Cytogenetics

1. Cytogenetics

2. Cytogenetics The study of chromosome and the related disease states caused by abnormal chromosome number and\or structure. Chromosomes : complex structures located in the cell nucleus, composed of DNA, histone and non-histone proteins, RNA, and polysacchairdes.

3. History of human cytogenetics “Dark Ages’’ ( prior to 1952 ) no. of chromosomes = 48. The “Hypotonic Era” started in 1952 no. of chromosomes = 46. The “Trisomy Period”. The “Banding Era”. The “Molecular Era”.

5. Cell Division – Meiosis I& II

6. Karyotype preparation

7. Chromosomes Banding

9. G-Banding/chromosome morphology

11. Chromosome Morphology

17. Changes in number, or sets, of chromosomes A) Polypoidy – change in complete sets of chromosomes (3n, 4n, etc) plants > animals. B) Aneuploidy – change in the no. of chromosomes nullisomy 2n-2 monosomy 2n-1 trisomy 2n+1 tetrasomy 2n+2 Gene dosage effect 1- Sex-chromosomal aneuploids . 2- Autosomal aneuploids .

21. Changes in structure of individual chromosome A) Chromosome rearrangements: Effects Deletion. pseudodominance dicentric chr. Duplication. gene dosage Inversion – paracentric positional pericentric Translocation. positional new linkage rearrangement B) Fragile-X Syndrome. C) Cancer/mutations.

32. Fragile-X-Syndrome

34.  Advantages 1- Enable the entire genome to be viewed at one time. 2- Suitable when a specific anomaly is suspected ( e.g. Philadelphia in CML ) and as a general diagnostic tool to detect additional chr. Abnormalities commonly seen in disease progression of CML. Disadvantages 1- Detect major structural abnormalities ( one band = 6mb of DNA ~ 150 genes ). 2- Labor intensive and highly dependent upon operator experience and skills.

35. Fluorescence in situ hybridization (FISH) Increased the sensitivity , specificity ,and resolution of chromosome analysis. Fluorescently labeled DNA probe ~40 kb.to detect or confirm gene or chromosome abnormalities that are beyond the resolution of routine cytogenetics. Metaphase FISH Interphase FISH

38. I. Microdeletion Syndromes Cri-du-chat (5p-). Miller-Dieker syndrome (7q11.23). Smith-Magenis syndrome (17p13.3). Steroid Sulfatase Deficiency (Xp22.3). DiGeorge/Velo-cardio-facial/CATCH-22/ Shprintzen Syndrome (22q11.2). Kallman Syndrome (Xp22.3). Williams Syndrome (7q11.23). Wolf-Hirsch horn (4p-). Prader-Willi/Angelman Syndrome (15q11.2-13). X-Linked Icthyosis (xp22.3). Retinoblastoma (13q14). Fluorescence in situ hybridization (FISH)

41. II. Trisomy Detection and Sex Determination Probes for chromosomes 13,18,21,X,Y and SRY.

43. III. Oncology Single Gene Probes( deletion or amplification) P58 CLK-1 Locus (1p36). D7S486 (7q31). Retinoblastoma (13q14). P53 (17p13.1). Her-2/ neu (17q11.2-q12).

44. Oncology-cont. Enumeration probes for all chromosomes Dual Color Translocation Probes. bcr/abl translocation t(9;22)(q34;q11.2). M-bcr/abl translocation t(9;22)(q34;q11.2). IGH/CCND1 translocation t(11;14)(q13;q32). PML/RARA translocation t(15;17)(q22;q21.1). TEL/AML1 translocation t(12;21)(p13;q22).

46. Fluorescence hybridization in situ(FISH) cont. Prenatal/Neonatal screening: Aneuploid detection by FISH for chromosomes 13,18,21,X,Y. Telomere Alteration Testing: Identify alterations in 7-10% of cases with MR and multiple congenital anomalies with mental retardation.

47. FISH-cont. Advantages: The resolution is better (L ~ 2mb). Can be applied to both dividing and non-dividing cells. The tech. is straightforward. Hybridization with multiple probes enable detection of translocation products. Can identify a range of mutations. Monitor recurrent or residual disease in BMT pt.

48. FISH-cont. Disadvantages: Cannot detect small mutations. Miss Uniparental disomy. Miss Inversions. Probes are not yet commercially available for all chromosomal regions.

49. Spectral Karyotyping (SKY) and Multiple Fluorescence In Situ Hybridization(M-FISH) Simultaneous visualization of all human (or mouse) Chromosomes in different colors. A combination of five fluorochromes to paint all 22 autosomes,and the X and the Y. then analyzed based on their particular emission spectra.

53. Spectral Karyotype of human chromosomes

54. Ewing Sarcoma

55. SKY-cont. Advantages: Mapping of chromosomal breakpoints. Detection of subtle translocations. Identification of marker chromosomes,homogeneously staining regions,and double minute chromosomes. Characterization of complex rearrangements. Disadvantages: Very expensive equipments. The technique is labor intensive. Dose not detect structural rearrangements within a single chromosome. Low resolution (up to 15 mb ). Specific, not a screening method.

56. Genomic Comparative Hybridization(CGH) Fluorescent molecular tech. that identifies DNA gains,losses,and amplifications (mapping to)metaphase chromosomes. Based on quantitative two-color FISH (FITC for tumor DNA and TRITC for the normal DNA). Advantages: Require only genomic tumor DNA. Can be applied to fresh or frozen tissues,cell lines, and archival formalin-fixed paraffin-embedded samples.

61. Genomic Comparative Hybridization(CGH) Disadvantages: Cannot detect balanced abnormalities. Chromosomal copy changes < 10 mb. are not resolved. Copy no. changes < ½ of the analyzed cells are not detected.

62. Diagnostic Potential For Karyotype, FISH, and Chromosomal Micro- array Analysis (CMA) For Selected Disorders