UC Davis

Epistaxis and sudden death due to cardiac events are substantial concerns in the Thoroughbred racing industry and often cannot be traced to a specific cause. Hematologic health in Thoroughbreds can be influenced by both inherited and induced conditions. To date, there have been five inherited bleeding disorders identified in the horse that all directly or indirectly affect coagulation protein concentration or function. Four also occur in other species, but one, Atypical Equine Thrombasthenia (AET), has only been diagnosed in Thoroughbred horses. AET is caused by platelets that cannot be properly activated by thrombin stimulation and also do not bind fibrinogen as efficiently, preventing affected horses from clotting normally after vascular injury. In racing Thoroughbreds, inducible causes for changes in red blood cell mass also lead to hematologic changes through illicit doping with recombinant erythropoietin (rHuEPO). This thesis will therefore use genomic tools to investigate both inherited and inducible hematologic disturbances that occur in the Thoroughbred racehorse.

This thesis provides an overview of what is currently known about inherited equine bleeding disorders, identifies and investigates putative variants for AET, determines the optimal RNA sequencing library preparation for long non-coding RNA (lncRNA) studies in horses, and identifies transcriptomic markers of rHuEPO micro-dosing. The first chapter provides an overview of the current body of knowledge of inherited equine bleeding disorders. The second chapter describes a whole-genome variant analysis study that was performed using AET affected and control Thoroughbreds. Three associated variants that were to be located in or near genes expressed in platelets were identified for further study: SEL1L c.1810A>G p.Ile604Val, AL355838.1:g.26447375_26448962del, and VIPAS39:g.22685398_22685470del. Since the lncRNA AL355838.1 was identified, we sought to determine the best RNA sequencing library preparation method in the third chapter. Poly-A+ selection was determined to be better overall for identifying lncRNA as compared to rRNA-depletion. AL355838.1:g.26447375_26448962del was subsequently excluded as it was not expressed in platelets. Based on molecular and functional studies, neither SEL1L c.1810A>G p.Ile604Val or VIPAS39:g.22685398_22685470del were conclusively identified as causative or excluded as putative variants and remain priorities for further investigation into the cause of AET. Lastly, chapter four identifies three transcriptomic markers (C13H16orf54, PUM2 and CHTOP) of rHuEPO micro-dosing using RNA sequencing (RNA-seq) of dosed and control Thoroughbreds that were on an exercise protocol comparable to the workload of a racehorse. However, reverse transcription quantitative PCR was not sensitive enough to replicate the differences observed with RNA-seq and thus, these are not suitable biomarkers to develop a commercial test to detect illicit rHuEPO micro-doping in racehorses.

Future studies should focus on further clarifying the functional effects of SEL1L c.1810A>G p.Ile604Val and VIPAS39:g.22685398_22685470del in platelets to determine the causative variant for AET. Additional work is also required to identify alternative methods of quantifying mRNA that are feasible as a commercial test or investigating the corresponding protein concentrations of these candidate genes to determine if identification of illicit drug dosing can be performed at a protein level.

This thesis also includes an addendum on the characterization of a novel heritable degenerative axonopathy identified in Quarter Horse foals. The disease was first identified by veterinarians who noted the close relation of the affected foals. This study, along with the studies on AET and rHuEPO biomarkers, emphasizes the important role that genomics can play in veterinary medicine to improve equine wellness while highlighting the value of collaborations between veterinarians and researchers.