Overview of Clinical Cytogenetics

UNIT 8.1

Patrick R. Gonzales,1 Andrew J. Carroll,1 and Bruce R. Korf2

1

Cytogenetics Laboratory, Department of Genetics, University of Alabama at Birmingham,

Birmingham, Alabama
2
Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama

Chromosome analysis is one of the first approaches to genetic testing and

remains a key component of genetic analysis of constitutional and somatic genetic disorders. Numerical or unbalanced structural chromosome abnormalities
usually lead to multiple congenital anomalies. Sometimes these are compatible
with live birth, usually resulting in severe cognitive and physical handicaps;
other times they result in miscarriage or stillbirth. Chromosome rearrangements
also occur as somatic changes in malignancies. Identification of constitutional
chromosomal anomalies (anomalies present in most or all cells of the body
and/or the germline) can provide important information for genetic counseling.
In this unit, we introduce chromosomal microarray analysis (CMA), which is
a relatively recent addition to cytogenetic technologies, and has become the
recommended first-tier testing method for patients with developmental delay,
intellectual disability, autism, and/or multiple congenital anomalies. We also
discuss non-invasive prenatal testing/screening (NIPTS), which uses circulating cell-free fetal DNA (cfDNA) from maternal plasma to rapidly screen for
autosomal and sex-chromosome aneuploidies. Cytogenetic analysis of tumors
is helpful in diagnosis and in monitoring the effects of treatment. The protocols
in this chapter cover the clinical study of chromosomes in nonmalignant tissues.

C 2016 by John Wiley & Sons, Inc.
Keywords: cytogenetics r chromosomes r microarray r FISH r prenatal r
postnatal r constitutional
How to cite this article:
Gonzales, P.R., Carroll, A.J. and Korf, B.R. 2016. Overview of clinical
cytogenetics. Curr. Protoc. Hum. Genet. 89:8.1.1-8.1.13.
doi: 10.1002/0471142905.hg0801s89

INDICATIONS FOR ANALYSIS

Chromosome analysis can be performed
prenatally to provide information about the developing fetus or postnatally to aid in diagnosis of individuals with congenital anomalies,
mental retardation, or recurrent miscarriage.

Indications for Prenatal Studies

Abnormal biochemical screening

Maternal serum -fetoprotein (AFP) testing has been used for many years to screen for
open neural tube defects in the fetus (Wald and
Cuckle, 1979). Low levels of AFP have been
found to be associated with an increased risk of
Down syndrome as well (Cuckle et al., 1984;
Merkatz et al., 1984; DiMaio et al., 1987).
In addition, testing of maternal serum levels of unconjugated estriol (UE3) and human

chorionic gonadotrophin (hCG) adds to the

sensitivity and specificity of detection of Down
syndrome (Wald et al., 1988; Canick, 1990;
Haddow et al., 1992). UE3 tends to be decreased and hCG increased if the fetus has
Down syndrome. Currently, second trimester
maternal quad screening using maternal serum
AFP, hCG, UE3, and inhibin-A yields an 80%
detection rate for Down syndrome and 60% for
trisomy 18 at a 5% false positive rate. Similarly, first trimester screening for hCG, plasma
protein A, and fetal nuchal translucency yields
an 85% detection rate for Down syndrome and
90% for trisomy 18 at a 5% false positive rate
(Hardisty and Vora, 2014). Maternal serum
screening can be used to identify women under
age 35 years who are at increased risk of carrying a fetus with Down syndrome, and may
identify women over age 35 years whose risk

Current Protocols in Human Genetics 8.1.1-8.1.13, April 2016

Published online April 2016 in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/0471142905.hg0801s89
C 2016 John Wiley & Sons, Inc.
Copyright 

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is low. Trisomy 18 is also associated with abnormal values of these tests (low AFP, UE3,
and hCG), although the a priori likelihood of
trisomy 18 is lower than that of trisomy 21.

Abnormal ultrasound results

It is common to perform one or more ultrasound examinations during pregnancy to monitor fetal growth and well being. When anomalies are detected, cytogenetic studies may be
performed to determine if a chromosome abnormality is present. Specific ultrasound findings, such as increased thickness of nuchal
skin folds or femur-length shortening, have
been correlated with an increased risk of a fetus having Down syndrome (Benacerraf et al.,
1987; Winston and Horger, 1988; Grist et al.,
1990; Nyberg et al., 1990; Lockwood et al.,
1991). Most malformations detected by ultrasound are not specific for a particular chromosome anomaly but provide a general indication
for examining fetal chromosomes. Before 20
weeks gestation, examination usually involves
amniocentesis. After 22 weeks, there may be
insufficient time to grow and analyze amniotic fluid cells, but fetal blood can be obtained
by umbilical vein sampling under ultrasound
guidance. This can be useful even late in pregnancy when termination may no longer be an
option, because accurate diagnosis of a fetal
problem can help with planning for the childs
medical needs.

Advanced maternal age

Overview of
Clinical
Cytogenetics

Historically, the most common reason to

pursue prenatal cytogenetic study was advanced maternal age. Currently, this indication is modified with an individualized risk
assessment based on maternal age, gestational
age, and the aforementioned ultrasonographic
and biochemical markers (Tabor and Alfirevic, 2010). Risk of fetal trisomy increases
with maternal age. This is particularly well
documented for trisomy 21 but has also been
demonstrated for trisomies 13 and 18 (Trimble and Baird, 1978; Hook et al., 1983; Hook,
1990). The risk of trisomy 21 (Down syndrome) rises steeply after age 35 years, when
the risk of bearing a liveborn child with this
syndrome is 1 in 324, compared with a risk of
1 in 1000 to a 20-year-old mother. The risk of
Down syndrome is higher at 15 to 18 weeks
gestation, when amniocentesis is usually performed, than at birth, because of the high likelihood of miscarriage of a trisomic fetus. The
risk of trisomy at this gestational age (1:270)
approximately matches the risk of miscarriage
due to amniocentesis (1:200); therefore, it is

common obstetrical practice to discuss prenatal diagnostic options with pregnant women
aged 35 years or older. The existence of a
weak paternal age effect on the incidence of
trisomy has been suggested, but supportive evidence is less convincing (Hook, 1987; Stene
and Stengel-Rutkowski, 1987).

Family history
A number of factors in a family history may
indicate increased risk of a fetal chromosome
abnormality. For example, one parent may
have a balanced translocation. Such rearrangements are usually ascertained in the parent of a
child with an unbalanced karyotype and multiple congenital anomalies. A balanced translocation carrier is at increased risk of producing
unbalanced gametes and having a child with
congenital anomalies (Daniel et al., 1989). The
risk differs depending on the rearrangement,
but prenatal detection of such a chromosome
rearrangement is usually straightforward.
It is important to be alert to the possibility
of a chromosome rearrangement if there is a
family history of a child with a chromosome
anomaly. This applies even if the propositus
has Down syndrome, as a small proportion of
individuals with Down syndrome have translocation of chromosome 21 to another chromosome. If a couple seeks counseling and a relative has Down syndrome, records should be
sought to determine whether the Down syndrome was due to free trisomy or translocation. If such information cannot be obtained,
chromosomal analysis should be done on the
parent whose relative is affected to rule out
a balanced translocation. A couple who has a
child with trisomy has an empiric risk of recurrence of 1% (Lister and Frota-Pessoa, 1980).
This risk exceeds the risk of amniocentesis,
so such couples should be offered prenatal
testing.

Recurrent miscarriage
Balanced chromosome rearrangements can
lead to the production of gametes with unbalanced karyotypes. Most embryos with unbalanced karyotypes are nonviable and are spontaneously aborted during the first trimester
(Boue et al., 1973). A history of recurrent early
pregnancy loss may indicate the presence of a
balanced rearrangement in one of the parents.
Miscarriage is relatively common in the general population, so one or two miscarriages do
not indicate a high likelihood of a balanced
rearrangement. In couples with two or more
spontaneous abortions, the frequency of chromosome anomalies in one parent is 4% to 5%

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and may be higher if the couple also has one

or more normal liveborn children (indicating
that obstetrical problems are less likely to have
caused the miscarriages; Schwartz and Palmer,
1983; Castle and Bernstein, 1988).

Abnormal non-invasive prenatal

testing/screening
Non-invasive prenatal testing/screening
(NIPTS; UNIT 8.15, Dharajiya et al., 2015) is
a recently implemented screening method for
fetal aneuploidy, whereby circulating cell-free
fetal DNA (cfDNA) is extracted from maternal plasma, made into genomic libraries for
next-generation sequencing and amplified via
PCR, then hybridized to a sequencing flowcell.
The resulting sequence data are computationally trimmed for sequencing read quality, and
normalized for relative copy-number analysis.
Current technologies typically assess the sex
chromosomes and select autosomes (e.g., 13,
18, and 21) for aneuploidy, based on a statistically significant increase in cfDNA from
those chromosomes relative to others in the
sample. Detection rates up to 99% for Down
syndrome, 96.8% for trisomy 18, and 92% for
trisomy 13, with a <1% false-positive rate for
each aneuploidy, have been reported. NIPTS
is also available clinically to detect abnormal
copy number in select microdeletion syndrome
regions (Hardisty and Vora, 2014). The high
sensitivity and specificity of NIPTS, and speed
of analysis to detect fetal aneuploidy, is poised
to supplant invasive prenatal screening methods. In the near future, whole-genome, highresolution prenatal analysis is likely (Aypar
et al., 2013).

Indications for Postnatal Studies

Multiple congenital anomalies
Most chromosome abnormalities have profound effects on fetal development and lead
to multiple congenital anomalies. A number of well-characterized syndromes correspond with the more common chromosome
anomalies (Schinzel, 2001; Borgaonkar, 2011;
Gersen and Keagle, 2013); the best known is
Down syndrome (trisomy 21). Clinical recognition of a chromosomal syndrome should
prompt cytogenetic study of the child. In many
cases, however, the chromosomal imbalance
is relatively small and no specific syndrome
is recognized, either because no syndrome has
been described or because the anomaly is very
rare. There is no single rule that will guarantee detection of all persons with chromosome rearrangements. One approach is to per-

form cytogenetic studies in cases where two or

more congenital anomalies, not related to one
another as cause and effect, are present and a
nonchromosomal etiological diagnosis cannot
be made.
Occasionally, when a child with congenital anomalies is born, it is learned that prenatal cytogenetic studies were performed and
interpreted to have normal results. A higherresolution chromosome analysis (or microarray study) may be warranted, and can be
obtained from lymphocyte DNA from the newborn rather than from either chorionic villus
material or amniotic fluid cells. Such an analysis may detect subtle chromosome anomalies
that have been missed prenatally. Also, a clinician may identify a syndrome that can guide
the cytogeneticist to focus on a particular chromosome for especially close scrutiny. This also
applies to individuals whose chromosomes
were studied postnatally in past years, as cytogenetic technology has improved markedly
during the last decade.

Mendelian chromosomal syndromes

Special cytogenetic studies or molecular
analyses are increasingly being used to elucidate chromosomal syndromes. Examples include fragile X detection and studies for microdeletion syndromes such as Prader-Willi,
Angelman, or DiGeorge syndromes (Table
8.1.1). Fragile X testing is undertaken using a DNA-based approach, such as that
described in UNIT 9.5 (Basehore and Friez,
2014). A number of Mendelian syndromes
cause increased chromosome breakage, including Fanconi anemia, Blooms syndrome,
and ataxia telangiectasia. Techniques for analysis of chromosome breakage in Fanconi anemia are provided in UNIT 8.7 (Auerbach, 2015),
and for analysis of sister chromatid exchange,
a marker of Blooms syndrome, in UNIT 8.6
(German and Alhadeff, 1994).

MOLECULAR CYTOGENETIC
METHODS
Fluorescence In Situ Hybridization
(FISH)
Fluorescence in situ hybridization (FISH;
Knoll and Lichter, 2005) is routinely
employed in the clinical cytogenetics laboratory. Probes for specific DNA sequences are
used to identify specific chromosome regions,
and are also used to detect microdeletions.
A number of microdeletion syndromes have
been identified. These syndromes are defined
clinically, and FISH studies with appropriate
probes can be used to detect the deletions for

UNIT 4.3,

Clinical
Cytogenetics

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laboratory diagnosis (UNIT 8.10, Kashork et al.,

2010).
Subtelomeric microdeletions have been
identified in individuals with developmental
impairment who usually do not have clinical signs of a specific genetic syndrome
(Slavotinek et al., 1999). These deletions can
be detected using FISH or by molecular analysis (UNIT 8.11, Martin and Ledbetter, 2015).
In addition to the use of site-specific DNA
probes, FISH can be used with sets of probes
labeled with fluorochromes so that different
chromosomes fluoresce with distinct colors.
Approaches to spectral karyotyping using M-FISH or SKY systems are described
in UNIT 4.9 (Lee et al., 2000). This approach is useful clinically to detect subtle chromosome rearrangements that may escape detection by conventional banding (Jalal
and Law, 1999). It is also helpful in the
elucidation of complex rearrangements involving multiple chromosomes and in the
study of marker chromosomes (Haddad et al.,
1998). Comparative genomic hybridization
(CGH; UNIT 4.6, DeVries et al., 1995) has
also been helpful in the study of complex
or subtle chromosome rearrangements (Levy
et al., 1998). Currently, most cytogenetic testing for microdeletions/microduplications of
subtelomeric and syndrome regions, in patients without a clinically distinct phenotype, is now performed by chromosomal microarray analysis (CMA; Miller et al., 2010;
UNIT 8.12, Miller et al., 2012; UNIT 8.13, Zahir
and Marra, 2015). However, FISH is still the
preferred method for confirmation of CMA
results in a proband, for parental testing to determine inheritance of copy-number variants
(CNV), and for analysis of a suspected syndrome region suggested by patient phenotype
(UNIT 8.10, Kashork et al., 2010).

Chromosomal Microarray Analysis

(CMA)

Overview of
Clinical
Cytogenetics

Chromosomal microarray analysis (CMA)

is a relatively recent addition to cytogenetic
testing labs, and has become the first-tier testing algorithm for patients with developmental delay, intellectual disability, autism, and/or
multiple congenital anomalies (MCA), where
a distinct phenotype indicative of a known genetic syndrome is not readily apparent (Miller
et al., 2010; South et al., 2013). Diagnostic
yields for CMA testing of such patients range
from 15% to 20%, while a G-banded karyotype
alone is 3% (Miller et al., 2010).
CMA denotes multiple microarray-based
technologies within this unit, including

microarray-based comparative genomic hybridization (array CGH or aCGH; UNIT 8.12,

Miller et al., 2012), single nucleotide polymorphism (SNP)-based microarrays (UNIT 8.13,
Zahir and Marra, 2015), or combined
CGH+SNP arrays (UNIT 8.12, Miller et al.,
2012). For CGH or combined CGH+SNP
arrays, genomic DNA (gDNA) is extracted
from peripheral blood, skin fibroblasts, tissue,
or amniocytes from a patient, along with a
cytogenetically normal, gender-matched control sample. The patient gDNA and control
gDNA are digested with restriction enzymes,
labeled with contrasting fluorescent cyanine
dyes (Cy5 and Cy3), and co-hybridized to a
microarray containing thousands of oligonucleotide probes. Unlike the two-color system
used in CGH arrays, SNP-based microarrays
(UNIT 8.13, Zahir and Marra, 2015) employ fluorescent labeling of the patient sample only,
and copy number is inferred by comparison
to a reference dataset. With most current platforms, microarray oligos are designed to cover
the entire euchromatic human genome at regularly spaced intervals, and provide denser coverage of genes conferring human genetic disease.
After hybridization, the microarray is
washed, scanned, and the ratios of the contrasting fluorescent dyes for each microarray oligo
probe are analyzed with image-visualization
software. Differences in copy number between
the patient and control samples are determined
based on the differential hybridization of the
fluorescently labeled gDNAs to the microarray probes. The copy-number ratios for each
oligo probe are converted to a log2 scale,
and the oligos are plotted based on their genomic positions along each of the 24 chromosomes. Copy-number aberrations are segmented based on averaged log2 ratios, with
deletions and duplications/amplifications of
genomic segments determined by the analysis software. Along with copy-number assessment, SNP arrays and combined CGH+SNP
arrays also provide genotype information using restriction-sensitive SNP oligo probes.
These arrays are helpful to determine uniparental disomy (UPD), as well as regions
of absence of heterozygosity (AOH), providing a degree of suspected consanguinity,
and delineating genomic regions of possible
morbid genes containing autosomal recessive
alleles.
CMA analysis is able to detect mosaicism
reliably down to 20% to 30% (Miller et al.,
2010; Gersen and Keagle, 2013) for segmental copy-number changes, and to a lower

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threshold with whole-chromosome aneuploidies, and with SNP-based microarrays. However, CMA is unable to detect truly balanced
chromosomal rearrangements, or low-level
mosaicism, as only robust copy-number information is provided (Miller et al., 2010; Bi et al.,
2013). Repetitive regions of the genome not
represented on the array (pericentromeric regions, heterochromatin) are also not analyzed.
Because CMA analysis is not able to determine
the structural consequences of copy-number
changes, in circumstances with low-level mosaicism, complex structural rearrangements,
and balanced translocations, G-banded karyotype and FISH are indicated (Miller et al.,
2010; Bi et al., 2013).

TISSUE SOURCES
Prenatal Analysis
Chorionic villus sampling
This procedure is usually performed at 10
to 12 weeks gestation. Fetal tissue is obtained
by insertion of a biopsy catheter either through
the cervix or transabdominally. The latter approach may be associated with a lower rate
of infectious complications. The fetal sample must be visually inspected and maternal
tissue dissected away prior to analysis. Cytogenetic studies can be performed on spontaneously dividing trophoblast cells or mesenchymal cells grown in culture (UNIT 8.3, Breman and Patel, 2012). The former permits very
rapid turnaround (as fast as 24 to 48 hr) but carries a risk of finding chromosomal anomalies
in some cells that may not be present in the fetus (Sachs et al., 1990). Such mosaicism is less
common in cultured chorionic villus cells and
still less so in amniotic fluid cells. Findings
from direct trophoblast analysis should be followed up with a study of cultured cells. Chorionic villus tissue can also be used as a source
of fetal DNA for molecular genetic studies.
The major advantage to chorionic villus
sampling is the ability to obtain fetal tissue
early in pregnancy. If termination of pregnancy is contemplated, the earlier termination
procedure is less traumatic and allows the patient to maintain privacy about the pregnancy.
Procedure-related complications, however, are
higher than with amniocentesis (1% compared with 0.5% for amniocentesis; Rhoads
et al., 1989). Concern has been raised about
distal limb reduction defects and anomalies of
the lower jaw and tongue in children born from
pregnancies studied by chorionic villus sampling (CVS; Burton et al., 1992; Schloo et al.,

1992). Currently, CVS is not recommended

before 10 weeks gestational age to reduce the
risks of these complications (Tabor and Alfirevic, 2010).

Amniocentesis
Amniocentesis is usually performed between 15 and 18 weeks of gestation, and not
recommended before 15 weeks as the risk of
talipes equinovarus in the fetus is significant
(Tabor and Alfirevic, 2010). The rate of complications is comparable to CVS, at 0.5% to
1%. Fetal tissue obtained by this means must
be grown in culture for cytogenetic analysis,
which usually requires 1 to 2 weeks (UNIT
8.4, Miron, 2012). Two approaches to amniotic fluid cell culture are in wide use. Some
laboratories grow cells at low density on coverslips for several days, allowing individual cells
to grow into colonies, and then prepare cells
for analysis directly on these slips. Analysis
of cells in multiple colonies is helpful in distinguishing chromosome anomalies that arise
during the culturing process from those that
occur in the fetus. If all cells from all colonies
are abnormal, the fetus is likely to be abnormal. If all cells from two or more colonies
are abnormal, but other colonies are normal, it
is likely that the fetus is mosaic with a normal
and an abnormal cell line. If only one colony is
abnormal, it is possible that the fetus has lowlevel mosaicism or that a single cell may have
acquired an abnormality in vitro. If only some
cells of a single colony are abnormal, the mosaicism (referred to as pseudomosaicism) is
likely to have arisen in vitro (Wilson et al.,
1989).
The alternative culture system is to grow
cells in culture flasks. In this case, analysis of
individual colonies is not possible, and evidence of true fetal mosaicism will consist of
finding the same abnormality in cells from two
or more independent cultures.
Rapid prenatal cytogenetic diagnosis can
be achieved with FISH (ACMG, 2000; UNIT
4.3, Knoll and Lichter, 2005). Chromosomespecific cloned DNA sequences are fluorescently labeled and hybridized to interphase nuclei. A discrete fluorescent spot in each nucleus
should be observed representing each chromosome for which the probe is hybridized;
therefore trisomy results in three spots and
monosomy in only one spot. This approach
offers very rapid turnaround time (24 to 48
hr) but has some limitations. Clinical use has
focused only on detecting trisomy or monosomy for a few specific chromosomes: X, Y,
13, 18, and 21. Chromosome rearrangements,

Clinical
Cytogenetics

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Table 8.1.1 ACMG Recommendations for Cytogenetic Analysis of Pre- and Postnatal Samplesa

Tissue

Count

Analyze

Karyotype

Amniotic fluid (flask

method)

20 cells from 2
cultures

5 cells

2 cells (or 1 per cell

line if mosaic)

Amniotic fluid (in situ

method)

15 cells from 15

coloniesb from 2
cultures

5 cells, each from

different colonies

2 cells (or 1 per cell

line if mosaic)

Chorionic villus (direct) Not recommended for

exclusive use
Chorionic villus
(culture, flask method)

20 cells from 2 cultures 5 cells

Chorionic villus (in situ 15 cells from 15

method)
coloniesb

2 cells (or 1 per cell

line if mosaic)

5 cells, each from

different colonies

2 cells (or 1 per cell

line if mosaic)

Chorionic villus
(culture plus direct)

20 cells with 10 from

cultured prep

5 cells

2 cells (or 1 per cell

line if mosaic)

Constitutional (e.g.,
lymphocyte, fibroblast)

20 cells (30 if
sex-chromosome
abnormality is
suspected)

5 cells

2 cells (or 1 per cell

line if mosaic)

a Source:
b For

ACMG (2009).
in situ studies, if 15 cells are not available, 10 must be used.

such as unbalanced translocations, are not

detected (Schwartz, 1993; ACMG, 2000).

Postnatal Analysis

mortem or in material from an abortus). It can

also be helpful if mosaicism for a chromosome
anomaly is suspected and lymphocyte chromosomes are found to be normal. In a few
instances, fibroblasts have been found to harbor a chromosomal abnormality not present
in peripheral blood, most notably an isochromosome for the short arm of chromosome 12
(Speleman et al., 1991). Fibroblasts may also
be used as a source of tissue for enzyme or
DNA studies.

Peripheral blood lymphocytes

Genomic DNA (gDNA)

Peripheral blood lymphocytes are the most

common source of tissue for postnatal chromosome analysis (UNIT 4.1, Bangs and Donlon, 2005). T lymphocytes are stimulated to
divide in culture in the presence of phytohemagglutinin. After 72 to 96 hr, mitotic cells
are collected by inhibition of the spindle with
a drug such as colchicine, are harvested, and
then spread onto slides. The technique is simple and reliable and yields chromosomes with
good morphological preservation.

For CMA studies, genomic DNA can be extracted from the prior cell types and utilized for
copy-number analysis (UNIT 8.12, Miller et al.,
2012).

Umbilical venous blood sampling

Fetal blood can be sampled at 19 weeks

gestation by umbilical vein sampling. Lymphocytes are then grown in culture for cytogenetic analysis (UNIT 4.1, Bangs and Donlon,
2005) or used as a source of fetal DNA.

Fibroblasts

Overview of
Clinical
Cytogenetics

Fibroblasts obtained by skin or other tissue biopsy can be grown in culture and used
as a source of dividing cells for chromosome
analysis (UNIT 8.5, Levy et al., 2009). This approach is used in cases where peripheral blood
may be impossible to obtain (such as post-

Analytical Considerations
Number of cells
Recommendations of the American College of Medical Genetics and Genomics
(ACMG) are presented in Table 8.1.1. Counts
are defined as the number of centric chromosomes per metaphase cell. Analyzed cells are
banded chromosomes evaluated either through
the microscope or on digitized images or
photographs.
Cell counting should include noting any numerical and structural aberrations seen. Cells
that are fully karyotyped may be included in

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the number of cells analyzed (the distinction

is that karyotyping involves arranging the individual chromosome pairs in a standard order
from a photograph).

Staining methods
Different laboratories employ different routine staining methods (see UNIT 4.2, Schreck
and Dist`eche, 1994; and Benn and Perle, 1992,
for staining protocols; abbreviations defined in
the references are used below). GTG-banding
is the most commonly employed, although
some laboratories use QFQ- or R-banding.
Laboratories also must set a standard level of
resolution for routine analysis. Level of resolution is usually expressed in terms of banding
levelthat is, an estimate of the total number of bands visible in the entire haploid chromosome complement (Josifek et al., 1991). A
standard level of resolution for routine analysis is 400 to 550 bands. Identification of
some microdeletion syndromes requires substantially higher resolution, e.g., 800 bands.
High-resolution analysis can be accomplished
in a variety of ways. Usually extended chromosomes are prepared by synchronization
of cells and harvesting at prometaphase or
treatment with an intercalating agent such as
ethidium bromide.
Special stains are used to aid in the identification of chromosome rearrangements. These
techniques include C-banding, NOR-staining,
T-banding, replication banding, and sister
chromatid differentiation. Recently, FISH has
been added to the armamentarium for precise
chromosome identification (UNIT 4.3, Knoll and
Lichter, 2005). Specific DNA probes can help
to identify a rearranged chromosome or reveal
submicroscopic deletions.

Image capture
Images of chromosomes were traditionally
recorded by photomicrography, but the use of
computers has greatly modernized cytogenetic
analysis. Chromosome analysis may be done
through the microscope or by the use of digital images with karyotyping analysis software
on desktop computers. A number of digitalimage-capture devices are available (UNIT 4.4,
McNamara et al., 2005). These devices, which
can substantially speed the counting and analysis of chromosomes and provide a digital photograph of chromosomes, are currently in use
in most cytogenetic laboratories.

Reporting
Reports of cytogenetic results usually include at least the following elements:

r Patients name and identifying information

r Referring clinicians name

r Referring diagnosis
r Source of material analyzed
r Date specimen received and date of report
r Banding technique(s) used
r Level of banding resolution achieved
r Number of cells (colonies and cultures)
counted

r Number of cells (colonies and cultures)

analyzed

r Modal chromosome number

r Sex-chromosome complement
r Description of aneuploid cells
r Description of structurally abnormal
chromosomes

r Final impression with karyotype described with International System for

Human Cytogenic Nomenclature (ISCN)
nomenclature (ISCN, 2013)
r Clinical interpretation

CHROMOSOME ANOMALIES
Introduction
This section will describe major types of
chromosome anomalies. Detailed guides to
chromosome nomenclature can be found in
APPENDIX 4C and ISCN (2013). Specific syndromes will not be covered here; several
books describe the clinical features of major chromosomal anomalies (de Grouchy and
Turleau, 1984; Schinzel, 2001; Borgaonkar,
2011; Gersen and Keagle, 2013).

Normal Chromosome Complement

Somatic cells normally contain 46 chromosomes: 22 pairs of autosomes plus XY in
males or XX in females. Karyotypes consistent
with chromosomally normal males are written
46,XY and 46,XX for chromosomally normal
females.

Numerical Abnormalities
Polyploidy
Tetraploidy occurs rarely as a constitutional
anomaly, probably because of early embryonic
lethality. Triploidy is common in early spontaneous miscarriages but may also be compatible with live birth. Triploid fetuses have the
karyotype 69,XXX or 69,XXY, or 69,XYY. A
69,YYY conceptus lacking an X chromosome
is nonviable. Some liveborn triploids are mosaics with a triploid and a diploid cell line, e.g.,
46,XX/69,XXX.

Clinical
Cytogenetics

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pericentric inversion

paracentric inversion

duplication

terminal
deletion

interstitial
deletion

isochromosome

ring chromosome

Figure 8.1.1 Examples of structural abnormalities of chromosomes, including pericentric and

paracentric inversion, duplication, interstitial and terminal deletion, isochromosome, and ring chromosome.

Trisomy
All possible chromosome trisomies have
been seen in the products of spontaneous abortions, but only a few (trisomies 8, 13, 18, 21,
X, and Y) occur with appreciable frequency
in liveborns. Trisomy is assumed to occur
by nondisjunction during meiosis or mitosis.
A trisomic karyotype is indicated using the
form 47,XY,+21 (male with trisomy 21). Sexchromosome aneuploidy is denoted 47,XXX
(triple X syndrome) or 47,XXY (Klinefelter
syndrome).

Monosomy
Overview of
Clinical
Cytogenetics

Most monosomies are nonviable. The one

notable exception is Turner syndrome, in
which there is a single X chromosome and

no Y. Turner syndrome with monosomy X is

indicated 45,X.

Structural Abnormalities
Chromosome structure abnormalities include a wide variety of different forms, as described below and depicted in Figure 8.1.1.

Deletion
Deletions can be either terminal or interstitial. Interstitial deletions involve two breaks
in the chromosome, loss of material, and reunion of the broken ends. Most deletions are
probably interstitial as all chromosome ends
must contain a telomere. The clinical effects
of deletion are due to monosomy for the
deleted chromosome material; or nullisomy,

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in the case of deletions of part of the X chromosome in males. A number of syndromes

have been discovered in which very small, frequently submicroscopic chromosome regions
are deleted (see Table 8.1.1). Symptoms are
presumed to be due to monosomy for a group
of contiguous genes subject to dosage or imprinting effects. Detection of such deletions
often requires high-resolution cytogenetic or
molecular genetic study.

Duplication
In these abnormalities a segment of chromosome material is duplicated, usually in tandem with the original sequence. The duplicated region may have the same orientation as
the original sequence (direct duplication) or
may be repositioned by 180 (inverted duplication). The phenotype is due to trisomy for
the duplicated region.

Inversion
Here a segment of chromosome material is
inverted 180 with respect to its normal orientation. This involves two sites of breakage and
subsequent reunion. If the inversion includes
the centromere it is referred to as pericentric; otherwise it is paracentric. Inversions
may be associated with a clinical phenotype if
the site of breakage occurs in a gene leading
to disruption of the gene sequence or dysregulation of a gene(s), but often do not result in
an abnormal phenotype. Inversions can however lead to unbalanced recombinant chromosomes. During meiosis, pairing between homologous chromosomes in which one is inverted leads to formation of an inversion loop.
If a cross-over occurs within the loop, the
products contain duplications of some chromosome regions and deletions of others. In a
paracentric inversion, cross-overs result in dicentric or acentric fragments that tend to be
unstable at mitosis. The consequences of having an inversion therefore can be formation of
chromosomally unbalanced gametes, leading
to recurrent miscarriage or in some instances
the birth of offspring with congenital anomalies. Pericentric inversion of chromosome 9 is
a common heteromorphic variant of no known
clinical significance (Lubs et al., 1977). Another common pericentric inversion involves
chromosome 2 and is likewise not usually associated with an adverse outcome (MacDonald
and Cox, 1985).

Isochromosome
Isochromosomes contain two copies of
short-arm or long-arm material separated by

a centromere and are thought to arise by misdivision of the centromere. They result in
trisomy for the duplicated arm and monosomy for the missing arm, with associated
phenotypic consequences.

Ring chromosome
A ring chromosome arises by breakage of
both the short and long arms and subsequent
fusion of the broken ends. Rings may exert
phenotypic effects by two mechanisms. First,
material is lost from the two arms, leading
to monosomy for these regions. Second, the
ring tends to be unstable during cell division:
it may be lost from some cells, leading to
monosomy, while in other cells a sister chromatid exchange within the ring may result in a
double-sized ring and consequent trisomy. As
a result, individuals with ring chromosomes
may have many cell populations with different
constitutions with respect to the ring. Specific
phenotypic effects vary widely with the chromosome involved and amount of chromosome
material lost.

Translocation
Translocation (Fig. 8.1.2) involves the exchange of segments from one chromosome to
another, usually in a reciprocal fashion. In a
balanced reciprocal translocation, breaks occur in each chromosome and the broken segments are exchanged between the two chromosomes so that no material is lost or gained. A
carrier for a balanced reciprocal translocation
may be asymptomatic, although some individuals manifest a phenotype related to disruption or dysregulation of a gene(s) at the site of
breakage. During meiosis, the chromosomes
involved in a balanced reciprocal translocation
pair in a quadrivalent configuration. Depending on how the chromosomes separate at the
first meiotic division, gametes may receive a
normal chromosome complement, one including the balanced translocation chromosomes,
or an unbalanced chromosomal complement.
The latter leads to partial trisomy and monosomy, which may result in miscarriage or multiple congenital anomalies.
The acrocentric chromosomes (13, 14, 15,
21, and 22) can engage in translocations with
one another in which fusions in repetitive sequences in the short arms result in dicentric
chromosomes (referred to as Robertsonian
translocations; Fig. 8.1.2). If a parent who has
a Robertsonian translocation involving chromosome 21 produces a gamete with both the
translocated chromosome and the normal 21,
this may lead to trisomy 21 in the offspring.

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Robertsonian
translocation

reciprocal
translocation

Figure 8.1.2 Examples of a Robertsonian translocation between chromosomes 14 and 21 and

a balanced reciprocal translocation between chromosomes 2 and 8.

Insertion
This is a form of translocation in which material is deleted from one chromosome and inserted into another. The inserted material can
be in its normal orientation or inverted relative to the centromere. Partial trisomies can result after meiotic segregation of chromosomes
carrying an insertion.

Complex rearrangements
Translocations or insertions can involve
more than two chromosomes. This occurs particularly with acquired chromosome abnormalities such as those observed in tumors
(Korf, 2010).

Mosaicism
The presence of two cell lines where chromosomal anomalies may be found in some but
not all cells of an individual is a condition
referred to as mosaicism. This results from
occurrence of the chromosome change during
embryonic development. The clinical sequelae
depend on the nature of the anomaly and the
proportion and distribution of cells affected.
In some instances, abnormal cell lines may be
confined to extraembryonic membranes. Effects of such mosaicism on fetal well being
are under study (Kalousek et al., 1992).

Uniparental disomy (UPD)

Overview of
Clinical
Cytogenetics

Uniparental disomy (UPD) is a phenomenon in which both copies of a chromosome are inherited from one parent. In most
cases, this probably results from an initial
nondisjunction event leading to trisomy, and

subsequent nondisjunction leaving two copies

of a chromosome from the same parent. It may
also occur if a parent who has a Robertsonian translocation chromosome transmits both
the translocation and one of the normal chromosomes involved in the translocation to the
same gamete. If the normal chromosome from
the other parent is subsequently lost in the
embryo, uniparental disomy results. Although
uniparental disomy does not lead to genetic
imbalance, some genes are differentially expressed in early development depending on
whether they are inherited from mother or father; this phenomenon is referred to as imprinting. Uniparental disomy can therefore
cause imbalance of the expression of imprinted
genes. The full list of imprinted genes is not
known, but there is good evidence for imprinting of genes on chromosomes 7, 11, and 15.
There is also some evidence favoring imprinting for genes on chromosomes 2, 6, 14, 16,
20, and X (Ledbetter and Engel, 1995). Uniparental disomy should be investigated when
mosaicism is found for any of the above chromosomes by prenatal testing or when a fetus inherits a Robertsonian translocation involving chromosome 15. This can be tested
by examining the inheritance of DNA polymorphisms from both parents. Several specific
syndromes have been found to result from
imbalance of imprinted genese.g., PraderWilli, Angelman, and Beckwith-Wiedemann
syndromesbut the full extent of developmental consequences of imbalance is unknown
(Hall, 1990).

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