CYTOGENETICS

1. CYTOGENETICS Dr.S.Chakravarty MD

4. Karyotyping • Metaphase stage • Maximally Condensed • Photographed under the microscope , a 23 pairs of chromosomes in atypical somatic cell • Arranged according to size • G-banding –Mitotic chromosomes are partially digested with trypsin (to digest some associated protein ) and stained with Giemsa , a dye that binds DNA

7. Type of Chromosomes • Metacentric – centromere in the middle • Sub metacentric – centromere b/w middle and the ends (4) • Acrocentric – cetromere at the end(13,14, 15 , 21 , 22) • Robersonian Translocations

8. Common Symbols used • 1-22 : AUTOSOME NUMBERS • X,Y : SEX CHROMOSOMES • (+) or (-) : EXTRA OR MISSING CHROMOSOMES • q : long arm • p : short arm • t : translocation • Del : deletion

9. Euploidy • When a cell has multiples of 23 chromosomes • Most cells are diploid 46 chromosomes • Gametes are haploid • Triploid :- 69 chromosomes • if two sperms fertilize an ova :LETHAL, 1 in 10,000 • Tetraploid :- 92 Chromosomes: LETHAL

10. Aneuploidy • Gain or Loss of a specific chromosome • Most commonly due to non-disjunction of chromosomes

11. Most monosomies of autosomes are lethalDo not exit !

12. XO  Turner’s syndrome • Short stature • Webbed neck • Low set ears • Amenorrhoea • Sterility

13. Klienfelter’s disease • XXY

14. Disjunction during meiosis Meiosis 2 Meiosis 1

15. Non disjunction in Meiosis 1

16. Non disjunction in Meiosis 2

17. Remember in such a case other offsprings are generally normal as this is an ACCIDENT !!

18. Reciprocal Translocation • Exchange of genetic material between non-homologous chromosomes Balanced translocations :- All the genetic information is present

19. 95% of DNA is junk. Only 5% codes for proteins • Very less chances that an important gene is deleted !

20. TRANSLOCATION INVOLVING ONCOGENES • t (9 :22) PHILADELPHIA CHROMOSOME – CHRONIC MYELOGENOYUS LEUKEMIA (c-abl) • t (8: 14) Burkitt’s Lymphoma (c-myc) • t (15:17) Acute myelogenous leukemia (retinoid receptor α)

21. Consequences of a Reciprocal Translocation

22. Robertsonian Translocations Loss of p arms

23. Consequences of a Robertsonian Translocation

24. Robertsonian translocation and Down’s syndrome • Trisomy 21 with 46 CHROMOSOMES • 46XY , -14 + t (14q; 21q)

25. Robertsonian translocation and Down’s syndrome • 5% cases are because of such translocations • Recurrence risk for trisomy 21 due to non-disjunction of chromosomes in meiosis is very low • Recurrence risk for trisomy 21 due to Robertsonian translocation is higher. • Males 1-2 % Females :- 10-15%(reasons not known )

27. DELETIONS

28. CRI-DU-CHAT • 46 XY del (5p) or 46XX del (5p) • HI PITCHED CAT LIKE CRY • MENTAL RETARDATION • MICROCEPHALY • CONGENITAL HEART DISEASE

29. INVERSIONS • TWO BREAKS ARE INSERTED IN THE SAME LOCATION PERICENTRIC – CENTROMERE INCLUDED PARACENTRIC - CENTROMERE NOT INCLUDED

30. RING CHROMOSOME • DELETION ON BOTH TIPS OF A CHROMOSOME

31. Few cases of Turner’s syndrome occurs because of this. • (NORMAL 46 XX minus one X chromosome due to Ring Chromosome formation = 46X0 )

32. ISOCHROMOSOME • Chromosome divides in an axis perpendicular to its normal axis  ISOCHROMOSOME • TWO COPIES OF ONE ARM BUT NO COPY OF THE OTHER Some cases of Turner’s syndrome

33. ADVANCES IN MOLECULAR CYTOGENETICS • FISH • SPECTRAL KARYOTYPING

34. FISH analysis of chromosomes: Fluorescent IN SITU hybridization • PROCUDURE :- • Denature the chromosomes • Denature the probe • Hybridization • Fluorescence staining • Examine slides or store in the dark Metaphase spread chromosomes stained with DAPI, a fluorescing stain that specifically binds double stranded DNA

35. Eg.FISH tests • Di GEORGE’S SYNDROME • Expose DAPI-stained chromosomes to mixture of fluorescent probes • green = control probe for chromosome 22 • red = probe for DiGeorge region on long arm of chromosome 22 • DOWN’S SYNDROME • Expose DAPI-stained chromosomes to mixture of fluorescent probes • Green- chromosome 9 • Red – probe for Chromosome 21 Di George’s syndrome

36. Expose DAPI-stained metaphase chromosomes to fluorescent probes The most commonly performed FISH does not detect all chromosomal abnormalities; this FISH test specifically looks at chromosomes 21,22, 18, 13, X and Y.  This enables the FISH test to detect most of the common chromosomal abnormalities, particularly Down syndrome. green = chromosome 22 red = control probe for centromere of the X chr & another probe for end of chromosome X Chromosomes to fluorescent probes that highlight chromosomes X,Y,22,13, 21 & 18 green = chromosome 13 red = chromosome 21 aqua = chromosome 18 green = X chromosome red= Y chromosome

37. SPECTRAL KARYOTYPING • Karyotyping using five different fluorescent probes that hybridize differentially to different sets of chromosomes. • Special cameras and image processing software • Every chromosome painted

38. Question 1 • A 26 year old woman has produced two children with Down syndrome and she had two miscarriages. What is the best explanation ? • A. First cousin has Down syndrome • B. Her husband is 62 years old • She carries a reciprocal translocation involving chromosome 14 and 18 • She carries a robertsonian translocation involving chromosomes 14 and 21 • She was exposed to multiple Xrays in childhood

39. Thank you