogenic disorder of SZ, showed association of rs4846049 in MTHFR
(p=0.04) & rs17881908 in GCLM (p= 0.05 ) with this condition. Further
regression analysis of the genotype data with neurocognitive measures in a
subset (cases n=152; controls n=290) of the study cohort, showed significant
association of nine SNPs (p< 0.05) with different domains of cognition.
Based on this moderately powered study, the contribution of miRNA
binding site SNPs in candidate genes to SZ and to TD seems negligible.
However, their promising contribution to cognitive parameters warrants
additional investigations.
P92
Understanding insulin resistance pathophysiology in PCOS: a genetic
approach
Srabani Mukherjee
1*
, Nuzhat Shaikh
1
, Roshan Dadachanji
1
, Nalini Shah
2
,
Anushree Patil
1
1
National Institute for Research in Reproductive Health, J.M. Street, Parel,
Mumbai- 400012, India;
2
Department of Endocrinology, Seth GS Medical
College, Mumbai- 400012, India
E-mail: srabanimuk@yahoo.com
Polycystic ovary syndrome (PCOS) is a common endocrine disorder which
affects women in their childbearing years, characterized by chronic
anovulation, hyperandrogenemia, hyperinsulinemia and obesity. It is
frequently associated with an increased risk for insulin resistance with long
term health implications like type 2 diabetes mellitus and atherosclerosis
disease. However, the aetiopathogenesis of this syndrome still remains
elusive. The familial clustering of women with PCOS suggests that heredity
is implicated in the origin of this syndrome. PCOS is now considered a
polygenic trait that might result from the interaction of various susceptible
and protective genomic variants along with the influence of environmental
factors. A candidate gene based approach for investigating association of
genes involved in insulin resistance such as INSR, PPARg, and PON1 with
PCOS and its related phenotypes in Indian women has been undertaken.
Clinical and biochemical parameters to evaluate PCOS associated traits
related to metabolic and hyperandrogenemia were also assessed in all the
participants. A polymorphism in insulin receptor gene (INSR), C/T His1058
showed association with PCOS only in lean women and also with
hyperandrogenemia and hyperinsulinemia traits. Peroxisome proliferator
activated receptor gamma (PPARg) is a transcription factor that regulates
glucose, lipid homeostasis and intracellular insulin signaling. The most
common variant Pro12Ala of PPARg which has a role in preserving insulin
sensitivity showed significant association with decreased PCOS
susceptibility while the other His447His polymorphism along with former
was significantly associated with lower insulin related traits in women with
PCOS. Paraoxonase 1 (PON1) gene encodes an antioxidant enzyme which
protects against LDL oxidation and thus prevents atherosclerosis. Serum
paraoxonase levels were significantly reduced in our PCOS group. A coding
region L55M SNP showed association with PCOS. The study establishes
that in Indian women, variations in genes related to insulin resistance do
have an influence on the pathophysiology of PCOS.
P93
Do variations in Insulin-like Factor 3 (INSL3) gene affect PCOS
susceptibility?
Nuzhat Shaikh
1*
, Nalini Shah
2
, Srabani Mukherjee
1
1
National Institute for Research in Reproductive Health, J.M. Street, Parel,
Mumbai- 400012, India;
2
Department of Endocrinology, Seth GS Medical
College, Mumbai- 400012, India
Polycystic ovary syndrome (PCOS) is a multigenic complex disorder causing
metabolic and gynecologic dysfunctions in women of reproductive age. Its
Page 47 of 59
cardinal features include hyperandrogenemia, chronic anovulation, insulin
resistance and hyperinsulinemia. Women with PCOS are at increased risk to
develop long term health implications like endometrial cancer, T2DM and
cardiovascular diseases. Insulin-like factor 3 (INSL3), also known as relaxin-
like factor (RLF), is a member of the relaxin-like hormone family. Relaxin and
INSL3 are peptide hormones with a number of important physiological roles
in reproduction, regulation of extracellular matrix turnover, and
cardiovascular function. INSL3 is a theca cell-secreted paracrine factor which
regulates androgen production and has been implicated a role in follicle
selection and resulting ovulation. In women, it is produced in lower
amounts by ovarian theca and luteal cells, and circulating levels are
increased in women with polycystic ovarian syndrome. This suggests that
INSL3 may have a modulatory role in ovarian function. The INSL3 gene is
localized on chromosome 19 and comprises of two exons and an intron.
Novel and known variations in both exonic regions were investigated to
understand their influence in PCOS pathophysiology. Genotyping of these
polymorphisms were carried out by direct sequencing in 150 women with
PCOS and 150 normal menstruating women. Clinical, biochemical and
hormonal parameters were also assessed in these women. Analysis revealed
association of db6523 SNP in exon 1 and rs1003887 polymorphism of exon
2 with susceptibility to PCOS. Other exonic region polymorphisms failed to
show any association with PCOS pathophysiology.
P94
Role of TNF a in the etiopathogenesis of PCOS: a clinical, biochemical
and molecular genetic study
Sujatha Thathapudi
1*
, Vijayalakshmi Kodati
1
, Ahuja Yog Raj
1
, Uma Addepally
2
,
Anuradha Katragadda
3
, Qurratulain Hasan
4,5
1
Vasavi Medical Research Centre, Hyderabad, India;
2
Jawaharlal Nehru
Technological University, Hyderabad, India;
3
Anu Test tube baby centre,
Hyderabad, India;
4
Kamineni Academy of Medical Sciences and Research
Centre, Hyderabad, India;
5
Hyderabad Science Society, Hyderabad, India
E-mail: sthathapudi@ymail.com
Background: Polycystic ovarian syndrome (PCOS) is one of the most
common endocrine conditions affecting women of reproductive age with a
prevalence of approximately 5-10 % worldwide. PCOS is characterized by
hyperandrogenism, irregular menstrual cycles, polycystic ovaries, insulin
resistance, obesity particularly abdominal phenotype. We evaluated the role
of TNF a in PCOS patients and compared to age matched healthy controls.
Methods: 204 women clinically diagnosed with PCOS and 204 healthy
women controls in the age group of 17 to 35 years were evaluated. PCOS
were subdiveded into Obese and lean based on BMI (< 25 or > 25 kg/m
2
).
Clinical, biochemical characteristics of PCOS and molecular study of TNF alpha
gene C850T polymorphism were studied.
Results: Significant differences were observed in PCOS phenotypes and
controls. All the PCOS including subtypes had elevated WC, W/H, Fasting
Insulin, HOMA-IR, LH, LH/FSH, TG, and decreased HDL than Controls
(P<0.0001). Patients had elevated serum TNF a, Free and Total testosterone,
Androstenedione and DHEA when compared to controls (P <0.05). We have
demonstrated an association between TNF a C850T polymorphism and
PCOS. Frequency of T allele was 93.5% in PCOS and 73.5% in controls (OR
5.1863, CI 3.2893 to 8.1773 and P value <0.0001). TT genotype confers a
fivefold high risk for PCOS in our population (OR 5.7, CI 3.4673 to 9.3724 and
P value <0.0001).
Conclusion: TNF a contributes to the clinical , biochemical manifestations
of PCOS, and C850T TNF a gene polymorphism is associated with PCOS
and could be used as a relevant molecular marker to identify women with
risk of developing PCOS in our population.
P95
Assessment of MBL2 gene polymorphism and lipid peroxidation in
Chronic Obstructive Pulmonary Disease (COPD)
Aarti Sharma
1
, Gursatej Gandhi
1
, Jatinder Singh
2
, Sukhdev Singh
2
, Manpreet Kaur
1*
1
Department of Human Genetics, Guru Nanak Dev University, Amritsar, India;
2
Department of Molecular Biology & Biochemistry, Guru Nanak Dev
University, Amritsar, India
E-mail: dr.manpreetdhuna@gmail.com
Background: Chronic Obstructive Pulmonary Disease (COPD) is fourth
leading cause of death worldover. It has been defined as a state
characterized by airflow obstruction due to inflammatory reaction. Various
innate and adaptive immune system molecules are involved in
pathogenesis of COPD. Mannose-binding lectin (MBL) is a Ca
2+
dependent
collagenous lectin which intervene immune response by inhibiting
pathogen activity. Point mutations, in codon 54 and 57 of exon 1 of
the MBL2 have been reported to affect the serum levels of MBL. Aim of
the present study was to investigate the association of MBL2 gene
polymorphism with severity and susceptibility towards COPD.
Methodology: 129 COPD patients and 90 age- and sex-matched controls
were recruited for the study. Genomic DNA was isolated from blood
samples. PCR-RFLP of codon 54 and 57 of the MBL2 were studied using
enzymes BanI and MboII respectively. In addition to this, serum MDA
concentrations were evaluated by TBA-TCA-HCl method. Genotypic
distribution was compared by odds ratio statistics using medcal software.
Differences in MDA concentrations were analyzed by studentst test using
SPSS version 18.0 (IL, USA, and Chicago).
Results: The genotypic frequencies of codon 54 in COPD patients were
significantly higher (p< 0.05) than that of controls. GG genotype was
found to be more prevalent in cases (OR= 3.402; CI= 1.14-10.10; p< 0.05).
However, there was no significant difference in genotypic distribution for
codon 57 of MBL2 gene. Serum MDA concentrations were significantly
(p< 0.001) higher in patients (9.002.906) as compare to controls
(6.312.361).
Conclusion: The results of the present study revealed that MBL2
polymorphism may be involved in pathogenesis of COPD.
P96
Role of IL-6/JAK/STAT pathway in inducing vascular insulin resistance
Aswath Balakrishnan
*
, Kapaettu Satyamoorthy, Manjunath B Joshi
Division of Biotechnology, School of Life Sciences, Manipal University,
Manipal, India
Introduction: Insulin resistance is a hall mark of metabolic disorders.
Studies have demonstrated that inflammatory regulator interleukin-6 (IL-6)
plays an important role in disruption of IR/Akt/eNOS signaling pathway
resulting vascular insulin resistance. Accumulating evidences suggests a
significant role of epigenetic mechanisms such as DNA methylation in
progression of metabolic disorders. Hence the present study aimed to
understand the role of epigenetic mechanisms involved during IL-6
induced vascular insulin resistance and its consequences in cardiovascular
diseases.
Materials and methods: Human umbilical vein endothelial cells (HUVEC)
and Human dermal microvascular endothelial cells (HDMEC) were used for
this study. Endothelial cells were treated in presence or absence IL-6
(20ng/ml) for 36 hours and followed by insulin (100nM) stimulation for 15
minutes. Levels of phosphorylated- and total Akt served as readout for
insulin resistance. To investigate changes in DNA methylation, cells were
treated with or without neutrophil conditioned medium (NCM) as a
physiological source of inflammation or IL-6 for 36 hours. Genomic DNA
was processed for HPLC analysis for methyl cytosine content and cell
lysates were analyzed for DNMT1 (DNA (cytosine-5)-methyltransferase 1)
and DNMT3A (DNA (cytosine-5)-methyltransferase 3A) levels using
immunoblotting.
Results: Endothelial cells stimulated with insulin exhibited an increase in
phosphorylation of Akt
ser 473
in serum free conditions but such insulin
response was not observed in cells treated with IL-6, suggesting chronic
exposure of endothelial cells to IL-6 leads to insulin resistance. HPLC
analysis for global DNA methylation resulted in decreased levels of
methyl cytosine in cells treated with pro-inflammatory molecules (both
by NCM and IL6) as compared to 3.2% in untreated control to 2% in
treated. Kinetic studies depicted a transient increase DNA methylation
at 24 hours which was followed by steep decrease at 36 hours.
Subsequently, analysis in cells treated with IL-6 showed a significant
decrease in DNMT1 levels but not in DNMT3A. Other pro-inflammatory
marker such as TNF-a did not exhibit such changes. Interestingly we
also observed Akt phsophorylation refelcetd DNMT1 changes suggesting
plausible role of PI3K/Akt signaling axis in regulation of DNMT1
expression.
Page 48 of 59
Conclusion: Taken together our study suggests that IL-6 induces vascular
insulin resistance and involvement of epigenetic changes.
P97
In silico docking studies for designing potent anti-diabetic derivatives
of swertiamarin with enzyme HMG COA reductase
Jayshil Bhatt
2*
, Hitesh Vaidya
1
, Varun Khanna
2
, Naisargee Patel
2
,
Ramesh Goyal
2
1
Memorial University of Newfoundland, St. Johns, Canada;
2
Institute of Life
Sciences, Ahmedabad University, Ahmedabad, India
Background: Swertiamarin, a secoiridoid glycoside is found in abundant
quantity in Enicostemma Littorale herb and is the main constituent
responsible for anti-diabetic and anti-obesity effects of the plant extract.
It has been reported to act on various enzymes and transcription factors
involved in glucose and lipid metabolism, including inhibition of the
enzyme HMG-CoA reductase which might be one of the mechanisms
responsible for the antihyperlipidaemic activity. However, owing to its
high water solubility, it has a low plasma half life; and thus we have
designed its derivatives which bind more efficiently with HMG CoA
Reductase.
Materials and methods: Docking was carried out using software namely
Autodock and Vina, and repeated at least thrice to minimise the error
and to confirm the repeatability. The obtained results were scored and
sorted on the basis of the binding energy. The Autodock docking was
only done on the inhibitor ligands that were taken from the crystal
structure to cross check the results of Molecular Operated Environment
[MOE] in terms of binding orientation. Attempts were also made to
evaluate in silico toxicity and identification of other possible targets for
the lead molecule swertiamarin.
Results: We could design 23 compounds which were docked into the
active site of the crystal structure of HMGR. The interactions of these
molecules were compared with the presently known inhibitors such as
atorvastatin and simvastatin. Based on the results of docking score a
number of potent ligands for the enzyme HMGR as compared to
swertiamarin were established. It was observed that the designed
molecules SWL18, SWL21, SWL22 showed better docking score (-60.74;
-61.43 and 55.68 respectively) and tight binding in pockets. Docking
score of these molecules were very similar to the atorvastatin and
simvastatin.
Conclusions: SWL18, SWL21, SWL22 possess tight binding with HMGR as
compared to swertiamarin, which suggest that these molecules could
have better HMGR inhibition.
PHARMACOGENETI CS
P98
Influence of CYP3A5 polymorphism on tacrolimus drug dosing in Indian
renal allograft recipients: initial experience
Mohan Patel
1*
, Manoj Gumber
1
, Vivek Kute
1
, Pankaj Shah
1
, Himanushu Patel
1
,
Hargovind Trivedi
1
, Aruna Vanikar
2
, Jayesh Sheth
3
1
Department of Nephrology and Clinical Transplantation, Institute of Kidney
Diseases and Research Center, Dr HL Trivedi Institute of Transplantation
Sciences, Ahmedabad, India;
2
Department of Pathology and
Immunohematology, IKDRC-ITS, Ahmedabad, India;
3
Foundation for Research
in Genetics and Endocrinology (FRIGE) Institute Of Human Genetics,
Ahmedabad, India
Background: Tacrolimus (Tac) is the mainstay of standard immuno-
suppressive regime in renal transplantation today. However, it needs regular
drug level monitoring in view of narrow therapeutic window to keep blood
level in therapeutic range. The objective of the study was to assess the
potential influence of a functional polymorphism in CYP3A5*3 gene on dose
requirements and trough blood levels of tacrolimus in renal transplant
patients
Materials & methods: This prospective observational study included 20
patients of end stage renal disease who underwent renal transplantion
and a follow up of 1 year at our hospital. All the patients were started on
standard immunosuppressive regime of Tacrolimus-Mycophenolate mofetil
along with steroids with a starting dose of Tac 0.08 mg/kg/day. Genotype
of CYP3A5 was studied in 20 patients requiring low dose of Tac. At 7
th
and
30
th
day, and 3 monthly after transplant, Tac dosages (mg/kg/d), were
adjusted based on blood levels and complications. Polymerase chain
reaction, followed by restriction fragment length polymorphism analysis
was used for genotyping CYP3A5*3 gene (A6986G) in intron 3.
Results: Out of 20 patients (17 males and 3 females) included in the study,
16 had live related renal transplantation and 4 had diseased donor renal
transplantation. Out of 20 patients 16 were found to have CYP3A5
homozygous status (A3986G polymorphism in CYP3A5*3 gene-wild allele)
and 4 with heterozygous status. Patients of CYP3A5 homozygous status had
mean Tac level of 16.84 ng/dL, median of 15.5 ng/dL (range- 13.2-25 ng/dL)
on 7
th
day of transplant. So Tac dose was reduced to achieve TDL and mean
Tac level at 30
th
day of transplant was 6.6 ng/dL and median of 7.4 ng/dL
(range- 5.5-12.5 ng/dL). At 6
th
month and 12
th
month of transplant, mean Tac
levels were 6.1 ng/dL and 5.9 ng/dL. In patients having CYP3A5 homozygous
status, mean and median Tac dose after 4 weeks was 0.03 mg/kg/day (range-
0.008 to 0.05 mg/kg/day) and mean creatinine was 1.17 mg/dL (range-0.8 to
1.4 mg/dL) after 30
th
day. Remaining 4 patients having CYP3A5 heterozygous
status were maintaining TDL at Tac dose of 0.06 mg/kg/day. Five patients
had graft biopsy during first 4 weeks of renal transplant showing acute
tubular necrosis (ATN) in three patients, acute cellular rejection in one patient
and cellular rejection with evidence of calcineurine inhibitor (CNI) toxicity in
one patient.
Conclusion: This study demonstrates the usefulness of CYP3A5 genotype
in transplant patients taking Tacrolimus-Mycophenolate mofetil and also
shows that majority of our patients carry mutant allele A3986G in
CYP3A5*3 gene.
P99
Multi- analytical approach: better predictor of pharmacogenetic based
clinical outcomes in breast cancer therapies
Sonam Tulsyan
Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical
Sciences, Lucknow, India
E-mail: sonam_tulsyan@yahoo.co.in
Background: Chemotherapeutic drug clinical outcomes are genetically
determined as there is a large heterogeneity in the response to, and toxicity
of, chemotherapeutic agents in breast cancer patients. Polymorphisms in
genes encoding Phase I Cytochrome P450 (CYP450) and NAD(P)H
dehydrogenase quinine (NQO1); phase II - glutathione-Stransferase (GST) and
methylene tetra hydrofolate reductase (MTHFR); phase III- p-glycoprotein
(ABCB1) and solute transporter (SLC22A16) drug metabolizing enzymes can
possibly predict clinical outcomes, and can be of prognostic significance in
breast cancer patients. The aim of this study was to determine the role of
genetic variations in drug metabolizing enzymes in predicting response and
toxicity in breast cancer patients, using multi-analytical approaches.
Materials & methods: Two hundred and four North Indian cases of
histology proven invasive breast carcinoma treated at the our institute
were genotyped for 17 polymorphisms by Polymerase Chain Reaction
(PCR) or PCR-Restriction Fragment Length Polymorphism (RFLP) or Taqman
allelic discrimination assay. All patients were treated with combination
chemotherapy containing an anthracycline drugepirubicin (62.70 %) or
doxorubicin (37.25%) along with cyclophosphamide and 5-fluorouracil.
Tumor response to NACT was recorded in 96 patients according to
Response Evaluation Criteria in Solid Tumors (RECIST). Toxicological
data was recorded according to National Cancer Institute- Common
Terminology Criteria for Adverse Events (NCI-CTCAE). The highest grade
toxicity that occurred in an individual patient during the course of
treatment was used for the analysis. Genetic variations were correlated
with response to NACT and chemo-toxicity using logistic regression
through SPSS software, version 17.0. Furthermore, higher-order gene-gene
interactions were determined through multifactor dimensionality reduction
(MDR) using software version 2.0 beta8.
Results: Heterozygous (CT) and variant (TT) genotype of ABCB1 1236C>T
polymorphism was found to be significant with breast cancer response to
NACT. Similarly, heterozygous (CT) genotype of ABCB1 1236C>T
Page 49 of 59
polymorphism was significantly associated with grade 2-4 anemia.
Furthermore, we found significant association of heterozygous genotype
(*1/*3) of CYP2C9*3 with grade 2-4 leucopenia and CT genotype of NQO1
polymorphism with dose delay/ reduction. However, none of the
polymorphisms were found to be statistically significant with grade 2-4
toxicity. On MDR analysis, ABCB1 1236C>T polymorphism yielded the
highest testing accuracy for response to NACT (CVT=0.61, CVC= 10/10, p
=0.0067). However, ABCB1 1236C>T, ABCB1 2677G>T/A, CYP2C19*2
combination of polymorphisms yielded the highest testing accuracy for
grade 2-4 anemia (CVT=0.66, CVC= 10/10, p < 0.0001). Similarly, the
above combination of polymorphisms provided the best interaction
model for overall toxicity as well (CVT=0.65, CVC= 10/10, p < 0.0001). For
dose delay/ reduction, NQO1 609 C>T polymorphism yielded the highest
testing accuracy (CVT=0.60, CVC= 9/10, p =0.0048). However, CYP2B6*9
polymorphism with the testing accuracy (CVT=0.43, CVC= 5/10) for grade
2-4 leucopenia did not achieve statistical significance (p= 0.1088).
Conclusions: Multi-analytical approaches may provide a better
prediction of pharmacogenetic based clinical outcomes in breast cancer
patients.
P100
Impact of KCNJ11, TCF7L2, SLC30A8, IGF2BP2, PPARG, SLC47A1, STK11,
HHEX, KCNQ1, CDKAL1, FTO, CYP2C9, ADIPOQ, CAPN10 gene
polymorphisms on risk of type 2 diabetes and therapeutic response to
sulfonylurea and metformin therapy
Nagaraja Phani
1*
, Padmalatha Rai
1
, Prabha Adhikari
2
, Shivashankara Nagri
3
,
Sydney DSouza
2
, Mundyat Gopinath
1
, Kapaettu Satyamoorthy
1
1
Division of Biotechnology, School of Life Sciences, Manipal University,
Manipal, India;
2
Department of Medicine, Kasturba Medical College, Manipal
University, Mangalore, India;
3
Department of Medicine, Kasturba Medical
College, Manipal University, Manipal, India
E-mail: mnphani1986@gmail.com
Type 2 Diabetes (T2D) represents a spectrum of metabolic disorders is
genetically heterogeneous disease with multiple genes located on
different chromosomes contributing to its susceptibility. Prevalence of
T2D has increased sharply in recent years with more than 300 million
people worldwide and 70 million in India. The application of molecular,
genomic knowledge will provide new opportunities to explore the
heterogeneity which patients clearly exhibit and provide a more accurate
understanding of individual patients. Single nucleotide polymorphisms
(SNPs) represent the most profuse form of genetic variation in humans
which are gaining increased popularity and emerging as a new
generation genetic markers. With several classes of drugs available to
treat T2D its clinical response exhibits significant variation among
individuals which exerts a huge impact on healthcare system and
economic burden due to diabetes treatment. Pharmacogenetic research
which assesses the role of genetic variants can be used in elucidating the
nature of these variants on differential drug response. In the current
study, we evaluated the association of KCNJ11, TCF7L2, SLC30A8, IGF2BP2,
PPARG, SLC47A1, STK11, HHEX, KCNQ1, CDKAL1, FTO, CYP2C9, ADIPOQ,
CAPN10 gene polymorphisms with T2D and response to sulfonylurea and
metformin using PCR-RFLP, TETRA-ARMS and DNA sequencing techniques.
Study subjects were 330 T2D patients who are on sulfonylurea (178) and
metformin (152) treatment with age and sex matched normal healthy
controls of south Indian origin. Allele frequencies, genotype and haplotype
distribution were analyzed by chi-squared test. Logistic regression analysis
was used to predict the effect of gene variants on treatment. Genegene
interactions were analyzed by generalized multifactor dimensionality
reduction method. Linkage disequilibrium between each pair of SNP loci,
were estimated and plotted with JLIN. We have also performed a
systematic review and Meta-Analysis of KCNJ11 rs5219 and PPARG
rs1801282 SNPs on South Asian populations to clarify inconsistency in
association results. Our analysis showed KCNJ11 rs5215, SLC30A8
rs1326634, TCF7L2 rs7903146 were associated with susceptibility to T2D.
Multivariate regression analysis showed that TCF7L2 rs12243326 TT
genotype, KCNJ11 rs5219 TT genotype were associated with response rate
to sulfonylurea treatment and STK11 rs741765 GG genotype, SLC30A8
rs1326634 TT genotype were associated with response rate to metformin
treatment.
POPULATI ON GENETI CS
P101
Effect of PPAR-g2 gene Pro12Ala and ADR-b3 gene Trp64AArg
polymorphism on glucose homeostasis in Type 2 diabetes subjects
from Western India
Ankna Shah
1*
, Frenny Sheth
1
, Avisek Majumder
1
, Bhavik Doshi
1
,
Navneet Shah
2
, Premal Thakor
3
, Rama Vaidya
4
, Jayesh Sheth
1
1
2
3
Gujarat
Diabetic Association, Ahmedabad-380007. India;
4
Medical Research Centre, Mumbai-400056. India
E-mail: anknashah@yahoo.co.in
Background: Several studies have shown the effect of Pro12Ala
polymorphism of PPAR-g2 on insulin sensitivity and Trp64Arg polymorphism
in ADR-b3 gene on obesity and insulin resistance in Type 2 Diabetic (T2D)
subjects. The present study was carried out to find the interaction of these
two gene polymorphisms and their combined effect on glucose
homeostasis (HbA1C) in T2D subjects.
Materials and methods: The present study comprises of 535 subjects
(including 235 T2D & 300 controls). Genotyping was carried out for the
above mentioned polymorphisms and glycosylated hemoglobin [HbA1C]
levels were analyzed for each subject. All T2D subjects were divided into
four groups according to their genotype. Group-I: 31 patients with Pro/
Pro and Trp/Arg genotype; Group-II: 159 patients with Pro/Pro and Trp/
Trp genotype; Group-III: 6 patients with Pro/Ala and Trp/Arg genotype;
and Group-IV: 39 patients with Pro/Ala and Trp/Trp phenotype.
Results: It was observed that 12Ala allele frequency was nearly equal in
T2D patients and controls (9.0% vs. 9.1%, p>0.05). 64Arg allele frequency
was 8.3% in T2D patients and 6.7% in controls (p>0.05). The mean HbA1C
level was lower in T2D patients with 12Ala allele compared to patients
homozygous for 12Pro allele (7.731.42% vs. 8.471.92%, p<0.02).
However, no significant difference in mean HbA1C levels was observed in
T2D patients with 64Arg allele compared to patients homozygous for
64Trp allele (8.501.81% vs. 8.311.88%, p>0.05). The mean HbA1C levels
were higher in Group-I (8.621.84%, p<0.0092), Group-II (8.471.94%,
p<0.001) and Group-III (8.261.71%, p>0.05) compared to grouPIV having
a mean HbA1C of 7.651.37%.
Conclusions: The protective effect of 12Ala allele is likely to be
diminished in Group-III T2D patients in the presence of 64Arg allele.
Polymorphisms of 64Arg and 12Pro alleles that is likely to play a role in
controlling glucose homeostasis by gene-gene interactions.
P102
Alarming findings about genomics of sudden cardiac arrest in India
Pankaj Mankad
*
, Srisanth Balan, Saleem Mohammed
Xcode Life Sciences, Chennai, 600 034, Tamil Nadu, India
E-mail: pankaj.mankad@tiscali.co.uk
Background: Over 10% of total deaths and 50% of cardiac deaths in India
are Sudden Cardiac Deaths (SCA) and occur at a young age of 5-8 years.
We analysed risk allele plus genotype frequencies and polymorphism
associations of 38 SNPs, confirmed in GWAS studies that increasing the risk
of SCA in 124 random people in the general population. The data were
compared with Caucasians reported in HapMap (n = 113).
Material and methods: Following DNA extraction from saliva, genotyping
was performed using Illumina golden gate genotyping assay. Allele and
genotype frequencies were determined by direct gene count method.
Results: Population Genomics indicated Odds Ratio of risk alleles as 1.62
(1.31 -1.92). The carrier rate of risk allele frequency was significantly higher
(p = 0.037 to p = 9.18E-37) in Indians compared to Caucasians in 56% of
SNPs in H-W equilibrium (15 of 27).6 SNPs showed statistically insignificant
comparatively lower risk allele frequency in Indians. Mean increase in higher
risk allele frequency SNPs was 2.4 fold (1.1 6.7). Frequency in Indians, for
the two SNPs with the strongest SCD risk correlate in the gene BAZ2B was 2
times (rs4665058) and 6 times (rs 174230) greater than in Caucasians.
Page 50 of 59
Personalized Genomics indicated that even the frequency of risk
homozygous genotype, resulting in the SCA threat in affected individuals
being the square of the risk allele hazard, was markedly different with
4.7% Caucasians carrying homozygous risk genotype versus 10.6% of
Indians (p<.0001). In 18 (out of 38) SNPs studied, the homozygous
genotype difference in Indians was more than double). Although
interplay of gene-gene interaction in individualized risk prediction is still
undefined, 59% (73 of 124) people carried between 10 to 19 risk alleles
and 41% (51 of 124) had between 20 to 30 risk alleles (median 19, range
10 -25), of the total maximum of 76.
Conclusions: This is the first report, highlighting extremely high genomic
propensity for Sudden Cardiac Arrest in Indians compared to the Caucasians.
In India, there is also a high propensity for risk towards homozygous
genotype and people carrying several risk alleles for SCA. At the population
level, this data reinforces an urgent need to train community in different
locations in basic resuscitation skills and at a personal level, it raises an
important point to test the vulnerable group for assessing the risk alleles for
better individualized preventive action plans could be advocated.
P103
Transmission Disequilibrium Test for quantitative traits based on
multiple sibs
Hemant S Kulkarni
*
, Saurabh Ghosh
Human Genetics Unit, Indian Statistical Institute, Kolkata, India
E-mail: hemant.statistics@gmail.com
Background: The Transmission Disequilibrium Test (TDT) is a well
established family-based test for genetic association. The advantage of TDT
over population-based association studies test, which is protected against
population stratification, and hence, an association finding can be attributed
to the presence of linkage. Quantitative traits (QT) are more informative on
within-genotype variability and hence, statistical tests for identifying genes
based on such traits tend to be more powerful compared to binary or
qualitative traits. The TDT is based on a trio design (two parents and one
offspring per family). However, if multiple sibs are present, TDT is only a
valid test for linkage. In our study, we have extended the test for
quantitative traits based on families with multiple offspring using a logistic
regression framework.
Materials and methods: We selected one offspring at random from each
family to compute the usual TDT statistic. We then repeated the process and
carry out separate permutation tests based on the mean and the maximum
values of the TDT statistics obtained over different replications. We
performed extensive simulations to evaluate the power of our proposed
tests under a wide spectrum of genetic parameters (allele frequencies, QTL
means and variances, extent of linkage disequilibrium between the QTL and
the marker locus) and probability models (normal and chi-squares). We also
compared the performance of the tests with a correction strategy using the
Benjamini-Hochberg threshold.
Results: We found that the test based on the mean TDT value is more
powerful than that based on the maximum value. However, in either case,
the power is higher compared to that obtained using the Benjamini-
Hochberg threshold. In general, the power decreases with increase in
heteroskedasticity at the QTL as well as the difference between the minor
allele frequencies at the two loci. The powers are marginally higher for the
chi-squares distribution compared to the normal distribution.
P104
Evaluation of MC4R [RS17782313, RS17700633], AGRP [RS3412352] and
POMC [RS1042571] polymorphisms with obesity in Northern India
Apurva Srivastava
1*
, Balraj Mittal
3
, Jai Prakash
2
, Neena Srivastava
1
1
Department of Physiology, King Georges Medical University, Lucknow, (U.P),
India;
2
Department of Pediatrics, King Georges Medical University, Lucknow,
(U.P), India;
3
Department of Medical Genetics, SGPGIMS, Lucknow, (U.P), India
E-mail: drneenasrivastava@hotmail.com
Background: Genetic variants of the melanocortin-4 receptor gene
(MC4R), agouti related protein (AGRP) and proopiomelanocortin (POMC)
are reported to be associated with obesity. Therefore, we examined MC4R
rs17782313, MC4R rs17700633, AGRP rs3412352 and POMC rs1042571 for
association with obesity in North Indian individuals.
Material and methods: The variants were investigated for association in
300 individuals with BMI30kg/m
2
and 300 healthy non-obese individuals
with BMI<30kg/m
2
. The genotyping were analyzed by Taqman probes.
The statistical analysis was performed by means of the software SPSS,
ver.19 and P 0.05 was considered statistically significant.
Results: The genotypes of MC4R rs17782313 and POMC rs1042571 were
significantly associated with obesity (BMI30kg/m
2
) (p=0.02; OR=1.7 and
p=0.01; OR=1.6 respectively). However, MC4R rs17700633 (p=0.001;
OR=0.55) was associated with low risk. AGRP rs3412352 (p=0.93; OR= 0.96)
showed no association with obesity (BMI30kg/m
2
) in North Indian
individuals.
Conclusions: The study provides the first report of association of MC4R
rs17782313 and POMC rs1042571 that these may have an effect on obesity
BMI30kg/m
2
but MC4R rs17700633 and AGRP rs34123523 may not have
any influence on obesity BMI30kg/m
2
in North Indian individuals.
P105
Population allele frequencies of disease associated SNPs in India: a
paradigm shift from HapMap
Pankaj Mankad, Srisanth Balan
*
, Saleem Mohammed
Xcode Life Sciences, 6B, Eldorado, Nungambakkam High Road,
Nungambakkam, Chennai, 600 034, India
Background: One of the challenges facing us in India, translating recent
discoveries of chronic disease associated SNPs into clinical domain for
prevention, is the lack of knowledge about the frequency of the
polymorphism in our population. Hence, using available HapMap data has
become a norm. However, it is anticipated that population genomics in
India could be different from the HapMap Caucasian population. Relative
disease risk prediction based on relevant SNPs, both for personalized
medicine and for population genetics, has little value without accurate
information of population allele frequencies. We report allele frequency of
384 SNPs directly related to chronic disease risk and metabolic traits in the
Indian population.
Materials and methods: We report the allele in a random sample of 146
individuals and compare them with the data reported in HapMap Caucasian
population (n = 112). Genotyping was performed using Illumina golden gate
genotyping assay following DNA extraction from saliva. Allele frequencies
were determined by direct gene count method.
Results: GWAS studies confirmed 384 SNPs to be associated with disease
risk (364) of Diabetes Type 1 and 2 (54 & 118 respectively), Coronary artery
disease(71), myocardial infarction(9), cardiac failure(24), sudden cardiac arrest
(38), atrial fibrillation(9), hypertension(18), obesity(10), metabolic syndrome(2)
and stroke(11); or were associated with metabolic traits (20). The master table
of their rs id, chromosomes, location and association is presented. Of the
384 SNPs, 44 were not in H-W equilibrium and were omitted. HapMap data
were not available for 13 SNPs. We are reporting their allele frequencyon the
Indian population for the first time. Of the remaining 307 disease association
SNPs, statistically significant difference (p<.05) from HapMap Caucasian
population was observed in 53% of them (164 of 307) and the difference of
>10% (considered major in population genetics) was found in 42% (130 of
307). Of the 20 metabolic association SNPs, 50% (10 of 20) had statistically
significant difference and in all of them it was >10%.
Conclusions: We are reporting the largest repository, documenting
disease and related SNP and allele frequencies in Indian population. We
have also highlighted clear differences with HapMap data and would
caution against indiscriminate use of HapMap for bench-to-bedside
application of genetic knowledge in our population.
P106
Genetic Variation in Intercellular Adhesion Molecule-1 (ICAM-1):
candidate gene in susceptibility to malaria in the Indian population
Anuroopa Gupta
*
, Harish Padh
Department of Cell and Molecular Biology, B.V. Patel Pharmaceutical
Education and Research Development (PERD) Centre, Ahmedabad-380 054,
Gujarat, India
Page 51 of 59
Background: Plasmodium falciparum malaria is the most severe form of
malaria causing morbidity and mortality worldwide. The key event in P.
falciparum pathogenesis is the sequestration of the infected erythrocytes to
the capillary endothelium, mediated by the receptor, Plasmodium
falciparum erythrocyte membrane protein-1 (PfEMP1). PfEMP1 has distinct
characteristics of binding to varied host ligands including Inter Cellular
Adhesion Molecule (ICAM-1). Polymorphisms in the gene encoding ICAM-1
influence the rate of progression or confer protection from or susceptibility
to malaria parasite infection. The objective of our study was to evaluate the
frequency of ICAM-1 SNP in exon 6 (rs5498, K469E) in the Indian population
and compare it with the global population influenced by the parasitic
infection.
Materials and methods: The genotyping for ICAM-1 SNP rs5498 was
performed by Restriction Fragment Length Polymorphism (RFLP) PCR.
Analysis of ICAM-1 variants was performed in 200 healthy subjects.
Results: The allelic frequency for the variant ICAM1-Aallele was 59% in
healthy control subjects. Chi square analysis reveals that it does not
follow the Hardy Weinberg Equilibrium. Comparison with literature data
indicates that frequency distribution in Indian population is similar to
German, Swedish and Danish populations while it is significantly different
from Finnish and Japanese populations. The genotypic frequency of AA
was 35%, AG was 49% and GG was 16% in healthy control subjects.
Conclusions: The frequency data from the above study will help in
understanding the role of genetic variations in ICAM-1 adhesion molecule in
the falciparum malaria pathogenesis in the Indian population. Upon
comparison, a noteworthy difference in the genotype frequency of Nigerian
children (p<0.001) was evinced. This data can be useful for predicting the
potential risk of malaria in world population.
P107
Genetic affinities of six populations of Manipur using a microsatellite
(STR) marker
Ahsana Shah
*
, Ruqaiya Hussain, Mohammad Afzal
Aligarh Muslim University, Aligarh, U.P., India
Background: The development of molecular genetic technology has led to
the discovery of large number of polymorphic loci in the human genome.
This has renewed the interest in investigating genomic diversity (and
affinity) between human populations with much more depth and clarity
than that was possible with the erstwhile traditional serological and
biochemical genetic markers. DNA markers are presently widely used in
gene mapping, forensic studies and information on population structure
and population genetics of regional and global populations. The study
attempts to understand the genetic structure and affinity among six
different populations of Manipur.
Materials and methods: The diversity of the studied populations was
examined by investigating the polymorphism for a STR locus (TPOX).
Unrelated individuals belonging to Muslims with different caste viz. Sheikh,
Syed, Pathan & Moghul; Hindu (Meitei) and tribal (Naga) were randomly
selected. Blood samples were collected after obtaining an informed
consent. The DNA was extracted using standard phenol chloroform
extraction method and then amplified with PCR using previously published
primers. The amplified PCR products were electrophoreses followed by
Silver staining.
Results: Different population containing allele with repeated number
varying from 5 -11 were detected. Discriminating Power Analysis (PD) lie
in the range from 0.444 to 0.889 and Matching Probability (MP) was
observed to vary from 0.111 to 0.556. Based on genetic distance,
dendrogram was constructed to depict the genetic affinities among the
six populations. The tree shows that the ethnic background of six
populations is different.
P108
Microsatellite variation and allele frequency distribution for (TPOX)
STRS locus in North Indian Muslim populations
Ruqaiya Hussain
*
, Mohammad Afzal
Human Genetics and Toxicology Lab., Section of Genetics, Department of
Zoology, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
Background: Autosomal short tandem repeats (STRs) have become the
most informative molecular markers because they are highly polymorphic
and multiallelic. The objective of the present work is to study the
genotypic polymorphisms, allele frequencies and forensic parameters at
highly polymorphic STR loci (TPOX) among four Muslim Populations of
North India: Pathan, Ansari, Saifi and Mansoori.
Materials and methods: The DNA was extracted from blood samples by
using the standard phenol-chloroform extraction method and then
amplified by PCR using specific primers for TPOX locus. The PCR products
were separated by electrophoresis on denaturing 6% polyacrylamide gel
and silver staining was done to resolve and observe different alleles. The
observed and expected heterozygosity (H
obs
and H
exp
) as well as forensic
and paternity indices including matching probability (MP), power of
discrimination (PD), power of exclusion (PE) and polymorphism
information content (PIC) were calculated using the Power Stats v.1.2
software for TPOX locus of the studied population. The POPGENE (v.32)
statistical package was used for analyzing allele frequency.
Results: The 6 to 10 different alleles were detected in the studied
populations with 8 allele repeats are most common in all four groups of
sub-population. The maximum expected heterozygosity was found in
Mansoori population (H
exp
= 0.867). The polymorphism information
content (PIC), has shown that this marker is highly informative for
Mansoori (0.67) and Ansari (0.63) population group.
Conclusion: In conclusion the interpopulation differentiation has been
found significantly to differ from each other (F
ST
= 16.37%). A dendrogram
was constructed using the Neighbor-joining (NJ) clustering method. The
dendrogram shows the low genetic distance between Pathan and Ansari
population groups. According to the statistical parameters, the combined
analysis of this TPOX STRs locus is a powerful tool for forensic personal
identification, paternity testing and population genetic studies in North
Indian Muslim Populations.
P109
Genetic variation of ITGB3 is associated with Autism Spectrum
Disorders (ASD) in South Indian children
Femina KMB Nair
1*
, PA Suresh
2
, Moinak Banerjee
1
1
Human Molecular Genetics laboratory, Rajiv Gandhi centre for
Biotechnology, Thiruvananthapura, India;
2
ICCONS, Shoranur, India
E-mail: femina@rgcb.res.in
Background: Autism spectrum disorders (ASDs) are a group of develop-
mental disabilities that can cause significant social, communication and
behavioral challenges in children. Genetic factors contribute significantly to
ASD. ITGB3 encodes integrin b3. This cell adhesion molecule has been
implicated as a modulator of serotonergic systems as well as in regulation of
synaptic plasticity and maturation. In the brain, integrin b3 couples to
integrin av to form a functional receptor, making integrin avb3 an interesting
target for regulation of neural 5-HT systems. The aim of this study was to
investigate the potential associations of single-nucleotide polymorphisms
(SNPs) of the integrin gene with Autism Spectrum Disorder (ASD).
Material and methods: Hundred and twenty five patients with ASD and
210 healthy volunteers were recruited. Four SNPs of Integrin genes were
analyzed by direct sequencing and polymerase chain reactionrestriction
fragment length polymorphism genotyping.
Results: We detected significant allelic and genotypic associations with
rs3809865 (Allelic and genotypic p value = 0.0089, 0.0044). Haplotypic
association involving risk allele was observed in two, three and four locus.
This 3UTR SNP would decrease/break or enhance/create miRNA-mRNA
binding sites and thus affect the expression of host genes in the brain.
Conclusions: Our finding identified the possible function of this SNP
locus, and provides the basis for subsequent functional research.
P110
Association of Genetic Polymorphisms in STAT 3, STAT 5b and GWAS
Identified PTPN22 Gene with Rheumatic Heart Disease
Usha Gupta
1*
, Avshesh Mishra
1
, Saurabh S. Rathore
1
, Snober S Mir
2
,
SK Agarwal
3
, Naveen Garg
4
, Balraj Mittal
1
1
Department of Genetics, Sanjay Gandhi Postgraduate Institute of Medical
Sciences (SGPGIMS), Lucknow, UP, India;
2
Department of Biotechnology,
Page 52 of 59
Integral University, Lucknow, UP, India;
3
Department of Cardiovascular and
Thoracic Surgery, Sanjay Gandhi Postgraduate Institute of Medical Sciences
(SGPGIMS), Lucknow, UP, India;
4
Department of Cardiology, Sanjay Gandhi
Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow, UP, India
Background: Rheumatic heart disease (RHD) is an inflammatory,
autoimmune disease, occurring as a consequence of group A streptococcal
infection complicated by rheumatic fever (RF). Cytokines are important
mediators of inflammatory and immune responses. JAK-STATs have been
demonstrated to be critical elements in signaling by certain families of
cytokines. GWAS has identified PTPN22 SNPs as non-HLA genetic variants to
be associated with susceptibility to autoimmune diseases. Based on these,
we looked for association of genetic variants of STAT 3, STAT 5B and GWAs
identified PTPN22 with RHD in North Indian population.
Methods and results: This case-control study included 400 RHD patients
and 200 controls. The polymorphisms were identified using RFLP/Taqman
probes. Statistical analysis was performed by using SPSS. We observed that
STAT3 CG and GG genotypes were significantly associated with RHD
(p=0.024 & p=0.027 respectively), STAT5b CT&TT genotypes were
significantly associated with RHD (p=0.001 & p=0.002 respectively) while
both the SNPs of PTPN22 gene did not show any association with RHD.
Further categorization of RHD patients into mitral valve disease (MVD) and
combined valve disease (CVD) subgroups revealed that STAT3 CG&GG
genotypes were associated with MVD and STAT5b CT&TT genotypes were
also associated with both MVD&CVD.
Conclusions: STAT3 & STAT5b gene polymorphisms may play an
important role in the pathogenesis of RHD but GWAS identified PTPN22
SNPs may not be associated with susceptibility of RHD.
P111
Role of cholecystokinin receptor-A gene polymorphism in development
of functional dyspepsia
Rajan Singh
*
, Balraj Mittal, Uday C Ghoshal
Departments of Gastroenterology and Genetics, Sanjay Gandhi Postgraduate
Institute of Medical Sciences (SGPGIMS), Lucknow-226014, India
E-mail: rajan.lubiotech@gmail.com
Background: Functional dyspepsia (FD) is characterized by epigastric pain,
burning, early satiety and post-prandial fullness in absence of organic or
metabolic causes. Cholecystokinin receptor-A (CCK-AR) is known to
modulate satiety signal and delay gastric emptying, which are associated
with FD. CCK-AR (rs1800857, T/C) polymorphism is associated with a
defective splicing of the primary transcript of CCK-AR mRNA, which may
result in the lower expression of the CCK-AR. Therefore, we evaluated the
role of genetic polymorphism of CCK-AR gene (rs1800857, T/C) in FD.
Material and methods: 237 consecutive patients with FD (Rome III) and
250 healthy controls (HC) were genotyped for CCK-AR gene polymorphism
(PCR-RFLP). Patients with FD were sub-classified into epigastric pain
syndrome (EPS), post-prandial distress syndrome (PDS) and EPSPDS overlap.
Results: Patients with FD [173 (73%) male, age 3812-y] were comparable
with HC [195 (78%) male, age 3712-y] with respect to age and gender.
26/237 (11%) had EPS, 55 (23.2%) PDS and 156 (65.8%) EPSPDS overlap.
Among 237 patients with FD, CC (variant) genotype of CCK-AR (rs1800857)
was infrequent among patients than HC [19 (8%) vs. 46 (18.4%) p=0.001,
odds ratio (OR) =0.36, 95% confidence interval (CI) =0.19-0.66]. However,
genotypes distribution was comparable among patients with different
subtypes of FD (p=0.44).
Conclusions: CC genotype of CCK-AR polymorphism is protective for FD.
EPSPDS overlap was common among patients with FD.
P112
Role of sarcomeric gene polymorphisms on left ventricular dysfunction
in coronary artery disease patients
Surendra Kumar
1*
, Avshesh Mishra
1
, Anshika Srivastava
1
, Naveen Garg
2
,
Surendra Kumar Agarwal
3
, Shantanu Pande
3
, Balraj Mittal
1
1
Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical
Sciences (SGPGIMS), Lucknow-UP, India;
2
Department of Cardiology, Sanjay
Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow-UP,
India;
3
Department of CVTS, Sanjay Gandhi Post Graduate Institute of
Medical Sciences (SGPGIMS), Lucknow-UP, India
E-mail: surendrakhedarcbt@gmail.com
Background: Coronary artery disease (CAD) is a major cardiac disease in
humans. Many CAD patients develop left ventricle dysfunction (LVD),
leading to congestive heart failure. Mutations in several genes including
those encoding sarcomeric proteins such as MYBPC3, TNNT2, and TTN are
common genetic cause of hereditary cardiac myopathies. An intronic 25-
bp deletion in MYBPC3 at 3 region is associated with dilated (DCM) and
hypertrophic (HCM) cardiomyopathies in Southeast Asia. We sought to
determine the role of MYBPC3 25bp, TNNT2 5bp and TNN 18bp ins/del
polymorphisms on LVD in CAD patients.
Methods and results: The study included 200 healthy controls and 988
consecutive patients with angiographically confirmed CAD. Among them,
253 with reduced ejection fraction (LVEF <45%) were categorized as having
LVD. MYBPC3 25bp, TNNT2 5bp and TNN 18bp ins/del polymorphisms were
determined by polymerase chain reaction. Our results showed that MYBPC3
25bp deletion was significantly associated with CAD as well as LVD (healthy
controls v/s CAD; p value = 0.003; OR=4.08, healthy controls v/s LVD;
p value < 0.0001; OR=6.67 and Non-LVD v/s LVD; p value = 0.031;
OR=1.67). The TNNT2 5bp and TNN 18bp polymorphisms were not found to
be associated with CAD (Pvalue=0.580, OR=0.88; Pvalue=0.795, OR=0.91;
respectively) or LVD (Pvalue=0.146, OR=1.35; Pvalue=0.935, OR=0.97
respectively) when compared to controls.
Conclusions: The frequency of MYBPC3 DW genotype and D allele was
associated with LVD implying that genetic variants of MYBPC3 encoding
mutant structural sarcomeric protein could increase susceptibility to left
ventricular dysfunction. Therefore, 25bp deletion in MYBPC3 may represent
a genetic marker for cardiac failure in CAD patients.
P113
A novel mutation in 3UTR of GJB2 gene in autosomal recessive
nonsyndromic sensorineural hearing loss in South Indian population
Maria sebastian
1*
, Praveena Davis
2
, Padmaja Ramdas
2
, Moinak Banerjee
1
1
Biotechnology, Kerala, India;
2
National Institute of Speech & Hearing, Kerala,
India
Background: Autosomal recessive nonsyndromic sensorineyral hearingloss
also known as DFNB causes 20% of all childhood deafness and may have
carrier rate as high as 2.8%. Fifty to eighty percent of DFNB cases causing
severe to profound hearing impairment, results from mutations in a single
gene, GJB2(DFNB1), that encodes the protein connexin 26(Cx26) located on
chromosome 13q11-12. Aim of this study was to explore the status of
reported pathogenic mutation as well as novel variants in South Indian
population.
Material and methods: Promoter region, exons and 3UTR of GJB2 gene
were screened to find mutations associated with autosomal recessive
nonsyndromic deafness from 50 families speaking Dravidian Malayalam
language. Primers were designed using primer Z software for sequencing
promoter, exons as well as 3UTR of GJB2 gene. Mutation surveyor
software was used to find changes in the sequence.
Results: Pathogenic mutations in the coding exon like W24X, R127H,
M163V were found in our study population. W24X mutation was the most
commonly found mutation causing Stop codon. Screening of 3UTR region
of GJB2 lead us to find a novel mutation in this region located 1031 bases
downstream of the gene causing a change from G to A. Bioinformatics
analysis using different miRNA prediction tool like Microsniper, mirSNP
suggest, this change causing a differential binding of miRna including hsa-
miR-924, hsa-mir-501-5p,hsa-mir-1225-3p,hsa-mir-558 and hsa-mir-615-3p.
Further it was revealed that this mutation indeed causes changes in
expression of this gene.
Conclusions: The present study could find a novel mutation in the 3UTR
region 1031bp downstream in GJB2. Bioinformatic analysis provides
evidence for functional implication of this mutation which might have a
role in pathogenicity of the disease. This study could also validate the
importance of reported mutations in our population with W24X to be the
common pathogenic mutation found in this population.
Page 53 of 59
P114
IL 10 gene -1082 G/A shows an associatoin with rheumatoid arthritis
patients of South Indian Population
EK Krishna Priya
1*
, Moinak Banerjee
2
, S Rajesh
3
, K Sasikala
1
1
Department of Zoology, Bharathiar University, Coimbatore;
2
Rajiv Gandhi
Center for Biotechnology, Trivandrum, India;
3
Department of Rheumatology,
KIMS Hospital, Trivandrum, India
Background: Rheumatoid arthritis (RA) is a systemic, chronic and inflamma-
tory joint disease of unknown etiology with genetic predisposition. This
disease is three times more frequent in women than men and is strongly
associated with the circulating auto-antibodies like rheumatoid factor (RF)
and anti cyclic peptide antibody (ACPA). The cytokine network dysfunction
plays a vital role in the pathogenesis of rheumatoid arthritis. The purpose of
the study was to understand the autoantibody specificities and its
association with Disease activity score Interleukin 10(-819 T/C, -592 A/C &
-1082 G/A) promoter polymorphisms in rheumatoid arthritis patients of
South Indian population.
Materials and methods: According to ACR criteria 1987, 168 rheumatoid
arthritis patients and 244 controls groups were selected as study population.
Genotyping of IL-10 promoter regions -819T/C and -592 A/C were
performed by PCR- Direct sequencing and -1082 G/A promoter
polymorphism by PCR- RFLP method.
Results: The results show that -1082 G/A promoter region shows a
significant association with rheumatoid arthritis patients in South Indian
population (genotypic p = 2 x 10
-4
& allelic p = 2 x 10
-5
). The disease
activity score were positively correlated with rheumatoid factor of RA
patients (p = 4 x 10
-2
).
Conclusions: The study concludes that the allele G of -1082 promoter
region predisposes susceptibility to Rheumatoid Arthritis. We also found a
strong association of positive rheumatoid factor with the disease
phenotype in RA patients.
P115
A case-control association study of K121Q and G/T Variants in ENPP1
and TCF7L2 gene with type 2 diabetes mellitus in North Indian Punjabi
Population
Basanti Barna
*
, Badaruddoza, Kawaljit Matharoo, Amarjeet Singh Bhanwer
Department of Human Genetics, Guru Nanak Dev University, Amritsar-
143005, Punjab, India
Background: The K121Q and G/T polymorphism of the ENPP1 and TCF7L2
genes respectively plays a significant role and found to be associated with
increased risk of T2DM in many worldwide populations. However, a very
few studies have been done for this association in North Indian Punjabi
populations. In the present cross section study K121Q polymorphism of
ENPP1 gene and G/T polymorphism of TCF7L2 gene were analysed to
determine the association of these genes with type 2 diabetic North Indian
Punjabi subjects.
Materials and methods: A total of 450 participants consisting of 239
T2DM and 211 healthy subjects were recruited for this study. Genomic DNA
was amplified by PCR-RFLP method. Anthropometric and physiometric
variables such as height, weight, Body Mass Index (BMI), Waist hip ratio
(WHR), Waist circumference (WC), Hip circumference (HC), biceps skinfold,
triceps skinfold, SBP, DBP, MBP, Pulse rate and pulse pressure were
measured using standard protocol. The Chi-square analyses were used to
test the significance difference in genotype, allele frequencies and Hardy-
Weinberg equilibrium deviation. Associations of genotypes with T2DM and
its corresponding Pvalues were calculated using Web-Assotest program.
Results: The results revealed that anthropometric and clinical
characteristics such as WHR, fasting and random glucose levels, SBP, DBP,
pulse rate and pulse pressure have significant (p<0.001) differences
between T2DM and control subjects. However, none of the anthropometric
and clinical characteristics have found significant difference with respect to
their genotypic distributions for both of genes except DBP for ENPP1
K121Q polymorphism.
Conclusions: The association analysis of the present results showed
K121Q variant of ENPP1 gene G/T variant of TCF7L2 gene have significant
association with type 2 diabetes with dominant and recessive model of
action respectively in North Indian Punjabi populations.
P116
A multifactorial dimensionality reduction model for gene
polymorphisms and environmental interaction analysis for the
detection of susceptibility for type 2 diabetic and cardiovascular
diseases
Badaruddoza
*
, Basanti Barna, Kawaljit Matharoo, Amarjeet Singh Bhanwer
Department of Human Genetics; Guru Nanak Dev University; Amritsar-
143005, Punjab, India
E-mail: doza13@yahoo.co.in
Background: The present study has presented a comparative gene-
environment interactions such as three genes (ENPP1-K121Q, TCF7L2-G>T
and GYS1 A1>A2) and six environmental factors (obesity and cardiovascular
related risk factors) for the detection of susceptibility for T2DM and CVD and
for the interpretation of epistasis involved in genetic studies of disease
susceptibility.
Materials and methods: The final sample size included 250 cases and
250 controls to focus on better methodological quality and a higher
statistical power analysis. All the interactions and approaches were carried
out using the methods of MDR.
Results: The MDR analysis showed the interaction between environmental
factor (SBP) and the genetic factor (ENPP1) for pooled and female T2DM
patients which indicated that SBP and TCF7L2 had significant contribution
on susceptibility to T2DM. The analysis showed that environmental factors
(BMI, WHR, WC, SBP, DBP and PR) and genetic factors (ENPP1-K121Q, TCF7L2
-G>T and GYS1 A1>A2) have identified risk factors and their interaction. All
these interactions were observed to be significant. The MDR method
showed all interaction models first to ninth order interactions for pooled
and male T2DM patients as significant for susceptibility of obesity. Whereas
in female T2DM patients, a first order (WHR) and third order (WHR * SBP *
ENPP1) have found a significant interaction for obesity. Both the genes
ENPP1 and TCF7L2 interacting with WHR and WC increase the susceptibility
of obesity many folds among T2DM patients and non-diabetic controls.
These results were also supported by dendrogram and interaction entropy
model. The three factor interaction model (BMI * SBP * ENPP1) for pooled
T2DM patients have been found significant for predicting hypertension in
T2DM patients whereas, in female T2DM patients all the interaction models
have been found as significant. However, third order model (SBP * TCF7L2 *
GYS1) have been found as a strong predictor for hypertension. In T2DM
male patients there has been no significant interaction observed for gene-
environment interaction although a seven factor model (BMI * WHR * WC *
SBP * PR * ENPP1 * TCF7L2) seems to be comparatively a good predictor for
hypertension.
Conclusions: The results showed that both the genes ENPP1 and TCF7L2
interacting with WHR and WC increase the susceptibility of obesity many
folds among T2DM patients and non-diabetic controls.
P117
Association of clock gene variants with Autism Spectrum Disorder in
South Indian population
Ann Mary Alex
1*
, PA Suresh
2
, Moinak Banerjee
1
1
Biotechnology, Kerala, India;
2
Institute for Communicative and Cognitive
Neuro Sciences, Kerala, India
Background: Autism Spectrum disorder is a group of neurodevelopmental
disorders that manifests in the first three years of life. Social impairments,
communication difficulties and repetitive/stereotyped behaviour are the
common symptoms of the spectrum. One of the major endophenotype
associated with the disease is circadian and sensory dysfunction. Circadian
dysfunction is mainly observed by difficulties in sleeping. Around 56-83% of
patients with ASD suffer from sleep problems. There is an endogenous
circadian clock that regulates the sleep and wakefulness, cognitive function,
systematic hormonal release and body temperature. We hypothesize that
Page 54 of 59
the genes functioning to maintain this molecular clock may be associated
with ASD either directly or by its transcriptional regulation of other genes.
Materials and methods: The study population was from the Malayalam
speaking population of Kerala. The patients were diagnosed and
characterized based on DSM-IV criteria. We selected 2 genes which are core
clock components, hCLOCK and PER3 due to its functional relevance. The
CLOCK gene is the first essential component of the mammalian clock and
was found to be associated with circadian rhythm sleep disorders. PER3
gene is implicated in delayed sleep phase syndrome and extreme diurnal
preference. Single Nucleotide Polymorphisms in both genes were studied
for association with the disease. Genotyping was done by sequencing and
PCR RFLP.
Results and conclusion: Genotypic and allelic frequencies of the SNPs
studied were analyzed to understand if there exists an association with the
disease. We could not find any association with the 8 polymorphisms
screened in hCLOCK gene in our population. However we were able to get
an association with a VNTR in PER3 gene, with the 5 repeat allele being
the risk allele. This is also the first report of PER3 gene being associated with
ASD.
P118
Skin miRNA profiling reveals differentially expressed miRNA signatures
from non-segmental vitiligo patients
Mohmmad Shoab Mansuri
1*
, Mala Singh
1
, Naresh C. Laddha
1
,
Mitesh Dwivedi
1
, Yogesh S. Marfatia
2
, Rasheedunnisa Begum
1
1
Department of Biochemistry, Faculty of Science, The M.S. University of
Baroda, Vadodara, Gujarat, India-390002;
2
Department of Skin and V.D.,
Faculty of Medicine, The M.S. University of Baroda, Vadodara, Gujarat, India-
390002
miRNAs are small conserved non-coding RNA molecules that post-
transcriptionally regulate gene expression by targeting the 3 UTR of
specific mRNA for degradation or translational repression. miRNAs have
been shown to be promising biomarkers for different diseases. At
present, the expression and function of miRNAs in human skin is largely
unknown. The aim of the present study was to detect the differentially
expressed miRNAs in non-segmental vitiligo (NSV) patients and to explore
the potential role of these miRNAs in vitiligo pathogenesis. We performed
the whole miRNA profiling of lesional as well as non-lesional skin from
four NSV patients and four healthy skin samples from controls using
TaqMan Low Density Array. Our results suggest that 38 miRNAs were
differentially expressed in the skin of patients compared to controls. We
identified 13 miRNAs which were significantly differentially expressed in
lesional skin of patients compared to healthy control skin. Further, 29
miRNAs were found to be significantly differentially expressed between
non-lesional skin of patients and healthy control skin. Interestingly, three
miRNAs were specifically down-regulated in the lesional skin compared to
non-lesional skin from patients with NSV. In conclusion, for the first time
the present study suggests the crucial role of differentially expressed
miRNAs in NSV patients from Gujarat.
P119
Gene copy number variation in Indian population and its implication in
health
Suhani Almal, Harish Padh
*
Cellular and Molecular Biology, B. V. Patel Pharmaceutical Education &
Research Development (PERD) Centre, Ahmedabad-380054, India
E-mail: hpadh@yahoo.com
Objectives: Copy number variations (CNV) are important source of
human genetic variation, found to be widely prevalent than what was
initially predicted. The involvement of CNVs in disease susceptibility and
drug response is well reported in other populations but has not been
studied to a larger extent in Indian population. The aim of the present
study was to evaluate the distribution of copy number variable genes in
Indian population and their link, if any, to health, disease and drug
response along with development of a comprehensive resource of CNV
frequency distribution among different populations.
Methods: A total of 100 280 healthy controls and in variable number of
patients from Indian population were genotyped. Genotyping of the copy
number (CN) variable genes was carried by PCR-based methodologies
(long range PCR, real-time PCR and PRT assay).
Results: An indicative correlation with disease susceptibility and significant
(p < 0.05) difference in the frequency distribution of the CNV variants was
observed in our study. MTUS1 deletion variant was found to be significantly
(p = 0.0207) associated with a decreased risk to breast cancer. A significant
association between CCL3L1 copy number and risk to HIV-1 was also
observed. The inter-ethnic comparison of CCL3L1 gene copy number
frequency demonstrated significant difference worldwide, being highest in
Africans. The common deletion frequency comprising LCE3B and LCE3C
genes was found to be highest in European and American populations. The
frequency of FCGR3B CN < 2 was found comparatively higher in Indians,
Americans and Africans as compared to European Caucasians. Thus, inter-
ethnic differences were observed among populations, highlighting varied
frequency distribution pattern based on their distinct geographical location.
Conclusion: This is our first attempt to create a comprehensive map of
the frequency distribution of CNVs and its association to disease
susceptibility in Indian population. This varied worldwide distribution can
thus be relevant from clinical or evolutionary point of view in future.
P120
Maternal gene polymorphisms of folate metabolism as genetic risk
factor for Down syndrome in North Indian population
Sushil Kumar Jaiswal
1*
, Ashok Kumar
2
, Vineeta Gupta
2
, Anjali Rani
3
,
Amit Kumar Rai
1
1
Centre for Genetic Disorders, Institute of Medical Sciences, Banaras Hindu
University, Varanasi-221005, India;
2
Department of Pediatric, Institute of
Medical Sciences, Banaras Hindu University, Varanasi-221005, India;
3
Department of Gynaecology, Institute of Medical Sciences, Banaras Hindu
University, Varanasi-221005, India
E-mail: sushil.biotech@yahoo.co.in
Down syndrome (DS), a chromosomal disorder has higher prevalence in
population occurring 1 in 700 live births. Recent reports have shown that
almost 92% of the DS children are born from young mothers, suggesting that
along with advanced maternal age some other risk factors are involved for
predisposition of mother to Down child. Polymorphism in genes involved in
folate metabolism as well as insufficient folic acid intake could result in
genomic instability, DNA hypomethylation and non-disjunction even resulting
in trisomy 21. In present study we compared the frequency of Thymidylate
Synthase (TYMS) 28 bp repeat polymorphism in 5UTR region, Cystathionine-
Beta-Synthase (CBS) 844ins68bp polymorphism and Solute Carrier (SLC) 19A1
G80A single nucleotide polymorphisms in 80 triads (mother, father and child)
and 77 matched control mothers in order to observe whether these variants
act as risk factors for DS. A significant association was observed for TYMS
5UTR 28 bp repeat with odds ratio 2.9 (95% CI 1.2-7.1, p=0.027). An
association which is very close to be significant was observed for SLC19A1
G80A with odds 2.01(95% CI 1.04-4.24, p=0.055). Heterozygosity for 68 bp
insertion at 844 in CBS showed significant association with odds ratio 10.5
(95% CI 1.29-85.1, p=0.019). Transmission disequilibrium test (TDT) for 28 bp
repeat polymorphism in TYMS gene presented more than four times greater
preferential transmission of maternal two repeats allele whereas paternal
three repeats allele had about 1.5 times higher rate of transmission. TDT for
SLC19 A1 G80A SNPs revealed preferential transmission of maternal A allele
more than two times greater as compared to G allele whereas paternal alleles
transmission didnt show much difference. The result shows that above three
polymorphism are significantly associated as a risk factor for predisposition of
mother to DS children in the North Indian population.
P121
Molecular basis of DYT1 and DYT6 primary dystonia in Indian patients
Subhajit Giri
1*
, Arindam Biswas
1
, Shyamal Kumar Das
2
, Kunal Ray
3
, Jharna Ray
1
1
S. N. Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, India;
2
Bangur Institute of Neurosciences & Psychiatry, Kolkata, India;
3
CSIR-Indian
Institute of Chemical Biology, Kolkata, India
Page 55 of 59
Background: Dystonia is the third most common movement disorder. It is
manifested by involuntary and sustained muscle contractions with frequent
twists and repetitive movement, abnormal posture and functional
impairment. Genetic factors play significant role for causing dystonia. Till
date twenty five loci (DYT1-DYT25) and sixteen genes have been reported
for dystonia. The present study reports the screening of TOR1A (DYT 1) and
THAP1 (DYT 6) among primary torsion dystonia patients of India.
Materials and methods: First, the most common GAG mutation (c. 904-
906/907-909 GAG; p. Glu302/303del) and the rs1801968 (p. Asp216His) in
TOR1A gene were screened following the published method (Naiya et al.,
2006). THAP1 was screened in 214 patients to identify mutations (mean age
of onset, 30.8 16.42 years) and 254 controls (mean age, 41.9 11.59 years).
All three exons and their flanking sequences including exon-intron
boundaries were screened by PCR , sequencing and/or RFLP analysis.
Results: A total of 321 patients were screened and GAG mutation was
identified in two brothers in a family suffering from primary generalized
dystonia. The analysis of rs1801968 (Asp216His) demonstrated that the minor
allele (216His) is significantly over represented in patients (p = 0.027; OR =
1.915; 95% CI = 1.058-3.451), suggesting a risk for the disease. On screening
THAP1 in 214 patients, a total of five nucleotide variants were identified
including a reported missense mutation (c.427 A>G; p.Met143Val), a novel
heterozygous deletion mutation (c. 208-209 del AA; p. K70VfsX15) in two
juvenile onset primary dystonia patients and a rare variant in 3 UTR region
(c. 1030 T>C) in a patient having Blepharospasm. In addition two SNPs,
(rs71521601 and rs111989331), were also found both in patients and controls.
The association study using rs111989331 (IVS2-87 A>G) demonstrate that the
major allele (A) can play as a risk factor (p = 0.001; OR = 1.977; 95% CI =
1.302-3.008) for primary dystonia.
Conclusion: Our preliminary results suggest that the TOR1A and THAP1
genes play significant role in the pathogenesis of Indian dystonia
patients. This is the first report on mutation detection in TOR1A and
THAP1 among Indian dystonia patients.
Acknowledgements: This study is funded by the Department of Science
& Technology (DST), Govt. of India, University Grant Commission (UGC),
Government of India.
P122
Role of Dopamine b Hydroxylase (DBH) in Parkinsons disease patients
of Indian population
Arunibha Ghosh
1
, Arindam Biswas
1
, Tamal Sadhukhan
1
, Shyamal Kumar Das
2
,
Jharna Ray
1*
1
S. N. Pradhan Centre for Neurosciences, University of Calcutta, Kolkata, India;
2
Burdwan Medical College & Hospital, Burdwan, India
E-mail: jharnaray@gmail.com
Background: Parkinsons disease is a neurodegenerative disease affecting
at least 1% of the population over age of 55. It is characterized by selective
loss of dopaminergic neurons in substantia nigra pars compacta and the
appearance of intracellular inclusions termed Lewy bodies. As the age
progresses, neurons in the other regions of brain are also degenerated.
Depletion of brain dopamine initiates aberrant motor activities including rest
tremor, rigidity, bradykinesia, postural instability. Apart from motor
symptoms, cognitive impairments, like depression, psychiatric illness, and
decreased mental ability are also observed in the patients. Genetic and non-
genetic components are believed to govern the pathogenesis of PD. Genes
in dopaminergic pathway & also dopamine synthesis, storage, binding,
metabolism are needed to be studied which seem to be major determinants
conferring differential risk of developing PD.
Methods: In this study, three reported SNPs, rs1611115 (T>C), rs1108580
(A>G), rs129882 (C>T) of DBH have been analyzed. For rs1611115 (T>C), 229
patients & 252 controls have been screened. A total of 298 patients & 276
control samples have been analyzed for rs129882 (C>T). A total of 378
patients & 253 control samples have been analyzed for rs 1108580 (A>G). All
these SNPs were screened by PCR , sequencing and/or RFLP analysis. Also,
DBH activity has been measured in plasma isolated from patients blood.
Results: For rs1611115 (T>C), the minor allele T is over represented in
control samples & pose a protection (p=0.043, OR=0.731, 95% CI=0.731
(0.538-0.847) for this disease. No significant association has been found for
rs1108580 (A>G). For rs129882 (C>T), the minor allele T is over represented
in patient pool & confers a risk (p=0.036, OR=1.322, 95% CI=1.012-1.727)
for this disease. DBH activity has been measured in plasma isolated from
patients blood. A correlation has been found between plasma DBH activity &
rs1611115.
Conclusion: This study suggests that DBH might have a role in
susceptibility of developing Parkinsons disease.
Acknowledgements: This study is funded by the Department of Science
& Technology (DST), Goverment of India, University Grant Commission
(UGC), Goverment of India.
P123
Beta thalassemia prevention in India: evaluation of socio- cultural
factors
Swati Chawla
*
, Rajnish Singh, VR Rao
Department of Anthropology , University of Delhi, India
E-mail: Swatichawla2011@gmail.com
Aim and background: There are approximately 240 million people
across the world who are heterozygous for Beta thalassemia and 200,000
affected homozygous are born annually.
Prevention is a big challenge.
But the situation in India is different. Social, cultural, and religious issues
are found to be closely intertwined with Beta thalassemia prevention. In
spite of 10,000 annual Beta thalassemic births, the situation here is not
well combated. Social stigma and negative attitude are largely understood
to be the bounding factors.
The present study examines the prevention issue in the high risk
community of India, analyzing their knowledge and perceptions in the
light of available health models.
Methods: A semi structured interview schedule was framed and imple-
mented in the rural and urban (Delhi) population of a high risk community i.e.
ARORAS and compared with the cosmopolitans (not at high risk).
Results: Correct knowledge of carriers is a very important criteria, which
is a major factor for the prevention of Beta thalassemia in a multi ethnic
country like in India.
Participants were found to carry positive attitude towards the public
perception of Beta thalassemia. In general, social discomfort were not a
serious issue, but acceptance of life partner with Beta thalassemia trait
was unacceptable among all the three populations but more among the
rural Aroras which showed, lack of knowledge among them.
The acceptability for prevention strategies and its implication was high
among all the three populations but rural Arora was found to be more
proficient towards premarital screening(80.8%) and prenatal diagnosis
(98.0%).
Conclusion: A lack of knowledge about the disorder, its manifestations,
survival rate, treatment availability, and psychosocial and cultural issues
have created barriers to optimal health care including disclosure of Beta
thalassemia status as well as to carrier testing. To overcome the problem
there is an urgent need to have knowledge about peoples perception to
find a common platform to harmonize various approaches for prevention.
PRENATAL DI AGNOSI S
P124
Prenatal diagnosis of Tay-Sachs disease: our institutional experience
Jayesh Sheth
1
, Mehul Mistri
1*
, Frenny Sheth
1
, Sarita Gupta
2
1
FRIGEs Institute of Human Genetics, FRIGE House, Jodhpur Gam road,
Satellite, Ahmedabad-380015, Gujarat, India;
2
M.S.University, Vadodara, India
Introduction: Tay-Sachs disease (TSD) is the second most common
storage disease after Guacher disease in the group of lipid storage
disorders in India. In absence of any therapeutic option, prenatal
diagnosis is the only way to prevent the disease burden.
Aims and objectives: The present investigation was undertaken to
provide cost effective prenatal diagnosis of TSD by enzyme based study
from cultured chorionic trophoblast (CT)/uncultured CV or cultured
amniotic fluid cells (AF) and further confirmation by molecular analysis of
HEXA gene.
Page 56 of 59
Material and methods: Prenatal diagnosis was carried out in six women
having confirmed index case with TSD was enrolled in this study. Enzyme
study and molecular analysis was carried from CT in 3 women at 11-13
weeks of gestational age while in remaining 3 women cultured AF cells
were used at 16 weeks of gestation. The b-Hexosaminidase-A activity was
carried out with synthetic substrates, 4-methylumbelliferyl-6-sulfo-N-
acetyl-beta-glucosaminide (4-MUGS) and DNA-based analysis was carried
out by the bidirectional sequencing of HEXA gene. Written informed
consent was obtained from all patients.
Results and discussion: Deficiency of b-Hexosaminidase-A enzyme activity
[21.7 nmol/hr/mg protein (NR: 2711621 nmol/hr/mg protein)] from CT cells
was detected in one case and mutation study observed homozygous
mutation of 4bp insertion at c.1277_1278insTATC in the CT same as index
case. Whereas, remaining 2 enzymatically normal fetuses were normal for
earlier identified index case mutations [Homozygous mutation D322N and
compound heterozygous mutations (D322N/c.1277_1278insTATC)
respectively]. The enzyme activity was carried out from cultured AF cells in
three women and deficiency of b-Hexosaminidase-A enzyme activity of 23.4
nmol/hr/mg protein (NR:271-2213.2 nmol/hr/mg protein) with homozygous
mutations E114K in one case; intermediate enzyme activity of 247.2 nmol/
hr/mg protein (~50% of mean) having heterozygous mutation D322N in one
case and normal enzyme activity (670.5 nmol/hr/mg protein) was detected
in one subject also showed absence of W485X mutation which was present
in the index case.
Conclusion: This study clearly demonstrates that for storage disorders like
Tay-Sachs enzyme analysis from cultured/ uncultured CV and cultured AF
provide highly reliable information for prenatal study and can be used as
with high sensitivity and specificity for prenatal diagnosis of the disease
where index case has confirmed diagnosis proven by enzyme activity or by
molecular analysis.
P125
Prenatal diagnosis of autosomal recessive osteopetrosis: a case report
Mehul Mistri
1
, Harsh Patel
1
, Tanmay Tanna
2*
, Chitra Ankleshwaria
1
,
Frenny Sheth
1
, Jayesh Sheth
1
1
Institute of Human Genetics, FRIGE House, Ahmedabad-15, Gujarat, India;
2
National Institute of Technology Warangal, Andhra Pradesh 506004, India
Background: Autosomal Recessive Osteopetrosis (ARO) or Malignant
Infantile Osteopetrosis (MIOP) is a congenital disorder characterized by
increased bone density on radiographs. It is known to be caused by
mutations that cause loss of function in the TCIRG1, CLCN7 or OSTM1 genes.
Classic symptoms include bone marrow failure, visual and hearing
impairment, pancytopenia and extramedullary hematopoiesis leading to
hepatosplenomegaly. The disease is lethal in infancy and the only curative
treatment known is Hematopoietic Stem Cell Transplantation (HSCT).
Prenatal diagnosis is done either by autoradiography or by identifying
disease causing mutation.
Case presentation: We present a family with consanguineous marriage
with a male child born at full term with an uneventful incident and normal
Apgar score. The child had normal milestones of development till two years
of age. After this, he developed symptoms such as low platelet, low
hemoglobin, splenomegaly and hydrocephalus. The patient was started with
fortnightly blood transfusions. Clinically he was suspected to have
osteopetrosis and investigated for mutations in TCIRG1 gene. Targeted exon
sequencing identified the patient to be homozygous for a novel nonsense
pathological mutation p.R426X (c.1276 C>T) in TCIRGI gene. He expired in
the course of treatment at the age of 2.5 years. Subsequently, both parents
were found to be heterozygous carriers for the same mutation. Prenatal
counseling was advised for any future pregnancies. In the subsequent
pregnancy, prenatal testing was done at 11 weeks of gestation using
uncultured chorionic villus sample (CVS) to investigate for the
aforementioned mutation. Analysis of genomic DNA obtained from
uncultured CV has shown heterozygous status for TCIRGI p.R426X (c.1276
C>T) mutation. The family was informed of the carrier status of the fetus
and their psychological trauma and stress was alleviated.
Conclusions: Prenatal Diagnosis of Malignant Infantile Osteopetrosis
(MIOP) through targeted sequencing of genomic DNA obtained from
uncultured CV can be carried out once the mutation has been identified
in an index case. This novel mutation also provides a new insight into the
molecular basis of the disease which can be utilized for molecular
diagnosis.
P126
Prenatal diagnosis of lysosomal storage disorders: our experience in
120 cases
Mehul Mistri, Nrupesh Oza
*
, Frenny Sheth, Jayesh Sheth
FRIGEs Institute of Human Genetics, FRIGE House, Ahmedabad-380015,
Gujarat, India
Background: Molecular study is considered to be the gold standard for
single gene disorders. For Lysosomal storage disorders (LSDs), plethora of
literature report indicates the use of lysosomal enzyme activity in uncultured
chorionic villus sample (CV), cultured chorionic villus (CT) or cultured
amniotic fluid cells (AF) as a reliable tool for prenatal diagnosis (PD) followed
by mutation study as a confirmation where mutation is known and/or
unequivocal enzyme activity is observed. Very few studies are available from
India with anecdotal reports using either molecular methods or enzyme
study in PD of LSDs. Nonetheless considering the reported prevalence of
1:5,000-7,000 live birth and limited availability of therapeutic option PD
remains the only preventable cure for storage disorders.
Aim: To establish prenatal diagnosis of LSDs and demonstrate the reliable
use of lysosomal enzyme study for prenatal diagnosis.
Material and method: One hundred twenty pregnancies with confirmed
index case of LSD were selected for lysosomal enzymes study from CV, CT
and AF. Written consent was obtained from guardian of the study subjects.
Results: Of 120 pregnancies, 57 (47.5%) were normal, 8 (6.66%) had an
intermediate enzyme values and rest 55 (45.8%) were found to be affected
with specific LSD. From the affected fetuses, 11 (9.1%) were affected with
MPS-I, 2 (1.7%) with MPS-II, 1 (0.8%) with MPS-IIIA, 1 (0.8%) with MPS-IIIB,
4 (3.4%) with MPS-IVA, 2 (1.7%) with MPS-VI, 7 (5.8%) with GM-1
gangliosidosis, 1 (0.8%) with Gaucher, 3 (2.5%) with Tay-sachs, 2 (1.7%) with
Sandhoff, 1 (0.8%) with NPD-A/B, 1 (0.8%) with MLD, 9 (7.5%) with Krabbe,
4 (3.4%) with Pompe, 2 (1.7%) with Batten and 4 (3.4%) with Mucolipidosis-
II/III. All affected fetuses have shown very low enzyme activity (~0-15% of
normal mean). We have also identified 8 (6.66%) pregnancies with
intermediate enzyme activity (~50% of mean) this includes 3 (2.5%) fetuses
were carrier with Sandhoff, 2 (1.7%) with Mucolipidosis-II/III, each one (0.8%
for each) with MLD, NPD-A/B and Tay-sachs disease. Except one case of
MPS-IVA who was found to have carrier (~30% enzyme activity) during
prenatal diagnosis was found to be affected with MPS-IVA after delivery.
Conclusions: Prenatal diagnosis of LSDs can be made with equal
sensitivity and specificity of the molecular study using CV, CT and AF.
Nonetheless carrier identification needs to be confirmed by molecular
analysis.
P127
Application of Chromosomal Microarray and Multiplex Ligation-
dependent Probe Amplification in prenatal diagnosis
Pankaj Sharma
1*
, Madhumita Roy Chowdhury
1
, Neerja Gupta
1
, Rashmi Shukla
1
, Shruthi Sudarshan
1
, Manju Ghosh
1
, Deepika Deka
2
, Madhulika Kabra
2
1
Genetics Unit, Department of Pediatrics, All India Institute of Medical
Sciences, New Delhi, India;
2
Department of Obstetrics & Gynecology, All
India Institute of Medical Sciences, New Delhi, India
Background: Chromosomal Microarray (CMA) and Multiplex Ligation-
dependent Probe Amplification (MLPA) are relatively newer techniques for
detecting cryptic copy number variations (CNVs). Here we are presenting
the data of eight families where CMA and MLPA were used for prenatal
diagnosis (PND) in view of their previous child with Intellectual disability/
developmental delay (ID/DD) and normal karyotype.
Methods: Families with ID/DD children were referred for genetic counseling
in Genetics Clinic, Department of Pediatrics, AIIMS. CNVs were studied in the
probands using Illumina Cyto-SNP12 chips and MLPA kits for subtelomeric
screening and microdeletion syndrome (P036 and P064) and PND by
chorionic villus biopsy or amniotic fluid was performed in cases with
pathogenic CNVs.
Page 57 of 59
Results: In eight families, CNVs of clear pathogenic significance were
identified using CMA and confirmed by MLPA in seven cases. The
probands had 2q32.3q33.3, 22q11.2, deletion in interstitial region while
subtelomeric CNVs were 9q34 deletion, 8p deletion and 12p duplication,
5p deletion and 8q duplication in two affected sibs, 7q deletion and 10q
duplication, 5p deletion and 6q duplication, 7q deletion and 20p
duplication respectively. PND by MLPA (six cases) and CMA(two cases)
were done. In six cases, fetus was found to be normal. In one case fetus
had 8p deletion and 12p duplication while in another, the fetus had 6q
deletion and 5p duplication.
Discussion: CMA and MLPA have clearly demonstrated an increased
resolution and improved detection rate of CNVs. CMA detects CNVs across
the genome at high resolution enabling precise breakpoints of the CNVs.
MLPA is a cost effective technique for developing countries but only limited
(upto 45) nucleic acid targets in the genome can be investigated. In our
settings it is an efficient technique for confirmation of CNVs and PND.
STEM CELLS
P128
Exposure to ionizing radiations can cause hazardous effects on
differentiation of human CD34+ hematopoietic stem cells
Angshuman Biswas
1*
, Asiti Sarma
2
, Subrata Kumar Dey
1
1
Stem Cell Research Laboratory, West Bengal University of Technology,
Kolkata 700 064, India;
2
Radiation Biology Lab, Inter University Accelerator
Centre, New Delhi- 110 067, India
E-mail: angshuman.in@gmail.com
Statement of purpose: The objective of this study is to investigate effects
of ionizing radiation on human hematopoietic stem cell differentiation.
Background: Despite being a strong mutagen, causing several genetic
and chromosomal aberrations, ionizing radiation in the form of gamma
rays and heavy ion radiation is becoming increasingly important in medical
therapies and cancer. Heavy ions especially the particle beam of carbon ion
are high energy radiations that is considered to be extremely hazardous
during occupational radiological emergencies, manned space missions,
high altitude flights, accidents casualties etc. Healthy blood cells in human
body arise by the differentiation of a very small population of pluripotent
hematopoietic stem cells that have the capacity of self renewal throughout
their lifespan. Effects of radiation whatsoever minimum on these unique
cells thus have greater impact on the human system in both long term
and short term scenario which might currently lead to an idea of a new
concept leading to cancer stem cells.
Methods: Umbilical cord blood collected in-utero was subjected to
immuno-magnetic enrichment, followed by flowcytometric estimation for
stem cell content. Isolated hematopoietic stem cells were then cultured into
specialized cytokine based medium and exposed to various doses of
ionizing radiation for investigations on CD34+ marker based flowcytometric
survival assay, differentiation & clonogenic potential by CFC assay,
genotoxicity and cytotoxicity.
Results and conclusion: Exposure to different doses of ionizing radiation
showed a marked difference in the survival of CD34+ stem cells in both
dose and time dependant manner. Differentiation of stem cells into their
adult progenitor cells were also altered with varying doses of radiation
treatment. Our findings show variations on response of the human CD34+
stem cells when exposed to ionizing radiation which is significantly
comparable to normal blood lymphocytes and cancerous K562 cells.
THERAPEUTI CS
P129
Impact of Vedic Chants Intervention Programme on Autistic Spectrum
Disorder
Kandasamy Dinesh Kumar
1*
, Sushruth Badhe
1,2
, Sathiyavedu Santhiya
1
1
Dept of Genetics, Dr ALM PG IBMS, University of Madras, Taramani Campus,
Chennai, India;
2
Midham charitable trust, Puduchery, India
E-mail: dineshkumar.genetics@gmail.com
Background: Autism Spectrum Disorder (ASD) is a developmental
disorder that affects the behavior and social communication of the child.
In India, awareness about Autism coupled with shortage of skilled
professionals poses severe constraints in management of children with
ASD. There arises a need to explore the avenues of alternate therapy in
the form of Group Therapy. A 44-bp insertion/deletion polymorphism in
the promoter region of the serotonin receptor gene 5-HTT gene
(5-HTTLPR) has been identified to modify the transcription rate of the gene
(Lesch et al., 1996; Greenberg et al., 1999). The short allelic variant has
been shown to reduce 5-HTT transcription resulting in diminished 5-HTT
levels and reduced serotonin (5-HTT) reuptake into human platelets. We
speculate the possibility of altered hormone levels (Melatonin and
Serotonin) in individuals with ASD.
As per earlier reports an association between 5-HTT gene polymorphism
and autism has been indicated. Recent evidences from clinical and
neuroscience research suggests an approach based on yogic principles
and meditative tools is worth experimenting in children with autism.
The same could be addressed in ASD individuals to ameliorate the drastic
effect of these hormones on ASD individuals.
Methods: A single group pre-/ post- test study design was employed.
With the aid of a social and behavioral assessment scale for ASD, fifteen
children with ASD between the age group of 7 - 14 years were evaluated
for assessing the effect of the Vedic Chants Intervention Program
recruited from special schools in and around Chennai. Chanting of select
Sanskrit Mantras by an experienced meditator were given as an auditory
stimulus in a closed room to the children for a period of 20 min daily at
a fixed time. Alterations in the endocrine profile (Melatonin and
Serotonin) were measured using ELISA Kits. Polymorphism in serotonin
transporter (5-HTT) is to be analyzed to correlate the endocrine findings.
Results: Study shows that there is a difference in the pre & post-test mean
achievement scores due to the Vedic Chants Intervention Program. Some of
the qualitative changes observed by the special educators were a reduction
in the hyper activity, self-biting and other common traits of ASD like head
nodding, hand flapping etc. This reflected in their class room sitting as well.
Some of the children were remembering certain lines of the mantras and
imitating the sound Om and responding to the Namaste gesture. Most of
the children including those with ADHD sat throughout the entire session.
The endocrine profile of the subjects was altered.
Conclusions: The auditory stimulus has the potential to provoke the
cognitive abilities of the children. Vedic Chants therapy might be one of
the efficient Group therapies for the management of children with ASD.
P130
Molecular basis of lysosomal storage disorders in India
Shweta P Kondurkar
1*
, Parag Tamhankar
1
, Pratima Kondurkar
1
, Ashwin Dalal
2
,
Jayesh Sheth
3
1
ICMR Genetic Research Centre (NIRRH), Mumbai, India;
2
Center for DNA
Fingerprinting and Diagnostics, Hyderabad, India;
3
Foundation for Research
in Genetics and Endocrinology, Ahmedabad, Gujarat, India
E-mail: shwetaprakashkondurkar@yahoo.com
Background: Lysosomal Storage Diseases(LSD) are a group of rare
recessive inherited metabolic disorders that result from the deficiency of
a single enzyme required for the metabolism of lipids, glycoproteins or
mucopolysaccharides. There is a lack of data on molecular basis of LSDs
in India . The current study involves molecular analysis of patients with
Tay Sachs disease(TSD) (HEXA gene), Sandhoff disease(SD) (HEXB gene),
Gaucher disease (GD) (GBA gene), GM1 gangliosidosis (GG) (GLB1 gene),
metachromatic leukodystrophy (MLD) (ARSA gene), and Pompe disease
(PD) (GAA gene).
Materials and methods: Patients presenting during the year 2011-2013
with characteristic clinical features of the above LSDs underwent specific
biochemical testing (leucocyte enzyme assay) followed by sequencing of the
respective gene. Enzyme assays performed include total hexosaminidase
and hexosaminidase B (TSD and SD), glucocerebrosidase (GD), beta
galactosidase (GG), arylsulphatase A (MLD) and alpha glucosidase (PD). The
molecular basis of disease in patients with enzyme deficiency was
confirmed by bidirectional Sanger sequencing covering all the exons and
exon-intron boundaries of the respective genes.
Page 58 of 59
Results: During the study period, 131 unrelated families across India were
studied. These included 47 families of TSD, 36 families with SD, 15 families
with GD, 8 families with GG, 15 families with MLD, and 10 families with PD.
Ninety two families (70. 2 %) families showed consanguinity. The age range
for patients was between 3 months to 7 years; however, one patient with
PD was adult (21 years). The youngest patient had PD. The study identified
218 mutant alleles in 119 patients. Only ten alleles were recurrent. Founder
mutation was identified in TSD patients from Gujarat (p.E462V) which will
help screening in patients from this state. Also in SD, the mutation hotspot
R284X represented 23.3 % of alleles (7/30). No mutations could be identified
in 12 patients and the second mutation could not be identified in 10
patients, despite being biochemically confirmed.
Conclusion: The study shows allelic heterogeneity in Indian patients with
LSDs. A molecular screening strategy for the common mutations could be
adopted for TSD and SD patients.
P131
Genome-wide analysis identifies common CNVs associated with
primary open angle glaucoma
Lalit Kaurani
1
, Mansi Vishal
2
, Dhirender Kumar
3
, Bharati Mehani
1
,
Charu Sharma
4
, Anchal Sharma
1,5
, Debasis Dash
3
, Jharna Ray
6
, Abhijit Sen
7
,
Kunal Ray
2,5
, Arijit Mukhopadhyay
1,5*
1
Genomics & Molecular Medicine, CSIR-Institute of Genomics & Integrative
Biology, Delhi, India;
2
Molecular & Human Genetics Division, CSIR-Indian
Institute of Chemical Biology, Kolkata, India;
3
G. N. Ramachandran
Knowledge Centre for Genome Informatics, CSIR-Institute of Genomics &
Integrative Biology, Delhi, India;
4
Mathematics Department, School of Natural
Sciences, Shiv Nadar University, Uttar Pradesh, India;
5
Academy of Scientific
and Innovative Research (AcSIR), Delhi, India;
6
S. N. Pradhan Centre for
Neurosciences, University of Calcutta, Kolkata, India;
7
Drishti Pradip Eye Clinic,
Kolkata, India
E-mail: arijit@igib.in
Background: Copy number variation (CNV) is one of the major factors
contributing to genomic diversity and diseases. Glaucoma is a major
neurodegenerative disease causing irreversible vision loss across the
globe. We wanted to analyze the impact of common CNVs in a genome-
wide scale in patients of primary open angle glaucoma (POAG) collected
from the West Bengal, India.
Method: Genome-wide data was generated on 364 POAG cases and 365
controls on Illumina 660W-Quad arrays and CNVs were called using
PennCNV. Copy number variant regions (CNVRs) were analyzed for
association. A publicly available dataset of POAG cohort of 866 cases and
495 controls from Caucasian origin (GLAUGEN study) was used as a
validation cohort. Representative CNVs were validated using real-time PCR.
Results: We analyzed genome-wide CNV from 1928 samples. After
association analysis we found 308 significantly associated (p<0.05) CNVRs
in the Indian data. These POAG associated CNVRs were enriched in
nervous system development. 113 CNVRs (37%) were significantly
associated with the Caucasian data set. These contain 5 genes previously
reported in eye diseases, namely, IDUA, FOXE3, NDUF7, PRPF6 and WNT3.
We also found 6 associated CNVRs in previously known glaucoma loci.
Conclusion: We have shown that common CNVRs are significantly
associated in both datasets irrespective of the population background. We
have also identified candidate genes/regions which are uniquely present in
POAG cases and absent in controls. Our data might provide new insights
into role of CNV in pathogenesis of POAG.
P132
Microarray based global transcriptome profiling reveals involvement of
non-Hsa21 genes and microRNAs in molecular mechanism of Down
syndrome pathogenesis
Ashutosh Pathak
*
, Divya Agarwal, Shubha R Phadke
Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of
Medical Sciences, Lucknow, India
E-mail: ashutoshsgpgi@gmail.com
Down syndrome (DS), the most frequent genetic disorder leading to mental
retardation is caused by partial/complete triplication of human chromosome
21 (Hsa21). The differential expression of genes located on extra
chromosome 21 is assumed responsible for phenotypic abnormalities but
this gene dosage hypothesis has not been assessed on genome-wide basis.
The gene expression patterns related to phenotypic abnormalities may
provide insights into their roles in DS pathogenesis. To analyze the
differential gene expression and understand the molecular mechanism
underlying pathogenesis of DS, we performed global gene expression
profiling in blood samples of 14 DS and 4 normal subjects using human
whole transcriptome microarray. The microarray analysis revealed total of
624 genes (195 upregulated and 429 down regulated) were differentially
expressed in DS patients as compared to control. Out of the genes present
on chromosome-21, a total of 210 genes were differentially expressed
ranging from 1.5 to 5 fold compared to normal individuals. Genes involved
in physiological pathways such as apoptosis and cell cycle regulation, signal
transduction, cell maturation, and immunity showed dysregulation. Several
genes localized on Hsa21 such as APP, SOD1, DYRK1A, COL6A1 showed
differential expression and the levels were conserved across all DS subjects.
Interestingly, several non-Hsa21 genes such as RCAN3 (Hsa1), ANK3 (Hsa10),
CDK17 (Hsa12) etc., having roles in cardiogenesis, signal transduction and
differentiation of neurons showed conserved levels of expression across the
DS subjects. Further to investigate the role of microRNAs (miRNAs) in
regulation of gene expression, global miRNA profiling was performed in 4
DS patients and 1 control using Affymetrix miRNA 3.0 array. Several Hsa21
miRNAs like miR-99a, let-7c, miR-125b-2, miR155, miR-802 showed
overexpression effecting the regulation of genes involved in DS
pathogenesis. Our results substantiate that involvement of non-Hsa21 genes
and miRNAs provide etiological basis for abnormal phenotypes during DS
pathogenesis. Our data lead to more systematic understanding of molecular
mechanism underlying DS pathogenesis and identification of miRNA based
therapeutic targets for better management of the disease.
Cite abstracts in this supplement using the relevant abstract number,
e.g.: Pathak et al.: Microarray based global transcriptome profiling
reveals involvement of non-Hsa21 genes and microRNAs in molecular
mechanism of Down syndrome pathogenesis. Molecular Cytogenetics
2014, 7(Suppl 1):P132
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