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Diabetes Research in the Era of Complete Genomes

Diabetes Research in the Era of Complete Genomes. American Diabetes Association June 17, 2002 Francis S. Collins, M.D., Ph.D. Fulfilling the Promise of Genomics for Better Health. Medical Genomics. Functional Genomics. Proteomics. Comparative Genomics.

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Diabetes Research in the Era of Complete Genomes

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

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

  3. 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

  4. FinlandUnitedStatesInvestigation OfNIDDM

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

  6. 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

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

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

  9. HNF4A  SLC2A10  PTPN1  Microsatellites 

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

  11. 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

  12. Allele Frequency Difference of Cases vs. Elderly Controls

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

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

  15. Fine mapping with SNPs Association identified Causative variant identified

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

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

  18. Fine mapping with SNPs Association identified Causative variant identified

  19. 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

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

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

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

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

  24. 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…

  25. 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…

  26. ~ 25 kb

  27. 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

  28. 2000 DNAs x 250,000 SNPs = 500,000,000 genotypes

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

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

  31. 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

  32. 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

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

  34. 2 DNAs x 250,000 SNPs = 500,000 genotypes

  35. Fine mapping with SNPs Association identified Causative variant identified

  36. http://genome.ucsc.edu

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

  38. 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

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

  40. 100% 50% Reference = HUMAN CAV2,1 MET CAPZA2 ST7 WNT2 GASZ CFTR CORTBP2 PipMaker: Human-Mouse Alignment

  41. MultiPIP Alignment: 12 Vertebrate Species

  42. 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

  43. The FUSION Research Team

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