Techniques to study complex genetic diseases

1. Techniques to study complex genetic diseases Dr. Nallur B. Ramachandra Principle Investigator Professor and Coordinator for M.Sc., Genetics Genomics Laboratory, DOS in Zoology University of Mysore Manasagangotri, Mysore – 570 006 nallurbr@gmail.com /nbruom@gmail.com

2. According to WHO • 140 million children were born every year • 5 million die in the 1st month of life in developing countries • 1/6 of the world population is suffering with genetic diseases

3. ~ 4-6% of all births result in defects • 20-30% of all infant deaths - genetic disorders • The distressing and disturbing truth in this sad saga is that there is no cure available to these diseases • Hence, the prevention of the inheritance of such diseases becomes absolutely essential

4. Genetic disorders • The problems are enormous • Causes physical misery and • psychological trauma to the patients and • to their family

5. Four categories: • Mendelian disorders • b) Chromosomal disorders • c) Multifactorial disorders • d) Nontraditional inheritance disorders • (www.merck.com/mrkshared/manual)

6. Online Mendelian Inheritance in Man (OMIM) A catalog of human genes and genetic disorders ~15,500 known

7. How Genetic Diseases Differ from Other Diseases • Predict recurrence risk in other family members • Predictive testing • Genetic variability and diversity • Correction of genetic defect may be possible

8. Humans are also very different On average two random people differ at 3 mbp ~1/1000. A few genes can have a big impact on appearance.

9. APPROACHES

10. Gene DNA Population Genome Individual Family (pedigree) Approach -1 Cell Chromosome

11. Approach - 2 • Transmission Genetics • Pedigree Analysis • Cytogenetics • Molecular Genetics • Population Genetics

12. Gene Genome DNA Methylation Epigenome Transcript Transcriptome Protein Proteome Approach – 3 Four Levels of Biomarkers

13. http://www.who.int/mediacentre/factsheets/fs317/en/index.htmlhttp://www.who.int/mediacentre/factsheets/fs317/en/index.html

14. 5 most common serious birth defects Congenital heart defects Neural tube defects The hemoglobin disorders Down syndrome Glucose-6-phosphate dehydrogenase deficiency March of demise birth defect foundation, 2006

15. Analysis • Prevalence • Pedigree • Chromosomes • Gene isolation • Single nucleotide polymorphisms • Changes in the protein • Whole genome scan • Counseling, management & treatment

16. Complex diseases undertaken 1) Chromosomal syndromes –DS,TS,KS 2) Congenital Heart diseases 3) Asthma 4) Dyslexia

17. Congenital Heart Disease (CHD)

18. Congenital Heart Disease (CHD) • Malformation of the heart or the large blood vessels of the heart • Constitute 1% of developmental abnormalities • Prevalence - 1.01 to 150 / 1000 livebirths • Mysore - 11.01/1000 livebirths Reamon-Buettner et al., 2007 BMC Med Genet.; 8: 38, Smitha et al.,2006, Indian Journal of Human Genetics, 12,1

19. A four chambered heart is necessary for the separation of oxygenated and deoxygenated blood. Garg, 2006,Cell Mol Life Sci, 63:1141-1148

20. www.americanheartassociation.org

21. Prevalence

22. Prevalence of pediatric disorders in Mysore Overall Prevalence = 1-2% live births

23. Prevalenceof CHD during 2000-2005 in three major hospitals of Mysore. Smitha et al., Ind J Hum Genet, 2005

24. PEDIGREE

25. A B I I 1 2 1 2 II II 1 2 3 4 7 9 1 2 3 4 5 6 7 5 6 III 1 2 3 4 5 6 7 8 III 1 3 4 5 6 IV 1 2 3 4 2 8 IV 1 2 3 Pedigree of consanguineous marriages between parents of CHD patients: (A) Uncle-niece marriage and(B) First-cousin marriage

26. Parental consanguinity~24% Smitha et al., Annals Hum Bio, 2006

27. CHROMOSOMES

29. Summary of CHD cases

30. CHD patients with chromosomal anomalies

31. Clinical feature of the patient with Isochromosome 18 • Hurried breathing, nasal discharge, • On-and-off fever and constipation • Body weight below the 5th percentile for age and sex • Short stature • Microcephaly • Depressed nasal bridge • Epicanthal folds • Low-set ears • High-arched palate, clinodactyly, and • Congenital Heart disease

32. i(18p) G-banded chromosomes of congenital heart disease patient with isochromosome 18p [47,XX,+i(18p)]: Metaphase showing the i(18p) (arrow). Smitha et al., J App Genet, 2006

33. 18p i(18p) 18p FISH plate showing chromosome 18p subtelomeric region on metaphase chromosome of the isochromosome 18p child. FISH probe identifies chromosome 18p (spectrum green),centromeric 18 (spectrum aqua),chromosome 11p (spectrum green) and chromosome 11q (spectrum orange). The labels 18p and arrow denote the 18p of the normal chromosome 18 (white) and isochromosome 18p (yellow) respectively.

34. Clinical feature of the patient with trisomy 21 • low set ears • flat occiput • depressed nasal bridge • mangoloid shunt • clinodactyly • simian crease • nonrestrictive ostium secundum (OS) type of ASD

35. 21 b a c 21 A. B. FISH image of the proband [47,XY,+21,t(9;13)(q34.1;q34)]:(A) Trisomy 21 (Spectrum red) and (B) Subtelomeric deletion on chromosome 9q and the translocation of the deleted region on subtelomeric region of chromosome 13q on metaphase chromosomes of the proband. (a) Normal chromosome 9q region (Spectrum red); (b) Chromosome 9 with subtelomeric deletion of 9q; (c) Chromosome 13 with subtelomeric region of chromosome 9 (Spectrum red).

36. Clinical feature of the patient -complex translocation • Complex CHD : • small sized muscular VSD • nonrestrictive ASD with OS type and • a large side PDA

37. A. B. G-banded chromosomes of the congenital heart disease patient with complex variant translocation among chromosomes 5, 9 and 13 [t(5;9;13)(q14;p24;q34.1)]: (A) Metaphase showing (i) del9q11 (arrow) and (ii) t(5;9;13)(q14;p24;q34.1), and (B) Karyotype of the above metaphase.

38. a e b d c FISH image showing the complex variant translocation between chromosomes 5q, 9p and 13q on metaphase chromosomes of the proband [46, XY, t(5;9;13)(q14;p24;q34.1)]:(a) Normal chromosome 5; (b) Abnormal chromosome 5 with part of 5q deleted; (c) Normal chromosome 9; (d) Abnormal chromosome 9 with the deleted part of chromosome 5q;(e) Chromosome 13 with the deleted subtelomeric region of chromosome 9p.

39. 9 9 G-banded chromosomes of the congenital heart disease patient with inversion in chromosome 9[inv(9)(p11-q13)]: Metaphase showing inv(9)(p11- q13) (arrow). Smitha et al., Int J Hum Genet, 2007

40. Karyotype and Metaphase of CHD patient with Patau syndrome- Trisomy 13

41. Clinical Features: Complex variant • Complex CHD small sized muscular VSD (3mm) • nonrestrictive ASD with OS type (7mm) • a large side PDA (6mm) • Anoxia amblyopia • Deafness

42. M-FISH image of congenital heart disease patient with complex variant translocation chromosomes 5, 9, 11 and13 [t(5;9;11;13)(q14;p24;p15;q33)] Smitha et al., Int J Hum Genet, 2010

44. Genes isolation

45. PROKARYOTIC GENES • EUKARYOTIC GENES • HUMAN GENES

46. GENE LENGTH Covers a spectrum of sizes 21bp to > 2000kb microRNA – 11bps - 21bps tRNA, snRNA genes – smallest 65-75bps The smallest protein coding gene- 406 bp –histone H4 The longest human gene so far discovereddystrophin gene- 2400 kb with 79 introns

47. Gene isolation strategy DNA LIBRARY PROBE HYBRIDIZATION PURIFICATION OF POSTIVE CLONE DNA SEQUENCING & ANALYSIS CONFIRMATION OF GENE USE AS UNIQUE MOLECULE / DRUG

48. Positional cloning • Microsatellite markers • RFLP markers • Chromosome walking • Chromosome jumping

49. Polymerase Chain Reaction …invented by Kary Mullis while cruising in a Honda Civic on Highway 128 from San Francisco to Mendocino, "It was quiet and something just went, Click!" Kary B. Mullis Nobel Laureate, 1993 Chemistry