Molecular genetic and epigenetic analysis of osteoarthritis risk at the WWP2 locus

Abstract

Osteoarthritis is a common multifactorial disease characterised by pathological changes to multiple joint tissues, primarily cartilage. Millions are affected yet no disease- modifying osteoarthritis drugs are available, highlighting a major clinically unmet need. Osteoarthritis is polygenic, with genome-wide association studies reporting over 100 associated variants. Most osteoarthritis-associated variants reside within noncoding regions of the genome, suggesting they confer pathogenicity via changes to gene expression. A quarter of osteoarthritis-associated variants co-localise with DNA methylation (DNAm) at CpG dinucleotides, forming methylation quantitative trait loci (mQTLs). mQTLs often act as functional intermediaries between risk variant and effector gene. This thesis investigates the osteoarthritis association signal rs34195470, mapping to the E3 ubiquitin ligase gene WWP2. Joint tissues from osteoarthritis patients were analysed to detect mQTLs at a putative enhancer. Epigenetic editing of the enhancer was used to establish causality between DNAm and WWP2 expression in a chondrocyte cell line. In-silico prediction of transcription factors (TFs) informed downstream TF-DNA interaction experiments. To establish the causal variant, reporter assays and CRISPR/Cas9 editing were employed. The osteoarthritis risk allele of rs34195470 correlated with increased DNAm levels of the enhancer in cartilage, marking a differentially methylated region (DMR). Epigenetic editing to increase DNAm at the DMR resulted in higher WWP2 expression. HIF-1α was predicted to bind the DMR, with downstream studies demonstrating this TF exclusively binds unmethylated DNA. rs34195470 exhibited allele-dependent regulatory function in the reporter assay. CRISPR/Cas9 deletion of rs111837947, a variant in high linkage disequilibrium with rs34195470, resulted in decreased WWP2 expression. Specific isoforms of WWP2 are regulatory targets of the rs34195470 association signal, modulated by the functional intermediary of DNAm at the DMR. Together, the work presented in this thesis provides a framework for the molecular characterisation of osteoarthritis genetic risk and the transition from associated variant to effector gene, revealing targets for future clinical intervention.