Differential gene expression in pre-race blood samples from racehorses with race-day fractures

Catastrophic musculoskeletal injuries (CMI) are a major concern in Thoroughbred horse racing, with identification of screening methods for horses at risk of injury being a top research priority. Postmortem exams have found the majority of these injuries to be associated with chronic accumulation of microdamage, which would theoretically produce localized and systemic changes detectable via biomarker analysis. Previous research using post-injury and post-race samples found increased messenger RNA (mRNA) expression of IGF1 and MMP2 and decreased expression of IL1RN in horses with CMI compared with controls. However, differences in mRNA expression immediately before injury, where there is no influence of exercise and/or the acute injury, have not been assessed. As such, to identify differentially expressed genes potentially associated with CMI, a total of 15,463 pre-race whole-blood samples were collected in Tempus Blood RNA Tubes over an 18-mo period. RNA sequencing of select samples was subsequently used to investigate transcriptional differences between 12 cases (fatal and nonfatal fractures) and 93 race-matched controls. Across all fractures, 31 genes were downregulated and 3 were upregulated. In phalanx and metacarpal fractures, there were 838 downregulated and 223 upregulated genes; in sesamoid fractures, there were 293 downregulated and 67 upregulated genes; and in hock and carpal fractures, there were 76 downregulated and 4 upregulated genes. CXCL1 and IL6 were downregulated in horses with long bone fractures, with these genes having been previously linked with adaptation to exercise. ANK2, a key regulator of bone mineralization that is increased during fracture healing, and SLIT3, a gene associated with production of fibrillar collagen, were downregulated in all fracture types and especially long bone fractures, consistent with a failure of proper bone remodeling before fracture. Integrative pathway analysis revealed a variety of pathway changes, including a downregulation of osteoclastic pathways in long bone and sesamoid fractures and of bone mineralization pathways in long bone fractures. These results continue to indicate that whole-blood mRNA samples may be useful in identifying racehorses with maladaptive bone remodeling responses, increasing their risk of fracture.