Genome-wide association study in Collaborative Cross mice revealed a skeletal role for Rhbdf2

Abstract

Osteoporosis, the most common bone disease, is characterized by a low bone mass and increased risk of fractures. Importantly, individuals with the same bone mineral density (BMD), as measured on two dimensional (2D) radiographs, have different risks for fracture, suggesting that microstructural architecture is an important determinant of skeletal strength. Here we took advantage of the rich phenotypic and genetic diversity of the Collaborative Cross (CC) mice. Using microcomputed tomography, we examined key structural parameters in the femoral cortical and trabecular compartments of male and female mice from 34 CC lines. These traits included the trabecular bone volume fraction, number, thickness, connectivity, and spacing, as well as structural morphometric index. In the mid-diaphyseal cortex, we recorded cortical thickness and volumetric BMD.\n\nThe broad-sense heritability of these traits ranged between 50 to 60%. We conducted a genome-wide association study to unravel 5 quantitative trait loci (QTL) significantly associated with 6 of the traits. We refined each locus by combining information obtained from the known ancestry of the mice and RNA-Seq data from publicly available sources, to shortlist potential candidate genes. We found strong evidence for new candidate genes, including Rhbdf2, which association to trabecular bone volume fraction and number was strongly suggested by our analyses. We then examined knockout mice, and validated the causal action of Rhbdf2 on bone mass accrual and microarchitecture.\n\nOur approach revealed new genome-wide QTLs and a series of genes that have never been associated with bone microarchitecture. This study demonstrates for the first time the skeletal role of Rhbdf2 on the physiological remodeling of both the cortical and trabecular bone. This newly assigned function for Rhbdf2 can prove useful in deciphering the predisposing factors of osteoporosis and propose new investigative avenues toward targeted therapeutic solutions.\n\nAuthor summaryIn this study, we used the novel mouse reference population, the Collaborative Cross (CC), to identify new causal genes in the regulation of bone microarchitecture, a critical determinant of bone strength. This approach provides a clear advantage in terms of resolution and dimensionality of the morphometric features (versus humans) and rich allelic diversity (versus classical mouse populations), over current practices of bone-related genome-wide association studies.\n\nOur genome-wide study revealed 5 loci significantly associated with microstructural traits in the cortical and trabecular bone. We found strong evidence for new candidate genes, in particular, Rhbdf2. We then validated the specific role of Rhbdf2 on bone mass accrual and microarchitecture using knockout mice. Importantly, this study is the first demonstration of a physiological role for Rhbdf2.