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CYTOGENETICS
Dr. Mary Ann Perle
I) Mitosis and metaphase chromosomes
A) Chromosomes are most fully condensed and clearly distinguishable during
mitosis.
B) Mitosis (M phase) takes 1 to 2 hrs and is divided into four stages: prophase,
metaphase, anaphase, and telophase. For cytogenetic purposes, chromosomes are
usually examined when they are in metaphase.
C) Structure of metaphase chromosomes.
1) At metaphase the chromosomes have been replicated. The doubled
chromosome consists of two sister chromatids joined at the centromere
through noncovalent protein-protein interactions.
2) Each sister chromatid is divided into two arms by the centromere.
Chromosomes may be classified by the position of the centromere.
(a) Metacentric - centromere is in the middle.
(b) Submetacentric - centromere is somewhat distant from the middle.
(c) Acrocentric - centromere is almost at the end. Chromosomes 13, 14, 15,
21 and 22 are acrocentric.
3) The short arm is referred to as the p arm (p for petite); the long arm is
referred to as the q arm. When arranging chromosomes in a karyotype, the p
arm is placed at the top.
II) Preparation of chromosome spreads.
A) A sample of cells is taken. These may be blood lymphocytes, fetal cells (from
amniotic fluid, chorionic villi or abortus material), tumor or bone marrow
aspirates, or other tissue.
B) The cells may be stimulated to divide, cultured and then blocked in metaphase.
C) Metaphase cells are swollen in a hypotonic solution, fixed with methanol/acetic
acid, dried onto microscope slides, stained and analyzed under the microscope.
III) Chromosome banding.
A) Three staining methods are commonly used.
1) G-banding - currently the most widely used method.
2) Q-banding - a fluorescent stain that gives the same banding pattern as Gbanding.
3) R-banding - gives the reverse of G-banding (dark G-bands appear light &
light G-bands appear dark).
B) The banding pattern is characteristic for each chromosome pair. An ideogram is
an idealized representation of the banding pattern; this is an international system.
1) Bands along each arm are numbered starting at the centromere.
2) By examining chromosomes at increased
resolution (e.g. observation of prophase or
prometaphase chromosomes), larger numbers of
bands can be visualized.
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Ideograms of Chromosome 7 at
450, 550 and 850 band level of
resolution (left to right)
3) Chromosomal polymorphisms – normal
variable regions without clinical significance
(a) Regions of constitutive heterochromatin are preferentially stained by Cbanding.
(b) Sizes of the stained polymorphic regions are useful markers for
identifying the origin of a particular chromosome (e.g. paternal, maternal,
or fetal).
(c) The q arm of the Y chromosome contains a highly polymorphic region of
genetically inactive material at the distal end.
IV) Nomenclature
A) A karyotype is described by listing, in this order:
1) Number of chromosomes followed by a comma
2) Sex chromosome constitution followed by a comma
3) Anomalies in number or morphology of chromosomes.
B) Examples:
1) Normal male: 46,XY; normal female: 46,XX
2) Klinefelter Syndrome: 47,XXY
3) Turner Syndrome: 45,X
4) Female with Down Syndrome: 47,XX,+21
5) Male with Angelman Syndrome (deletion between q11 and q13 on
chromosome 15): 46,XY,del(15)(q11q13). q11 is read as "q one one"; q13 is
read as "q one three".
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6) Female with a balanced reciprocal translocation: 46,XX,t(9;22)(q34;q11.2).
This example is the Philadelphia chromosome translocation seen in chronic
myelogenous leukemia (CML).
C) Commonly used cytogenetic nomenclature:
/
p
q
del
der
dup
i
ins
inv
r
t
ter
Diagonal line indicates mosaicism, e.g., 46/47 designates a mosaic with both a 46 chromosome and
47 chromosome cell line
Short arm of chromosome, "petite"
Long arm of chromosome
Deletion
Derivative, a structurally rearranged chromosome
Duplication
lsochromosome
Insertion
Inversion
Ring chromosome
Translocation
Terminal (may also be written as pter or qter)
+ or - Placed before the chromosome number, these symbols indicate addition (+) or loss (-) of a whole
chromosome, e.g., +21 indicates an extra chromosome 21, as in Down syndrome. Placed after the
chromosome number, these symbols indicate gain or loss of a chromosome part, e.g., 5p- indicates
loss of part of the short arm of chromosome 5. However, del(5)(p ) is the preferred nomenclature.
V) Structural chromosomal aberrations
A) A significant cause of morbidity and mortality.
1) About 0.7% of liveborns and half of spontaneous abortions contain a
chromosomal abnormality.
2) Abnormalities arising in somatic cells are significant in tumorigenesis.
B) Due to breakage and rejoining of one or more chromosomes, sometimes
accompanied by a gain or loss of genetic material.
C) Types of aberrations.
1) Deletions.
(a) Small losses of terminal regions or small interstitial deletions are most
typically seen. Example: Cri du Chat Syndrome ("5p- syndrome").
(b) The individual is monosomic for the deleted region; most large deletions
are incompatible with life.
2) Duplications and Insertions.
(a) Large duplications or insertions are rarely seen in liveborns.
(b) Commonly seen in neoplastic disorders.
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3) Isochromosomes
(a) Result from an abnormal division of the
centromere; the arms of two sister chromatids
are joined together.
(b) Important in sex chromosome abnormalities.
4) Inversions
(a) Usually do not affect the individual, but may
interfere with chromosome pairing during
meiosis: a cause of multiple spontaneous
abortions or abnormal liveborns.
(b) Pericentric inversions include the centromere.
(c) Paracentric inversions do not include the
centromere.
5) Translocations: Breaks occur in two or more
chromosomes that rejoin after exchange of segments.
(a) Reciprocal translocations
3 ↑
21↓
21↑
3 ↓
(i) Relatively common, about 1/1000 individuals.
(ii) Most are phenotypically normal. About 6-10% show an abnormal
phenotype due to either a small gain or loss of DNA or a position
effect. Abnormal phenotypes range from mild to severe.
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(iii) Carriers of balanced translocations are at increased risk for
producing unbalanced gametes.
(iv) Can lead to unbalanced translocations in the gametes.
Segregation
Pairing at meiosis
unbalanced
Adjacent 1
normal
balanced
Alternate
unbalanced
Adjacent-2
(b) Robertsonian translocation: exchange between two acrocentric
chromosomes (Chromosomes 13, 14, 15, 21or 22).
(i) The p arms are lost, and the two chromosomes fuse.
21 ↑
14 ↓
14
21
der(14;21)
(ii) Robertsonian translocations are balanced. Genes (primarily rRNA
genes) on the p arms are also present in genetically sufficient numbers
on the other acrocentric chromosomes.
(iii) A carrier of a Robertsonian translocation is phenotypically normal,
but is at risk for producing abnormal gametes. An increased rate of
spontaneous abortions or abnormal livebirths may be seen.
VI) Numerical chromosomal aberrations: Aneuploidy.
A) Gain of a chromosome [e.g. trisomy 21 (Down Syndrome) and XXY (Klinefelter
Syndrome)] or loss of a chromosome (Turner syndrome is a monosomy of the X
chromosome); monosomies of the autosomes are rare.
B) Nondisjunction is the most common mechanism leading to aneuploidy.
1) Nondisjunction in mitosis at an early cleavage division may lead to a
clinically significant mosaicism.
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2) Nondisjunction during meiosis I or II will lead to gametes with abnormal
chromosome number.
VII) Down Syndrome
A) Chromosomal abnormalities that may result in trisomy 21.
1) Nondisjunction (about 95%). About 80% of these are due to a maternal
error in meiosis I (most often) or meiosis II.
2) A Robertsonian translocation between 21q and the q arm of another
chromosome, usually 14. The parent carrying the translocation may have
normal children (some of whom may also be translocation carriers) and
children with Down Syndrome with a karyotype:
46,XX,+21,der(14;21)(q10;q10) (if female). You should convince yourself
that this is true (for help see Gelehrter et al Fig 8.15, p167).
3) A parent may also have a 21q21q translocation. The progeny will either
have Down syndrome or monosomy 21, which is not viable. Convince
yourself that this is true.
B) Increased incidence of Down Syndrome is associated with maternal age: about
1/952 live births at 30, about 1/385 at 35, about 1/100 at 40 and 1/30 at 45.
C) Determination of the cytogenetic basis of Down syndrome is important since a
parent and possibly other family members may be a carrier of a Robertsonian
translocation.
VIII) Other trisomies
A) Trisomy 18, seen in about 1/5,000 live births (also with a maternal age effect).
Most fetuses abort spontaneously and most liveborn children die within the
first few weeks due to severe cardiac and internal malformations. Those that
survive to a year and older show considerable growth and mental retardation.
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B) Trisomy 13, seen in about 1/8,000 live births (with a maternal age effect).
Most infants do not survive past the neonatal period.
IX) Molecular cytogenetics: Fluorescence in situ hybridization (FISH).
A) Methodology
1) Metaphase chromosomes or interphase cells are fixed onto a microscope
slide, the DNA is denatured and then hybridized with a probe that can be
detected by fluorescence. The chromosomes or hybridized regions are
observed microscopically.
2) Probes are usually generated from cloned DNAs derived from the
chromosomal region of interest.
B) Types of Probes
1) Centromere probes
(a) Isolated from repetitive DNA sequences located at or near centromeres.
The probes are specific for individual chromosomes.
(b) Useful for the detection of aneuploidy, and in the identification of
otherwise unidentifiable chromosomes. Can be used at interphase i.e.
when the mitotic yield is poor (as in cells from tumors or leukemias).
2) Whole chromosome probes - "chromosome painting".
(a) Mixtures of DNA probes that hybridize to regions along the entire
length of a specific chromosome.
(b) Useful for identifying translocations and marker chromosomes.
3) Unique sequence or locus specific probes.
(a) These probes are specific for individual loci or genes.
(b) Useful for the detection of microdeletion syndromes (e.g. DiGeorge,
Prader-Willi, Angelman syndromes).
(c) Useful for detection of specific leukemia translocations (e.g. CML,
APL) or gene amplification (breast cancer Her-2/neu gene).
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