Multi‐Tissue Epigenetic and Gene Expression Analysis Combined With Epigenome Modulation Identifies RWDD2B as a Target of Osteoarthritis Susceptibility

Abstract

Objective. Osteoarthritis (OA) is polygenic, with more than 90 risk loci currently mapped, including at the singlenucleotide polymorphism rs6516886. Previous analysis of OA cartilage DNA identified 6 CpG dinucleotides with methylation levels that correlated with the rs6516886 genotype, forming methylation quantitative trait loci (mQTLs). We undertook this study to investigate these mQTLs and to map expression quantitative trait loci (eQTLs) across joint tissues in order to identify a particular gene as a target of the rs6516886 association effect. Methods. Nucleic acids were extracted from the cartilage, fat pad, synovium, and peripheral blood from OA patients. Methylation of CpGs and allelic expression imbalance of potential target genes were assessed by pyrosequencing. A chondrocyte cell line expressing deactivated Cas9 (dCas9)-TET1 was used to directly alter CpG methylation levels, with effects on gene expression quantified by polymerase chain reaction. Results. Multiple mQTLs were detected, with effects strongest in joint tissues and methylation at CpG cg20220242 correlating most significantly with the rs6516886 genotype. CpG cg20220242 is located upstream of RWDD2B. Significant rs6516886 eQTLs were observed for this gene, with the OA risk-conferring allele of rs6516886 correlating with reduced expression. CpG methylation also correlated with allelic expression of RWDD2B, forming methylation-expression QTLs (meQTLs). Deactivated Cas9-TET1 reduction in the methylation of cg20220242 increased expression of RWDD2B. Conclusion. The rs6516886 association signal is a multi-tissue meQTL involving cg20220242 and acting on RWDD2B. Modulating CpG methylation reverses the impact of the risk allele. RWDD2B codes for a protein about which little is currently known. Further analysis of RWDD2B as a target of OA genetic risk will provide novel insight into this complex disease.

Genetic and Epigenetic Interplay Within a COLGALT2 Enhancer Associated With Osteoarthritis

“…The effect allele at OA-associated SNPs correlates with an increase in gene expression. The advent of CRISPR-Cas9 and subsequent development of the Cas9 toolbox has revolutionized targeted editing of the genome and epigenome (29). CRISPR-Cas9 deletion of the enhancer confirmed that COLGALT2 was a target gene, while gene expression of RGL1 and TSEN15, which flank COLGALT2, remained unchanged.…”

“…For demethylation of the enhancer using dCas9-ten-eleven translocation 1 (TET1), Tc28a2 cells that had been previously stably integrated with a gene expressing an inducible dCas9-TET1 construct were created and cultured as described by Parker et al (29). The 6 sequences targeting the enhancer (Supplementary Table 3, available on the Arthritis & Rheumatology website at http://onlin e libr ary.wiley.com/doi/10.1002/art.41738/ abstract) were ordered from IDT as gRNAs.…”

Osteoarthritis year in review: genetics, genomics, epigenetics

“…A T > A SNP at rs6516886, located in a region containing multiple genes, is associated with both KOA and HOA 21 . Parker et al demonstrated that the OA-associated T allele of rs6516886 correlated with increased methylation of neighbouring CpGs 22 . Differential allelic expression was identified for 3 genes in proximity to the SNP, but most notably for the gene RWDD2B, an RWD domainecontaining protein coding gene of unknown function, across multiple joint tissues, with additional evidence of correlation with local CpG methylation.…”

Insights into the molecular landscape of osteoarthritis in human tissues

“…Rs6516886 was found to be associated with a nearby methylation site (cg20220242, located upstream of RWDD2B ) across several tissues (cartilage, fat pad, synovium and peripheral blood) in osteoarthritis patients ( n = 348). They found that the risk allele ‘T’ of rs6516886 correlates with reduced expression of RWDD2B , which was reversed in a chondrocyte cell model by increasing the methylation levels of RWDD2B methylation [ 46 ].…”