Background: The use of osteoporosis medications has decreased, partly due to side-effects associated with their use. New therapies are required, and human genetics combined with functional follow-up offer a promising means to identify putative drug targets for osteoporosis treatment.
Aims: To identify drug targets for osteoporosis treatment using whole-exome sequencing (WES), genome-wide association studies (GWAS), CRISPR-Cas9, and murine knockout models.
Methods: WES was conducted in 42,263 white British UK-Biobank Study participants with estimated bone mineral density (eBMD) measures derived by heel quantitative ultrasound. Summary association results were synthesised with our recent eBMD GWAS involving 426,824 UK-Biobank participants. CRISPR-Cas9 mediated deletion of selected genes was used to assess the consequences of gene deletion in SaOS-2 osteoblast-like cell lines. Skeletal phenotype screening from knockout mice were used to validate the consequences of gene deletion in-vivo.
Results: WES identified 46 genes harbouring variants associated with eBMD (P<1x10-7). Notably, two rare variants with large eBMD-increasing effects were detected: CYP2A7 [β=3.5 standard deviations (SD) per risk allele (95%CI:4.8−2.2)], and C8orf33 [β=2.8 (3.9−1.8)]. Synthesis of WES and GWAS results revealed that 45/46 genes were located within 400kb of common eBMD GWAS associated variants. Focusing on CADM1, a cell adhesion molecule expressed in osteoblasts, we identified genetic variants associated with eBMD from WES (P=2x10-8) and GWAS (P=3x10-76). CADM1 editing in SaOS-2 cells suppressed protein abundance at the cell surface, and total protein expression, and resulted in elevated bone markers: RUNX2 (1.3-fold), COL1A1/COL1A2 (1.5-fold), ALPL (1.9-fold), and alkaline phosphatase activity (1.6-fold). Murine knockout models of Cadm1 presented with decreased femur bone mineral content (-3.9 SD), femur length (-4.5 SD) and maximum load to femur fracture (-2.0 SD) compared to litter-mate controls.
Conclusions: Population based WES identifies osteoporosis genes (and a putative drug target) that demonstrates important phenotypic consequences in CRISPR and murine model screens.