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.

Author Summary

Bone traits are highly dependent on genetic factors. To date, numerous genetic loci for bone mineral density (BMD) and only one locus for osteoporotic fracture have been previously identified to be genome-wide significant. Cortical bone has been reported to be an important determinant of bone strength; so far, no genome-wide association studies (GWAS) have been performed for cortical bone thickness (CBT) of the tibial and radial diaphysis or BMD at forearm, a skeletal site rich in cortical bone. Therefore, we performed two separated meta-analyses of GWAS for cortical thickness of the tibia in 3 independent cohorts of 5,878 men and women, and for forearm BMD in 5 cohorts of 5,672 individuals. We identified the 7q31 locus, which contains WNT16, to be associated with CBT and BMD. Four SNPs from this locus were then tested in 2,023 osteoporotic fracture cases and 3,740 controls. One of these SNPs was genome-wide significant, and two were genome-wide suggestive, for forearm fracture. Generating a mouse with targeted disruption of Wnt16, we also demonstrated that mice lacking this protein had substantially thinner bone cortices and reduced bone strength than their wild-type littermates. These findings highlight WNT16 as a clinically relevant member of the Wnt signaling pathway and increase our understanding of the etiology of osteoporosis-related phenotypes and fracture.