Genome-Wide Association Meta-Analysis of Cortical Bone Mineral Density Unravels Allelic Heterogeneity at the RANKL Locus and Potential Pleiotropic Effects on Bone

Abstract

Previous genome-wide association (GWA) studies have identified SNPs associated with areal bone mineral density (aBMD). However, this measure is influenced by several different skeletal parameters, such as periosteal expansion, cortical bone mineral density (BMD _C) cortical thickness, trabecular number, and trabecular thickness, which may be under distinct biological and genetic control. We have carried out a GWA and replication study of BMD _C, as measured by peripheral quantitative computed tomography (pQCT), a more homogenous and valid measure of actual volumetric bone density. After initial GWA meta-analysis of two cohorts (ALSPAC n = 999, aged ∼15 years and GOOD n = 935, aged ∼19 years), we attempted to replicate the BMD _C associations that had p<1×10 ⁻⁵ in an independent sample of ALSPAC children (n = 2803) and in a cohort of elderly men (MrOS Sweden, n = 1052). The rs1021188 SNP (near RANKL) was associated with BMD _C in all cohorts (overall p = 2×10 ⁻¹⁴, n = 5739). Each minor allele was associated with a decrease in BMD _C of ∼0.14SD. There was also evidence for an interaction between this variant and sex (p = 0.01), with a stronger effect in males than females (at age 15, males −6.77mg/cm ³ per C allele, p = 2×10 ⁻⁶; females −2.79 mg/cm ³ per C allele, p = 0.004). Furthermore, in a preliminary analysis, the rs1021188 minor C allele was associated with higher circulating levels of sRANKL (p<0.005). We show this variant to be independent from the previously aBMD associated SNP (rs9594738) and possibly from a third variant in the same RANKL region, which demonstrates important allelic heterogeneity at this locus. Associations with skeletal parameters reflecting bone dimensions were either not found or were much less pronounced. This finding implicates RANKL as a locus containing variation associated with volumetric bone density and provides further insight into the mechanism by which the RANK/ RANKL/ OPG pathway may be involved in skeletal development.

Author Summary

Previous studies that have identified genetic polymorphisms involved in bone density have used a technique that cannot differentiate between cortical and trabecular bone. We have carried out the first genome-wide association study using a bone scanning method that can differentiate between the constituent parts of bone. We found a genetic variant (rs1021188) near the RANKL gene that was associated with the density of cortical bone in the three cohorts that we studied (ranging in age from 15 to 78 years old). We also found that this variant may have a more prominent effect on cortical bone density in males than females. In addition, the minor C allele of rs1021188 was associated with higher circulating levels of free RANKL. Although the RANKL gene has been previously identified as being important for bone structure (albeit with a different SNP showing association), we show for the first time that this may be primarily due to its influence on the density of cortical bone, rather than the size of the bone or other bone features.