1. ULRIKE PETERS, FRED HUTCHINSON CANCER RESEARCH CENTER, UNIVERSITY OF WASHINGTON Fine-mapping of obesity GWAS loci using the Metabochip in PAGE (Population Architecture using Genetics and Epidemiology)

2. Design of Metabochip for anthropometric related traits  Anthropometric related MetaboChip content  Replication 13k SNPs for BMI, WHR, WC, height, % fat mass  Fine-mapping 41 regions, 26k SNPs

3. Current Study Population in PAGE and Collaborative Studies Studyn ARIC3.300 MEC3,900-5,300 WHI imputed6,300 WHI genotyped5,300 GenNet500 HyperGEN1,200 Total20,500-22,000

4. SUBMITTED TO PLOS GENETICS 2012 16q12.2/FTO Strongest GWAS finding for obesity-related traits

5. 16q12.2/FTO Association with BMI r 2 based on AAr 2 based on EA 1,529 SNPs across 650kb

6. Bioinformatic Characterization by Praveen Sethupathy, UNC  Candidate intronic regulatory elements: rs11642015, rs17817497, rs3751812, rs17817964, rs62033408, and rs1421085  Highly sequence-conserved elements among vertebrates: rs3751812 and rs1421085  Predicted to have allele-specific binding affinities for different transcription factors: rs11642015 ->Paired box protein 5 (PAX5) rs1421085 ->Cut-like homeobox 1 (CUX1), previously implicated in the transcriptional regulation of FTO (Stratigopoulos, J Biol Chem 2011)

7. Definition of Significance Levels  Different alpha-levels for different aims: A.Fine-mapping regions: 1.Fine-mapping of GWAS index SNPs Adjust only for SNPs that are correlated with GWAS index SNP at r 2 >0.2, >0.5, >0.8 in population that identified GWAS index SNP (mostly EA or Asian) Accounting for correlation among SNPs, e.g. by permutation or estimate # of bins 2.Search for second independent signals Adjust for all other SNPs in the fine-mapping region (excluding those included in #1) while accounting for correlation B.Replication/generalization C.Pleiotropy– or analysis across the Metabochip

8. FTO region with correlation in EA In total 88 SNPs are correlated at r 2 >0.2 with 9 GWAS index SNPs in EA (all dotes that are red, yellow, green or light blue) GWAS hit 1 GWAS hit 2 GWAS hit 3

9. Example FTO Region SNPCAF% change in BMI per coding alleleNominal pAdjusted P Beta estimate95%CI Fine-mapping of GWAS index SNP (# of independent tests = 30) rs620484020.121.13(0.51,1.74)2.4E-047.2E-03 rs116420150.111.09(0.47,1.7)4.9E-040.01 rs560946410.121.12(0.5,1.73)2.8E-048.4E-03 rs558727250.111.09(0.47,1.7)5.3E-040.02 rs14210850.121.11(0.49,1.72)3.0E-049.1E-03 Search for second independent signals (# of independent tests = 1,109) rs591092764.3E-031.00 rs116428414.8E-031.00 rs133308315.5E-031.00

10. Based on ~21,000 subjects (ARIC, HyperGEN, GenNet, MEC, WHI)

11. Summary for primary signals region# SNPsSNPMAFEffectP.valueadjusted Pr 2 in AAr 2 in EA 11p31.1 Toprs26135040.190.0072.65E-030.70 265GWASrs25689580.460.0010.740.110.32 rs28157520.460.0010.70.110.32 21p31.1 Toprs75531580.23-0.0074.56E-040.03 55GWASrs15141750.340.0020.320.310.93 31q25.286Top/GWASrs5438740.25-0.0121.01E-088.70E-07 42p25.3 Toprs1115934200.100.0155.80E-071.05E-04 181GWASrs65482380.12-0.0133.00E-060.640.94 rs75613170.24-0.0050.020.280.93 rs28671250.12-0.0121.13E-050.700.97 53p12.1 Toprs13755640.25-0.0073.05E-040.04 127GWASrs130788070.06-0.0010.80.000.30 63q27.2 Top/GWASrs76473050.41-0.0071.76E-040.0 62GWASrs98162260.210.0071.00E-030.360.85 74p12 Toprs3484950.35-0.0122.49E-071.22E-05 * 49GWASrs109383970.25-0.0081.07E-04 0.60 85q13.3 Toprs7676760.190.0121.03E-042.61E-02 253GWASrs21123470.500.0000.99 0.050.27 96p12.3 Toprs27444750.33-0.0071.03E-041.07E-02 104GWASrs9872370.11-0.0050.08 0.240.50 109p21.1 Toprs177703360.180.0087.12E-047.48E-02 105GWASrs109685760.17-0.0073.10E-03 0.890.99 1111p15.4 Toprs101285970.18-0.0143.41E-057.19E-03 211GWASrs49299490.40-0.0010.76 0.020.33

12. region# SNPsSNPMAFEffectP.valueadjusted Pr 2 in AAr 2 in EA 1211p14.1toprs15194800.25-0.0129.95E-091.18E-06 119GWASrs9259460.26-0.0020.35 0.120.97 toprs350706130.020.0322.47E-072.72E-05 (r2<0.1 with rs925946) 110GWASrs107676640.070.0173.99E-03 ** rs62650.05-0.0215.80E-07 0.330.38 1311p11.2 Toprs64858020.18-0.0108.93E-052.22E-02 249GWASrs108387380.100.0030.28 0.240.14 rs38173340.26-0.0010.52 0.040.07 1412q13.12 Toprs108759820.38-0.0040.042.52E+00 63GWASrs71388030.170.0020.49 0.340.79 1514q12 Toprs284015800.33-0.0030.091.80E+00 20GWASrs118476970.330.0020.3 0.510.92 1615q23 Toprs80251630.02-0.0189.91E-041.80E-01 182GWASrs22414230.37-0.0030.15 0.020.28 1716p12.3 Toprs47822820.22-0.0093.31E-055.27E-03 159GWASrs124449790.09-0.0030.31 0.010.47 1816p11.2 Toprs1156167840.12-0.0082.74E-034.52E-01 165GWASrs74986650.27-0.0030.160.040.36 rs73593970.090.0000.970.010.36 2018q21.32 Toprs129671350.270.0097.32E-061.41E-03 * 192GWASrs177823130.28-0.0083.87E-05 0.94 rs129701340.140.0081.84E-03 0.13 rs108717770.29-0.0071.26E-04 0.90 rs5713120.340.0030.16 0.25 2119q13.11 Toprs148100.15-0.0093.86E-041.00E-02 26GWASrs110847530.36-0.0030.27 0.220.62 rs299410.18-0.0060.01 0.671.00 224q24 Toprs1514110.25-0.0070.037.20E-01 24GWASrs131073250.010.0000.990.040.18

13. 11p14.1/BDNF,LIN7C,LGR4 Correlation based on EA with 2 different GWAS index SNPs

14. 11p14.1/BDNF,LIN7C,LGR4 Correlation based on AA with one GWAS index SNP and most significant SNP in the region

15. r 2 with GWAS hits region # SNPsSNPMAFEffectP.valueAdjusted Pr 2 in EAr 2 in AA 11p31.1862rs1148750570.03-0.02742.31E-040.20*<0.01 21p31.1255rs745436980.000.27362.85E-030.73<0.01 31q25.2580rs31313100.06-0.01554.66E-050.03 0.01 42p25.3727rs26839620.14-0.00962.16E-040.15<0.01 53p12.1507rs1146168540.01-0.0336.26E-040.32*<0.01 63q27.2242rs784196490.02-0.01698.95E-032.17<0.01<0.03 74p12240rs1168100970.25-0.00871.89E-054.53E-03*0.11 85q13.3647rs803246920.020.0239.80E-040.630.030.02 96p12.31330rs97848140.320.01225.05E-060.01*0.02 109p21.1189rs169131230.05-0.01626.63E-031.25<0.010.01 1111p15.4333rs766337990.040.01721.81E-040.06*0.03 1211p14.1354rs122841580.240.01171.51E-085.36E-06<0.01<0.2 1311p11.21280rs618957650.01-0.04891.06E-050.01<0.02<0.01 1412q13.12201rs1149565320.01-0.03290.012.30*<0.01 1514q12189rs669551070.050.01291.26E-030.240.010.02 1615q23835rs758216920.030.01675.02E-040.42*0.02 1716p12.3447rs116444320.170.01311.20E-075.36E-050.05<0.01 1816p11.2385rs344139220.030.02843.56E-040.14<0.04<0.01 2018q21.32903rs734456510.070.01362.34E-040.21*<0.2 2119q13.1188rs1169812380.00-0.11070.010.92<0.02<0.01 224q24238rs729229360.04-0.01781.28E-040.03*<0.01 Summary for secondary signals

16. Decisions for Next Paper(s)  Study populations Focus on AA, AA and Asian or multiethnic panel? Data freeze  Outcome Two separate papers for BMI and WHR/WC  Metabochip content Focus on fine-mapping regions or entire Metabochip content Note, some of the most significant findings are outside of the BMI regions, but require more complex follow up  Overall timing We need to be fast to avoid being scooped by other groups

17. Study population for next papers StudynAvailabilityInclude in next papers African Americans ARIC3.300YesX MEC3,900- 5,300 YesX WHI imputed6,300YesX WHI genotyped5,300YesX GenNet500YesX HyperGEN1,200YesX CARDIA~500No CHS800 BioVU~10,000No Hispanic WHI5,500Not cleaned SOL12,000Genotyping ongoing Asian MEC3500Genotyping ongoing WHI3500Genotyping ongoing ThaiChi10,000Yes? CLHNS1,000Yes?

18. Within HDL region # 3 rs6712203 is most significant SNP 1.7 x 10 -10 Correlation between BMI and HDL ~ 0.2

19. GWAS hit in HDL region #3 is rs10195252 BMI HDL lnBMI ~ SNP + HDL + age*sex + PC1 + PC2 HDL ~ SNP + BMI + age*sex + PC1 + PC2 Note results based on 11,792 subjects with HDL and BMI data (~55% of all with BMI in Manhattan plot)!

20. Extra slides

21. Example FTO region: Fine-mapping of GWAS index SNPs  1,529 SNP genotyped across 640kb region  Correlation with 9 index SNPs in CEU (EA) 1000 Genome Project pilot: r 2 >0.2 = 88 SNPs on Metabochip (r 2 >0.5 = 72; r 2 >0.8 = 59 SNPs)  Permute random normal distributed phenotype and run analysis of all 97 (88+9) SNPs 10,000 times to compute the # of independent tests =>30 Nominal p-value * number of independent test = multi- comparison adjusted p-value (e.g. 2.4E-04*30=7.2E-03) OR Alpha of 0.05 /# of independent test = multi-comparison adjusted alpha level (e.g. 0.05/30 = 0.002)

23.  1,529 SNP genotyped across 640kb Exclude 97 SNPs included in fine-mapping of GWAS index SNPs (1,529-97 = 1,432) Repeat permutation for all SNPs in entire region => 1109 independent tests Example FTO region: Search for second independent signals There are 1,432 SNPs that are not correlated with GWAS index SNPs in EA (r 2 <0.2, dark blue dots) These result in 1,109 independent tests

24. Exploration if most significant BMI locus is independent from HDL lnBMI ~ SNP + HDL + age*sex + PC1 + PC2 HDL ~ SNP + BMI + age*sex + PC1 + PC2 BMI HDL Note results based on 11,792 subjects with HDL and BMI data (~55% of all with BMI in Manhattan plot)!

25. Same as slide before but not mutually adjusted for HDL and BMI lnBMI ~ SNP + age*sex + PC1 + PC2 HDL ~ SNP + age*sex + PC1 + PC2 BMI HDL Note results based on 11,792 subjects with HDL and BMI data (~55% of all with BMI in Manhattan plot)!