Repetitive stress injury to bone during training can cause changes in the fetlock joints of Thoroughbred racehorses and lead to joint degeneration or be associated with future condylar fractures. CT imaging considered the “gold standard” for viewing early changes in bone is expensive, requires sedation and has limited availability. Identification of biomarkers in blood could provide less expensive, less-invasive alternatives for monitoring racehorses in the field to determine which horses require advanced imaging. This study used RNA sequencing to identify gene expression signatures that could be possible biomarkers of fetlock injury in Thoroughbred racehorses.
Blood samples were collected from six two-year-old Thoroughbred horses at 0 and 6 months during their first year of training: three with minimal evidence of fetlock pathology (H) and three that developed more severe fetlock pathology (P) at 6 months, diagnosed on CT imaging. RNA was extracted from WBCs and sequenced on an Illumina NextSeq 2000. Raw reads were mapped to the equine reference transcriptome (Ensembl; EquCab 3.0). Principal component analysis (PCA), differential gene expression (DGE) and gene set enrichment analysis (GSEA) were performed using R version 4.4.1 and Bioconductor, version 3.20.
43.86 +/- 2.84 (Mean +/-SD) million reads per sample were generated 72.3% +/- 0.6% reads per sample aligned to the reference genome and were summarized to 13,868 +/- 42 genes per sample. PCA revealed samples clustered by disease status (H/P) but not by time point. DGE found two genes were significantly upregulated, and six genes were significantly down regulated in P compared to H horses. GSEA revealed 30 gene sets that were differently enriched in P and H horses.
Additional studies are warranted to identify other genes of interest and to validate whether expression of the genes identified in this study can predict presence of fetlock pathology in a larger population of Thoroughbred racehorses.