Novel sequence variants associated with bone mineral density

Transcription

1 Supplementary Material: Novel sequence variants associated with bone mineral density Unnur Styrkarsdottir 1*, Bjarni V. Halldorsson 1,2, Solveig Gretarsdottir 1, Daniel F. Gudbjartsson 1, G. Bragi Walters 1, Thorvaldur Ingvarsson 3, Thorbjorg Jonsdottir 1, Jona Saemundsdottir 1, Steinunn Snorradóttir 1, Jacqueline R. Center 4, Tuan V. Nguyen 4, Peter Alexandersen 5, Jeffrey R. Gulcher 1, John A. Eisman 4, Claus Christiansen 6, Gunnar Sigurdsson 7, Augustine Kong 1, Unnur Thorsteinsdottir 1, Kari Stefansson 1* 1deCODE Genetics, Sturlugata 8, IS-101 Reykjavik, Iceland 2 Reykjavik University, Kringlan 1, IS-103 Reykjavik, Iceland 3 FSA University Hospital, Institution of Health Science, University of Akureyri, IS-600 Akureyri, Iceland 4Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst; St Vincent s Hospital and University of New South Wales, Sydney NSW 2010, Australia 5Center for Clinical and Basic Research A/S, DK-2750 Ballerup, Denmark 6 Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev, Denmark 7Department of Endocrinology and Metabolism, University Hospital, IS-108 Reykjavik, Iceland *Corresponding author: Kari Stefansson, decode Genetics, Sturlugata 8, IS-101 Reykjavik, Iceland; tel: , fax: , kari.stefansson@decode.is; Unnur Styrkarsdottir, decode Genetics, Sturlugata 8, IS-101 Reykjavik, Iceland; tel: , fax: , unnur.styrkarsdottir@decode.is 1

2 Supplementary Methods: Study populations: The Icelandic discovery sample set comprised 5,934 women and 931 men who had dualenergy X-ray absorptiometry (DEXA, Hologic QDR4500A) bone mineral density (BMD) measurements at the lumbar spine (L2-L4) and the hip ( values at the femoral neck, trochanter and intertrochanter region). There were 6,674 (5,759 women) with hip measurement and 6,862 (5,931 women) with spine measurement. Of this group, 1,200 women were selected for IlluminaHap300K genotyping because of their low BMD (<- 1.2 SD of age and weight corrected BMD) as a part of our osteoporosis genetic program. All others were genotyped on Illumina Hap300K through other disease projects. The Icelandic replication group was comprised of 2,252 women and 884 men. Information on diseases that influence BMD or medications that have an effect on BMD (e.g. Hormone Replacement Therapy or Corticosteroids use) was only available for 2,800 of the original discovery set and for 1,500 of the replication samples. No subjects with BMD values were excluded from the study. Most low-impact fractures were self-reported in a detailed questionnaire; reports of hip fractures were obtained from hospital records at Reykjavik s University Hospital or the General Hospital, Akureyri. Fractures of fingers, hands, toes, or feet were not included. Of the fracture cases used in this study (n=3,015) 1,902 (63%) had BMD measurement. The control group used in the fracture case control analysis (n= 29,089) were genotyped on Illumina Hap300K through other projects at decode. All participants gave informed consent and the study was approved by the Data Protection Commission of Iceland (DPC) and the National Bioethics Committee of Iceland. The Danish samples were derived from the Prospective Epidemiological Risk Factor (PERF study) 1. These are postmenopausal women (n = 3,884), in the age range years, taking part in a prospective epidemiological study and in various clinical trials for osteoporosis at the Center for Clinical and Basic Research, Copenhagen. Baseline DEXA- measurement (Hologic QDR2000) at the hip (total hip) and lumbar spine (L2-L4) was used for 3,884. Osteoporotic fractures (n=905) included self-reported low trauma fractures and vertebral fractures assessed by digital measurements of morphologic changes. 97% of the fracture cases also had a BMD measurement. The study was approved by the Ethics Committee of Copenhagen County and was in accordance with the principles of the Helsinki Declaration. The Australian cohort in this study is from the Dubbo Osteoporosis Epidemiology Study (DOES) 2, including 571 males and 920 females in the age range years. All are of Caucasian ethnicity. Bone mineral density was measured at the lumbar spine and the hip femoral neck (FNBMD) by DEXA (LUNAR DPX-L). Osteoporotic fractures (n=550) included low trauma fractures assessed by questionnaire and ascertained by reviewing all radiography reports. All fracture 2

3 cases also had a BMD measurement. The study was approved by the St. Vincent s Ethics Review Committee (Sydney), and all subjects gave written informed consent. Summary characteristics of the study populations Iceland discovery Iceland Replication Denmark Australia Men Women Men Women Women Men Women # Age (SD), year Age-range, year BMI (SD), kg/m2 Height (SD), cm Weight (SD), kg raw HipBMD (SD), g/cm HipBMDcorr., (SD) raw SpineBMD (SD), g/cm SpineBMDcorr., (SD) Genotyping: Samples for the genome-wide association study were assayed with the Infinium humanhap300 or humancnv370 SNP chips (Illumina). Three different versions of the chips were used, humanhap300, humanhap300-duo and humancnv370. The analysis was restricted to SNPs that had a call rate of 97% on each of the chip types and did not deviate from Hardy Weinberg equilibrium (P>10-7 ) on any chip type. Thus, the final analyses used 305,051 SNPs. A genome-wide significant for association using the full set of markers is therefore set as (=0.05/305,051) for one phenotype. The marker map from Illumina, HumanHap300_v1.0.0_SNP_nsSNP_annotation, was used for analysis of the non-synonymous SNPs 3

4 present on the chips. This map contains 7,339 SNPs, of which 6,980 passed our quality filters and were used in the analysis. All single-snp genotyping was carried out at decode Genetics on the Centaurus (Nanogen) platform3. The quality of each Centaurus SNP assay was evaluated by genotyping each assay in the CEU HapMap samples and comparing the results with the HapMap data. The Icelandic set contained 300 individuals that were genotyped on both the humancnv370 chip and with individual assays and were used for genotype comparisons. Assays with a mismatch rate >1.5% were not used, and a linkage disequilibrium (LD) test was used for markers known to be in LD. Standardization and Association analysis: Age and weight corrected bone mineral density was computed for each sex and population separately to have a mean 0 and standard deviation 1 (commonly known as weight corrected Z-scores in the bone field). Bone density increased linearly with weight in both genders. For women we modeled bone density to decrease linearly after the age of 45 while for men we modeled bone density to decrease linearly in all age ranges. For the Icelandic population regression was done against a randomly recruited reference group 4. In the Australian and Danish cohorts correction was done against the whole cohort 1,2. For each SNP, a linear regression, using the genotype as an additive covariate and standardized bone density as the response, was fitted to test for association. Each SNP was tested separately for its association with bone mineral density of the hip and for its association with bone mineral density of the lumbar spine. We tested the additive model against the full model (i.e. treating the genotype as a categorical variable) and no significant deviation from the additive model was detected (P>0.05 for all SNPs in Table 1). For association analysis of osteoporotic fractures a standard likelihood ratio statistics was used, as implemented in the NEMO software created at decode 5, to calculate two-sided s and odds ratio (OR) for each individual allele, assuming a multiplicative model for risk, i.e., that the risk of the two alleles a person carries multiply. Allelic frequencies, rather than carrier frequencies are presented for the markers. Our program, NEMO, was used for evaluating the measures of LD between individual markers. Results from multiple case-control groups were using a Mantel-Haenszel model in which the groups were allowed to have different population frequencies for alleles, haplotypes and genotypes but were assumed to have a common relative risk 6. The threshold for genome-wide significance was set at a of less than (=0.05/305,051) for each phenotype and the threshold for replication was set at a of less than 0.05 in the Danish and Australian replication sets. 4

5 Correction for relatedness and genomic control For the Icelandic discovery sample, the method of genomic control was used to adjust for the relatedness of the subjects 7. To do that, we transformed the (unadjusted) s, based on the t- tests, to 1-df chi-square statistics. The inflation factor λ for the chi-square statistics was estimated as 1.21 for hip BMD and 1.24 for spine BMD. The unadjusted chi-square statistics were divided by this inflation factor λ to obtain the adjusted test statistics. For the analysis an inflation factor for the test statistics was computed using simulations using our genealogical database: We created simulated genotypes by choosing uniformly at random a minor allele frequency as a multiple of 5% in the range 5% to 50%. We then randomly assign alleles to individuals born in the year 1700 or earlier. Individuals born after year 1700 were randomly assigned one of their parents genotypes. The s obtained from associating the BMD to these genotypes, assuming that the patients are unrelated, was converted to a corresponding 1 df chi-square statistic that gives the same. This chi-square statistic was then divided by the inflation factor to obtain the adjusted chi-square statistic and the adjusted. This inflation factor was estimated as λ = 1.22 for hip, 1.21 for spine and 1.24 for fractures. For comparison purposes we also simulated inflation factor for the original population, these were estimated as 1.17 (hip) and 1.16 (spine). The actual sample size divided by this inflation factor was used as an estimate of the effective sample size. An overall Z -score was calculated by summing the Z -scores weighted by the square root of the effective sample size, over all populations, and dividing by the square root of the sum of the sample sizes. An overall estimate of the effect per allele was calculated by weighting together the effects in each population by the population s effective sample size. Stratification analysis in the DOES cohort Because of a known, although very small, Asian component to the DOES cohort we performed a principle component analysis in order to investigate whether the observed effect on bone density could be due to stratification. The analysis was based on 78 independent SNPs, one from each considered region. We found that the first two principle components did not significantly associate with bone density, suggesting that even if there is stratification there is no substantial difference in bone density in the two sample sets. Further, no meaningful impact on the s was observed when we added these principle components as covariates Interaction between loci Possible interaction between the multiple variants considered in this publication was investigated by comparing a model which includes pairwise interaction terms for the multiple 5

6 variants presented in this paper to the additive model without the interaction terms. This interaction model did not provide a significantly better fit (P= 0.25 spine, P = 0.11 hip). Database Accession numbers GeneID accession numbers for the genes MARK3: 4140 (Unigene Hs.35828), SOST: 50964, Osx: , RANK: 8792, C17orf53: 78995, LRP4: 4038, ADAM19: 8728 and IBSP: References: 1. Bagger, Y.Z. et al. Osteoporos Int 17, (2006). 2. Nguyen, T.V., Sambrook, P.N. & Eisman, J.A. J Bone Miner Res 12, (1997). 3. Kutyavin, I.V. et al. Nucl. Acids Res. 34, e128- (2006). 4. Gudmundsdottir, S.L., Indridason, O.S., Franzson, L. & Sigurdsson, G. J Clin Densitom 8, 80-6 (2005). 5. Gretarsdottir, S. et al. Nat Genet 35, (2003). 6. Mantel, N. & Haenszel, W. J Natl Cancer Inst 22, (1959). 7. Devlin, B. & Roeder, K. Biometrics 55, (1999). 6

7 Supplementary Tables Supplementary Table 1 Results for SNPs in a genome-scan of hip bone mineral density. Part A) Results for the top 100 associated SNPs with hip bone mineral density and Part B) Results for the top 20 non-synonymous or potentially functional SNPs with hip bone density. The SNPs are listed as they are ranked in the genome-wide association scan of hip bone mineral density in the Icelandic discovery set. The allelic frequency is given for the Icelandic set, the corrected in the Icelandic discovery set, the in the set of the Danish (DK) samples and the Australian DOES cohort (AUS), the effect on hip bone density (BMD) in all the sets (Icelandic, discovery and replication, the Danish and the Australian) with the corresponding for all the sets. The odds ratios (OR) and s are shown for all the sets. The effect on spine bone density and are also shown for all the sets. The markers that were included in our previous report (NEJM) and those that reached the criteria of P < 0.1 in the first replication stage (P<0.1 DK1) are indicated. The estimated effect on bone density is expressed as standard deviations below the population average, per copy of the allele. All s are two-sided. The markers that failed in replication are indicated by a * and NA for for DK and AUS. The markers reaching genome-wide significance that were not included or did not reach genome-wide significance in our previous report are indicated by bold typesetting across the marker, the P-values for all markers reaching genome-wide significance level are indicated by red and bold typesetting, and those replicating in the Danish and Australian datasets by red typesetting. 7 A) Top 100 associated SNPs Allele marker Position_build36 Freq. discovery set for DK and AUS Effect in all sets P Value All sets OR low-trauma fracture lowtrauma fracture Effect on Spine BMD in all sets for Spine BMD G rs chr1_ E (-0.18, -0.10) 2.5E (1.08, 1.27) 1.4E (-0.16, -0.08) 2.4E-08 NEJM Comment A rs chr6_ E (-0.10, -0.04) 5.5E (1.01, 1.15) (-0.10, -0.04) 2.4E-06 P<0.1 DK1 C rs chr6_ E (-0.11, -0.05) 8.1E (1.00, 1.12) (-0.11, -0.06) 3.5E-09 NEJM A rs chr1_ E E (-0.18, -0.11) 1.2E (1.06, 1.22) 4.6E (-0.14, -0.07) 7.0E-09 NEJM A rs chr9_ E (-0.10, -0.03) 4.4E (1.00, 1.16) (-0.06, 0.01) 0.11 T rs chr6_ E (-0.11, -0.05) 1.3E (0.98, 1.10) (-0.11, -0.05) 2.2E-08 NEJM A rs chr9_ E (-0.09, -0.03) 2.6E (1.01, 1.14) (-0.06, 0.00) NEJM G rs chr4_ E (-0.07, -0.02) (0.95, 1.07) (-0.07, -0.01) NEJM G rs chr6_ E (-0.11, -0.06) 4.0E (1.00, 1.11) (-0.12, -0.06) 1.9E-10 NEJM G rs chr14_ E (-0.08, -0.02) (0.93, 1.07) (-0.07, 0.00) G rs chr19_ E (-0.15, -0.01) (0.89, 1.16) (-0.12, 0.11) 0.93 G rs chr9_ E (-0.09, -0.03) 4.7E (0.96, 1.10) (-0.04, 0.03) 0.78 T rs chr7_ E (-0.09, -0.03) 1.7E (1.03, 1.15) (-0.08, -0.02) 5.1E-04 P<0.1 DK1 T rs chr5_ E (-0.07, -0.01) (0.99, 1.13) (-0.08, -0.02)

8 C rs chr6_ E (-0.11, -0.06) 2.2E (1.00, 1.12) (-0.12, -0.06) 4.2E-09 P<0.1 DK1 A rs chr9_ E (-0.09, -0.02) 5.8E (0.96, 1.11) (-0.04, 0.03) 0.79 G rs chr3_ E (-0.10, -0.02) (0.96, 1.11) (-0.08, 0.01) 0.14 NEJM G rs chr1_ E E (-0.14, -0.07) 1.0E (1.02, 1.16) (-0.11, -0.04) 9.3E-06 P<0.1 DK1 G rs chr8_ E (-0.07, -0.02) (0.98, 1.09) (-0.05, 0.01) 0.13 G rs * chr15_ E-05 NA (-0.12, -0.05) 1.7E (0.96, 1.12) (-0.10, -0.02) C rs chr18_ E (-0.09, -0.01) (0.94, 1.10) (-0.12, -0.03) 6.8E-04 NEJM A rs chr6_ E (-0.11, -0.05) 1.5E (0.99, 1.12) (-0.13, -0.07) 1.6E-10 NEJM C rs chr20_ E (-0.07, -0.01) (0.98, 1.10) (-0.06, -0.00) NEJM G rs * chr6_ E-05 NA (-0.13, -0.05) 9.7E (1.00, 1.17) (-0.15, -0.06) 9.4E-07 A rs chr2_ E (-0.13, -0.03) (0.98, 1.19) (-0.09, 0.01) 0.13 NEJM G rs chr6_ E (-0.07, -0.01) (0.97, 1.09) (-0.09, -0.03) 2.5E-04 NEJM C rs chr10_ E (-0.09, -0.03) 3.8E (0.97, 1.08) (-0.08, -0.02) P<0.1 DK1 C rs chr2_ E (-0.09, -0.04) 1.6E (0.98, 1.09) (-0.06, -0.01) P<0.1 DK1 G rs chr4_ E (-0.10, -0.00) (0.93, 1.12) (-0.11, -0.01) C rs chr8_ E (-0.08, -0.01) (0.89, 1.02) (-0.05, 0.02) 0.38 T rs chr4_ E (-0.10, -0.00) (0.93, 1.15) (-0.11, -0.00) NEJM C rs * chr16_ E-05 NA (-0.37, -0.13) 4.7E (0.93, 1.52) (-0.24, 0.02) A rs chr19_ E (-0.10, -0.04) 3.5E (0.99, 1.13) (-0.08, -0.00) NEJM G rs chr19_ E (-0.27, -0.09) 7.3E (0.78, 1.16) (-0.17, 0.02) 0.11 T rs chr11_ E (-0.17, -0.04) (0.92, 1.20) (-0.12, 0.01) 0.12 A rs * chr6_ E-05 NA (-0.11, -0.03) (0.97, 1.10) (-0.10, -0.03) T rs chr6_ E E (-0.12, -0.06) 8.0E (0.97, 1.10) (-0.14, -0.08) 6.6E-13 NEJM T rs chr18_ E (-0.09, -0.03) 9.9E (1.02, 1.15) (-0.08, -0.02) 3.0E-04 G rs chrx_ E (-0.09, -0.03) 5.5E (0.95, 1.06) (-0.06, -0.01) P<0.1 DK1 A rs chrx_ E (-0.16, -0.02) (0.88, 1.16) (-0.13, 0.01) A rs chr13_ E (-0.10, -0.02) (1.00, 1.18) (-0.10, -0.02) A rs chr17_ E (-0.10, -0.05) 2.1E (1.01, 1.13) (-0.07, -0.02) P<0.1 DK1 A rs chr6_ E (-0.08, -0.02) 7.5E (0.97, 1.09) (-0.06, 0.00) T rs chrx_ E (-0.09, -0.04) 1.3E (0.99, 1.11) (-0.07, -0.01) P<0.1 DK1 T rs chr4_ E (-0.10, -0.00) (0.89, 1.08) (-0.11, 0.00) C rs chr3_ E (-0.08, -0.02) (0.97, 1.11) (-0.06, 0.01) 0.13 G rs chr6_ E (-0.10, -0.05) 2.2E (1.02, 1.13) (-0.11, -0.05) 9.5E-08 NEJM C rs chr13_ E (-0.10, -0.02) (1.01, 1.18) (-0.10, -0.02) G rs chr8_ E (-0.07, -0.01) (0.90, 1.04) (-0.04, 0.02) 0.57 C rs * chr9_ E-05 NA (-0.12, -0.04) 2.3E (0.95, 1.12) (-0.09, -0.01) T rs chr6_ E (-0.07, -0.01) (0.96, 1.09) (-0.05, 0.01)

9 A rs chr11_ E (-0.11, -0.05) 7.2E (1.01, 1.16) (-0.08, -0.01) P<0.1 DK1 T rs chr17_ E (-0.10, -0.05) 1.0E (1.04, 1.17) 6.1E (-0.08, -0.02) NEJM G rs chr15_ E (-0.09, -0.02) 9.5E (1.00, 1.13) (-0.06, 0.01) 0.23 G rs chr10_ E (-0.15, -0.04) 2.8E (0.98, 1.23) (-0.11, -0.00) T rs chr4_ E (-0.09, -0.02) (0.95, 1.09) (-0.06, 0.01) 0.12 A rs chr14_ E E (-0.11, -0.06) 1.8E (1.03, 1.15) (-0.08, -0.03) 2.3E-04 P<0.1 DK1 A rs chr3_ E (-0.08, -0.02) 3.9E (0.97, 1.08) (-0.05, 0.01) 0.11 G rs872593* chr3_ E-05 NA (-0.12, -0.04) 3.1E (0.99, 1.16) (-0.11, -0.03) 8.8E-04 G rs * chr9_ E-05 NA (-0.12, -0.04) 1.2E (0.96, 1.12) (-0.05, 0.03) 0.54 A rs chr4_ E (-0.07, -0.01) (1.00, 1.12) (-0.06, -0.00) G rs chr5_ E (-0.08, -0.01) (0.96, 1.10) (-0.06, 0.00) A rs * chr3_ E-05 NA (-0.11, -0.03) 3.1E (0.93, 1.09) (-0.06, 0.02) 0.31 C rs chr6_ E (-0.07, -0.01) (0.95, 1.08) (-0.05, 0.02) 0.29 A rs * chr14_ E-04 NA (-0.12, -0.04) 1.3E (1.01, 1.18) (-0.10, -0.01) C rs chr4_ E (-0.11, -0.03) 5.4E (0.94, 1.10) (-0.08, 0.00) NEJM G rs chr3_ E (-0.09, -0.03) 9.4E (1.00, 1.11) (-0.08, -0.02) 2.8E-04 P<0.1 DK1 T rs chr13_ E (-0.10, -0.02) (1.01, 1.19) (-0.10, -0.02) T rs chr12_ E (-0.09, -0.03) (0.96, 1.10) (-0.07, -0.00) T rs chr13_ E (-0.10, -0.02) (1.00, 1.18) (-0.10, -0.03) T rs chr8_ E (-0.10, -0.03) 1.6E (0.91, 1.06) (-0.07, 0.01) 0.13 T rs chr16_ E (-0.10, -0.01) (1.03, 1.28) (-0.09, 0.00) T rs chr6_ E (-0.09, -0.03) 4.1E (0.99, 1.10) (-0.09, -0.03) 2.3E-05 NEJM G rs * chr4_ E-04 NA (-0.13, -0.04) (0.93, 1.10) (-0.10, -0.01) G rs289061* chr12_ E-04 NA (-0.12, -0.04) 2.2E (0.99, 1.18) (-0.10, -0.01) T rs chr5_ E (-0.11, -0.03) 6.4E (1.01, 1.19) (-0.10, -0.02) G rs * chr6_ E-04 NA (-0.12, -0.04) 3.0E (1.03, 1.20) (-0.07, 0.01) 0.13 C rs chr18_ E (-0.15, -0.05) 1.5E (1.00, 1.24) (-0.12, -0.01) NEJM A rs chr17_ E (-0.10, -0.05) 2.2E (1.03, 1.15) (-0.08, -0.03) 2.1E-04 P<0.1 DK1 A rs chr6_ E (-0.09, -0.04) 4.4E (1.05, 1.17) 3.7E (-0.11, -0.05) 1.5E-08 NEJM C rs chr3_ E (-0.08, -0.03) 2.1E (1.00, 1.12) (-0.08, -0.02) 4.7E-04 P<0.1 DK1 C rs chr17_ E (-0.09, -0.03) 8.5E (0.98, 1.11) (-0.07, -0.01) A rs chr6_ E (-0.12, -0.05) 1.3E (0.99, 1.13) (-0.13, -0.06) 5.7E-08 NEJM G rs chr3_ E (-0.07, -0.02) 3.3E (0.99, 1.10) (-0.05, 0.00) P<0.1 DK1 C rs chr4_ E (-0.09, -0.03) 5.5E (0.94, 1.09) (-0.06, 0.01) 0.13 G rs chr18_ E (-0.09, -0.03) 3.1E (0.99, 1.13) (-0.09, -0.02) 5.8E-04 T rs chr19_ E (-0.08, -0.02) (1.05, 1.19) (-0.07, -0.01) T rs chr1_ E (-0.10, -0.03) 5.9E (0.99, 1.16) (-0.08, 0.00)

10 A rs405717* chr4_ E-04 NA (-0.10, -0.02) (1.04, 1.20) (-0.07, 0.02) 0.20 C rs chr11_ E (-0.13, -0.02) (0.99, 1.24) (-0.10, 0.01) 0.12 T rs chr1_ E (-0.09, -0.02) 9.1E (0.93, 1.06) (-0.05, 0.03) 0.54 C rs chr6_ E (-0.14, -0.03) (0.89, 1.11) (-0.10, 0.01) 0.12 T rs chr1_ E (-0.09, -0.02) 8.0E (0.92, 1.06) (-0.05, 0.03) 0.52 A rs chr22_ E (-0.08, -0.02) (0.97, 1.09) (-0.04, 0.03) 0.72 P<0.1 DK1 A rs563811* chr11_ E-04 NA (-0.11, -0.03) 4.0E (0.98, 1.15) (-0.08, 0.00) T rs chr6_ E (-0.07, -0.02) (1.01, 1.12) (-0.07, -0.01) P<0.1 DK1 A rs chr5_ E (-0.09, -0.03) 2.0E (0.99, 1.10) (-0.07, -0.01) P<0.1 DK1 C rs chr17_ E (-0.09, -0.03) 7.7E (1.00, 1.12) (-0.08, -0.02) P<0.1 DK1 G rs * chrx_ E-04 NA (-0.10, -0.03) 1.9E (1.02, 1.19) (-0.08, -0.01) G rs chr1_ E (-0.08, -0.02) (0.96, 1.10) (-0.07, -0.01) B) Top 20 non-synonymous SNPs or potentially functional SNPs Allele marker Gene - change Freq. discovery set for DK and AUS Effect on hip BMD in all sets P Value All sets OR low-trauma fracture lowtrauma fracture Effect on Spine BMD in all sets for Spine BMD A rs C6orf97-V604I E (-0.11, -0.05) 1.5E (0.99, 1.12) (-0.13, -0.07) 1.6E-10 NEJM C rs * RPGRIP1L-Q774R E-05 NA (-0.37, -0.13) 4.7E (0.93, 1.52) (-0.24, 0.02) T rs KANK4-A840V E (-0.10, -0.03) 5.9E (0.99, 1.16) (-0.08, 0.00) Comment A rs ADAM19-G284S E (-0.09, -0.03) 2.0E (0.99, 1.10) (-0.07, -0.01) P<0.1 DK1 T rs ADCY6-S674A E (-0.21, -0.05) (1.02, 1.45) (-0.19, -0.02) P<0.1 DK1 T rs * ZSWIM2-C265Y E-04 NA (-0.19, -0.06) 2.1E (0.89, 1.16) (-0.16, -0.02) T rs C17orf53-P126T E (-0.10, -0.04) 8.9E (0.98, 1.11) (-0.06, 0.00) P<0.1 DK1 G rs ZSWIM2-I302T E (-0.13, -0.02) (0.91, 1.14) (-0.13, -0.02) A rs FAM118A-H239R E (-0.08, -0.02) (0.97, 1.10) (-0.04, 0.03) 0.78 P<0.1 DK1 T rs CPN2-V536M E (-0.09, -0.03) 9.2E (0.99, 1.11) (-0.07, -0.01) P<0.1 DK1 T rs ERLIN1-V291I E (-0.07, -0.02) 5.6E (0.99, 1.10) (-0.07, -0.02) P<0.1 DK1 G rs OR5G1P E (-0.11, -0.02) (1.11, 1.34) 5.3E (-0.10, -0.01) P<0.1 DK1 A rs CPN2-A305T E (-0.09, -0.03) 9.2E (0.99, 1.12) (-0.07, -0.01) P<0.1 DK1 C rs TBC1D2-T241P E (-0.08, -0.02) 3.1E (0.98, 1.10) (-0.06, -0.00) P<0.1 DK1 A rs IBSP-G195E (-0.09, -0.03) 4.6E (0.99, 1.11) (-0.09, -0.03) 9.1E-05 P<0.1 DK1 A rs * WDR60-R273Q NA (-0.10, -0.03) (1.01, 1.19) (-0.09, -0.01) C rs WDSOF1-R47S (-0.08, -0.01) (0.95, 1.09) (-0.04, 0.02) 0.49 T rs LTBP (-0.07, -0.01) (0.97, 1.08) (-0.06, 0.00) P<0.1 DK1 T rs * ZNF645-D166E NA (-0.22, -0.06) (0.78, 1.11) (-0.13, 0.04) 0.29 C rs LRP4-I1086V (-0.09, -0.04) 2.1E (1.02, 1.14) (-0.06, -0.00) P<0.1 DK1 10

11 Supplementary Table 2. Results for SNPs in a genome-scan of spine bone mineral density. Part A) Results for the top 100 associated SNPs with hip bone mineral density and Part B) Results for the top 20 non-synonymous or potentially functional SNPs with hip bone density. The SNPs are listed as they are ranked in the genome-wide association scan in the Icelandic discovery set. The allelic frequency is given for the Icelandic set, the corrected in the Icelandic discovery set, the in the set of the Danish (DK) samples and the Australian DOES cohort (AUS), the effect on spine bone density (BMD) in all the sets (Icelandic, discovery and replication, the Danish and the Australian) with the corresponding for all the sets. The odds ratios (OR) and s are shown for all the sets. The effect on hip bone density and are also shown for all the sets. The markers that were included in our previous report (NEJM) and those that reached the criteria of P < 0.1 in the first replication stage (P<0.1 DK1) are indicated. The estimated effect on bone density is expressed as standard deviations below the population average, per copy of the allele. All s are two-sided. The markers that failed in replication are indicated by a * and NA for for DK and AUS. The markers reaching genome-wide significance that were not included or did not reach genome-wide significance in our previous report are indicated by bold typesetting across the marker, the P-values for all markers reaching genome-wide significance level are indicated by red and bold typesetting, and those replicating in the Danish and Australian datasets by red typesetting. A) Top 100 associated SNPs Allele marker Position_build36 Freq. discovery set for DK and AUS Effect in all sets P Value All sets OR low-trauma fracture lowtrauma fracture Effect on Hip BMD in all sets T rs chr13_ E E (-0.15, -0.09) 1.5E (0.96, 1.08) (-0.10, -0.04) 1.2E-06 NEJM A rs chr6_ E (-0.11, -0.05) 1.5E (1.05, 1.17) 3.7E (-0.09, -0.04) 4.4E-06 NEJM T rs chr6_ E (-0.11, -0.05) 2.2E (0.98, 1.10) (-0.11, -0.05) 1.3E-08 NEJM C rs chr6_ E (-0.11, -0.06) 3.5E (1.00, 1.12) (-0.11, -0.05) 8.1E-09 NEJM T rs chr6_ E (-0.08, -0.02) (0.95, 1.07) (-0.06, -0.00) NEJM C rs chr6_ E (-0.11, -0.06) 3.9E (0.98, 1.09) (-0.09, -0.04) 6.9E-07 NEJM A rs chr13_ E (-0.13, -0.07) 3.0E (0.91, 1.04) (-0.11, -0.05) 7.9E-07 NEJM T rs chr6_ E E (-0.14, -0.08) 6.6E (0.97, 1.10) (-0.12, -0.06) 8.0E-10 NEJM G rs chr6_ E (-0.12, -0.06) 1.9E (1.00, 1.11) (-0.11, -0.06) 4.0E-10 NEJM A rs chr8_ E E (-0.14, -0.08) 1.4E (1.00, 1.11) (-0.11, -0.06) 6.9E-10 NEJM G rs * chr6_ E-06 NA (-0.15, -0.06) 9.4E (1.00, 1.17) (-0.13, -0.05) 9.7E-06 G rs chr2_ E (-0.10, -0.04) 3.5E (1.06, 1.19) 8.6E (-0.07, -0.01) NEJM for Hip BMD Comment C rs chr8_ E E (-0.14, -0.09) 1.1E (1.00, 1.11) (-0.12, -0.07) 3.1E-13 P<0.1 DK1 A rs chr6_ E (-0.10, -0.04) 2.4E (1.01, 1.15) (-0.10, -0.04) 5.5E-07 P<0.1 DK1 G rs chr2_ E (-0.10, -0.03) 3.6E (1.04, 1.17) (-0.07, -0.02) NEJM A rs chr18_ E (-0.09, -0.03) 2.0E (0.93, 1.05) (-0.07, -0.02) NEJM C rs chr11_ E (-0.13, -0.04) 2.0E (0.93, 1.13) (-0.07, 0.01)

12 A rs chr8_ E (-0.08, -0.02) (0.96, 1.06) (-0.05, 0.01) 0.17 NEJM T rs chr12_ E (-0.08, -0.01) (0.93, 1.07) (-0.07, -0.01) G rs chr12_ E (-0.07, -0.01) (0.93, 1.05) (-0.07, -0.02) G rs chr6_ E (-0.10, -0.04) 3.1E (1.00, 1.12) (-0.08, -0.03) 2.6E-05 NEJM A rs chr12_ E (-0.11, -0.05) 1.3E (0.99, 1.11) (-0.08, -0.02) 5.6E-04 NEJM A rs chr21_ E (-0.13, -0.05) 6.4E (0.99, 1.15) (-0.08, -0.00) NEJM C rs chr8_ E E (-0.14, -0.08) 2.1E (1.01, 1.12) (-0.12, -0.06) 1.5E-11 NEJM C rs chr9_ E (-0.15, -0.04) (0.84, 1.06) (-0.13, -0.03) NEJM G rs chr6_ E (-0.09, -0.03) 2.5E (0.97, 1.09) (-0.07, -0.01) NEJM C rs chr18_ E (-0.09, -0.03) 7.3E (0.93, 1.05) (-0.07, -0.02) NEJM G rs chr8_ E (-0.08, -0.02) (0.91, 1.04) (-0.06, 0.00) T rs chr16_ E (-0.10, -0.04) 2.8E (0.96, 1.10) (-0.07, -0.01) T rs chr16_ E (-0.10, -0.03) 4.3E (0.96, 1.10) (-0.07, -0.00) T rs * chr14_ E-05 NA (-0.19, -0.07) 1.5E (1.07, 1.36) (-0.14, -0.03) A rs chr3_ E (-0.10, -0.03) 1.2E (0.97, 1.12) (-0.06, 0.00) A rs chr8_ E (-0.12, -0.06) 5.2E (0.97, 1.08) (-0.09, -0.04) 5.3E-07 P<0.1 DK1 C rs chr10_ E (-0.08, -0.02) (0.97, 1.08) (-0.09, -0.03) 3.8E-05 P<0.1 DK1 C rs chrx_ E (-0.05, 0.01) (0.93, 1.04) (-0.05, 0.01) 0.18 NEJM C rs * chr10_ E-05 NA (-0.13, -0.05) 2.5E (1.00, 1.17) (-0.11, -0.03) 6.3E-04 A rs chr12_ E (-0.10, -0.04) 4.6E (0.96, 1.07) (-0.07, -0.01) NEJM T rs238256* chr13_ E-05 NA (-0.14, -0.05) 1.3E (0.94, 1.11) (-0.06, 0.03) 0.47 A rs chr2_ E (-0.09, -0.02) 7.1E (0.93, 1.04) (-0.05, 0.02) 0.39 P<0.1 DK1 T rs chr13_ E E (-0.15, -0.08) 5.4E (1.00, 1.15) (-0.07, -0.00) C rs chr9_ E (-0.12, -0.04) 9.7E (0.99, 1.15) (-0.09, -0.01) NEJM T rs chr5_ E (-0.12, -0.03) 7.5E (0.94, 1.13) (-0.10, -0.02) C rs chrx_ E (-0.09, -0.01) (0.91, 1.07) (-0.05, 0.02) 0.37 C rs * chrx_ E-05 NA (-0.15, -0.05) 4.2E (0.95, 1.16) (-0.10, -0.01) A rs chr14_ E (-0.12, -0.04) 6.1E (1.00, 1.16) (-0.11, -0.03) 1.7E-04 NEJM G rs chr2_ E (-0.09, -0.01) (0.94, 1.09) (-0.07, 0.00) G rs * chr19_ E-05 NA (-0.15, -0.05) 4.3E (1.08, 1.28) 1.3E (-0.08, 0.01) 0.13 C rs chr3_ E (-0.08, -0.02) 5.9E (0.94, 1.05) (-0.04, 0.02) 0.48 NEJM A rs chr14_ E (-0.12, -0.04) 1.3E (1.02, 1.20) (-0.10, -0.02) NEJM G rs chr3_ E (-0.09, -0.02) (0.93, 1.07) (-0.08, -0.01) P<0.1 DK1 A rs chr9_ E (-0.11, -0.03) 3.7E (0.98, 1.14) (-0.08, -0.00) NEJM C rs chr17_ E (-0.06, -0.01) (0.97, 1.08) (-0.04, 0.01) 0.24 P<0.1 DK1 C rs chr11_ E (-0.11, -0.04) 1.0E (0.93, 1.06) (-0.07, -0.00) NEJM T rs chr13_ E (-0.13, -0.05) 1.5E (0.91, 1.04) (-0.12, -0.05) 2.6E-06 12

13 C rs * chr16_ E-05 NA (-0.13, -0.04) 5.9E (1.00, 1.17) (-0.10, -0.02) A rs * chrx_ E-05 NA (-0.12, -0.05) 1.5E (0.93, 1.09) (-0.10, -0.03) 5.6E-04 C rs chr2_ E (-0.08, -0.02) 6.4E (1.05, 1.18) 1.5E (-0.05, 0.00) NEJM G rs chr14_ E (-0.08, -0.02) 5.7E (1.01, 1.13) (-0.08, -0.03) 1.5E-05 P<0.1 DK1 C rs chr18_ E (-0.12, -0.03) 6.8E (0.94, 1.10) (-0.09, -0.01) NEJM T rs chr14_ E (-0.11, -0.02) (1.03, 1.22) (-0.08, 0.00) A rs chr2_ E (-0.09, -0.02) (0.94, 1.08) (-0.06, 0.00) G rs chr3_ E (-0.10, -0.03) 6.2E (0.93, 1.06) (-0.05, 0.01) 0.23 NEJM G rs chr8_ E (-0.09, -0.02) (0.89, 1.03) (-0.06, 0.01) 0.19 G rs chr10_ E (-0.12, -0.03) 5.2E (0.97, 1.14) (-0.09, -0.00) P<0.1 DK1 C rs chrx_ E (-0.07, -0.01) (0.93, 1.08) (-0.04, 0.03) 0.72 G rs chr21_ E (-0.13, -0.04) 4.6E (0.90, 1.09) (-0.06, 0.04) 0.59 A rs chr6_ E (-0.13, -0.07) 1.6E (0.99, 1.12) (-0.11, -0.05) 1.5E-08 NEJM T rs chr2_ E (-0.10, -0.03) 1.4E (0.96, 1.10) (-0.07, -0.00) T rs chr17_ E (-0.09, -0.03) 1.6E (0.94, 1.07) (-0.07, -0.00) T rs chr8_ E (-0.10, -0.03) 2.1E (0.94, 1.08) (-0.08, -0.01) G rs chr5_ E (-0.15, -0.03) (0.93, 1.16) (-0.14, -0.03) P<0.1 DK1 A rs chr12_ E (-0.13, -0.02) (0.91, 1.15) (-0.08, 0.02) 0.31 C rs chr7_ E (-0.16, -0.06) 1.0E (0.92, 1.11) (-0.14, -0.05) 9.3E-05 P<0.1 DK1 C rs chr6_ E (-0.10, -0.05) 1.4E (1.00, 1.12) (-0.11, -0.06) 4.9E-10 NEJM C rs chr20_ E (-0.06, 0.00) (0.91, 1.03) (-0.05, 0.02) 0.36 NEJM G rs chr1_ E (-0.16, -0.08) 2.4E (1.08, 1.27) 1.4E (-0.18, -0.10) 2.5E-12 NEJM T rs chr10_ E (-0.08, -0.03) 1.6E (1.01, 1.13) (-0.08, -0.03) 2.9E-05 P<0.1 DK1 C rs chr6_ E (-0.12, -0.06) 4.2E (1.00, 1.12) (-0.11, -0.06) 2.2E-09 P<0.1 DK1 T rs chr8_ E (-0.11, -0.05) 7.9E (0.99, 1.11) (-0.10, -0.04) 1.7E-06 P<0.1 DK1 G rs chr1_ E (-0.09, -0.01) (0.92, 1.09) (-0.07, 0.00) C rs * chr13_ E-05 NA (-0.15, -0.05) 1.6E (0.89, 1.08) (-0.11, -0.02) C rs chr4_ E (-0.12, -0.04) 8.4E (0.91, 1.07) (-0.09, -0.01) T rs * chr12_ E-05 NA (-0.12, -0.04) 2.0E (0.94, 1.10) (-0.06, 0.02) 0.24 C rs * chr14_ E-04 NA (-0.14, -0.04) 3.4E (0.87, 1.06) (-0.09, 0.00) A rs * chr6_ E-04 NA (-0.10, -0.03) (0.97, 1.10) (-0.11, -0.03) 1.6E-04 T rs * chrx_ E-04 NA (-0.12, -0.04) 7.0E (0.95, 1.12) (-0.08, -0.00) G rs chr18_ E (-0.09, -0.02) (0.93, 1.07) (-0.07, -0.01) T rs chr5_ E (-0.12, -0.03) (0.94, 1.13) (-0.10, -0.02) G rs * chr19_ E-04 NA (-0.16, -0.05) 1.2E (1.03, 1.26) (-0.07, 0.03) 0.38 C rs chr5_ E (-0.07, -0.01) (0.96, 1.10) (-0.06, 0.00) G rs * chr12_ E-04 NA (-0.12, -0.04) 2.1E (1.02, 1.19) (-0.10, -0.02)

14 T rs chr21_ E (-0.07, -0.01) (1.01, 1.13) (-0.05, 0.01) 0.21 NEJM G rs chr21_ E (-0.07, -0.01) (0.99, 1.12) (-0.07, -0.01) P<0.1 DK1 C rs chr16_ E (-0.08, -0.02) (0.97, 1.11) (-0.06, 0.00) G rs chr21_ E (-0.09, -0.02) (0.97, 1.12) (-0.09, -0.02) 7.0E-04 G rs13320 chr1_ E (-0.06, 0.01) (0.98, 1.09) (-0.06, 0.01) 0.17 T rs chr2_ E (-0.09, -0.02) 9.9E (0.93, 1.06) (-0.05, 0.01) 0.22 T rs chr2_ E (-0.16, -0.05) 1.7E (0.93, 1.16) (-0.12, -0.02) T rs chrx_ E (-0.07, -0.00) (0.91, 1.05) (-0.07, -0.00) T rs chr7_ E (-0.08, -0.01) (0.95, 1.10) (-0.06, 0.00) P<0.1 DK1 B) Top 20 non-synonymous SNPs or potentially functional SNPs Allele marker Gene - change Freq. discovery set for DK and AUS Effect on spine BMD in all sets P Value All sets OR low-trauma fracture lowtrauma fracture Effect on Hip BMD in all sets G rs SPTBN1-intron E (-0.10, -0.04) 3.5E (1.06, 1.19) 8.6E (-0.07, -0.01) NEJM A rs C6orf97-V604I E (-0.13, -0.07) 1.6E (0.99, 1.12) (-0.11, -0.05) 1.5E-08 NEJM G rs TDRD10-I215V E (-0.11, -0.03) 5.1E (0.89, 1.06) (-0.07, 0.00) T rs * CD300LF-R218Q E-04 NA (-0.14, -0.04) 7.4E (0.91, 1.10) (-0.07, 0.02) 0.25 C rs MED6-intron E (-0.13, -0.03) 9.2E (0.92, 1.13) (-0.10, -0.01) for Hip BMD Comment C rs10788 SERGEF-E457G E (-0.14, -0.03) (0.99, 1.23) (-0.13, -0.02) P<0.1 DK1 T rs1528 SERGEF-E429K E (-0.13, -0.03) (0.98, 1.19) (-0.12, -0.03) P<0.1 DK1 A rs ESPL1-A25D E (-0.09, -0.03) 6.7E (0.97, 1.12) (-0.06, 0.00) A rs BAT2-V1895L (-0.11, -0.02) (1.02, 1.21) (-0.10, -0.01) NEJM T rs LOC A96V (-0.10, -0.04) 3.9E (1.00, 1.12) (-0.08, -0.02) 5.4E-04 P<0.1 DK1 T rs ERLIN1-V291I (-0.07, -0.02) (0.99, 1.10) (-0.07, -0.02) 5.6E-04 P<0.1 DK1 A rs * ANKRD12-P1735S NA (-0.16, -0.03) (0.95, 1.22) (-0.12, 0.01) C rs FMN1-E239G (-0.07, -0.01) (1.01, 1.13) (-0.05, 0.00) P<0.1 DK1 T rs PTPLAD2-A36T (-0.07, -0.01) (0.94, 1.06) (-0.08, -0.02) P<0.1 DK1 A rs RASSF4-G88R (-0.09, -0.01) (0.95, 1.10) (-0.05, 0.01) 0.22 P<0.1 DK1 G rs conserved region (-0.06, 0.00) (0.95, 1.09) (-0.05, 0.02) 0.36 C rs KIF18A-I735V (-0.07, -0.01) (0.94, 1.05) (-0.05, 0.01) 0.13 P<0.1 DK1 C rs9898 HRG-S204P (-0.07, 0.00) (0.96, 1.11) (-0.06, 0.00) C rs RGNEF-Q284P (-0.06, -0.00) (0.96, 1.06) (-0.06, -0.01) P<0.1 DK1 A rs ITGA11-L471M (-0.16, -0.02) (0.97, 1.31) (-0.12, 0.02)

15 Supplementary Table 3. Comparison of current and previous results for previously reported loci. Part A), markers from genome-wide significant regions in our previous publication (Styrkarsdottir et. al., NEJM 2008), Part B), suggestively associated loci reported in the same publication, and Part C), the LRP5 marker that was reported genome-wide significant by Richard et. al. (Lancet 2008) and vanmeurs et. al. (JAMA 2008). Data for this marker is only for the Icelandic discovery set. The s of association in the previous publication and in the current dataset are shown, and the effect on bone density in all sets in the current study for hip and spine bone density. All the markers shown replicate nominally in the Danish and Australian sets for either hip or spine bone denstiy or both. Markers that were not included in previous publication but reach genome-wide significance (part A) and replicate nominally in the current dataset are marked as new 15 Allele marker A) 1p36.12 Frequency P Value Previous Study Hip P Value Current Study Effect in all sets P Value Previous Study Spine P Value Current Study Effect in all sets (-0.18, - A rs ) (-0.14, -0.07) (-0.18, - G rs ) (-0.16, -0.08) (-0.14, - G rs NA ) NA (-0.11, -0.04) new 6p21.32 MHC T rs * q ESR (-0.08, ) (-0.13, -0.06) C rs T rs G rs A rs A rs C rs NA C rs A rs NA ) (-0.10, -0.05) , ) (-0.14, -0.08) (-0.12, ) (-0.12, -0.06) (-0.11, ) (-0.13, -0.07) (-0.11, ) (-0.13, -0.06) new (-0.12, ) NA (-0.12, -0.06) new (-0.11, ) (-0.11, -0.06) (-0.09, - 8q OPG 0.04) NA (-0.12, -0.06) new (-0.09, -

16 C rs NA A rs C rs ) NA (-0.14, -0.09) new (-0.12, ) (-0.14, -0.08) (-0.11, ) (-0.14, -0.08) (-0.12, - 13q RANKL T rs NA (-0.07, ) NA (-0.15, -0.08) new T rs ** ) (-0.19, -0.13) (-0.13, - T rs ) (-0.15, -0.09) (-0.10, - B) 2p16.2 G rs q RANK (-0.07, ) (-0.10, -0.04) A rs A rs NA G rs C rs NA G rs ) (-0.09, -0.04) (-0.10, - 11p LRP4 0.05) NA (-0.08, -0.01) new (-0.11, ) (-0.06, 0.01) (-0.11, ) NA (-0.06, -0.00) new (-0.09, ) (-0.06, 0.00) (-0.09, - 12q Osx (SP7) A rs (-0.08, ) (-0.11, -0.05) C) 11q LRP5 T rs *** (-0.07, 0.04) (-0.13, -0.02) * not among the top100 or the top20 ns SNPs in the current scan **not on the IlluminaHap300 chip *** data for the discovery group only 16