Genome-wide association study for early-onset and morbid adult obesity identifies three new loci in European populations

Transcription

1 Genome-wide association study for early-onset and morbid adult obesity identifies three new loci in European populations David Meyre 1, Jérôme Delplanque 1, Jean-Claude Chèvre 1, Cécile Lecoeur 1, Stéphane Lobbens 1, Sophie Gallina 1, Emmanuelle Durand 1, Vincent Vatin 1, Franck Degraeve 1, Christine Proença 1, Stefan Gaget 1, Antje Körner 2, Peter Kovacs 3, Wieland Kiess 2, Jean Tichet 4, Michel Marre 5, Anna-Liisa Hartikainen 6, Fritz Horber 7, Natascha Potoczna 7, Serge Hercberg 8, Claire Levy-Marchal 9, François Pattou 10, Barbara Heude 11, Maithé Tauber 12, Mark I. McCarthy 13, 14, 15, Alexandra I. F. Blakemore 16, Alexandre Montpetit 17, Constantin Polychronakos 17, Jacques Weill 18, Lachlan J. M. Coin 19, Julian Asher 16, Paul Elliott 19, Marjo- Riitta Jarvelin 19,20, Sophie Visvikis-Siest 21, Beverley Balkau 11, Rob Sladek 17, David Balding 19, Andrew Walley 16, Christian Dina 1, Philippe Froguel 1,16 1 CNRS 8090-Institute of Biology, Pasteur Institute, Lille, France 2 University Hospital for Children & Adolescents, University of Leipzig, Germany 3 Interdisciplinary Centre for Clinical Research, University of Leipzig, Germany 4 IRSA, La Riche, France 5 Department of endocrinology, diabetology, nutrition, Bichat-Claude Bernard University Hospital, Assistance Publique des Hôpitaux de Paris, Paris, France, and INSERM U695, Université Paris 7, Paris, France 6 Department of Clinical Sciences/ Obstetrics and Gynecology, University of Oulu, University of Oulu, Finland 7 Klinik Lindberg, Winterthur, and University of Berne, Switzerland 8 UMR U557 INSERM, U1125 INRA, CNAM, Université Paris 13, CRNH IdF, F Bobigny, France 9 INSERM, U690, Paris, FR-75019, France and Université Paris Diderot, Paris, FR cedex 13, France 10 INSERM U859, CHRU Lille, Lille North of France University, France 11 INSERM U780, Villejuif, F-94807; University Paris-Sud, Orsay, F-91405, France 12 INSERM U563, Children's Hospital, CHU Toulouse, France 13 Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, United Kingdom 14 Wellcome Trust Centre for Human Genetics, University of Oxford, UK 15 Oxford NIHR Biomedical Research Centre, Oxford, UK 16 Section of Genomic Medicine, Hammersmith Hospital, Imperial College London, United Kingdom 17 McGill University and Genome Quebec Innovation Centre, Montreal, Canada 18 Pediatric Endocrine Unit, Jeanne de Flandre Hospital, Lille, France 19 Department of Epidemiology and Public Health, Imperial College London, United Kingdom 20 Institute of Health Sciences, University of Oulu, Department of Child and Adolescent Health, National Public Health Institute, Biocenter Oulu, University of Oulu, Finland 21 INSERM Cardiovascular Genetics team, CIC 9501, Nancy, France

2 Supplementary figure 1. Negative Log 10 of for single marker association analysis with three affection binary traits: childhood obesity under the additive (A1), dominant (A2) and recessive (A3) models; adult obesity under the additive (B1), dominant (B2) and recessive (B3) models; pooled childhood/adult obesity under the additive (C1), dominant (C2) and recessive (C3) models. Supplementary figure 1-A1 Supplementary figure 1-A2

3 Supplementary figure 1-A3 Supplementary figure 1-B1

4 Supplementary figure 1-B2 Supplementary figure 1-B3

5 Supplementary figure 1-C1 Supplementary figure 1-C2

6 Supplementary figure 1-C3

7 Supplementary figure 2. Regional plots of chromosomes 10, 16 and 18 using the GWA adult samples (N=1,426). On the x axis is chromosomal position in kilobases (NCBI build 36) and on the y axis is the for association (expressed as log 10 ). The imputed data signals are shown in red and the directly genotyped signals in blue. Estimated recombination rates (taken from HapMap) are plotted in grey to reflect the local LD structure around the associated SNP. Genes annotations (represented in green) were taken from the University of California Santa Cruz genome browser. Supplementary figure 2A

8 Supplementary figure 2B Supplementary figure 2C

9 Population GWA with familial obesity lean with familial obesity lean N total (female) Status Age[years] (mean; SD) BMI [m/kg²] (mean; SD) BMI Zscore (mean; SD) 685 (375) cases ± ± ± (353) controls ± ± ± (548) cases ± ± ± (550) controls ± ± ± 0.49 confirmation studies obese 519 (307) cases ± ± ± 1.07 lean young 566 (311) controls ± ± ± 0.62 obese 135 (102) cases ± ± ± 1.01 lean 794 (565) controls ± ± ± 0.69 German obese 377 (189) cases ± ± ± 0.55 German lean 731 (379) controls ± ± ± 1.00 Swiss obese 1,036 (769) cases ± ± ± 0.92 Swiss randomly selected 320 (-) controls adult general population (DESIR) 4,417 (2,225) population ± ± ± 0.94 Finnish childhood general population (NFBC 1986) 5,291 (2,677) population ± ± ± 0.99 Supplementary table 1. Description of samples genotyped with the genome-wide SNP array (GWA) and the populations used for confirmation studies.

10 SNP Chrom. Position (nucleotides) Risk allele Major allele MAF (case) MAF (ctrl) OR P FDR Q-value Nearest gene Study Genetic model Selection rs chr G A x x10-5 FLJ45872 Adult obesity DOM Statistics rs chr G A x x10-4 A2BP1 Adult obesity ADD Statistics rs chr C C x10-7 6x10-3 PTER Adult obesity ADD Statistics rs chr G A x10-7 6x10-3 DKFZp434O0320 Adult obesity DOM Statistics rs chr T G x10-7 1x10-2 KIAA1429 Childhood/adult obesity ADD Statistics rs chr G A x10-7 1x10-2 C10orf112 Adult obesity DOM Statistics rs chr C C x10-8 2x10-2 EVL Childhood obesity DOM Statistics rs chr T G x10-7 2x10-2 RET Adult obesity DOM Statistics rs chr A C x10-7 2x10-2 LOC Adult obesity ADD Statistics rs chr C A x10-6 4x10-2 SMAD3 Childhood/adult obesity ADD Statistics rs chr C A x10-6 4x10-2 NRXN3 Childhood/adult obesity DOM Statistics rs chr A A x10-6 4x10-2 PRL Childhood/adult obesity DOM Statistics rs chr G G x10-7 5x10-2 RP5-875H10.1 Childhood obesity DOM Statistics rs chr G A x10-7 5x10-2 TMEM195 Childhood obesity REC Statistics rs chr G T x10-6 5x10-2 KIAA1155 Childhood/adult obesity DOM Statistics rs chr A A x10-6 5x10-2 NPC1 Adult obesity ADD Statistics rs chr C T x10-6 5x10-2 C10orf53 Adult obesity REC Statistics rs chr C C x10-6 6x10-2 LOC Adult obesity ADD Stat/biology rs chr A G x10-6 6x10-2 COL23A1 Childhood/adult obesity ADD Stat/biology rs chr G A x10-6 6x10-2 CALCR Adult obesity REC Stat/biology rs chr T G x10-6 8x10-2 SEC11L3 Childhood/adult obesity ADD Stat/biology rs chr C T x10-6 8x10-2 BHLHB2 Childhood/adult obesity REC Stat/biology rs chr C C x10-6 8x10-2 LOC Adult obesity DOM Stat/biology rs chr T T x10-6 8x10-2 FLJ34870 Adult obesity ADD Stat/biology rs chr T C x LOC Childhood/adult obesity ADD Stat/biology rs chr A G x MAF Adult obesity REC Stat/biology rs chr G G x SFRP1 Adult obesity DOM Stat/biology rs chr G G x FNDC3B Childhood/adult obesity REC Stat/biology rs chr A G x LOC Childhood obesity ADD Stat/biology rs chr C C x KBTBD9 Adult obesity DOM Stat/biology rs chr C C x SMOC1 Childhood/adult obesity ADD Stat/biology rs chr T T x CXCR4 Childhood obesity ADD Stat/biology rs chr T C x HLA-DQA2 Childhood obesity ADD Stat/biology rs chr G G x LOC Childhood obesity ADD Stat/biology rs chr G G x LOC Childhood obesity ADD Stat/biology rs chr C C x LOC Childhood obesity ADD Stat/biology rs chr T C x ASTN2 Childhood/adult obesity ADD Stat/biology Supplementary table 2. Thirty-seven SNPs selected for stage 2 confirmation studies from the GWA study of three affection binary traits (childhood obesity, adult obesity, pooled childhood/adult obesity) using both additive, dominant and recessive models. Association studies were carried out using PLINK.

11 obese lean young obese lean Swiss obese SNP Geno count HW MAF Geno count HW MAF Geno count HW MAF Geno count HW MAF Geno count HW MAF rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs

12 Swiss randomly selected German obese German lean adult general population Finnish childhood general population SNP Geno count HW MAF Geno count HW MAF Geno count HW MAF Geno count HW MAF Geno count HW MAF rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs rs Supplementary table 3. Raw genotypic data of stage 2 confirmation studies

13 SNP Nearest.gene (ADD) a (DOM) a (REC) a (ADD) b (DOM) b (REC) b overall (ADD) c overall (DOM) c overall (REC) c rs ENPP1 1.8e e e e e-01 NA 3.5e e-02 NA rs CNR1 1.0e e-00 NA 4.1e e-02 NA 9.8e e-01 NA rs PTER 7.9e e-03 NA 6.4e e-02 NA 5.0e e-04 NA rs SMAD3 8.5e e-01 NA 8.8e e-02 NA 7.7e e-02 NA rs COL23A1 8.1e e e e e-01 NA 9.5e e-01 NA rs EVL 5.2e e-02 NA 1.4e e-02 NA 4.7e e-02 NA rs C10orf53 8.2e e e e e e e e e-01 rs SEC11L3 4.2e e-02 NA 7.7e e-01 NA 9.7e e-01 NA rs FTO 3.5e e e e e e e e e-13 rs MAF 1.1e e e e e e e e e-08 rs KBTBD9 2.0e e e e e e e e e-01 rs MC4R 4.9e e e e e e e e e-04 rs ENPP1 2.3e e e e e e e e e-01 rs NPC1 7.7e e e e e e e e e-04 rs FLJ e e-01 NA 9.7e e-01 NA 9.1e e-01 NA rs CXCR4 9.4e e e e e e e e e-01 rs CALCR 8.2e e e e e e e e e-01 rs MC4R 4.7e e-03 NA 1.0e e-03 NA 5.8e e-04 NA rs TMEM e e e e e-03 NA 1.0e e-04 NA rs ASTN2 1.2e e-01 NA 9.3e e-01 NA 8.0e e-01 NA rs PRL 7.8e e e e e e e e e-04 rs A2BP1 5.3e e e e e e e e e-02 (95% C.I.) (ADD) d ( in (ADD) d 1.514) 1.1e ( ) 3.3e ( ) 5.9e ( ) 8.9e ( ) 2.4e ( ) 2.7e ( ) 9.3e ( ) 2.4e ( ) 1.8e ( ) 3.1e ( ) 1.5e ( ) 2.9e ( ) 2.0e ( ) 1.0e ( ) 4.5e ( ) 2.1e ( ) 7.0e ( ) 1.2e ( ) 3.4e ( ) 2.3e ( ) 6.2e ( ) 5.4e-02

14 SNP Nearest.gene (ADD) a (DOM) a (REC) a (ADD) b (DOM) b (REC) b overall (ADD) c overall (DOM) c overall (REC) c rs CTNNBL1 8.8e e-01 NA 9.1e e-01 NA 9.5e e-01 NA rs LOC e e e e e e e e e-01 rs6232 PCSK1 6.7e e-02 NA 1.9e e-01 NA 8.4e e-02 NA rs6234 PCSK1 8.3e e e e e e e e e-04 rs CNR1 9.2e e e e e e e e e-01 rs LOC e e e e e e e e e-01 rs RP5-875H e e e e e-01 NA 5.0e e-02 NA rs LOC e e-01 NA 5.6e e-03 NA 1.4e e-02 NA rs LOC e e e e e e e e e-01 rs LOC e e e e e e e e e-02 rs INSIG2 9.5e e e e e e e e e-01 rs LOC e e-01 NA 9.0e e-01 NA 8.4e e-01 NA rs ENPP1 7.2e e e e e e e e e-02 rs BHLHB2 8.1e e-01 NA 8.4e e-01 NA 8.6e e-01 NA rs HLA-DQA2 8.1e e e e e e e e e-02 rs DKFZp434O e e e e e e e e e-02 (95% C.I.) (ADD) d ( in (ADD) d 1.517) 8.4e ( ) 6.4e ( ) 3.5e ( ) 4.8e ( ) 3.2e ( ) 1.2e ( ) 2.6e ( ) 7.3e ( ) 8.1e ( ) 1.7e ( ) 6.0e ( ) 4.1e ( ) 1.5e ( ) 9.1e ( ) 3.0e ( ) 3.6e-01

15 SNP rs rs rs rs rs rs rs rs rs rs rs rs rs Nearest.gene ENPP1 CNR1 PTER SMAD3 COL23A1 EVL C10orf53 SEC11L3 FTO MAF KBTBD9 MC4R ENPP1 German (95% C.I.) (ADD) e in German (ADD) e ( ) 5.3e ( ) 1.0e ( ) 1.1e ( ) 1.7e ( ) 6.3e-01 (ADD) f ( in (ADD) f 1.75) 2.7e ( ) 1.4e ( ) 4.4e ( ) 1.2e ( ) 1.9e ( ) 9.2e-01 1 (0-Inf) 1.0e ( ( ) 2.7e ) 3.1e ( ) 2.6e ( ) 1.7e ( ) 6.5e ( ) 7.3e ( ) 4.3e ( ) 6.3e ( ) 6.9e ( ) 1.0e ( ) 2.0e ( ) 2.8e ( ) 8.7e ( ) 4.5e-01 Swiss (95% C.I.) (ADD) g ( in Swiss (ADD) g 0.957) 2.1e ( ) 7.4e ( ) 3.6e ( ) 9.6e ( ) 6.9e ( ) 4.4e ( ) 2.2e ( ) 1.4e ( ) 1.5e ( ) 1.7e ( ) 5.9e ( ) 4.1e ( ) 1.3e-02 Per allele change in BMI in (ADD) h in (ADD) h ( ) 4.9e ( ) 9.6e ( ) 9.8e ( ) 7.9e ( ) 9.7e ( ) 3.5e ( ) 2.5e ( ) 7.4e ( ) 1.9e ( ) 3.2e ( ) 2.6e ( ) 1.3e ( ) 2.6e-01 Per allele change in BMI (ADD) i ( in (ADD) i 0.061) 7.8e ( ) 7.8e ( ) 6.1e ( ) 3.3e ( ) 4.4e ( ) 8.4e ( ) 6.9e ( ) 2.2e ( ) 7.5e ( ) 1.3e ( ) 2.0e ( ) 1.3e ( ) 8.4e-01

16 SNP rs rs rs rs rs rs rs rs rs rs rs rs6232 rs6234 rs Nearest.gene NPC1 FLJ45872 CXCR4 CALCR MC4R TMEM195 ASTN2 PRL A2BP1 CTNNBL1 LOC PCSK1 PCSK1 CNR1 German (95% C.I.) (ADD) e in German (ADD) e ( ) 3.1e ( ) 9.1e ( ) 9.9e ( ) 5.8e ( ) 2.0e ( ) 6.4e ( ) 5.2e ( ) 2.4e ( ) 8.4e ( ) 4.9e ( ) 4.6e ( ) 1.2e ( ) 5.6e ( ) 8.3e-01 (ADD) f ( in (ADD) f 1.085) 1.7e ( ) 9.7e ( ) 7.2e ( ) 8.7e ( ) 6.3e ( ) 4.2e ( ) 4.4e ( ) 2.2e ( ) 3.9e ( ) 5.4e ( ) 2.3e ( ) 5.1e ( ) 5.2e ( ) 5.0e-01 Swiss (95% C.I.) (ADD) g ( in Swiss (ADD) g 0.939) 8.6e ( ) 1.3e ( ) 7.4e ( ) 9.8e ( ) 9.4e ( ) 1.1e ( ) 1.6e ( ) 3.0e ( ) 5.0e ( ) 1.5e ( ) 3.2e ( ) 5.2e ( ) 8.7e ( ) 1.8e-01 Per allele change in BMI in (ADD) h ( in (ADD) h 0.006) 9.5e ( ) 4.6e ( ) 6.0e ( ) 7.2e ( ) 5.5e ( ) 3.7e ( ) 8.0e ( ) 5.1e ( ) 5.0e ( ) 9.5e ( ) 2.1e ( ) 8.3e ( ) 5.3e ( ) 5.9e-01 Per allele change in BMI (ADD) i ( in (ADD) i 0.001) 5.4e ( ) 8.1e ( ) 3.7e ( ) 5.9e ( ) 5.9e ( ) 1.5e ( ) 3.4e ( ) 4.6e ( ) 4.1e ( ) 2.2e-01 0 ( ) 9.9e ( ) 1.2e ( ) 2.4e ( ) 3.5e-01

17 SNP rs rs rs rs rs rs rs rs rs Nearest.gene LOC RP5-875H10.1 LOC LOC LOC INSIG2 LOC ENPP1 BHLHB2 German (95% C.I.) (ADD) e in German (ADD) e ( ) 8.1e ( ) 1.5e ( ) 2.2e ( ) 6.2e ( ) 2.2e ( ) 7.8e ( ) 4.0e ( ) 7.1e ( ) 1.2e-01 rs HLA-DQA2 1 ( ) 1.0e-00 rs DKFZp434O ( ) 9.3e-01 (ADD) f ( in (ADD) f 1.488) 3.9e ( ) 7.1e ( ) 5.1e ( ) 3.9e ( ) 8.7e ( ) 7.1e ( ) 5.7e ( ) 4.3e ( ) 9.6e ( ) 7.4e ( ) 9.5e-01 Swiss (95% C.I.) (ADD) g in Swiss (ADD) g ( ) 5.0e ( ) 2.4e ( ) 2.3e ( ) 7.8e ( ) 6.4e ( ) 9.9e ( ) 1.0e ( ) 6.2e ( ) 1.8e ( ) 2.7e ( ) 7.9e-02 Per allele change in BMI in (ADD) h in (ADD) h ( ) 8.0e ( ) 9.3e ( ) 2.8e ( ) 3.6e ( ) 7.7e ( ) 1.1e ( ) 3.7e ( ) 6.6e ( ) 1.3e ( ) 2.8e ( ) 6.2e-01 Per allele change in BMI (ADD) i in (ADD) i ( ) 1.7e ( ) 4.8e ( ) 2.9e ( ) 5.4e ( ) 2.7e ( ) 5.9e ( ) 5.2e ( ) 6.6e ( ) 6.8e ( ) 1.8e ( ) 9.2e-01

18 SNP Nearest.gene rs ENPP1 rs CNR1 rs PTER rs SMAD3 rs COL23A1 rs EVL rs C10orf53 rs SEC11L3 rs FTO rs MAF rs KBTBD9 rs MC4R rs ENPP1 (DOM) d ( in (DOM) d 1.468) 3.7e ( ) 5.2e ( ) 7.1e ( ) 9.6e ( ) 9.6e ( ) 2.7e ( ) 5.9e ( ) 2.6e ( ) 4.7e ( ) 1.9e ( ) 6.2e ( ) 1.8e ( ) 1.3e-01 German (DOM) e ( in German (DOM) e 1.838) 2.2e ( ) 9.9e ( ) 8.7e ( ) 3.0e ( ) 4.7e-01 (DOM) f ( in (DOM) f 1.707) 5.2e ( ) 1.2e ( ) 3.4e ( ) 1.3e ( ) 2.9e ( ) 8.7e-01 1 (0-Inf) 1.0e ( ) 2.6e ( ) 3.5e ( ) 8.1e ( ) 4.2e ( ) 5.6e ( ) 6.4e ( ) 4.6e ( ) 6.3e ( ) 6.8e ( ) 3.6e ( ) 2.0e ( ) 4.4e ( ) 1.1e ( ) 6.4e-01 Swiss (DOM) g ( in Swiss (DOM) g 0.971) 3.0e ( ) 2.0e ( ) 3.5e ( ) 7.8e ( ) 1.2e ( ) 3.9e ( ) 9.2e ( ) 2.1e ( ) 2.1e ( ) 1.1e ( ) 6.6e ( ) 5.4e ( ) 3.3e-02 Per allele change in BMI in (DOM) h in (DOM) h 0.02 ( ) 5.4e ( ) 7.8e ( ) 8.3e ( ) 7.4e ( ) 8.5e ( ) 3.3e ( ) 5.3e ( ) 7.4e ( ) 1.1e ( ) 2.8e ( ) 6.5e ( ) 8.0e ( ) 2.8e-01

19 SNP rs rs rs rs rs rs rs rs rs rs rs rs6232 rs6234 rs Nearest.gene NPC1 FLJ45872 CXCR4 CALCR MC4R TMEM195 ASTN2 PRL A2BP1 CTNNBL1 LOC PCSK1 PCSK1 CNR1 (DOM) d in (DOM) d ( ) 1.5e ( ) 5.0e ( ) 1.0e ( ) 9.7e ( ) 1.2e ( ) 1.6e ( ) 3.1e ( ) 2.0e ( ) 1.2e ( ) 1.0e ( ) 3.3e ( ) 3.3e ( ) 2.0e ( ) 1.7e-01 German (DOM) e ( in German (DOM) e 1.093) 2.0e ( ) 7.7e ( ) 7.0e ( ) 7.6e ( ) 2.0e ( ) 1.4e ( ) 4.2e ( ) 5.2e ( ) 9.7e ( ) 6.4e ( ) 5.9e ( ) 1.5e ( ) 3.8e ( ) 7.1e-01 (DOM) f ( in (DOM) f 1.094) 1.4e ( ) 5.7e ( ) 2.7e ( ) 6.9e ( ) 6.3e ( ) 5.5e ( ) 4.7e ( ) 4.2e ( ) 2.1e ( ) 5.9e ( ) 3.7e ( ) 4.3e ( ) 7.4e ( ) 4.0e-01 Swiss (DOM) g ( in Swiss (DOM) g 0.98) 3.5e ( ) 5.8e ( ) 1.2e ( ) 4.4e ( ) 5.7e ( ) 4.9e ( ) 1.9e ( ) 6.0e ( ) 1.6e ( ) 9.1e ( ) 4.5e ( ) 4.4e ( ) 5.1e ( ) 4.0e-01 Per allele change in BMI in (DOM) h in (DOM) h ( ) 6.9e ( ) 5.1e ( ) 5.2e-01 0 ( ) 9.9e ( ) 5.5e ( ) 2.8e ( ) 9.0e ( ) 2.6e ( ) 5.7e ( ) 1.6e ( ) 1.7e ( ) 8.9e ( ) 7.5e ( ) 1.4e-01

20 SNP rs rs rs rs rs rs rs rs rs rs rs Nearest.gene LOC RP5-875H10.1 LOC LOC LOC INSIG2 LOC ENPP1 BHLHB2 HLA-DQA2 DKFZp434O0320 (DOM) d ( in (DOM) d 1.025) 7.7e ( ) 2.3e ( ) 8.9e ( ) 3.3e ( ) 6.0e ( ) 8.4e ( ) 3.9e ( ) 6.5e ( ) 8.2e ( ) 6.8e ( ) 1.7e-01 German (DOM) e ( in German (DOM) e 1.343) 7.8e ( ) 1.7e ( ) 3.4e ( ) 9.3e ( ) 2.1e ( ) 7.0e ( ) 7.2e ( ) 4.4e ( ) 2.5e ( ) 7.2e ( ) 9.4e-01 (DOM) f ( in (DOM) f 1.499) 8.9e ( ) 4.4e ( ) 4.3e ( ) 9.2e ( ) 1.3e ( ) 2.5e ( ) 5.9e ( ) 5.1e ( ) 8.2e ( ) 2.3e ( ) 6.9e-01 Swiss (DOM) g ( in Swiss (DOM) g 1.149) 3.7e ( ) 7.5e ( ) 1.6e ( ) 5.7e ( ) 4.0e ( ) 7.0e ( ) 2.7e ( ) 9.5e ( ) 3.0e ( ) 1.8e ( ) 3.5e-02 Per allele change in BMI in (DOM) h in (DOM) h ( ) 2.3e ( ) 9.4e ( ) 3.9e ( ) 2.9e ( ) 9.6e ( ) 1.8e ( ) 3.9e ( ) 3.6e ( ) 1.7e ( ) 5.6e ( ) 8.4e-01

21 SNP Nearest.gene rs ENPP1 rs CNR1 rs PTER rs SMAD3 rs COL23A1 rs EVL rs C10orf53 rs SEC11L3 rs FTO rs MAF rs KBTBD9 rs MC4R rs ENPP1 rs NPC1 rs FLJ45872 Per allele change in BMI in (95% C.I.) (DOM) i in (DOM) i ( ) 7.2e ( ) 7.1e ( ) 6.7e ( ) 4.7e ( ) 3.9e ( ) 4.8e ( ) 3.7e ( ) 3.8e ( ) 2.1e ( ) 2.7e ( ) 4.2e ( ) 1.7e ( ) 7.3e ( ) 3.4e ( ) 6.7e-01 (REC) d in (REC) d ( ) 1.4e ( ) 5.1e ( ) 3.2e ( ) 6.4e ( ) 5.5e ( ) 2.1e ( ) 2.6e ( ) 1.2e ( ) 9.5e ( ) 5.8e ( ) 5.0e-01 German (REC) e in German (REC) e ( ) 1.0e ( ) 1.3e ( ) 6.0e ( ) 1.8e ( ) 3.4e ( ) 1.7e ( ) 9.6e ( ) 8.1e ( ) 1.8e ( ) 7.6e ( ) 8.0e-01 (REC) f in (REC) f ( ) 8.1e ( ) 2.1e ( ) 3.5e ( ) 1.1e ( ) 2.7e ( ) 3.0e ( ) 3.3e ( ) 5.4e-01 Swiss (REC) g ( in Swiss (REC) g 1.243) 1.7e ( ) 3.1e ( ) 1.1e ( ) 8.2e ( ) 2.1e ( ) 2.6e ( ) 4.0e ( ) 6.3e ( ) 7.6e ( ) 5.3e ( ) 2.8e-02

22 SNP rs rs rs rs rs rs rs rs rs rs6232 rs6234 rs rs rs rs Nearest.gene CXCR4 CALCR MC4R TMEM195 ASTN2 PRL A2BP1 CTNNBL1 LOC PCSK1 PCSK1 CNR1 LOC RP5-875H10.1 LOC Per allele change in BMI in (95% C.I.) (DOM) i in (DOM) i ( ) 4.1e ( ) 7.7e ( ) 5.9e ( ) 3.6e ( ) 3.3e ( ) 2.8e ( ) 4.1e ( ) 2.6e ( ) 7.5e ( ) 8.2e ( ) 4.6e ( ) 3.9e ( ) 1.4e ( ) 7.2e ( ) 7.5e-01 (REC) d in (REC) d ( ) 3.9e ( ) 4.8e ( ) 2.2e ( ) 6.4e ( ) 8.2e ( ) 4.9e ( ) 1.6e ( ) 8.5e ( ) 5.9e ( ) 7.4e ( ) 4.0e-01 German (REC) e ( in German (REC) e 1.275) 6.3e ( ) 2.2e ( ) 9.0e ( ) 8.8e ( ) 4.1e ( ) 4.3e ( ) 7.7e ( ) 3.3e ( ) 9.4e ( ) 4.7e-01 (REC) f ( in (REC) f 0.67) 9.2e ( ) 8.6e ( ) 2.1e ( ) 3.3e ( ) 2.2e ( ) 3.4e ( ) 9.2e ( ) 1.3e-01 Swiss (REC) g ( in Swiss (REC) g 1.096) 1.7e ( ) 2.6e ( ) 1.2e ( ) 1.1e ( ) 1.0e ( ) 2.7e ( ) 3.5e ( ) 3.5e ( ) 1.2e ( ) 9.2e ( ) 3.1e ( ) 2.7e-01

23 SNP rs rs rs rs rs rs rs rs Nearest.gene LOC LOC INSIG2 LOC ENPP1 BHLHB2 HLA-DQA2 DKFZp434O0320 Per allele change in BMI in (95% C.I.) (DOM) i in (DOM) i ( ) 2.6e ( ) 3.6e ( ) 7.1e ( ) 5.5e ( ) 7.4e ( ) 8.5e ( ) 5.8e ( ) 9.3e-01 (REC) d in (REC) d ( ) 5.0e ( ) 2.4e ( ) 4.3e ( ) 7.4e ( ) 8.1e ( ) 7.7e ( ) 3.1e-01 German (REC) e in German (REC) e 1.11 ( ) 4.8e ( ) 6.3e ( ) 1.0e ( ) 6.7e ( ) 5.6e ( ) 8.0e ( ) 4.4e ( ) 9.4e-01 (REC) f ( in (REC) f 2.059) 1.8e ( ) 1.9e ( ) 8.0e ( ) 5.2e ( ) 3.4e ( ) 2.1e-01 Swiss (REC) g ( in Swiss (REC) g 1.333) 8.8e ( ) 5.1e ( ) 5.7e ( ) 2.5e ( ) 2.0e ( ) 8.8e ( ) 7.2e-01

24 SNP Nearest.gene Per allele change in BMI in (REC) h in (REC) h Per allele change in BMI in (REC) i in (REC) i rs ENPP ( ) 6.0e ( ) 9.3e-01 rs CNR ( ) 6.3e ( ) 8.0e-01 rs PTER ( ) 3.4e ( ) 6.4e-01 rs SMAD ( ) 9.1e ( ) 2.2e-01 rs COL23A ( ) 6.5e ( ) 9.8e-01 rs EVL ( ) 8.8e ( ) 4.3e-03 rs C10orf ( ) 2.0e ( ) 7.5e-01 rs SEC11L ( ) 8.8e ( ) 9.5e-02 rs FTO ( ) 1.2e ( ) 5.7e-04 rs MAF ( ) 6.1e ( ) 1.7e-01 rs KBTBD ( ) 5.7e ( ) 1.4e-01 rs MC4R ( ) 3.9e ( ) 9.1e-02 rs ENPP ( ) 5.1e ( ) 8.3e-01 rs NPC ( ) 1.3e ( ) 3.8e-01 rs FLJ ( ) 5.5e ( ) 4.3e-01 rs CXCR ( ) 9.2e ( ) 5.2e-01 rs CALCR ( ) 4.9e ( ) 5.6e-01 rs MC4R rs TMEM ( ) 9.3e ( ) 5.7e-02 rs ASTN ( ) 5.0e ( ) 8.2e-01 rs PRL ( ) 6.4e ( ) 2.4e-02 rs A2BP ( ) 5.5e ( ) 8.2e-01 rs CTNNBL ( ) 1.2e ( ) 3.7e-01 rs LOC ( ) 8.7e ( ) 4.8e-01 rs6232 PCSK ( ) 4.4e ( ) 4.3e-01 rs6234 PCSK ( ) 3.7e ( ) 1.5e-01 rs CNR ( ) 3.1e ( ) 5.3e-01 rs LOC ( ) 6.7e ( ) 5.3e-01 rs RP5-875H ( ) 5.4e ( ) 2.1e-01 rs LOC ( ) 2.7e ( ) 1.7e-02 rs LOC ( ) 6.5e ( ) 8.6e-01 rs LOC ( ) 4.2e ( ) 3.4e-01 rs INSIG ( ) 2.1e ( ) 5.9e-01 rs LOC ( ) 7.0e ( ) 7.2e-01 rs ENPP ( ) 3.8e ( ) 6.6e-01 rs BHLHB ( ) 3.1e ( ) 3.8e-01 rs HLA-DQA ( ) 1.6e ( ) 4.3e-01 rs DKFZp434O ( ) 3.7e ( ) 5.7e-01 Supplementary table 4. Detailed presentation of stage 2 confirmation studies for the 38 SNPs that passed stage 2 quality control. ADD, DOM, REC are abbreviations for additive, dominant and recessive models. Results are presented for all three genetic models. NA: not available. cohorts included 1) 519 obese and 566 lean young of origin 2) 377 obese and 731 lean of German origin 3) 135 obese and 794 lean of origin 4) 1,036 obese and 320 randomly selected of Swiss origin 5) a general population of 5,291 Finnish 6) a general population of 4,417 (cohort 8 was used to evaluate the effect of SNPs on BMI in

25 adulthood f ). Associations between SNPs and obesity status were performed using logistic regression. The effect of SNPs on BMI variation was tested using linear regression. A global stage 2 meta-analysis was calculated in childhood (cohorts 1, 2, 5) a, in adulthood (cohorts 3, 4, 6) b and overall (cohorts 1, 2, 3, 4, 5, 6) c. Analyses were also estimated separately in each cohort. Odds ratios were calculated for cohorts 1 d, 2 e, 3 f, 4 g. The effect of SNPs on BMI was evaluated in cohorts 5 h and 6 i.

26 SUPPLEMENTARY ONLINE METHODS Genome-wide association samples We studied 663 obese, recruited by the CNRS UMR8090 and another 22 obese who were patients of Toulouse Children s Hospital along with 655 normal-weight selected from the STANISLAS study and 30 lean from the Fleurbaix- Laventie Ville Santé II study. We included 695 obese recruited by the CNRS UMR8090 and the Department of Nutrition of Paris Hotel Dieu Hospital along with 731 control subjects selected from the D.E.S.I.R. (Data from the Epidemiological Study on the Insulin Resistance syndrome) prospective study. Confirmation samples The samples for confirmation of associations with early-onset obesity included 519 obese (122 recruited by the CNRS UMR8090 and 397 by the Saint Vincent de Paul Paris hospital), 114 lean from the Fleurbaix-Laventie Ville Santé II study, 452 young adult control subjects from the Haguenau study, 377 obese German and 731 lean German from Leipzig and, for a general population cohort, 5,291 subjects of the Northern Finland Birth Cohort 1986 (NFBC 1986). Other samples for confirmation included 135 obese who were patients of the CHRU Lille hospital, 794 lean subjects from the SUVIMAX study, 1,036 obese Swiss from Zurich and 320 anonymous healthy Swiss blood donors from CHUV, Lausanne (for whom we have no phenotypic data). We also studied 4,417 additional subjects from the D.E.S.I.R. general population cohort. All participants gave written informed consent, and all local ethics committees approved the study protocol. Phenotyping In the genome-wide association study, we used the 97 th age- and sex-specific percentile of BMI 1 from a reference population 2, as a threshold for childhood obesity. Obese were selected on the basis of having evidence of familial obesity (at least one obese first degree relative (sibling, parents) in the pedigree; BMI 97 th percentile in, BMI 30 kg/m² in ) and age lower than 18 years. Control were selected as having a BMI < 90 th percentile for gender and age (threshold for being overweight) 1. Obese were selected if they exceeded the threshold of class III obesity (BMI 40 kg/m²) and showed evidence of familial obesity (at least one severely obese first degree relative in the pedigree; BMI 35 kg/m²) and

27 being 18 years old. Control were selected for having BMI < 25 kg/m² during a 9-year follow-up (measurements at time 0, 3, 6, 9 years). In confirmation studies, we again used the 97 th age- and sex-specific national percentile of BMI 1 from reference populations, as a threshold for childhood obesity. Control were selected as having a BMI < 90 th percentile for gender and age. Obesity status in was defined by BMI 30 kg/m². The whole obese adult confirmation sample included 8.4% of class I moderately obese subjects (30 BMI < 35 kg/m²), 18% of class II severely obese subjects (35 BMI < 40 kg/m²) and 73.6% of class III morbidly obese subjects (BMI 40 kg/m²). adult controls were selected with a BMI < 25 kg/m². In both stages, carriers of monogenic pathogenic MC4R mutations were excluded from the obese subject groups. Genotyping and quality control In GWA stage 1, 2,335 individuals were genotyped using the Illumina Human CNV370-Duo array. Genotyping was performed using 750ng of genomic DNA following the manufacturer s protocols (Illumina Inc., USA). In addition, 523 lean adult controls previously genotyped using the Illumina HAP300 array 3 were included in the analysis. We identified 311,398 SNPs shared by the two arrays. 2,552 SNPs were discarded due to extreme Hardy-Weinberg disequilibrium in the control samples (P < 0.001), low genotyping calling rates (<95%) or low minor-allele frequencies (<1%). We retained 308,846 SNPs for analysis and the average call rate was 97.7%. Two individuals were genotyped twice using the Illumina Human CNV370-Duo array and the concordance rate was 99.75%. Genotypes were called using the Illumina s BeadStation genotyping solutions, based on the GenCall software application to automatically cluster, call genotypes, and assign confidence scores. The GenCall application incorporates a clustering algorithm (GenTrain) and a calling algorithm. The genotyping of SNPs in confirmation stage 2 was done using the Applied Biosystems SNPlex technology based on the Oligonucleotide Ligation Assay (OLA) combined with multiplex PCR target amplification ( A quality control measure was included by using specific internal controls at each step of the assay (according to the manufacturer s instructions) and 14 SNPs failed during this process. Seven SNPs (rs , rs413693, rs , rs , rs , rs , rs ) were discarded during the SNPlex design, and 7 SNPs (rs , rs , rs299575, rs , rs , rs , rs227416) did not pass the genotyping quality control. Quality control criteria in stage 2 were: individual SNP call rates 95%, Hardy-Weinberg equilibrium in the control samples (P 0.005). Allelic discrimination was performed by capillary electrophoresis analysis using an Applied Biosystems 3730xl DNA