Cytogenetic Analysis

ISCNDr. Ruslan Bayramov MDMedical Genetics, Erciyes University2015

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.

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

•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.

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.

•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.

• Subsequent developments in cancer cytogeneticshave 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.

• 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.

Ford reported in 1959 that Turner syndrome

was usually associated with a 45, X

chromosome complement

and

Jacobs and Strong foundthat Klinefelter syndrome had a 47, XXY

• 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.

•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.

•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.

•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.

• 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.

• Even before chromosome denaturation was being used to produce various banding patterns, Caspersson

et al. independently discovered that quinacrinecompounds 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.

• 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 cytologicpreparations 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).

• 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.

• 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).

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

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.

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.

Normal Variable Chromosome FeaturesVariation 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.

• 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.

Variation in Number and PositionThe 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.

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.

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.

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 Robertsoniantranslocation inherited from the father. Because the father has the same

karyotype. This has been interpreted to be uniparental disomy.

• 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]

Reference:

1. Emery & Rimoin’s Principles and Practice of Medical Genetics (6th Ed.)

2. ISCN 2013