Dr. Ruslan Bayramov MDMedical Genetics, Erciyes University2015Cytogenetics 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 2003Array-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.HistoryHuman 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 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.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, Xchromosome complement and Jacobs and Strong foundthat Klinefelter syndrome had a 47, XXYThe 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 23 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 involvedthe 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 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.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). 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 ANALYSIS1. 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 andTHE NORMAL HUMAN KARYOTYPEThe International System for Human Cytogenetic Nomenclature (ISCN) was established in 1978.1. p (petit) for the short arm and q for the long arm2.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.heteromorphismsIt 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 c