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Genetic Dissection of Human Diseases

The Hebrew University of Jerusalem. Genetic Dissection of Human Diseases. Ariel Darvasi. Genetic strategies for gene discovery. Genetic association. Linkage analysis. Whole-genome scans. Candidate genes. Linkage analysis. A1A2 A3A4. Affected sib pairs. A1A3 A1A3.

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Genetic Dissection of Human Diseases

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

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

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

  4. Association Analysis aa AA AA aa AA aa Controls Cases aa AA AA aa AA aa

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

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

  7. Sample Size Required With the Case-Control Design: Na= number of cases Nc= number of controls Pa,Pc= disease allele frequency in the affected and control population respectively.

  8. Sample Size Required: Numerical Example

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

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

  11. Average LD

  12. D’=1 r2=1 Proportion of high LD SNP pairs

  13. The Advantage of Homogeneity

  14. The Advantage of Homogeneity

  15. LD calculated within ‘haplotype blocks’

  16. 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 LD in 10 selected regions (r2) 105kb with at least 7 SNPs in 3 populations. ASH CC AA

  17. Combinations testedSelecting 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. 1/r2=2 Number of SNPs required for whole-genome scan Random haplotypes 80,000 SNPs (2.8 SNPs per 105kb) Random SNPs 120,000 SNPs (4.2 SNPs per 105kb) Optimal haplotypes 70,000 SNPs (2.4 SNPs per 105kb) Optimal SNPs 100,000 SNPs (3.5 SNPs per 105kb)

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

  21. DNA Pooling for SNP allelotyping Controls Cases

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

  23. 1 . 00 0 . 80 0 . 60 0 . 40 0 . 20 0 . 20 0 . 40 0 . 60 0 . 80 1 . 00 RFLP SE=0.04

  24. 1 . 00 0 . 80 0 . 60 0 . 40 0 . 20 0 . 20 0 . 40 0 . 60 0 . 80 1 . 00 Pyrosequencing SE=0.02

  25. 1 . 00 0 . 80 0 . 60 0 . 40 0 . 20 0 . 20 0 . 40 0 . 60 0 . 80 1 . 00 SBE SE=0.03

  26. 1 . 00 0 . 80 0 . 60 0 . 40 0 . 20 0 . 20 0 . 40 0 . 60 0 . 80 1 . 00 MS SE=0.02

  27. 1 . 00 0 . 80 0 . 60 0 . 40 0 . 20 0 . 20 0 . 40 0 . 60 0 . 80 1 . 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. Schizophrenia Family Studies Average lifetime morbid risks for developing schizophrenia (Riley & McGuffin, 2000)

  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 4 3 . 5 100 KB 3 2 . 5 Z Score 2 1 . 5 1 0 . 5 0 Chromosomal Location

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

  34. Individual Genotyping Results N P-value Sex Con Scz Genotype Allele GG SNP rs165688 F 706 262 0.25 0.30 0.90 M 2264 458 0.027 0.041 0.0074 All 0.041 0.024 0.023 rs165599 - 5 - 6 - 6 F 1380 263 1.0x10 9.1x10 6.8x10 M 3519 461 0.203 0.104 0.090 - 5 - 5 - 5 All 3.0x10 8.8x10 7.2x10 rs737865 F 685 250 0 .012 0.014 0.43 - 4 M 2164 464 0.0011 0.0083 2.3x10 - 4 All 1.6x10 - 4 3.6x10-4 4.8x10

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

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

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

  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. Summary 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

  44. Acknowledgements HUJI IDgene Clinical CollaborationsAvraham 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  Sagiv Shifman Ester Inbar Meira Sternfeld Tami Mendelbaum Anat Horowitz Shoshi Berger Gal Romano Anne Pisante Benjamin Yakir Mira Korner 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 Technology Companies Qiagen Genomics Inc.

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