Diabetes Research in the Era of Complete Genomes

1. Diabetes Research in the Era of Complete Genomes American Diabetes Association June 17, 2002 Francis S. Collins, M.D., Ph.D.

7. Fulfilling the Promise of Genomics for Better Health Medical Genomics Functional Genomics Proteomics Comparative Genomics

8. Positional cloning of a gene for a highly penetrant Mendeliandisorder is now straightforward –but tracking genetic susceptibility factors for non-Mendelian disorders continues to be vexing

9. FinlandUnitedStatesInvestigation OfNIDDM

10. Study design • Linkage study using affected sibling pairs (ASPs) • In the presence of linkage, siblings show excess sharing • Additional relatives collected to determine haplotypes

11. Study sample • Collected from Finland • Founder population • High participation rate FUSION 1: 580 families n = 3584 FUSION 2: 275 families n = 877 Elderly controls n = 231

12. Linkage analysis Positive LOD score identified Fine mapping with SNPs Association identified Causative variant identified

13. Genome scan linkage results Ghosh et al (2000) AJHG 67:1174

14. HNF4A  SLC2A10  PTPN1  Microsatellites 

15. Linkage analysis Positive LOD score identified Fine mapping with SNPs Association identified Causative variant identified

16. Mass in Daltons GACCTGGAGCCCCCACC 5430.5 GACCTGGAGCCCCCACCC 5703.7 GACCTGGAGCCCCCACCTG 6047.9 Primer extension massspectrometry Primer extension reactions designed to generate different sized products C T primer

17. Allele Frequency Difference of Cases vs. Elderly Controls

18. Association is much more powerful than linkage to identify common susceptibility variants N. Risch and S. Merikangas Science 273: 1516-1517, 1996

19. Linkage analysis Positive LOD score identified Fine mapping with SNPs Association identified Causative variant identified

20. Fine mapping with SNPs Association identified Causative variant identified

21. -log10(p-value) -log10(p-value) Genome scan association results Ghosh et al (2000) AJHG 67:1174

22. A cluster of markers on chromosome 22 shows association with T2DM -log10(p-value) kilobases

23. Fine mapping with SNPs Association identified Causative variant identified

24. Whole Genome Association Approach to Common Disease • Identify all 10 million common SNPs • Collect 1000 cases and 1000 controls • Genotype all DNAs for all SNPs

25. 2000 DNAs x 10,000,000 SNPs = 20,000,000,000 genotypes

26. Current high throughput SNP genotyping methods • DNA chips • Beads/fiberoptics • Fluorescent single base extension • Pyrosequencing • Mass spectrometry • TaqMan • Invader • Etc., etc.

27. Shortcut #1: Use haplotypes to reduce number of SNPs that have to be genotyped

28. Sequence from chromosome 7 GAAATAATTAATGTTTTCCTTCCTTCTCCTATTTTGTCCTTTACTTCAATTTATTTATTTATTATTAATATTATTATTTTTTGAGACGGAGTTTCACTCTTGTTGCCAACCTGGAGTGCAGTGGCGTGATCTCAGCTCACTGCACACTCCGCTTTCC/TGGTTTCAAGCGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGTCACACACCACCACGCCCGGCTAATTTTTGTATTTTTAGTAGAGTTGGGGTTTCACCATGTTGGCCAGACTGGTCTCGAACTCCTGACCTTGTGATCCGCCAGCCTCTGCCTCCCAAAGAGCTGGGATTACAGGCGTGAGCCACCGCGCTCGGCCCTTTGCATCAATTTCTACAGCTTGTTTTCTTTGCCTGGACTTTACAAGTCTTACCTTGTTCTGCCTTCAGATATTTGTGTGGTCTCATTCTGGTGTGCCAGTAGCTAAAAATCCATGATTTGCTCTCATCCCACTCCTGTTGTTCATCTCCTCTTATCTGGGGTCACA/CTATCTCTTCGTGATTGCATTCTGATCCCCAGTACTTAGCATGTGCGTAACAACTCTGCCTCTGCTTTCCCAGGCTGTTGATGGGGTGCTGTTCATGCCTCAGAAAAATGCATTGTAAGTTAAATTATTAAAGATTTTAAATATAGGAAAAAAGTAAGCAAACATAAGGAACAAAAAGGAAAGAACATGTATTCTAATCCATTATTTATTATACAATTAAGAAATTTGGAAACTTTAGATTACACTGCTTTTAGAGATGGAGATGTAGTAAGTCTTTTACTCTTTACAAAATACATGTGTTAGCAATTTTGGGAAGAATAGTAACTCACCCGAACAGTGTAATGTGAATATGTCACTTACTAGAGGAAAGAAGGCACTTGAAAAACATCTCTAAACCGTATAAAAACAATTACATCATAATGATGAAAACCCAAGGAATTTTTTTAGAAAACATTACCAGGGCTAATAACAAAGTAGAGCCACATGTCATTTATCTTCCCTTTGTGTCTGTGTGAGAATTCTAGAGTTATATTTGTACATAGCATGGAAAAATGAGAGGCTAGTTTATCAACTAGTTCATTTTTAAAAGTCTAACACATCCTAGGTATAGGTGAACTGTCCTCCTGCCAATGTATTGCACATTTGTGCCCAGATCCAGCATAGGGTATGTTTGCCATTTACAAACGTTTATGTCTTAAGAGAGGAAATATGAAGAGCAAAACAGTGCATGCTGGAGAGAGAAAGCTGATACAAATATAAATGAAACAATAATTGGAAAAATTGAGAAACTACTCATTTTCTAAATTACTCATGTATTTTCCTAGAATTTAAGTCTTTTAATTTTTGATAAATCCCAATGTGAGACAAGATAAGTATTAGTGATGGTATGAGTAATTAATATCTGTTATATAATATTCATTTTCATAGTGGAAGAAATAAAATAAAGGTTGTGATGATTGTTGATTATTTTTTCTAGAGGGGTTGTCAGGGAAAGAAATTGCTTTTTTTCATTCTCTCTTTCCACTAAGAAAGTTCAACTATTAATTTAGGCACATACAATAATTACTCCATTCTAAAATGCCAAAAAGGTAATTTAAGAGACTTAAAACTGAAAAGTTTAAGATAGTCACACTGAACTATATTAAAAAATCCACAGGGTGGTTGGAACTAGGCCTTATATTAAAGAGGCTAAAAATTGCAATAAGACCACAGGCTTTAAATATGGCTTTAAACTGTGAAAGGTGAAACTAGAATGAATAAAATCCTATAAATTTAAATCAAAAGAAAGAAACAAACTA/GAAATTAAAGTTAATATACAAGAATATGGTGGCCTGGATCTAGTGAACATATAGTAAAGATAAAACAGAATATTTCTGAAAAATCCTGGAAAATCTTTTGGGCTAACCTGAAAACAGTATATTTGAAACTATTTTTAAA Three variants are present

29. These three variants could theoretically occur in 8 different haplotypes …C…A…A… …C…A…G… …C…C…A… …C…C…G… …T…A…A… …T…A…G… …T…C…A… …T…C…G…

30. But in practice, only two are observed …C…A…A… …C…A…G… …C…C…A… …C…C…G… …T…A…A… …T…A…G… …T…C…A… …T…C…G…

31. ~ 25 kb

33. A Haplotype Map of Human Variation • Goal is to define all common haplotypes in the human genome • Genome-wide association studies can then be done with a haplotype tag set of about 250,000 SNPs • Pilot studies underway to determine how many populations to sample, and best strategy for defining haplotype blocks

34. 2000 DNAs x 250,000 SNPs = 500,000,000 genotypes

35. Shortcut #2: Use DNA pooling to greatly reduce amount of genotyping

36. Creating DNA Pools Case DNA Samples Control DNA Samples “Case” Pool “Control” Pool

37. Testing performance of methods for determining allele frequency differences in pools • Tested 16 non-optimized SNPs with: • MALDI-TOF mass spec • Pyrosequencing • Fluorescent primer extension • Each assay carried out 8 – 16 times • Each method tested on three pools with n > 100 • Diabetics • Spouses • Elderly non-diabetics

38. G A Diabetic Probands 38% A G Spouse Controls 34% A A G Elderly Non-diabetic Controls 30% A A MALDI-TOF Mass Spectrometric Detection of Allele Frequencies in Pooled DNA Samples

39. Frequency Difference from Pooled Genotypes (MALDI-TOF)

40. 2 DNAs x 250,000 SNPs = 500,000 genotypes

41. Fine mapping with SNPs Association identified Causative variant identified

42. http://genome.ucsc.edu

43. Fulfilling the Promise of Genomics for Better Health Medical Genomics Functional Genomics Proteomics Comparative Genomics

44. A sample of ongoing public large scale animal genome sequencing projects Mouse – now at 7X Rat – now at 4X Tetraodon – now at 6X Fugu – now at 5X Zebrafish – now at 2X Ciona intestinalis – now at 10X Ciona savignyi – now at 12X C. briggsae – now at 10X

45. The Mouse Genome Sequencing Consortium The Sanger Institute Washington University Whitehead Institute NHGRI Wellcome Trust www.ensembl.org

46. 100% 50% Reference = HUMAN CAV2,1 MET CAPZA2 ST7 WNT2 GASZ CFTR CORTBP2 PipMaker: Human-Mouse Alignment

47. MultiPIP Alignment: 12 Vertebrate Species

48. Diabetes Research in the Era of Complete Genomes The major predisposing genes for type 1 and type 2 diabetes should be identified in the next 5 – 7 years

50. The FUSION Research Team