Genes Associated With Fracture Risk in Thoroughbred Horses Have Novel Roles in Osteogenesis

Bone fractures in Thoroughbred racehorses are a major welfare problem. Genetic factors contribute to fracture risk. Cell models have previously identified 112 differentially expressed genes in bone-forming osteoblasts derived from horses at high and low genetic risk of fracture. However, 42 of these genes have no published role in bone. In this study, we identified novel roles for a subset of these genes in bone formation. Twenty-six of the 42 genes were expressed in Saos2 cells during basal culture and/or after 21 days of osteogenic culture. Five of these genes (ADSSL1, CABP1, ENO2, SPARCL1 and UCP2) were then stably overexpressed and knocked down, and their effect on osteogenesis was measured. Gene overexpression resulted in significant decreases in Saos2 cell viability and decreased expression of osteogenic genes under basal cell culture, but after 21 days of osteogenic culture there were few significant changes in osteogenic gene expression, collagen deposition or matrix mineralisation. Knockdown of SPARCL1 resulted in total cell death, whereas knockdown of ADSSL1, CABP1, ENO2 and UCP2 resulted in decreased cell viability but limited significant changes in osteogenic gene expression under basal cell culture. However, following osteogenic culture, gene knockdown induced widespread changes in osteogenic gene expression, decreased collagen deposition and increased matrix mineralisation. ADSSL1, CABP1, ENO2 and UCP2 were all expressed at significantly lower levels in osteoblasts from genetically high-risk horses. Taken together, this work demonstrates novel roles for fracture-associated genes in bone formation and matrix mineralisation suggesting these processes may be altered in genetically susceptible horses.