Mediation of the Same Epigenetic and Transcriptional Effect by Independent Osteoarthritis Risk–Conferring Alleles on a Shared Target Gene, COLGALT2

Abstract

Objective. Over 100 DNA variants have been associated with osteoarthritis (OA), including rs1046934, located within a linkage disequilibrium block encompassing part of COLGALT2 and TSEN15. Here, we used human foetal cartilage, cartilage from arthroplasty patients, and a chondrocyte cell model to determine the target gene(s) at the locus and the mechanism of action. Methods. Genotyping and methylation array data of cartilage DNA (n=87) were used to determine if rs1046934 genotype associated with differential DNA methylation at proximal CpGs. Results were replicated in arthroplasty (n=132) and foetal (n=77) cartilage DNA using pyrosequencing. Allelic expression imbalance (AEI) measured effect of genotype upon COLGALT2 and TSEN15 expression. Reporter gene assays and epigenetic editing determined the functional role of regions harbouring differentially methylated CpGs. In silico analyses complemented these experiments.Results. Three differentially methylated CpGs residing within regulatory regions were detected, two of which replicated. AEI was detected for COLGALT2 and TSEN15, with associations between expression and methylation for COLGALT2. Reporter assays confirmed that the CpGs are in chondrocyte enhancers with epigenetic editing directly linking methylation with COLGALT2 expression. Conclusion.COLGALT2 is a target of this OA locus. We previously characterised another OA locus, marked by rs11583641, that independently targets COLGALT2. rs1046934, like rs11583641, mediates its effect by modulating expression of COLGALT2 via methylation changes to CpGs located in enhancers. The SNPs, CpGs and enhancers are distinct between the loci but the effect on COLGALT2 is the same. COLGALT2 is the target of independent OA risk loci sharing a common mechanism of action.

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

“…We have reported that for several OA SNPs the association with CpG methylation observed in arthroplasty cartilage also occurs in foetal cartilage, implying that a proportion of OA genetic risk may be functionally active during skeletogenesis [ 33 , 34 ]. We therefore assessed whether the rs34195470 mQTL was detectable in foetal cartilage.…”

“…This implies that DNAm may be an intermediate between the genetic risk signal and changes in gene expression, with the causal variant altering methylation of the enhancer which then alters expression of the target gene. DNAm as a functional intermediate of genetic risk is a relatively common phenomenon [ 26 – 28 ] and we have previously used associating DNAm signatures to experimentally characterize OA risk loci and identify target genes of risk-conferring alleles [ 29 – 34 ]. These experiments have been conducted on cartilage from arthroplasty patients and on cartilage from human foetal samples, the latter used to assess whether OA risk alleles have functional impact during joint development [ 33 , 34 ].…”

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

“…We have reported that for several OA SNPs the association with CpG methylation observed in arthroplasty cartilage also occurs in foetal cartilage, implying that a proportion of OA genetic risk may be functionally active during skeletogenesis [29,30]. We therefore assessed whether the rs34195470 mQTL was detectable in foetal cartilage.…”

“…This implies that DNAm may be an intermediate between the genetic risk signal and changes in gene expression, with the causal variant altering methylation of the enhancer which then alters expression of the target gene. DNAm as a functional intermediate of genetic risk is a relatively common phenomenon [22][23][24] and we have previously used associating DNAm signatures to experimentally characterize OA risk loci and identify target genes of risk-conferring alleles [25][26][27][28][29][30]. These experiments have been conducted on cartilage from arthroplasty patients and on cartilage from human foetal samples, the latter used to assess whether OA risk alleles have functional impact during joint development [29,30].…”

Epigenetic mechanisms of osteoarthritis risk in human skeletal development

“…The integration of DNAm data into genetic studies, such as the statistical fine mapping of GWAS signals, has identified the co-localisation of methylation quantitative trait loci (mQTLs) with genetic risk signals 8,[11][12][13] . This interplay between DNA sequence and CpG methylation status 14 has further been shown to underpin tissue-specific molecular mechanisms of gene expression within the joint 15,16 .…”