1. Chromosomal abnormalitilies & Clinical Cytogenetics Mohamad Nusier M.D., Ph.D.

3. Cytogenetics Is a study of chromosomes and their abnormalities Chromosome abnormalities are responsible for a significant proportion of genetic diseases, occurring in approximately 1 of every 150 live birth They are the leading known cause of both mental retardation and pregnancy loss Chromosome abnormalities are seen in 50% of first-trimester and 20% of second-trimester spontaneous abortions

4. Cytogenetic Techniques The use of spindle poisons, such as colchicine and colcemid, that arrest dividing cells in metaphase The use of a hypotonic solution, which causes swelling of cells, rupture of the nucleus and better separation of individual chromosomes The use of staining materials that are absorbed differentially by different parts of chromosomes, thus producing the characteristic bands that help to identify individual chromosomes

5. Analysis of chromosomes Collecting a living tissue (usually blood) Culturing the tissue for amount of time Adding colcemid to produce metaphase arrest Harvesting the cells Placing the cell sediment on a slide Rupturing the cell nucleus with a hypotonic saline solution Staining with a designated nuclear stain Photographing the metaphase “spread” of chromosomes on the slide

6. Cytogenetic Nomenclature Karyotype : Is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. The term is also used for the complete set of chromosomes in a species, or an individual organism.appearancechromosomesnucleuseukaryoticcellspecies Karyotypes describe the number of chromosomes, and what they look like under a light microscope. Attention is paid to their length, the position of the centromeres, banding pattern, any differences between the sex chromosomes, and any other physical characteristics. The preparation and study of karyotypes is part of cytogenetics.microscopecentromeressex chromosomescytogenetics

7. Cytogenetic Nomenclature Chromosomes are further classified according to the position of the centromere: –Metacentric: centromere occurs near the middle of the chromosome –Submetacentric: centromeres somewhere between the middle and the tip of the chromosome –Acrocentric: centromere near the tip of the chromosome –Telocentric: centromere at the end of the chromosome Telomere: the tip of each chromosome p: the short arm of a chromosome (petite) q: the long arm of a chromosome

8. A comparison of centrometric location

9. The human karyotype The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes.autosomalsex chromosomes Normal karyotypes for females contain two X chromosomes and are denoted 46,XX; males have both an X and a Y chromosome denoted 46,XY.femalesX chromosomesmalesY chromosome Any variation from the standard karyotype may lead to developmental abnormalities.

10. Healthy Human Female Karyotype

11. Different characteristics of karyotypes are usually observed and compared Differences in basic number of chromosomes. Differences in absolute sizes of chromosomes. Differences in relative size of chromosomes (can only be caused by segmental interchange of unequal lengths). Differences in the position of centromeres. This is brought about by translocations.centromerestranslocations Differences in number and position of satellites, which (when they occur) are small bodies attached to a chromosome by a thin thread. Differences in degree and distribution of heterochromatic regions. Heterochromatin stains darker than euchromatin, indicating tighter packing, and mainly consists of genetically inactive repetitive DNA sequences.heterochromaticeuchromatin In conclusion: A full account of a karyotype may therefore include the number, type, shape and banding of the chromosomes, as well as other cytogenetic information.

12. Variation is often found Between the sexes Between the germ-line and soma (between gametes and the rest of the body)germ-linesomagametes Between members of a population (chromosome polymorphism)chromosome polymorphism Geographical variation between racesGeographical variationraces Mosaics or otherwise abnormal individuals.Mosaics

13. Cytogenetics employs several techniques to visualize different aspects of chromosomes

14. Chromosome banding It facilitates the correct identification of individual chromosomes Helps in the detection of deletions, duplications and other structural abnormalities.

15. Chromosome-banding Techniques Giemsa banding (G-banding): is obtained with Giemsa stain following digestion of chromosomes with trypsin. It yields a series of lightly and darkly stained bands - the dark regions tend to be heterochromatic, late-replicating and AT rich. The light regions tend to be euchromatic, early-replicating and GC rich.Giemsatrypsin This method will normally produce 300-400 bands in a normal, human genome.human genome

16. Karyogram of human male using Giemsa stainingGiemsa

17. Human G-bands

18. Chromosome-banding Techniques…cont Quinacrine banding (Q-banding): is a fluorescent pattern obtained using quinacrine for staining. The pattern of bands is very similar to that seen in G-banding.fluorescentquinacrine Reverse banding (R-banding): is the reverse of G-banding (the R stands for "reverse"). The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).

19. Human Q-bands

20. Human R-bands

21. Chromosome-banding Techniques…cont Staining certain portions of the chromosome –C-banding: stains the constitutive heterochromatin which usually lies near the centromere –Nucleolar organizing region stain (NOR stains): staining highlights the satellites and stalks of acrocentric chromosomes High-resolution banding: staining the chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation

22. Human C-bands

24. Chromosome Banding The major bands on each chromosome are numbered, 14q32 refers to the second band in the third region of the long arm of chromosome 14. Sub-bands are designated by decimal points following the band number (14q32.3 is the third sub-band of band 2)

26. Other Techniques Fluorescence in situ hybridization (FISH): Is a technique in which a labeled probe is hybridized to metaphase, prophase, or interphase chromosomes. Can be used to test for missing or additional chromosomal material as well as chromosome rearrangement.

27. Fluorescence in-situ hybridization (FISH)

28. Scheme of the principle of the FISH Experiment to localize a gene in the nucleus.

29. Fluorescence in-situ hybridization (FISH)

30. FISH: Urothelial cells marked with four different probes

31. A metaphase cell positive for the bcr/abl rearrangement (associated with chronic myelogenous leukemia using FISH. The chromosomes can be seen in blue. The chromosome that is labeled with green and red spots (upper left) is the one where the rearrangement is present. chronic myelogenous leukemia

32. Interphase cells positive for a chromosomal t(9;22) rearrangement.

33. Other Techniques…cont Spectral karyotyping: Utilize varying combinations of five different fluorescent probes in conjunction with special cameras and image- processing software so that each chromosome is uniquely colored, used mostly to detect chromosome rearrangements.

34. Spectral karyogram of a human female

35. Spectral human karyotype