Scott G. Wilson

Bone mineral density, osteoporosis, and osteoporotic fractures: a genome-wide association study

Summary Background Osteoporosis is diagnosed by the measurement of bone mineral density, which is a highly heritable and multifactorial trait. We aimed to identify genetic loci that are associated with bone mineral density. Methods In this genome-wide association study, we identified the most promising of 314 075 single nucleotide polymorphisms (SNPs) in 2094 women in a UK study. We then tested these SNPs for replication in 6463 people from three other cohorts in western Europe. We also investigated allelic expression in lymphoblast cell lines. We tested the association between the replicated SNPs and osteoporotic fractures with data from two studies. Findings We identified genome-wide evidence for an association between bone mineral density and two SNPs (p<5×10−8). The SNPs were rs4355801, on chromosome 8, near to the TNFRSF11B (osteoprotegerin) gene, and rs3736228, on chromosome 11 in the LRP5 (lipoprotein-receptor-related protein) gene. A non-synonymous SNP in the LRP5 gene was associated with decreased bone mineral density (rs3736228, p=6·3×10−12 for lumbar spine and p=1·9×10−4 for femoral neck) and an increased risk of both osteoporotic fractures (odds ratio [OR] 1·3, 95% CI 1·09–1·52, p=0·002) and osteoporosis (OR 1·3, 1·08–1·63, p=0·008). Three SNPs near the TNFRSF11B gene were associated with decreased bone mineral density (top SNP, rs4355801: p=7·6×10−10 for lumbar spine and p=3·3×10−8 for femoral neck) and increased risk of osteoporosis (OR 1·2, 95% CI 1·01–1·42, p=0·038). For carriers of the risk allele at rs4355801, expression of TNFRSF11B in lymphoblast cell lines was halved (p=3·0×10−6). 1883 (22%) of 8557 people were at least heterozygous for these risk alleles, and these alleles had a cumulative association with bone mineral density (trend p=2·3×10−17). The presence of both risk alleles increased the risk of osteoporotic fractures (OR 1·3, 1·08–1·63, p=0·006) and this effect was independent of bone mineral density. Interpretation Two gene variants of key biological proteins increase the risk of osteoporosis and osteoporotic fracture. The combined effect of these risk alleles on fractures is similar to that of most well-replicated environmental risk factors, and they are present in more than one in five white people, suggesting a potential role in screening. Funding Wellcome Trust, European Commission, NWO Investments, Arthritis Research Campaign, Chronic Disease Research Foundation, Canadian Institutes of Health Research, European Society for Clinical and Economic Aspects of Osteoporosis, Genome Canada, Genome Quebéc, Canada Research Chairs, National Health and Medical Research Council of Australia, and European Union.

Twenty bone-mineral-density loci identified by large-scale meta-analysis of genome-wide association studies

Bone mineral density (BMD) is a heritable complex trait used in the clinical diagnosis of osteoporosis and the assessment of fracture risk. We performed meta-analysis of five genome-wide association studies of femoral neck and lumbar spine BMD in 19,195 subjects of Northern European descent. We identified 20 BMD loci that reached genome-wide significance (GWS; P < 5 × 10−8), of which 13 map to regions not previously associated with this trait: 1p31.3 (GPR177), 2p21 (SPTBN1), 3p22 (CTNNB1), 4q21.1 (MEPE), 5q14 (MEF2C), 7p14 (STARD3NL), 7q21.3 (FLJ42280), 11p11.2 (LRP4, ARHGAP1, F2), 11p14.1 (DCDC5), 11p15 (SOX6), 16q24 (FOXL1), 17q21 (HDAC5) and 17q12 (CRHR1). The meta-analysis also confirmed at GWS level seven known BMD loci on 1p36 (ZBTB40), 6q25 (ESR1), 8q24 (TNFRSF11B), 11q13.4 (LRP5), 12q13 (SP7), 13q14 (TNFSF11) and 18q21 (TNFRSF11A). The many SNPs associated with BMD map to genes in signaling pathways with relevance to bone metabolism and highlight the complex genetic architecture that underlies osteoporosis and variation in BMD.

Meta-analysis of genome-wide association data identifies two loci influencing age at menarche.

We conducted a meta-analysis of genome-wide association data to detect genes influencing age at menarche in 17,510 women. The strongest signal was at 9q31.2 (P = 1.7 × 10−9), where the nearest genes include TMEM38B, FKTN, FSD1L, TAL2 and ZNF462. The next best signal was near the LIN28B gene (rs7759938; P = 7.0 × 10−9), which also influences adult height. We provide the first evidence for common genetic variants influencing female sexual maturation.

An Integration of Genome-Wide Association Study and Gene Expression Profiling to Prioritize the Discovery of Novel Susceptibility Loci for Osteoporosis-Related Traits

Osteoporosis is a complex disorder and commonly leads to fractures in elderly persons. Genome-wide association studies (GWAS) have become an unbiased approach to identify variations in the genome that potentially affect health. However, the genetic variants identified so far only explain a small proportion of the heritability for complex traits. Due to the modest genetic effect size and inadequate power, true association signals may not be revealed based on a stringent genome-wide significance threshold. Here, we take advantage of SNP and transcript arrays and integrate GWAS and expression signature profiling relevant to the skeletal system in cellular and animal models to prioritize the discovery of novel candidate genes for osteoporosis-related traits, including bone mineral density (BMD) at the lumbar spine (LS) and femoral neck (FN), as well as geometric indices of the hip (femoral neck-shaft angle, NSA; femoral neck length, NL; and narrow-neck width, NW). A two-stage meta-analysis of GWAS from 7,633 Caucasian women and 3,657 men, revealed three novel loci associated with osteoporosis-related traits, including chromosome 1p13.2 (RAP1A, p = 3.6×10−8), 2q11.2 (TBC1D8), and 18q11.2 (OSBPL1A), and confirmed a previously reported region near TNFRSF11B/OPG gene. We also prioritized 16 suggestive genome-wide significant candidate genes based on their potential involvement in skeletal metabolism. Among them, 3 candidate genes were associated with BMD in women. Notably, 2 out of these 3 genes (GPR177, p = 2.6×10−13; SOX6, p = 6.4×10−10) associated with BMD in women have been successfully replicated in a large-scale meta-analysis of BMD, but none of the non-prioritized candidates (associated with BMD) did. Our results support the concept of our prioritization strategy. In the absence of direct biological support for identified genes, we highlighted the efficiency of subsequent functional characterization using publicly available expression profiling relevant to the skeletal system in cellular or whole animal models to prioritize candidate genes for further functional validation.

Meta‐Analysis of Genome‐Wide Scans Provides Evidence for Sex‐ and Site‐Specific Regulation of Bone Mass

Several genome‐wide scans have been performed to detect loci that regulate BMD, but these have yielded inconsistent results, with limited replication of linkage peaks in different studies. In an effort to improve statistical power for detection of these loci, we performed a meta‐analysis of genome‐wide scans in which spine or hip BMD were studied. Evidence was gained to suggest that several chromosomal loci regulate BMD in a site‐specific and sex‐specific manner.

Comparison of Genome Screens for Two Independent Cohorts Provides Replication of Suggestive Linkage of Bone Mineral Density to 3p21 and 1p36

Low bone mineral density (BMD) is a major risk factor for osteoporotic fracture. Studies of BMD in families and twins have shown that this trait is under strong genetic control. To identify regions of the genome that contain quantitative trait loci (QTL) for BMD, we performed independent genomewide screens, using two complementary study designs. We analyzed unselected nonidentical twin pairs (1,094 pedigrees) and highly selected, extremely discordant or concordant (EDAC) sib pairs (254 pedigrees). Nonparametric multipoint linkage (NPL) analyses were undertaken for lumbar spine and total-hip BMD in both cohorts and for whole-body BMD in the unselected twin pairs. The maximum evidence of linkage in the unselected twins (spine BMD, LOD 2.7) and the EDAC pedigrees (spine BMD, LOD 2.1) was observed at chromosome 3p21 (76 cM and 69 cM, respectively). These combined data indicate the presence, in this region, of a gene that regulates BMD. Furthermore, evidence of linkage in the twin cohort (whole-body BMD; LOD 2.4) at chromosome 1p36 (17 cM) supports previous findings of suggestive linkage to BMD in the region. Weaker evidence of linkage (LOD 1.0-2.3) in either cohort, but not both, indicates the locality of additional QTLs. These studies validate the use, in linkage analysis, of large cohorts of unselected twins phenotyped for multiple traits, and they highlight the importance of conducting genome scans in replicate populations as a prelude to positional cloning and gene discovery.

WNT16 influences bone mineral density, cortical bone thickness, bone strength, and osteoporotic fracture risk.

We aimed to identify genetic variants associated with cortical bone thickness (CBT) and bone mineral density (BMD) by performing two separate genome-wide association study (GWAS) meta-analyses for CBT in 3 cohorts comprising 5,878 European subjects and for BMD in 5 cohorts comprising 5,672 individuals. We then assessed selected single-nucleotide polymorphisms (SNPs) for osteoporotic fracture in 2,023 cases and 3,740 controls. Association with CBT and forearm BMD was tested for ∼2.5 million SNPs in each cohort separately, and results were meta-analyzed using fixed effect meta-analysis. We identified a missense SNP (Thr>Ile; rs2707466) located in the WNT16 gene (7q31), associated with CBT (effect size of −0.11 standard deviations [SD] per C allele, P = 6.2×10−9). This SNP, as well as another nonsynonymous SNP rs2908004 (Gly>Arg), also had genome-wide significant association with forearm BMD (−0.14 SD per C allele, P = 2.3×10−12, and −0.16 SD per G allele, P = 1.2×10−15, respectively). Four genome-wide significant SNPs arising from BMD meta-analysis were tested for association with forearm fracture. SNP rs7776725 in FAM3C, a gene adjacent to WNT16, was associated with a genome-wide significant increased risk of forearm fracture (OR = 1.33, P = 7.3×10−9), with genome-wide suggestive signals from the two missense variants in WNT16 (rs2908004: OR = 1.22, P = 4.9×10−6 and rs2707466: OR = 1.22, P = 7.2×10−6). We next generated a homozygous mouse with targeted disruption of Wnt16. Female Wnt16−/− mice had 27% (P<0.001) thinner cortical bones at the femur midshaft, and bone strength measures were reduced between 43%–61% (6.5×10−13<P<5.9×10−4) at both femur and tibia, compared with their wild-type littermates. Natural variation in humans and targeted disruption in mice demonstrate that WNT16 is an important determinant of CBT, BMD, bone strength, and risk of fracture.

Age-Related Changes in Thyroid Function: A Longitudinal Study of a Community-Based Cohort

CONTEXT In cross-sectional studies, serum TSH concentrations increase with age. This has not been examined longitudinally, and it is uncertain whether the TSH increase reflects healthy aging or occult thyroid failure. METHODS We measured serum TSH, free T(4), thyroid peroxidase, and thyroglobulin antibodies in 1100 participants in the 1981 and 1994 Busselton Health Surveys and derived a reference group of 908 individuals without thyroid disease or thyroid antibodies. We examined changes in thyroid function longitudinally and, in 781 participants, explored associations with the CAPZB polymorphism rs10917469. RESULTS At 13 yr follow-up, mean serum TSH increased from 1.49 to 1.81 mU/liter, a change in mean TSH (ΔTSH) of 0.32 mU/liter [95% confidence interval (CI) 0.27, 0.38, P < 0.001], whereas mean free T(4) concentration was unchanged (16.6 vs. 16.6 pmol/liter, P = 0.7). The TSH increase was most marked in the elderly, such that gender-adjusted ΔTSH increased by 0.08 mU/liter (95% CI 0.04, 0.11) for each decade of baseline age. People with higher baseline TSH values had proportionally smaller increases in TSH, with each additional 1.0 mU/liter of baseline TSH associated with a 0.13 mU/liter decrease (age and gender adjusted) in ΔTSH (95% CI 0.09, 0.16). The ΔTSH did not differ significantly by CAPZB genotype. CONCLUSIONS Aging is associated with increased serum TSH concentrations, with no change in free T(4) concentrations. The largest TSH increase is in people with the lowest TSH at baseline. This suggests that the TSH increase arises from age-related alteration in the TSH set point or reduced TSH bioactivity rather than occult thyroid disease.

A Meta-Analysis of Thyroid-Related Traits Reveals Novel Loci and Gender-Specific Differences in the Regulation of Thyroid Function

Thyroid hormone is essential for normal metabolism and development, and overt abnormalities in thyroid function lead to common endocrine disorders affecting approximately 10% of individuals over their life span. In addition, even mild alterations in thyroid function are associated with weight changes, atrial fibrillation, osteoporosis, and psychiatric disorders. To identify novel variants underlying thyroid function, we performed a large meta-analysis of genome-wide association studies for serum levels of the highly heritable thyroid function markers TSH and FT4, in up to 26,420 and 17,520 euthyroid subjects, respectively. Here we report 26 independent associations, including several novel loci for TSH (PDE10A, VEGFA, IGFBP5, NFIA, SOX9, PRDM11, FGF7, INSR, ABO, MIR1179, NRG1, MBIP, ITPK1, SASH1, GLIS3) and FT4 (LHX3, FOXE1, AADAT, NETO1/FBXO15, LPCAT2/CAPNS2). Notably, only limited overlap was detected between TSH and FT4 associated signals, in spite of the feedback regulation of their circulating levels by the hypothalamic-pituitary-thyroid axis. Five of the reported loci (PDE8B, PDE10A, MAF/LOC440389, NETO1/FBXO15, and LPCAT2/CAPNS2) show strong gender-specific differences, which offer clues for the known sexual dimorphism in thyroid function and related pathologies. Importantly, the TSH-associated loci contribute not only to variation within the normal range, but also to TSH values outside the reference range, suggesting that they may be involved in thyroid dysfunction. Overall, our findings explain, respectively, 5.64% and 2.30% of total TSH and FT4 trait variance, and they improve the current knowledge of the regulation of hypothalamic-pituitary-thyroid axis function and the consequences of genetic variation for hypo- or hyperthyroidism.

Metabolomic markers reveal novel pathways of ageing and early development in human populations

Background Human ageing is a complex, multifactorial process and early developmental factors affect health outcomes in old age. Methods Metabolomic profiling on fasting blood was carried out in 6055 individuals from the UK. Stepwise regression was performed to identify a panel of independent metabolites which could be used as a surrogate for age. We also investigated the association with birthweight overall and within identical discordant twins and with genome-wide methylation levels. Results We identified a panel of 22 metabolites which combined are strongly correlated with age (R2 = 59%) and with age-related clinical traits independently of age. One particular metabolite, C-glycosyl tryptophan (C-glyTrp), correlated strongly with age (beta = 0.03, SE = 0.001, P = 7.0 × 10−157) and lung function (FEV1 beta = −0.04, SE = 0.008, P = 1.8 × 10−8 adjusted for age and confounders) and was replicated in an independent population (n = 887). C-glyTrp was also associated with bone mineral density (beta = −0.01, SE = 0.002, P = 1.9 × 10−6) and birthweight (beta = −0.06, SE = 0.01, P = 2.5 × 10−9). The difference in C-glyTrp levels explained 9.4% of the variance in the difference in birthweight between monozygotic twins. An epigenome-wide association study in 172 individuals identified three CpG-sites, associated with levels of C-glyTrp (P < 2 × 10−6). We replicated one CpG site in the promoter of the WDR85 gene in an independent sample of 350 individuals (beta = −0.20, SE = 0.04, P = 2.9 × 10−8). WDR85 is a regulator of translation elongation factor 2, essential for protein synthesis in eukaryotes. Conclusions Our data illustrate how metabolomic profiling linked with epigenetic studies can identify some key molecular mechanisms potentially determined in early development that produce long-term physiological changes influencing human health and ageing.