Dr. Nasser A Elhawary Professor of Medical Genetics

1. Chromosomal Abnormalities Dr. Nasser A Elhawary Professor of Medical Genetics

2. 2 Chromatides Centromere A chromosome distincts into 2 “sister chromatides”. They are linked together via a centromere

3. Tjio & Levan reported it is 46 chrom. in 1956 Human Chromosomes… • Contains DNA and Protein • 46 chromosomes • Autosomes: Pairs 1-22 • Sex chromosmes Human metaphase

4. Identification of Chromosomes… • The chr’s are present in pairs ‘homologs’ (one from female, the other from male). • Diploid cells (2n): Cells that contain pairs of homologous chr’s. • Haploid cells (n): Certain cells (sperm/egg gametes) contain only one copy of each chr. • At fertilization, the fusion of haploid gametes together produces a cell carrying the diploid no. of chr’s (zygote).

5. Classification of Chromosomes • The centromere(1ry constriction) divides the chr into 2 arms (petit short ‘p’-arm & long ‘q’-arm). • The location of centromere in each chr is characteristic for a given chromosome. • ‘Metacentric’: a chromosome with a centrally placed centromere. • ‘Submetacentric’: a chromosome with a centromere close to one end than the other. • ‘Acrocentric’: a chr with the centromer placed very close to one end.

6. Classification of Chromosomes…

7. Chromosome Preparation

8. Chromosome Banding… • Human chromosomes are arranged in groups from A to G. • Each group is defined by chromosomal size and centromere location. • In 1960s, new staining procedures were developed that resulted in banded chromosomes (‘dark’ and ‘light’). • Banding protocols: G-banding, R-banding, Q-banding, C-banding, etc.

9. Chromosome Banding… A karyptype of G-banded Human Chromosomes

10. R-banding…

11. Q-banding

12. C-banding…

13. What is the importance of high resolution banding?

14. Identification of Chromosomes… • Each arm is subdivided into numbered regions starting at the centromere. • Thus, any region can be identified by a descriptive address such as Xq27.3/ FMR1 gene

15. Karyotype Analysis… The banding pattern of the chromosomes in the human karyotype.

16. Banding chromosomes… G-Banding using trypsin and Giemsa stain the chromatin in 2 main forms: • Euchromatin: stains ‘light’ and consists of genes which are actively expressed. • Heterochromatinstains ‘dark’ and is made up largely of inactive unexpressed repetitive DNA.

17. Molecular Cytogenetics • Fluorescence In-Situ Hybridization (FISH): It is ability of a portion of ssDNA (i.e. probe) to anneal with its complement-ary target sequence on: i) a metaphase chromosome, ii) interphase nucleus or iii) extended chromatin fiber.

18. FISH… • FISH is widely used in clinical diagnostic purposes. • In FISH, the DNA probe is labeled with a fluorochrome + patient’s chrom visualized using fluorescent microscope.

19. Types of FISH 1- Centromeric probes. 2- Chromosome-specific unique-sequence probes. 3- Telomeric probes. 4- Whole chromosome paint probes.

20. Types of FISH… 1- Centromeric probes FISH of interphase nuclei with Centromeric probes for chromosomes 18.X and Y: showing 3 signals consistent with trisomy 18.

21. 2- Unique sequence probes are useful for identifying tiny submicroscopic deletions & duplications. Also, use of interphase FISH probe to identify HER2 over-expression in breast tumors. Metaphase image: Chromosome band 7q11.23 showed deletion associated with Williams syndrome. Normal chrom. has 2 signals (green) for the control probe and the ELN gene probe signal (orange), but the deleted chrom. shows only the control probe signal

22. Types of FISH… Telomeric probes • Telomeric probes have been used for identifying tiny ‘cryptic’ subtelomeric abnormalities, e.g. deletions, translocations.

23. Types of FISH… 3- Whole-chrom paint probes • These consists of a cocktail of probes obtained from different parts of a particular chromosome. • When this mixture of probes is used together in a single hybridization, the entire chromosome fluoresces (i.e. is “painted”). • Useful in rearrangements (e.g. translocations) & additional chromosomal materials.

24. Types of FISH… Whole-chrom paint probes Chrom painting showing a reciprocal translocation involving chrom 3 (red) & 20 (green).

25. Types of FISH… Whole-chrom paint probes • M-FISH or Spectral karyotyping (SKY) uses pools of whole human chrom. paint probes to provide a multicolor human karyotype. Each homologous chrom. shows a unique color. • These are useful to detect chromosomal rearrangements (e.g. deletions, trans-locations), ring chrom, …

26. Types of FISH… Whole-chrom paint probes M-FISH showing complex chromosome rearrangement involving chromosomes 4, 8, 13, 18, and 21

27. Comparative Genomic Hybridization • CGH was originally developed to overcome the difficulty of obtaining good-quality metaphase preparations from solid tumors. • CGH enables the detection of regions of allele loss and gene amplification. • Tumor ‘test’ DNA is labeled with green paint & control normal DNA with red paint. • The two samples are mixed and hybridized com-petitively to normal metaphase chromosomes. • watch the green-to-red ratio????

28. Comparative Genomic Hybridization CGH Analysis showing areas of gene amplification & reduction (del) in tumor DNA. DAPI: diamidophenylindole FITC: Fluorescein isothiocyanate

29. CGH… • CGH extended to include the analysis of single cells for prenatal diagnosis following whole-genome amplification. • CGH limits for more than 10Mb for losses and 2Mb for gains. • Microarray or array CGH is likely to replace metaphase CGH.

30. Chromosomal Abnormalities

31. Numerical Abnormalities • Aneuploidy: Loss or gain one or more chromosome • Polyploidy: Addition of one or more complete haploid sets (69, 92 chromosomes). • Monosomy: Loss of a single chromosome (Turner syndrome: 45,X0) • Trisomy: gain of a single chromosome (e.g. Down syndrome: 47,XY,+21).

32. Aneuploidy.. Numerical abnormality… Turner syndrome Monosomy Down syndrome Trisomy 21

33. A Karyotype of triploidy cell, 69,XXX

34. Mechanism of Trisomy

35. Structural Abnormalities • Translocation: Transfer a genetic material from one chromosome to another. • Reciprocal translocation: Breakage in 2 chromosomes and exchanged. • Robertsonian translocation: Breakage close to centromeres of acrocentric chrom. (e.g. Down syndrome)

36. Translocation

37. Origin of translocation in mother giving Down syndrome

38. Structural abnormalities… 4- Deletions: loss of part of a chromosome (e.g. Wolf syndrome ‘4p-’, cri du chat syndrome ‘5p-’). 5- Insertions: a segment of one chromosome is inserted into another chromosome. 6- Inversions: a two-break rearrangement in a chrom. which re-inserted in the inverted positions. 7- Ring chromosome: 2 breaks leaving sticky ends which re-unit to give ring chromosome. 8- Isochromosomes: from loss of one arm of the chrom. with duplication of the other.

39. Cri du chat syndrome (5p–) http://en.wikipedia.org/wiki/Cri_du_chat

40. Insertion

41. Ring chromosome (1926)

42. Inversion: A) Pericentric inversion B) Paracentric inversion

43. Mosaicism & Chimerism • Mosaic: Presence of two or more cell lines in an individual, but derived from the same zygote. • Mosaic produces from non-disjunction in an early embryonic mitotic division (mosaic trisomy 21). • Chimerism: Presence of two or more cell lines in an individual, derived from more than one zygote. • Blood Chimeras & Dispermic chimeras.

44. Mosaicism & Chimersim mechanism