While North American Thoroughbred race-day injuries have decreased in recent years, further improvements are needed. Blood-based transcriptomic biomarkers offer a potential adjunct to imaging and wearable technologies for identification of horses at risk for orthopedic injury. Building on prior work identifying differentially expressed genes (DEGs) in catastrophically injured racehorses, this study aimed to characterize whole-blood gene expression profiles in horses sustaining race-day fractures versus matched controls.
Pre-race blood samples were collected in Tempus® Blood RNA Tubes during routine pre-race total CO₂ testing (~30 min pre-race) at three racetracks (Santa Anita, Los Alamitos, and Del Mar) between February 2022 and October 2023, resulting in the collection of 15,463 samples. Within this sample set, 13 horses with race-day fractures and 68 race-matched, non-injured controls met the study’s inclusion criteria. Total RNA was extracted and sequenced, resulting in an average of 61.4M paired-end reads/sample.
Initial principal component analysis revealed injury-specific clustering. DEGs between injured and non-injured horses were identified and analyzed via Ingenuity Pathway Analysis, Weighted Gene Co-expression Network Analysis (WGCNA), and Database for Annotation, Visualization, and Integrated Discovery (DAVID). Of the 295 DEGs that were identified as potential biomarker targets, 23 appeared repeatedly across functional analyses and/or exhibited large expression differences between groups. From these 23 genes, seven genes were prioritized based on biological relevance to bone physiology and/or fold changes between injured and non-injured horses. A five-gene panel (CAV1, DKK3, IL17A, RGS5, and TNFSF15) had 100% sensitivity for third metacarpal (MC3) fractures while a separate four-gene panel (IL17A, IL13, RGS5, and HTR6) correctly identified 75% of proximal sesamoid bone (PSB) fractures, with both panels having approximately 95% specificity. Distinct transcriptomic patterns for MC3 and PSB fractures were also noted. These findings support the feasibility of whole-blood mRNA profiling for pre-race injury risk assessment and warrant further validation in larger cohorts.