Presenter Esther Palomino Lago
Authors Esther Palomino Lago (1), Debbie Guest (1)
Affiliations 1. Royal Veterinary College
Presentation Type Talk
Abstract
Catastrophic fractures are the most common type of injury are the main cause of euthanasia on the racecourse. Bone fracture is a complex condition caused by environmental and genetic factors.
Using a polygenic risk score (PRS) for fracture we generated induced pluripotent stem cells (iPSCs) from horses at high and low genetic risk and differentiated them into osteoblasts-like cells. RNA sequencing performed on the osteoblasts revealed 112 differentially expressed genes (DEGs).
Whole Genome Sequencing (WGS) on 7 catastrophic fracture cases and 7 controls identified 12,224,941 DNA variants across all genomes. These were filtered based on their segregation between cases and controls, predicted consequence, location in the genome, and minor allele frequency (MAF) across breeds. 474 candidate variants were then genotyped using a custom-designed MassARRAY (Agena Bioscience) in 155 fracture cases and 206 controls. This identified 25 variants significantly associated with fracture, 11 were upstream of six different genes and 14 were mis-sense in 10 different genes. 64% of variants were located on chromosome 18, which was previously found to be associated with fracture, but no variants associated with the DEGs. Four of the variants were protective and 21 conferred risk. Four of the mis-sense variants were in ICA1L and epistatic analysis revealed that they interact with all the significant upstream variants. Three of these variants are predicted to have a deleterious effect on the ICA1L protein.
Primary osteoblasts from Thoroughbred were isolated and used to generate immortalized osteoblast by constitutive expression of human telomerase reverse transcriptase (hTERT) and Simian Vacuolating Virus 40 large T antigen (SV40LT) to provide an in vitro tool to study gene and variant function. Knockdown of ICA1L reduced cell viability and bone formation demonstrating a novel role for this gene in osteoblasts. Â Luciferase assays performed in immortalized osteoblasts revealed significant differences in reporter gene expression between alleles when analysing small regions (<120bp) flanking some of the variants.
Assay for Transposase-Accessible Chromatin (ATAC)Â sequencing on iPSC-derived osteoblasts from horses at high and low genetic risk and in immortalized osteoblasts during osteogenic culture is currently being performed to combine with our RNA seq and WGS to help to prioritize further variants for follow up. This will enable us to refine our PRS to increase its accuracy in the identification of high and low risk horses to allow informed breeding and targeted monitoring of bone health.