Cytogenetics

2005

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.

History of human cytogenetics 

´Dark Ages·· ( prior to 1952 ) The ´Hypotonic Eraµ started in 1952
no. of chromosomes = 46.

no. of chromosomes = 48.  

 

The ´Trisomy Periodµ. The ´Banding Eraµ. The ´Molecular Eraµ.

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Cell Division ± Meiosis I& II .

Karyotype preparation .

mostly repetitive AT-rich DNA R-banding Variety of techniques Chromosome arms.Chromosomes Banding Type Q-banding Stain Quinacrine Area Stained Chromosome arms. distinct fluorescent banded pattern for each chromosome. chromosomes 7. highly repetitive. 9. same as Q-banding pattern except single additional band near centromere of chromosomes 1 and 16 Reverse banding pattern of that observed with Q. and 15 have medium-sized bands. 10.or Gbanding G-banding Giemsa Chromosome arms. 16. mostly unique GC-rich DNA C-banding Variety of techniques Centromere region of each chromosome and distal portion of Y chromosome. Distinct banded pattern for each chromosome. size of C-bands highly variable from person to person . and Y. mostly repetitive AT-rich DNA Effect Under UV light. mostly AT-rich DNA Largest bands usually on chromosomes 1.

Non-Banded Karyotype .

G-Banding/chromosome morphology .

Ideograms .

Chromosome Morphology .

Normal Karyotype .

Low/High Resolutions Karyotype 18 7 .

Q-Banding .

C-Banding .

R-Banding .

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

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Inversion ² paracentric pericentric Translocation. . Duplication. pseudodominance dicentric chr. C) Cancer/mutations.Changes in structure of individual chromosome A) Chromosome rearrangements: Effects     Deletion. gene dosage positional positional new linkage rearrangement B) Fragile-X Syndrome.

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Fragile-X-Syndrome .

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

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

FISH .Metaphase.

Interphase-FISH .

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

Di-George Syndrome .

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Trisomy Detection and Sex Determination Probes for chromosomes 13.II.Y and SRY.21.18.X. .

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

22)(q34. . PML/RARA translocation t(15. TEL/AML1 translocation t(12.Oncology-cont.21)(p13. Enumeration probes for all chromosomes Dual Color Translocation Probes.q11.1). IGH/CCND1 translocation t(11. M-bcr/abl translocation t(9.2).2).q21. bcr/abl translocation t(9.q32).17)(q22.q22).14)(q13.22)(q34.q11.

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Telomere Alteration Testing: Identify alterations in 7-10% of cases with MR and multiple congenital anomalies with mental retardation. .Y.Fluorescence hybridization in situ(FISH) cont.X.21.18. Prenatal/Neonatal screening: Aneuploid detection by FISH for chromosomes 13.

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

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

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. .

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Spectral Karyotype of human chromosomes .

Ewing Sarcoma .

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

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

Comparative Genomic Hybridization(CGH) .

Comparative Genomic Hybridization(CGH) .

Genomic Comparative Hybridization(CGH) .

Comparative Genomic Hybridization(CGH) .

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

Diagnostic Potential For Karyotype.array Analysis (CMA) For Selected Disorders Condition Locus studied Karyotype Disease specific FISH Telomere FISH CMA Aneuploidy Large deletions. FISH. translocation of large segments Cryptic Rearrangements of telomeres 1p36 deletion Wolf-Hirschhorn Cri-du-chat Williams-Beuren Prader-Willi Angelman Miller-Dieker lissencephaly Smith-Magenis Velocardiofacial/DiGeorage 1 various various ~100% ~100% Not detected Not detected Detected by karyotype Detected by karyotype ~100% >95% >95% >95% Not detected Not detected Not detected Some detected Not detected Not detected ~100% Karyotype better for present ~100% for unbalanced ~99% ~99% ~99% ~99% ~70% ~70% >90% >95% >95% various 1p36.2 7q11. large dupllications.2 22q11.3 4p16.2 Not detected Few Most Most Almost none Unreliable Unreliable Few Some Rarely Not detected ~99% ~99% ~99% ~99% ~70% ~70% >90% >95% >95% . and Chromosomal Micro.3 17p11.3 5p15.2 15q11-q13 15q11-q13 17p13.

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