cytogenetics iscn

1. Cytogenetic Analysis
ISCNDr. Ruslan Bayramov MD
Medical Genetics, Erciyes University
2015

2. Cytogenetics traditionally refers to the study of
chromosomes by microscopy following the
application of banding techniques, permitting
identification of abnormalities of chromosome
number, loss or gain of chromosomal material or
positional changes.

3. The current field is a hybrid of microscopic and
molecular based technologies.
-Fluorescence in situ hybridization (FISH), first introduced in the
mid-1980s
-Array-based genomic analysis or chromosome microarray
analysis (CMA), introduced in the 1990s and brought into
clinical practice beginning around 2003

4. •Array-based techniques generally
identify imbalances resulting in
deletions or duplications.
•There are cases of balanced translocations,
inversions or ring chromosomes that can only be
identified currently by standard chromosome
analysis.

5. History• Human chromosomes were probably first observed by Arnold in
1879 by dividing tumor.
• In 1921, Painter demonstrated in testis sections the presence of
an additional small Y chromosome. Although he assumed that 48
was the correct chromosome number in both sexes, it is
interesting to note that in his 1921 paper he states that he could
count only 46 chromosomes in the clearest mitotic figures.
• In a paper published in 1923, Painter predicted the existence of
individuals with unusual combinations of sex chromosomes, in
particular intersexes with an XXY sex chromosome complement.
No one seems to have tested this idea until 1942, when
Severinghaus described an XY sex chromosome constitution in an
XY female.

6. •Pathologic disorders might be due to abnormalities of
chromosome number and structure have been
suggested first by Theodor Boveri. He described his
theory on the origin of cancer from chromosomal
aberrations in a classic monograph published in 1914.
Then, 46 years later, the first specific chromosome
abnormality associated with malignancy was
described, namely, the Philadelphia chromosome in
chronic myeloid leukemia.

7. •Subsequent developments in cancer cytogenetics
have fully confirmed the role of chromosome
aberrations in the pathogenesis of cancer and
established cytogenetic analysis as an essential
component in classification and prognosis.
•With regard to constitutional chromosome
abnormalities, Waardenburg in 1932 was one of the
first to suggest that Down syndrome might be due to a
numeric chromosome aberration resulting from
nondisjunction.

8. • Human cytogenetics became a practical proposition with the
discovery by Tjio and Levan (1956) that the correct
chromosome number was 46 and not 48.
• Levan had earlier introduced into plant cytogenetics the use
of colchicine to arrest and accumulate mitoses at
metaphase, and he knew about the effect of hypotonic
solutions to separate individual chromosomes from one
another by pretreatment before fixation.
• The hypotonic technique had been discovered independently
by three scientists, Hsu (1952)in the United States, Makino
and Nishimura (1952) in Japan, and Hughes (1952) in
England. Apparently, both Hsu and Makino made the
discovery fortuitously, after mistakenly adding hypotonic
instead of isotonic salt solution during the washing stage
before fixation.

9. Ford reported in 1959 that Turner syndrome
was usually associated with a 45, X
chromosome complement
and
Jacobs and Strong found
that Klinefelter syndrome had a 47, XXY

10. •The important conclusion from these studies was that human
sex differentiation was determined by the Y chromosome and
not by the number of X chromosomes.
•There followed intense activity worldwide to determine
whether other dysmorphic conditions were due to
chromosomal abnormalities visible under the microscope.
•Trisomies 13 and 18 were quickly identified, followed by
several instances of sex chromosomal mosaicism,
translocation Down syndrome, and the deletion of the short
arm of chromosome 5, which causes the Cri du chat
syndrome.

11. •Early technical developments was the
introduction of phytohemagglutinin, which
allowed chromosome preparations to be made
within 2–3 days from peripheral blood samples.
This reagent was originally used to clear red
cells from preparations of lymphocytes, but it
was found that T lymphocytes underwent
transformation and division under its influence.

12. • When colchicine was used to accumulate lymphocytes
in metaphase during short-term culture, air-dried
drop preparations of metaphase chromosomes could
be made far superior to any previous method.
•The simplicity of the technique, which is still in use
almost unchanged, has undoubtedly been responsible
for the widespread application of chromosome
analysis through out the world and for the growth of
human cytogenetics as a diagnostic procedure in
clinical medicine.

13. •In the 1960s, individual chromosomes were
identified by characteristics such as total
length, centromere index (length of short
arm divided by total length), the presence of
heterochromatic regions.

14. • These studies revealed considerable normal variation in chromosome
size and centromere position, much of which was heritable and of no
clinical significance.
• Only chromosomes 1, 2, 3, 9, 16, and the Y chromosome could be
identified with certainty in any one metaphase by standard techniques.
• Chromosomal heteromorphisms mainly involved
- the centromeres of chromosomes 1, 9, 16 (and occasionally
chromosomes 3 and 4);
-the short arms and satellites of chromosomes 13, 14, 15, 21, and 22;
-and the distal heterochromatic region of the long arm of the Y
chromosome.

15. •Even before chromosome denaturation was being
used to produce various banding patterns, Caspersson
et al. independently discovered that quinacrine
compounds that intercalate in DNA could produce
bright fluorescent bands visible along the
chromosome using fluorescence microscopy. The
quinacrine bands(Q bands) were at first more
reproducible than those produced by denaturing and
Giemsa staining but yielded virtually the same
banding pattern and were equally useful for
chromosome identification.

16. •In 1970 two new techniques were introduced that
have had a major impact on modern cytogenetic
analysis.
-The first was the demonstration by Pardue and Gall
that isotopically labeled DNA probes could be annealed
to complementary DNA sequences in cytologic
preparations of chromosomes made by standard
techniques, a procedure referred to as in situ
hybridization (ISH).
-Pardue and Gall also noted that when the denatured
chromosomes were stained by Giemsa, the paracentric
regions were preferentially stained (C bands).

17. • The growing emphasis on timely management of patients and the detection of
chromosome abnormalities beyond the resolution of the light microscopy in recent
years has led to the development of targeted molecular cytogenetic techniques freed
from the cell culturing and lengthy protocols used for the preparation of high-quality
metaphase spreads. These new technological advances allow quantitative evaluation of
the chromosomal content and include methods suchas
-quantitative FISH and
-polymerase chain reaction(Q-PCR),
-comparative genomic hybridization (CGH) and
- array-CGH,
- These methods allow higher resolution chromosome analysis and at the same time are
more amenable to automation and high through put of the samples than traditional
methods.

18. • CGH was the next major advance in genomic analysis, and provided a
tool to determine the amount of DNA in specific genomic regions
(there by diagnosing deletions or duplications) on a molecular level.
• CGH paved the way for array-based CGH, in which DNA from patient
and control is co-hybridized against DNA that has been spotted on an
array. With array-CGH, the choice of which clones to place on the
array lies in the hands of the investigator and can range from selected
clones covering specific regions of the genome to a tiling path of the
entire genome.
• The array can utilize large pieces of DNA such as inserts of human
DNA into bacterial artificial chromosomes(BACs), smaller DNA
fragments (oligonucleotides) or can utilize polymorphic regions of the
genome such as single nucleotide polymorphisms (SNPs).

19. THE INDICATIONS FOR CYTOGENETIC ANALYSIS
• 1. Confirmation or exclusion of the diagnosis for known chromosomal
syndromes.
• 2. Intellectual disability or developmental delay with or without dysmorphic
features.
• 3. Autism spectrum disorders.
• 4. Congenital anomalies.
• 5. Abnormalities of sexual differentiation and development.
• 6. Infertility/subfertility.
• 7. Recurrent miscarriages or stillbirth.
• 8. Pregnancies shown to be at risk of aneuploidy from the results of maternal
serum screening or fetal ultrasound scanning.
• 9. Neoplastic conditions for which the identification of specific chromosomal
aberrations may be valuable in diagnosis and

20. THE NORMAL HUMAN KARYOTYPE
• The International System for Human Cytogenetic Nomenclature (ISCN) was
established in 1978.
1. p (petit) for the short arm and q for the long arm
2.The main landmarks of each chromosome are the centromere, cen, and the
end of the arm, pter for the short arm and qter for the long arm.
3.The most striking of the bands are the remaining landmarks, and these divide
the arm into distinct regions. Each region is further subdivided into bands and
sub-bands. Thus, band Xp21.2 is to be found in the short arm of the X
chromosome in region 2, band1, and sub-band 2. The shorthand for the
exchange of chromosome fragments between 7p21.2 and, for example,9q34.1
in a female individual would be given as:
46,XX, t (7;9) (p21.2;q34.1), where t, translocation and the semicolon is used to
separate the chromosomes and break points.

21. heteromorphisms
• It is important to recognize and distinguish this normal variation from
the abnormal chromosomal rearrangements that are clinically
significant.
• The most striking of these variations, or heteromorphisms, occur:
1. at the centromeric regions of chromosomes 1, 9, and 16,
2. at the short arms of chromosomes 13, 14,15, 21, and 22, and
3. at the distal end of the long arm of the Y chromosome.

22. Normal Variable Chromosome Features
Variation in Length :
• heterochromatic segments (h),
• stalks (stk) or
• satellites (s)
should be distinguished from increases or decreases in arm length as
a result of other structural alterations by placing a plus (+) or minus (-)
sign after the symbols h, stk or s following the appropriate
chromosome and arm designation.

23. • 16qh+ Increase in length of the heterochrormatin on the long arm of chromosome 16.
• Yqh- Decrease in length of the heterochromatin on the long arm of the Y chromosome.
• 21ps+ Increase in length oft he satellite on the short arm of chro•mosome 21.
• 22pstk+ Increase in length of the stalk on the short arm of chromosome 22.
• 13cenh+pat Increase in length of the centromeric heterochromatin of the chromosome 13
inherited from the father.
• 1 qh-, 13cenh+, 22ps+ Decrease in length of the heterochromatin on the long arm of
chromosome 1, increase in length of the centromeric heterochrormatin on chromosome
13, and large satellites on chromosome 22.
• 15 ccnh+mat, l 5ps+pat Increase in length of the centromeric heterochromatin on the
chromosome 15 inherited from the mother and large satellites on the chromosome 15
inherited from the father.
• 14cenh+pstk+ps+ Increase in length of the centromcric heterochromatin, the
stalk, and the size of satellites on the same chromosome 14.

24. Variation in Number and Position
The same symbols as described above are used to describe variation in position of
heterochromatic segments, satellite stalks, and satellites.
• 22pvar Variable presentation of the short arm of chromosome 22
• 17ps Satellites on the short arm of chromosome 17.
• Yqs Satellites on the long arm of the Y chromosome.
• 9phqh Heterochrormatin in both the short and the long arms of chromosome 9
• 9ph Heterochromatin only in the short arm of chromosome 9.
• 1q41h Heterochromatic segment in chromosome 1 at band 1q41.

25. In contrast, the common population
inversion variants are specified by
their euchrormatic breakpoints.
•inv(9)(p12q13) Pericentric inversion on chromosome 9.
•inv(2)(p11.2q13) Pericentric inversion on chromosome 2.

26. chimerism
• 46,XY[3]//46,XX[17]
Three cells from the male recipient were identified along with 17 cells
from the female donor.
• 46,XY,t(9;22)(q34;q11.2)[4]//46,XX[16]
Four recipient cells showing a 9;22 translocation were identified along
with 16 donor cells.
• //46,XX[20]
All 20 cells were identified as derived from the female donor.
• 46,XY[20]//
All 20 cells were identified as derived from the male recipient.

27. Uniparental Disomy
• Uniparental disomy, abbreviated upd, example detected by microarray
analysis.
• 46,XY,upd(15)mat
Male karyorype showing uniparental disorny for a maternally derived
chromosome 15.
• mos 47,XX,+21[23]/46,XX,upd(21)pat[7]
Mosaic female karyotype consisting of one cell line with uniparetal disomy for a
paternally derived chromosome 21, identified in 7 cells, and the other with
trisormy 21 identified in 23 cells. Note that the trisormic cell line is listed first
since it is larger.
45,XY,upd der(13;13)(q10;q10)pat
• A male karyotype with a single chromosome 13 that is a Robertsonian
translocation inherited from the father. Because the father has the same
karyotype. This has been interpreted to be uniparental disomy.

28. • 46, XX 46, XY
• inv(2) del(4) r( 18)
• t(X;3) t(2;5)
• ins(5;2)
• 47,XX+ 21, 45,XY- 7, +der(24p+,5q-)
• mos 45,X/46,XX chi 46,XX/46,XY.
• mos 47,XY+21/46,XY mos 47,XXY/46,XY
• mos 45,X[15]/47,XXX[10]/46,XX[23]

29. Reference:
1. Emery & Rimoin’s Principles and Practice of Medical Genetics (6th Ed.)
2. ISCN 2013