Plenary Poster 29th Australian and New Zealand Bone and Mineral Society Annual Scientific Meeting 2019

Identification of drug targets for osteoporosis: Evidence from whole-exome sequencing in 42,263 individuals, CRISPR-Cas9 and murine models (#90)

John P. Kemp 1 2 , Laetitia Laurent 3 , Sirui Zhou 3 4 , Albena Pramatarova 5 , Vincenzo Forgetta 3 , Peter I. Croucher 6 , Graham R. Williams 7 , Duncan J.H. Bassett 7 , David Goltzman 8 , Brent J. Richards 3 5 9 , David M. Evans 1 2
  1. University of Queensland Diamantina Institute, University of Queensland, Wooloongabba, QLD, Australia
  2. Medical Research Council Integrative Epidemiology Unit, University of Bristol, Bristol, Avon, United Kingdom
  3. Lady Davis Institute, Jewish General Hospital, McGill University, Montréal, Québec, Canada
  4. Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montréal, Québec, Canada
  5. McGill University , Genome Quebec Innovation Centre, Montréal, Québec, Canada
  6. Garvan Institute of Medical Research, University of New South Wales, Sydney, New South Wales, Australia
  7. Molecular Endocrinology Laboratory, Department of Medicine, Imperial College London, London, Middlesex, United Kingdom
  8. Research Institute of the McGill University Health Centre, McGill University, Montréal, Québec, Canada
  9. Department of Medicine, McGill University, Montréal, Québec, Canada

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.