Collaborative cross mice in a genetic association study reveal new candidate genes for bone microarchitecture

Roei Levy, Richard F. Mott, Fuad A. Iraqi, Yankel Gabet

The microstructure of trabecular bone is a composite trait governed by a complex interaction of multiple genetic determinants. Identifying these genetic factors should significantly improve our ability to predict of osteoporosis and its associated risks. Genetic mapping using collaborative cross mice (CC), a genetically diverse recombinant inbred mouse reference panel, offers a powerful tool to identify causal loci at a resolution under one mega base-pairs, with a relatively small cohort size. Here, we utilized 31 CC lines (160 mice of both sexes in total) to perform genome-wide haplotype mapping across 77,808 single-nucleotide polymorphisms (SNPs). Haplotype scans were refined by imputation with the catalogue of sequence variation segregating in the CC to suggest potential candidate genes. Trabecular traits were obtained following microtomographic analysis, performed on 10-μm resolution scans of the femoral distal metaphysis. We measured the trabecular bone volume fraction (BV/TV), number (Tb.N), thickness (Tb.Th), and connectivity density (Conn.D). Heritability of these traits ranged from 0.6 to 0.7. In addition there was a significant (P < 0.01) sex effect in all traits except Tb.Th. Our haplotype scans yielded six quantitative trait loci (QTL) at 1 % false discovery rate; BV/TV and Tb.Th produced two proximal loci each, on chromosome 2 and 7, respectively, and Tb.N and Conn.D yielded one locus on chromosomes 8 and 14, respectively. We identified candidate genes with previously-reported functions in bone biology, and implicated unexpected genes whose function in bone biology has yet to be assigned. Based on the literature, among the genes that ranked particularly high in our analyses (P < 10(-6)) and which have a validated causal role in skeletal biology, are Avp, Oxt, B2m (associated with BV/TV), Cnot7 (with Tb.N), Pcsk6, Rgma (with Tb.Th), Rb1, and Cpb2 (with Conn.D). Other candidate genes strongly suggested by our analyses are Sgcz, Fgf20 (associated with Tb.N), and Chd2 (with Tb.Th). We have demonstrated for the first time genome-wide significant association between several genetic loci and trabecular microstructural parameters for genes with previously reported experimental observations, as well as proposing a role for new candidate genes with no previously characterized skeletal function.