1. Genome-wide association studies Misha Kapushesky Slides: Johan Rung, EBI St. Petersburg Russia 2010

4. Recombination

5. Linkage disequilibrium Two markers on the genome are inherited together more often than would be expected by chance This leads to high correlation between nearby markers in its haplotype block

6. Haplotypes and genotype tagging

8. Study power 1 2 3 4 1 2 3 4 A B Cases Controls

10. How many SNPs should be tested? Studies of small regions revealed linkage disequilibrium blocks in which common SNPs are highly correlated (usually <10,000–30,000 base pairs in African populations or 30,000–50,000 base pairs in the newer European and Asian populations) (22). This motivated the HapMap Project (www.hapmap.org [12]), which has validated approximately 4 million SNPs, including 2.8 million of the estimated 10 million common SNPs in major world populations, while creating competition among biotechnology companies to develop high-throughput genotyping technologies. Sequencing and genotyping studies showed that sets of 500,000 (European populations) to 1,000,000 (African populations) SNPs could &quot;tag&quot; (serve as proxies for) approximately 80% of common SNPs (23).

17. Molecular vs disease phenotypes Molecular phenotypes can give more precise information about disease state

22. Genomic control

23. Eigenstrat

25. Imputation Wu et al, Nat. Genet. 41, 991-995, 2009

26. Montreal GWAS

28. Type 2 diabetes

32. Multi-stage GWAS

33. Study design Stage 1: Genome-wide scan - 392,365 SNPs French (N=1,376) 679 cases, 697 controls Focused Stage 2 - 16,273 SNPs French (N=4,977) 2,245 cases, 2,732 controls Focused Stage 3 - 28 SNPs Danish (N=7,698) 3,334 cases, 4,364 controls Stage 4: population effect study - 1 SNP (rs2943641) Population based study samples French (N=3,351), Finnish (N=5,183), Danish (N=5,824) CASE-CONTROL T2D ASSOCIATION QT ASSOCIATION IN POPULATIONS Fast-track confirmation - 57 SNPs French (N=5,511) 2,617 cases, 2,894 controls Previously published, Nature, Feb 2007 Fasting glucose Normoglycemic individuals Stage 1: French (N=654) Stage 2: rs560887 (N=9,353) Previously published, Science, May 2007 Stage 1: Genome-wide scan - 392,365 SNPs French (N=1,376) 679 cases, 697 controls Focused Stage 2 - 16,273 SNPs French (N=4,977) 2,245 cases, 2,732 controls Fast-track confirmation - 57 SNPs French (N=5,511) 2,617 cases, 2,894 controls Previously published, Nature, Feb 2007 Stage 1: Genome-wide scan - 392,365 SNPs French (N=1,376) 679 cases, 697 controls Focused Stage 2 - 16,273 SNPs French (N=4,977) 2,245 cases, 2,732 controls Fasting glucose Normoglycemic individuals Stage 1: French (N=654) Stage 2: rs560887 (N=9,353) Previously published, Science, May 2007 Fast-track confirmation - 57 SNPs French (N=5,511) 2,617 cases, 2,894 controls Previously published, Nature, Feb 2007 Stage 1: Genome-wide scan - 392,365 SNPs French (N=1,376) 679 cases, 697 controls Focused Stage 2 - 16,273 SNPs French (N=4,977) 2,245 cases, 2,732 controls Fasting glucose Normoglycemic individuals Stage 1: French (N=654) Stage 2: rs560887 (N=9,353) Previously published, Science, May 2007 Fast-track confirmation - 57 SNPs French (N=5,511) 2,617 cases, 2,894 controls Previously published, Nature, Feb 2007 Stage 1: Genome-wide scan - 392,365 SNPs French (N=1,376) 679 cases, 697 controls Focused Stage 2 - 16,273 SNPs French (N=4,977) 2,245 cases, 2,732 controls Stage 1: Genome-wide scan - 392,365 SNPs French (N=1,376) 679 cases, 697 controls Focused Stage 2 - 16,273 SNPs French (N=4,977) 2,245 cases, 2,732 controls Focused Stage 3 - 28 SNPs Danish (N=7,698) 3,334 cases, 4,364 controls Stage 4: population effect study - 1 SNP (rs2943641) Population based study samples French (N=3,351), Finnish (N=5,183), Danish (N=5,824) Fast-track confirmation - 57 SNPs French (N=5,511) 2,617 cases, 2,894 controls Previously published, Nature, Feb 2007 Stage 1: Genome-wide scan - 392,365 SNPs French (N=1,376) 679 cases, 697 controls Focused Stage 2 - 16,273 SNPs French (N=4,977) 2,245 cases, 2,732 controls Fast-track confirmation - 57 SNPs French (N=5,511) 2,617 cases, 2,894 controls Previously published, Nature, Feb 2007 Stage 1: Genome-wide scan - 392,365 SNPs French (N=1,376) 679 cases, 697 controls Focused Stage 2 - 16,273 SNPs French (N=4,977) 2,245 cases, 2,732 controls Fast-track confirmation - 57 SNPs French (N=5,511) 2,617 cases, 2,894 controls Previously published, Nature, Feb 2007 Stage 1: Genome-wide scan - 392,365 SNPs French (N=1,376) 679 cases, 697 controls Focused Stage 2 - 16,273 SNPs French (N=4,977) 2,245 cases, 2,732 controls Fasting glucose Normoglycemic individuals Stage 1: French (N=654) Stage 2: rs560887 (N=9,353) Previously published, Science, May 2007 Fast-track confirmation - 57 SNPs French (N=5,511) 2,617 cases, 2,894 controls Previously published, Nature, Feb 2007 Stage 1: Genome-wide scan - 392,365 SNPs French (N=1,376) 679 cases, 697 controls Focused Stage 2 - 16,273 SNPs French (N=4,977) 2,245 cases, 2,732 controls

36. Stage 1 results

38. Results SNP Chr Position pMAX Closest gene rs7903146 10 114748339 1.5 x 10 -34 TCF7L2 rs13266634 8 118253964 6.1 x 10 -8 SLC30A8 rs1111875 10 94452862 3.0 x 10 -6 HHEX rs7923837 10 94471897 7.5 x 10 -6 HHEX rs7480010 11 42203294 1.1 x 10 -4 LOC387761 rs3740878 11 44214378 1.2 x 10 -4 EXT2 rs11037909 11 44212190 1.8 x 10 -4 EXT2 rs1113132 11 44209979 3.3 x 10 -4 EXT2

39. SLC30A8 Chimienti et al. Biometals 18:313

40. HHEX KIF11 HHEX IDE D' 0 0.2 0.4 0.6 0.8 1

41. HHEX controls pancreatic development Habener Endocrinology 146:1025 Hex homeobox gene-dependent tissue positioning is required for organogenesis of the ventral pancreas. Bort (2004) Heart induction by Wnt antagonists depends on the homeodomain transcription factor Hex. Foley (2005) The homeobox gene Hex is required in definitive endodermal tissues for normal forebrain, liver and thyroid formation. Martinez Barbera (2000)

43. QC Exclusion criterion Samples Call rate < 95% 27 Continental stratification 296 Sex mismatch 64 Related individuals 70 Total 457 Chromosome SNPs Failed HWE Failed MAF Successful TOTAL 16,360 48 43 16,273

44. EIGENSTRATcorrection filters for MAF, HWE, call rate filters for MAF, HWE, call rate and r 2

45. Results - stage 1 vs stage 2

46. Results - taking out known loci

51. Oral Glucose Tolerance Test

52. Metabolic traits 1 Metabolic trait Cohort rs2943641 P add P dom P rec C/C C/T T/T Age NFBC 1986 16 16 16 DESIR 47.1 ± 9.8 47.5 ± 9.9 47.6 ± 10.1 INTER99 44.9 ± 7.9 45.4 ± 7.8 45.2 ± 7.6 Sex NFBC 1986 1062/1092 1153/1208 322/346 DESIR 645/728 728/812 216/222 INTER99 776/942 974/1070 307/354 BMI (kg/m 2 ) NFBC 1986 21.3 ± 3.8 21.3 ± 3.7 21.1 ± 3.5 0.24 0.43 0.21 DESIR 24.5 ± 3.7 24.4 ± 3.5 24.4 ± 3.4 0.55 0.63 0.61 INTER99 25.6 ± 3.9 25.4 ± 4.1 25.7 ± 4.2 0.57 0.094 0.24 Fasting plasma glucose (mmol/l) NFBC 1986 5.13 ± 0.41 5.14 ± 0.40 5.13 ± 0.41 0.77 0.62 0.90 DESIR 5.21 ± 0.44 5.20 ± 0.42 5.18 ± 0.43 0.05 0.32 0.07 INTER99 5.31 ± 0.40 5.31 ± 0.41 5.33 ± 0.39 0.66 0.93 0.32 Fasting serum insulin (pmol/l) NFBC 1986 78.7 ± 48.6 76.8 ± 44.5 71.7 ± 32.1 0.001 0.03 0.0009 DESIR 50.6 ± 32.9 48.4 ± 29.7 49.1 ± 29.1 0.05 0.003 0.76 INTER99 38.8 ± 24.7 36.4 ± 21.9 37.6 ± 23.3 0.018 0.0043 0.49

53. Metabolic traits 2 HOMA-B NFBC 1986 141 ± 95.1 136 ± 80.1 131 ± 91.6 0.006 0.05 0.009 DESIR 109 ± 87.0 103 ± 64.8 108 ± 92.2 0.16 0.006 0.24 INTER99 75.2 ± 65.6 68.3 ± 42.2 71.0 ± 49.9 0.005 0.0011 0.32 HOMA-IR NFBC 1986 2.52 ± 1.63 2.47 ± 1.58 2.29 ± 1.06 0.007 0.07 0.005 DESIR 1.95 ± 1.35 1.86 ± 1.20 1.88 ± 1.17 0.03 0.004 0.95 INTER99 1.54 ± 1.00 1.44 ± 0.89 1.49 ± 0.95 0.026 0.0058 0.59 Insulin 30’ INTER99 300 ± 183 277 ± 172 281 ± 169 0.0019 8.1 x 10 ‑4 0.14 Insulin 120’ 176 ± 138 163 ± 127 162 ± 124 0.0059 0.011 0.057 AUC insulin 22000 ± 13800 20300 ± 12900 20500 ± 12700 6.9 x 10 ‑4 2.2 x 10 ‑4 0.12 Glucose 30’ 8.19 ± 1.53 8.17 ± 1.56 8.22 ± 1.50 0.72 0.34 0.55 Glucose 120’ 5.51 ± 1.11 5.51 ± 1.11 5.47 ± 1.15 0.54 0.99 0.23 AUC glucose 182 ± 101 181 ± 102 180 ± 99.5 0.44 0.48 0.59 AUC insulin / AUC glucose 32.5 ± 17.4 30.1 ± 16.2 30.6 ± 16.1 6.0 x 10 ‑4 1.6 x 10 ‑4 0.13 CIR 1140 ± 4210 1000 ± 1130 1000 ± 1060 0.045 0.066 0.17 ISI 0.151 ± 0.095 0.16 ± 0.098 0.156 ± 0.096 0.026 0.0058 0.59 Disp. Index (CIR * ISI) 180 ± 1610 147 ± 220 143 ± 174 0.73 1.0 0.50

54. IRS1 locus - rs2943641

56. rs2943641 - IRS1 protein association

57. rs2943641 - IRS1 protein association rs2943641 CC rs2943641 CT rs2943641 TT P Add P Dom P Rec n (male/female) 74 (35/39) 88 (51/37) 28 (10/18) Age (years) 42.5 ± 17.1 43.5 ± 16.9 43.2 ± 17.6 BMI (kg/m 2 ) 25.0 ± 3.8 24.9 ± 3.9 25.3 ± 4.1 0.3 0.7 0.2 R d insulin clamp (mg/kg FFM /min) 10.4 ± 3.5 11.0 ± 3.2 11.7 ± 3.7 0.2 0.2 0.4 D i (x 10 ‑7 ) 1.7 ± 1.1 1.8 ± 1.3 1.8 ± 1.1 0.8 0.8 0.9 IRS-1 protein basal (AU) 296.7 ± 167.7 314.0 ± 155.1 413.1 ± 227.6 0.03 0.3 0.009 IRS-1 protein insulin (AU) 276.6 ± 143.6 280.9 ± 156.4 313.3 ± 147.9 0.3 0.7 0.2 IRS-1-associated PI3K activity basal (AU) 25.0 ± 12.6 26.6 ± 15.4 30.1 ± 17.2 0.3 0.4 0.4 IRS-1-associated PI3K activity insulin (AU) 47.1 ± 29.9 56.6 ± 32.1 72.2 ± 41.3 0.001 0.02 0.002

59. Paper Rung et al., Nature Genetics, 41, 1110-1115, 2009

63. A B G B E F D A C

64. Redundancy