Specific isoforms of the ubiquitin ligase gene WWP2 are targets of osteoarthritis genetic risk via a differentially methylated DNA sequence

Abstract

Abstract Background Transitioning from a genetic association signal to an effector gene and a targetable molecular mechanism requires the application of functional fine-mapping tools such as reporter assays and genome editing. In this report, we undertook such studies on the osteoarthritis (OA) risk that is marked by single nucleotide polymorphism rs34195470 and which maps to functional candidates WWP2 and microRNA-140 (miR-140). Methods Nucleic acids were extracted from adult OA (arthroplasty) and foetal cartilage. Samples were genotyped and DNA methylation (DNAm) quantified by pyrosequencing at 16 CpG dinucleotides located within a putative enhancer. CpGs were tested for transcriptional regulatory effects using a chondrocyte cell line and reporter gene assay. DNAm was altered using epigenetic editing, with the impact on gene expression determined using RT-qPCR. In silico analysis complemented laboratory experiments. Results rs34195470 genotype associates with differential methylation of the CpGs, forming a methylation quantitative trait locus (mQTL). The mQTL is more pronounced in adult versus foetal cartilage. The differential methylation acts as a transcriptional regulatory intermediate between risk allele and level of WWP2 expression by targeting the full-length and N-terminal transcript isoforms of the gene. Conclusions As far as we are aware, this is the first experimental demonstration of an OA association signal targeting specific transcript isoforms of a gene. WWP2 encodes a ubiquitin ligase, with its isoforms encoding proteins with varying substrate specificities, including for components of the TGFb signaling pathway. Future analysis should focus on the substrates regulated by the WWP2 isoforms that are the targets of the genetic risk.