Genome-wide association study identifies variants in TMPRSS6 associated with hemoglobin levels.

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1 Supplementary Online Material Genome-wide association study identifies variants in TMPRSS6 associated with hemoglobin levels. John C Chambers, Weihua Zhang, Yun Li, Joban Sehmi, Mark N Wass, Delilah Zabaneh, Clive Hoggart, Henry Bayele, Mark I McCarthy, Leena Peltonen, Nelson B Freimer, Surjit K Srai, Patrick H Maxwell, Michael JE Sternberg, Aimo Ruokonen, Gonçalo Abecasis, Marjo-Riitta Jarvelin, James Scott, Paul Elliott, Jaspal S Kooner.

2 Supplementary methods Populations London Life Sciences Prospective Population Study The London Life Sciences Prospective Population (LOLIPOP) study is an ongoing population based cohort study of ~30,000 men and women, aged years, recruited from the lists of 58 General Practitioners in West London, United Kingdom. 1 Recruitment was by post, and the response rate averaged 62%. All individuals completed an interviewer-administered questionnaire which collected data on medical history, family history, current prescribed medication, cardiovascular risk factors and self reported ethnicity. Individuals classified as Indian Asian ancestry ( Indian Asians ) reported having all 4 grandparents born on the Indian subcontinent. Individuals classified as Europeans identified themselves as white and born in Europe. Physical assessment included anthropometric measurements (height, weight, waist, hip) and blood pressure. Blood was collected after an 8 hour fast for full blood count and biochemical analyses. For the current study, genome-wide association data were available for 1,599 Europeans, and 9,685 Indian Asians. All participants gave written consent including for genetic studies. The study is approved by the local Research Ethics Committee. North Finland Birth Cohort of 1966 The North Finland Birth Cohort of 1966 (NFBC1966) was designed to study factors affecting preterm birth, low birth weight and subsequent morbidity and mortality. Participants comprise 12,068 mothers and their 12,231 births, in the provinces of Oulu and Lapland during At age 31, all individuals still living in the Helsinki area or Northern Finland were asked to participate in a detailed biological and medical examination (n=6,007) including measurement of fasting glucose. For the current study, genotype and hemoglobin measurements were available for 4,717 NFBC1966 participants (2,259 males and 2,458 females). Study methods and characteristics of participants have been described previously. 2 The University of Oulu ethics committee approved the study. Molecular modelling The structure of the TMPRSS6 serine protease domain was modelled using the Phyre structure prediction server. 3 The model is based on Matriptase-1, the most closely related homologue with reported crystal structure. Matriptase-1 catalytic residues and binding site were mapped onto TMPRSS6 using the alignment generated by Phyre.

3 Supplementary references 1. Chambers,J.C. et al. Common genetic variation near MC4R is associated with waist circumference and insulin resistance. Nat. Genet. 40, (2008). 2. Sabatti,C. et al. Genome-wide association analysis of metabolic traits in a birth cohort from a founder population. Nat. Genet. 41, (2009). 3. Kelley,L.A. & Sternberg,M.J. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, (2009). 4. Uda,M. et al. Genome-wide association study shows BCL11A associated with persistent fetal hemoglobin and amelioration of the phenotype of beta-thalassemia. Proc. Natl. Acad. Sci. U. S. A 105, (2008).

4 Supplementary Table 1. Characteristics of Europeans and Indian Asians studied. Data are presented as mean (SD) for continuous, and as % for categorical traits. Europeans (NFBC1966) Europeans (LOLIPOP) Indian Asians (LOLIPOP) N= 4,717 1,599 9,685 Age (yrs) (9.8) 53.6 (10.8) Male gender (%) Weight (kg) 72.7 (14.9) 84.8 (16.8) 77.1 (13.7) Body mass index (kg/m 2 ) 24.7 (4.2) 27.2 (4.3) 27.1 (4.3) Hypertension (%) Type-2 diabetes (%) Hemoglobin (g/dl) 14.2 (1.3) 14.4 (1.6) 14.5 (1.5) MCV (fl) NA 90.3 (4.7) 86.6 (6.4) Creatinine (micromol/l) 97 (25) 92.6 (35.3) 92.3 (30.8) GWA platform Illumina 317K Affymetrix 500K (N=878), Perlegen (N=721) Illumina 610K (N=6988), Illumina 300K (N=2109), Perlegen (N=588)

5 Supplementary Table 2. Characteristics of Europeans and Indian Asians in the replication sample. Data are presented as mean (SD) for continuous, and as % for categorical traits. Europeans Indian Asians N= 5,187 6,721 Age (yrs) 52.4 (11.8) 46.4 (10.3) Male gender (%) Weight (kg) 79.4 (16.8) 72.8 (14.3) Body mass index (kg/m 2 ) 27.4 (5.1) 27.6 (4.8) Hypertension (%) Type-2 diabetes (%) Hemoglobin (g/dl) 14.5 (1.4) 13.7 (1.7) RBC ( /L) 4.75 (0.44) 4.88 (0.52) MCV (fl) 90.8 (5.2) 84.5 (6.6) MCH (pg) 30.6 (1.9) 28.3 (4.4) MCHC (pg/l) 33.7 (1.0) 33.4 (1.2) WBC ( 10 9 /L) 6.9 (6.6) 6.9 (1.7) PLT ( 10 9 /L) 255 (63) 280 (67) Creatinine (micromol/l) 91.0 (24.8) 83.1 (14.1)

6 Supplementary Table 3. Association rs in TMPRSS6 and of rs near HFE with hemoglobin and other red cell parameters in the replication sample. Results are presented as mean (SD) change per copy of allele A, from regression analysis using additive genetic model and with adjustment for age and gender, and are for Europeans and Indian Asians separately and combined. rs (G A) Europeans Indian Asians Combined Effect P Effect P Effect P Hemoglobin (g/dl) (0.02) 4.3x (0.02) 1.4x (0.02) 2.4x10-17 MCV (fl) (0.10) 1.1x (0.11) 3.0x (0.08) 8.0x10-15 MCH (pg) (0.04) 1.4x (0.08) 1.0x (0.05) 6.1x10-13 MCHC (pg/l) (0.02) 1.2x (0.02) 1.8x (0.01) 5.5x10-10 RBC ( /L) 0.00 (0.01) (0.01) (0.01) 0.64 WBC ( 10 9 /L) (0.04) (0.03) (0.02) 0.36 PLT ( 10 9 /L) 2.35 (1.24) (1.12) (0.83) 0.15 rs (G A) Hemoglobin (g/dl) 0.11 (0.03) 1.8x (0.04) 1.0x (0.03) 8.7x10-6 MCV (fl) 0.82 (0.14) 3.6x (0.21) 1.69x (0.12) 8.6x10-13 MCH (pg) 0.37 (0.05) 4.9x (0.14) 4.8x (0.07) 3.3x10-7 MCHC (pg/l) 0.11 (0.03) 1.6x (0.04) 4.2x (0.02) 7.3x10-7 RBC ( /L) (0.01) (0.01) (0.01) 5.1x10-3 WBC ( 10 9 /L) 0.06 (0.05) (0.05) (0.04) 0.10 PLT ( 10 9 /L) 0.32 (1.68) (2.10) (1.33) 0.72 MCV mean cell volume; MCH mean cell hemoglobin; MCHC mean cell hemoglobin concentration; RBC red blood cell count; WBC white blood cell count; PLT platelet count. Frequencies of minor allele (A) of rs855791: Europeans 43%, Indian Asians 52%, P=3.5x10-33 ; rs198846: Europeans 16%, Indian Asians 8%, P=6.9x10-81.

7 Supplementary Table 4. SNPs associated with hemoglobin levels at P<5x10-8 in combined analysis of European and Indian Asian genome-wide data. SNP Chr Position Locus Alt Effect N Europeans P= Indian Asians P= Combined analysis P= rs TMPRSS6 A G x x x10-13 rs TMPRSS6 T C x x x10-13 rs TMPRSS6 A G x x x10-13 rs TMPRSS6 A G x x10-10 rs TMPRSS6 A G x x10-10 rs TMPRSS6 A G x x10-10 rs TMPRSS6 T C x x10-10 rs TMPRSS6 T G x x10-10 rs HFE A G x x x10-8 rs HFE A G x x10-8 rs TMPRSS6 C G x x10-8 rs TMPRSS6 A C x x10-8 rs TMPRSS6 T C x x10-8 rs HFE T C x x x10-8 rs HFE T G x x10-8 rs TMPRSS6 A G x x10-8 rs TMPRSS6 T C x x10-8 rs HFE C G x x10-8

8 Supplementary Figure 1. QQ plot of the association test results with hemoglobin levels, in Europeans (Panel 1a), Indian Asians (Panel 1b) and in combined analysis (Panel 1c) of the genome-wide association study. The red line shows the expected distribution under the null hypothesis of no association at any locus, and the green line the 95% confidence limits of the null hypothesis distribution. Panel 1a Europeans (lambda=1.04) Panel 1b Indian Asians (lambda=1.06)

9 Panel 1c Combined analysis (lambda=1.06)

10 Supplementary Figure 2. Signal-Intensity (Manhattan) plot showing the association of SNPs with hemoglobin levels in Europeans (Panel 2a), in Indian Asians (Panel 2b) and in combined analysis (Panel 2c). Panel 2a Europeans 10 -Log 10 (P) TMPRSS Chromosome Panel 2b Indian Asians 10 -Log 10 (P) TMPRSS Chromosome

11 Panel 2c Combined -analysis 12 TMPRSS6 -Log 10 (P) 10 8 HFE Chromosome

12 Supplementary Figure 3. Genomic context of TMPRSS6 region on chromosome 11. Pairwise linkage disequilibrium is shown between genotyped SNPs with MAF 0.05 amongst Europeans (NFBC sample, N=4,717, upper panel) and Indian Asians (Illumina 610 sample, N=6,988, lower panel), using Haploview s standard color scheme.

Supplementary Figure 1: Attenuation of association signals after conditioning for the lead SNP. a) attenuation of association signal at the 9p22.

Supplementary Figure 1: Attenuation of association signals after conditioning for the lead SNP. a) attenuation of association signal at the 9p22.32 PCOS locus after conditioning for the lead SNP rs10993397;