Identification of a global gene expression signature associated with the genetic risk of catastrophic fracture in iPSC-derived osteoblasts from Thoroughbred horses
Bone fractures are a significant problem in Thoroughbred racehorses. The risk of fracture is influenced by both genetic and environmental factors. To determine the biological processes that affected in genetically susceptible horses, we utilised polygenic risk scoring to establish induced pluripotent stem cells (iPSCs) from horses at genetically high and low risk. RNA-sequencing on iPSC-derived osteoblasts revealed 112 genes that were significantly differentially expressed. 43 of these genes have known roles in bone, 27 are not yet annotated in the equine genome and 42 currently have no described role in bone. However, many of the proteins encoded by the known and unknown genes have reported interactions. Functional enrichment analyses revealed that the differentially expressed genes were overrepresented in progresses regulating the extracellular matrix and pathways known to be involved in bone remodelling and bone diseases. Gene set enrichment analysis also detected numerous biological processes and pathways involved in glycolysis that had a higher expression in the iPSC-osteoblasts from horses with low polygenic risk scores for fracture.Therefore, the differentially expressed genes may be relevant for maintaining bone homeostasis and contribute to fracture risk. A deeper understanding of the consequences of mis-regulation of these genes and the identification of the DNA variants which underpin their differential expression may reveal more about the molecular mechanisms which are involved in equine bone health and fracture risk. Overall design: Skin fibroblasts that were used in this study had previously been scored for their polygenic risk for fracture and were selected to represent horses with low and high risk scores. Equine iPSC clones were derived in either duplicate or triplicate for each sample and have previously been characterized for their ability to form embryoid bodies, express pluripotency markers, and differentiate into derivatives of endoderm, ectoderm and mesoderm. All of the iPSC lines used in this study were differentiated into osteoblasts capable of producing a mineralised matrix and expressing osteoblast associated genes. RNA-sequencing was performed on 14 lines of iPSC-osteoblasts derived from six different horses (three with a low PRS (L1-3) and three with a high PRS (H1-H3) for fracture).