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

Abstract

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.

Figure 1. SNP rs2707466 regional association plot of the discovery genome-wide meta-analysis of cortical thickness.

Circles show GWA meta-analysis p-values, with different colors indicating varying linkage disequilibrium with rs2707466 (diamond).

Figure 2. The genome-wide meta-analysis with cortical thickness according to sex.

Figure 3. Scatter plots of the observed association of 7q31 locus with forearm BMD.

The P values of SNPs (shown as −log10 values in y-axis, from the genome-wide single-marker association analysis using the linear regression model) are plotted against their map position (b36) (x-axis). The color of each SNP spot reflects its r2 with rs2908004. Missense SNPs are plotted as triangles, and other SNPs are plotted as circles.

Figure 4. Forest plots of association of top SNPs for forearm fracture.

Figure 5. Decrease of bone strength of Wnt16 knockout mice at femur and tibia.

In Femur group, the sample size are 23 wide type (WT) mice and 13 knock out (KO) mice, and in Tibia group, the sample size are 12 WT mice and 9 KO mice. The P values for each group are shown in the figure.