1. Genetic Dissection of Human Diseases Ariel Darvasi The Hebrew University of Jerusalem

2. Genetic strategies for gene discovery Genetic association Linkage analysis Candidate genes Whole-genome scans

3. Linkage analysis A1A2 A3A4 A1A3 IBD=2 Affected sib pairs (Identical by descent)

4. Association Analysis Cases Controls AA aa

5. Linkage Disequilibrium (LD) Mapping SNPs Functional SNP Genes

6. Genotypic Relative Risk (GRR) dd DD diagnogenomicist Disease penetrance: 1%4% GRR=4.0

7. Sample Size Required With the Case- Control Design: N a = number of cases N c = number of controls P a,P c = disease allele frequency in the affected and control population respectively.

8. Sample Size Required: Numerical Example

9. Genetic strategies for gene discovery Linkage analysis Candidate genes Whole-genome scans Genetic association * Population selection (outbred/inbred)? * How many SNPs? * How to select the SNPs? * How to genotype the SNPs (DNA pooling)?

10. A random sample of different chromosomal areas 1 Mb - 16 SNPs 300 Kb 150 Kb 300 Kb 150 Kb 5 or 15 Kb Each SNP was genotyped on: - 90 Caucasians - 90 Afro-Americans - 90 Ashkenazi Jews

11. Average LD

12. Proportion of high LD SNP pairs D ’ =1r 2 =1

13. The Advantage of Homogeneity

15. LD calculated within ‘haplotype blocks’

16. LD in 10 selected regions (r 2 ) ASH CC AA 105kb with at least 7 SNPs in 3 populations. 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

17. Combinations tested Selecting 1,2,3,4 or 5 SNPs/100kb with: Single SNP analysis: –Random SNP selection –Optimal SNP selection Haplotype analysis: –Random SNP selection –Optimal SNP selection

18. Random and optimal selection for single SNP and haplotype analysis

19. Optimal haplotypes 70,000 SNPs (2.4 SNPs per 105kb) Random haplotypes 80,000 SNPs (2.8 SNPs per 105kb) Optimal SNPs 100,000 SNPs (3.5 SNPs per 105kb) Random SNPs 120,000 SNPs (4.2 SNPs per 105kb) 1/r 2 =2 Number of SNPs required for whole- genome scan

20. Common Disease AlleleRare Disease Allele Rare Variants (<10%) Affecting Complex Traits

21. DNA Pooling for SNP allelotyping Cases Controls

22. RFLP, Restriction fragment length Polymorphism Pyrosequencing Single base-pair extension (SBE) Mass Spectrometry (MS) TaqMan Evaluating DNA Pooling Technologies

23. 0.20 0.40 0.60 0.80 1.00 0.200.400.600.801.00 RFLP SE=0.04

24. 0.20 0.40 0.60 0.80 1.00 0.200.400.600.801.00 Pyrosequencing SE=0.02

25. 0.20 0.40 0.60 0.80 1.00 0.200.400.600.801.00 SBE SE=0.03

26. 0.20 0.40 0.60 0.80 1.00 0.200.400.600.801.00 MS SE=0.02

27. 0.20 0.40 0.60 0.80 1.00 0.200.400.600.801.00 TaqMan SE=0.02

28. An Example: Dissecting the genetic basis of schizophrenia

29. Main Symptoms of Schizophrenia Positive Symptoms: Delusions, hallucinations, disorganized speech, unusual behavior, agitation. Negative Symptoms: Lack of emotion, inability to speak, lack of motivation, no pleasure from fun activities, slow movements. Symptoms Involving Thoughts: Decreased attention span and memory, difficulty making decisions. Mood: Depression, unpleasant feelings, hopelessness, low self-esteem.

30. Average lifetime morbid risks for developing schizophrenia (Riley & McGuffin, 2000) Schizophrenia Family Studies

31. Gene Discovery in Schizophrenia Choosing a genomic interval and creating a high density SNP map in that region

32. High throughput scanning of all SNPs in DNA pools 0 0.5 1 1.5 2 2.5 3 3.5 4 Chromosomal Location Z Score 100KB

33. Pooled genotyping of additional SNPs at the COMT locus COMT gene

34. 3.6x10 -4 4.8x10 -4 Individual Genotyping Results

35. G-----------G-----------G Haplotype P-value = 9.5 x 10 -8 SNPs: rs165688 rs737865 rs16599 Haplotype Analysis MalesFemales 13.5%32.2%PAR The G-G-G risk haplotype also has the strongest effect on mRNA expression levels (Bray et al. 2003)

36. Candidate Functional SNPs Point deletion (C/-) 3’ UTR SNP (C/T) near Estrogen Response Element in promoter

37. Is COMT a reasonable susceptibility gene for schizophrenia? Is it reasonable to expect a sex- specific effect of COMT? COMT and Schizophrenia

38. Few facts on COMT COMT catalyzes one of the major degradation pathways of dopamine (and other catecholamine transmitters) COMT is involved in the metabolism of catechol estrogens. Estrogen down regulates COMT transcription

39. Gender Differences in Schizophrenia Age of Onset Men: early 20s Women: mid to late 20s Symptoms Men: have more negative symptoms Women: have more depressive symptoms and paranoia Course of illness Woman schizophrenic patients have a more benign course of illness than men

40. COMT Knockout Mice In homozygous males: 2-3 fold increase in the amount of dopamine in the frontal cortex Homozygous females demonstrate increased anxiety in a dark/light exploratory model Heterozygous males exhibit increased aggressive behavior

41. The Estrogen Connection According to the estrogen theory, women are protected to some extent against schizophrenia between puberty and menopause by their relatively high physiological estrogen production during this phase.

42. Estrogen Theory  Estrogen levels have significant effects on the mental state of schizophrenic women  Women have a second peak of illness onset after age 45, with a more severe course of illness  Estrogen was tested with success as a therapeutic agent in schizophrenia  The protective effect of estrogen seems to contribute to some of the gender differences in schizophrenia: age of onset, severity and response to neuroleptic treatment

43. Science has reached today the point where complete dissection of the genetic basis of common diseases is an achievable challenge This will lead to the identification of disease related biochemical pathways which in turn will lead to novel and efficient therapies Summary

44. Naomi Zak Michal Bornstein Efrat Lev-Lehman Becky Houry Galit Hershko Guy Amit Ilana Blech Irina Barsky Michal Millo Svetlana Lobovsky Svetlana Shpiudlez Sari Lubin Vardit Ben-Dor Erella Kenoshi Dvora Rubinow Sagiv Shifman Ester Inbar Meira Sternfeld Tami Mendelbaum Anat Horowitz Shoshi Berger Gal Romano Anne Pisante Benjamin Yakir Mira Korner Acknowledgements Clinical Collaborations Avraham Weizman Haim Y. Knobler Nimrod Grisaru Leon Karp Moshe Kotler Ilya Reznik Richard Schiffer Eilat Shinar Baruch Spivak Rael D. Strous Marnina Swartz- Vanetik Stanford Neil Risch Technology Companies Qiagen Genomics Inc. IDgene HUJI