Principles of Clinical Cytogenetics

Introduction to Cytogenetics

Clinical Indications for Chromosome Analysis

Chromosome Identification
 Clinical cytogenetics is the study of chromosomes,
their structure and their inheritance, as applied to
the practice of medical genetics.
 It has been apparent for nearly 50 years that
chromosome abnormalities—microscopically
visible changes in the number or structure of
chromosomes—could account for a number of
clinical conditions that are thus referred to as
chromosome disorders.
 Directing their focus to the complete set of
chromosome material, cytogeneticists were the
first to bring a genome-wide perspective to
medical genetics.
 Today, chromosome analysis—now with
dramatically improved resolution and precision
at both the cytological and genomic levels—is
an increasingly important diagnostic procedure
in numerous areas of clinical medicine.
 Chromosome disorders form a major category of
genetic disease.
 They account for a large proportion of all
reproductive wastage, congenital malformations,
and mental retardation and play an important
role in the pathogenesis of malignant disease.
 Specific chromosome abnormalities are
responsible for hundreds of identifiable
syndromes that are collectively more common
than all the mendelian single-gene disorders
together.
 Cytogenetic disorders are present in nearly 1% of
live births, in about 2% of pregnancies in women
older than 35 years who undergo prenatal
diagnosis, and in fully half of all spontaneous
first-trimester abortions.
 We discuss the general principles of clinical
cytogenetics and the various types of numerical
and structural abnormalities observed in human
karyotypes.
 

• To be examined by chromosome analysis for routine

clinical purposes, cells must be capable of growth and
rapid division in culture.
• The most readily accessible cells that meet this
requirement are white blood cells, specifically T
lymphocytes.

• To prepare a short-term culture that is suitable for

cytogenetic analysis of these cells, a sample of peripheral
blood is obtained, usually by venipuncture, and mixed with
heparin to prevent clotting.
• The white blood cells are collected, placed in tissue
culture medium, and stimulated to divide.
• After a few days, the dividing cells are arrested in
metaphase with chemicals that inhibit the mitotic
spindle, collected, and treated with a hypotonic solution
to release the chromosomes.
• Chromosomes are then fixed, spread on slides, and
stained by one of several techniques, depending on the
particular diagnostic procedure being performed.
• They are then ready for analysis.
• The determination of what approaches
are most appropriate for particular
diagnostic or research purposes is a
rapidly evolving area, as the resolution,
sensitivity, and ease of chromosome and
genome analysis increase.  
Chromosome analysis is indicated as a routine
diagnostic procedure for a number of specific
phenotypes encountered in clinical medicine,
there are also some nonspecific general
clinical situations and findings that indicate a
need for cytogenetic analysis:
• Problems of early growth and development.
Failure to thrive, developmental delay, dysmorphic
facies, multiple malformations, and mental
retardation are frequent findings in children with
chromosome abnormalities, although they are not
restricted to that group.
 Unless there is a definite non-chromosomal
diagnosis, chromosome analysis should be
performed for patients presenting with a
combination of such problems.
 

• Stillbirth and neonatal death.

 The incidence of chromosome abnormalities is much
higher among stillbirths (up to approximately 10%) than
among live births (about 0.7%).
 It is also elevated among infants who die in the neonatal
period (about 10%).
 Chromosome analysis should be performed for all
stillbirths and neonatal deaths that might have a
cytogenetic basis to identify a possible specific cause or,
alternatively, to rule out a chromosome abnormality as
the reason for the loss.

 In such cases, karyotyping (or other comprehensive

ways of scanning the genome) is essential for accurate
genetic counseling and may provide important
information for prenatal diagnosis in future pregnancies
• Fertility problems.

 Chromosome studies are indicated for women

presenting with amenorrhea and for couples with
a history of infertility or recurrent miscarriage.

 A chromosome abnormality is seen in one or the

other parent in a significant proportion (3% to 6%)
of cases in which there is infertility or two or
more miscarriages.
• Family history.
A known or suspected chromosome
abnormality in a first-degree relative is an
indication for chromosome analysis under
some circumstances.
• Neoplasia.

 Virtually all cancers are associated with one or

more chromosome abnormalities.
 Chromosome and genome evaluation in the
appropriate tissue sample (the tumor itself, or
bone marrow in the case of hematological
malignant neoplasms) can provide useful
diagnostic or prognostic information.
• Pregnancy in a woman of advanced age.

 There is an increased risk of chromosome

abnormality in fetuses conceived by women
older than about 35 years.
 Fetal chromosome analysis should be offered as a
routine part of prenatal care in such pregnancies.
 Although ideal for rapid clinical analysis, cell
cultures prepared from peripheral blood have
the disadvantage of being short-lived (3 to 4
days).

 Long-term cultures suitable for permanent

storage or molecular studies can be derived
from a variety of other tissues.
 Skin biopsy, a minor surgical procedure, can
provide samples of tissue that in culture
produce fibroblasts, which can be used for a
variety of biochemical and molecular studies
as well as for chromosome and genome
analysis.

 White blood cells can also be transformed in

culture to form lymphoblastoid cell lines that
are potentially immortal.
 Bone marrow can be obtained only by the relatively
invasive procedure of marrow biopsy, but it has the
advantage of containing a high proportion of
dividing cells, so that little if any culturing is
required.
 Its main use is in the diagnosis of suspected
hematological malignant neoplasms.
 Its disadvantage is that the chromosome
preparations obtained from marrow are relatively
poor, with short, poorly resolved chromosomes that
are more difficult to analyze than are those from
peripheral blood.
 Fetal cells derived from amniotic fluid (amniocytes) or
obtained by chorionic villus biopsy can also be cultured
successfully for cytogenetic, genomic, biochemical, or
molecular analysis.

 Chorionic villus cells can also be analyzed directly,

without the need for culturing.
 Molecular analysis of the genome can be
carried out on any appropriate clinical material,
provided that good-quality DNA can be
obtained.

 Cells do not have to be dividing for this purpose,

and thus it is possible to perform tests on tissue
and tumor samples, for example, as well as on
peripheral blood.
 The 24 types of chromosome found in the
human genome can be readily identified at the
cytological level by a number of specific
staining procedures.
 There are three commonly used staining
methods that can distinguish among human
chromosomes.
 Chromosomes stained by Giemsa banding (G
banding), the most common method used in
clinical laboratories.

 Other procedures used in some laboratories or

for specific purposes include the following: Q
Banding This method requires staining with
quinacrine mustard or related compounds and
examination by fluorescence microscopy.
 The chromosomes stain in a specific pattern of bright
and dim bands (Q bands), the bright Q bands
corresponding almost exactly to the dark bands seen
after G banding.
 Q banding, as well as C banding, is particularly useful
for detecting occasional variants in chromosome
morphology or staining, called heteromorphisms.
 These variants are generally benign and reflect
differences in the amount or type of satellite
DNA sequences at a particular location along a
chromosome.
R Banding If the chromosomes receive special
treatment (such as heating) before staining, the
resulting dark and light bands are the reverse of
those produced by G or Q banding and are
accordingly referred to as R bands.
Especially when regions that stain poorly by G or
Q banding are examined, R banding gives a
pattern that is easier to analyze than that given by
G or Q banding.
It is the standard method in some laboratories,
particularly in Europe.
Figure 5-1 is an ideogram of the banding
pattern of a set of normal human
chromosomes at metaphase, illustrating the
alternating pattern of dark and light bands
used for chromosome identification.

The pattern of bands on each chromosome is

numbered on each arm from the centromere
to the telomere, as shown in detail in Figure
5-2 for several chromosomes.
By use of this numbering system, the location of
any particular band as well as the DNA sequences
and genes within it and its involvement in a
chromosomal abnormality can be described
precisely and unambiguously.
Human chromosomes are often classified by the
position of the centromere into three types that
can be easily distinguished at metaphase (see Fig.
5-1): metacentric chromosomes, with a more or
less central centromere and arms of approximately
equal length;
submetacentric chromosomes, with an off-center
centromere and arms of clearly different lengths;
and acrocentric chromosomes, with the
centromere near one end.
A potential fourth type of chromosome,
telocentric, with the centromere at one end and
only a single arm, does not occur in the normal
human karyotype, but it is occasionally observed
in chromosome rearrangements and is a common
type in some other species.
The human acrocentric chromosomes
(chromosomes 13, 14, 15, 21, and 22) have small,
distinctive masses of chromatin known as satellites
attached to their short arms by narrow stalks
(secondary constrictions).
The stalks of these five chromosome pairs contain
hundreds of copies of genes encoding ribosomal
RNA (the major component of ribosomes) as well
as a variety of repetitive sequences.