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

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 (SNP) rs34195470 (A > G). The OA risk-conferring G allele of this SNP associates with increased DNA methylation (DNAm) at two CpG dinucleotides within WWP2. This gene encodes a ubiquitin ligase and is the host gene of microRNA-140 (miR-140). WWP2 and miR-140 are both regulators of TGFß signaling. METHODS: Nucleic acids were extracted from adult OA (arthroplasty) and foetal cartilage. Samples were genotyped and DNAm quantified by pyrosequencing at the two CpGs plus 14 flanking CpGs. 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 at 14 of the 16 CpGs in OA cartilage, forming a methylation quantitative trait locus (mQTL). The mQTL is less pronounced in foetal cartilage (5/16 CpGs). The reporter assay revealed that the CpGs reside within a transcriptional regulator. Epigenetic editing to increase their DNAm resulted in altered expression of the full-length and N-terminal transcript isoforms of WWP2. No changes in expression were observed for the C-terminal isoform of WWP2 or for miR-140. 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. The WWP2 isoforms encode proteins with varying substrate specificities for the components of the TGFß signaling pathway. Future analysis should focus on the substrates regulated by the two WWP2 isoforms that are the targets of this genetic risk.

Identification of TMEM129, encoding a ubiquitin-protein ligase, as an effector gene of osteoarthritis genetic risk.

BACKGROUND: Osteoarthritis is highly heritable and genome-wide studies have identified single nucleotide polymorphisms (SNPs) associated with the disease. One such locus is marked by SNP rs11732213 (T > C). Genotype at rs11732213 correlates with the methylation levels of nearby CpG dinucleotides (CpGs), forming a methylation quantitative trait locus (mQTL). This study investigated the regulatory activity of the CpGs to identify a target gene of the locus. METHODS: Nucleic acids were extracted from the articular cartilage of osteoarthritis patients. Samples were genotyped, and DNA methylation was quantified by pyrosequencing at 14 CpGs within a 259-bp interval. CpGs were tested for enhancer effects in immortalised chondrocytes using a reporter gene assay. DNA methylation at the locus was altered using targeted epigenome editing, with the impact on gene expression determined using quantitative polymerase chain reaction. RESULTS: rs11732213 genotype correlated with DNA methylation at nine CpGs, which formed a differentially methylated region (DMR), with the osteoarthritis risk allele T corresponding to reduced levels of methylation. The DMR acted as an enhancer and demethylation of the CpGs altered expression of TMEM129. Allelic imbalance in TMEM129 expression was identified in cartilage, with under-expression of the risk allele. CONCLUSIONS: TMEM129 is a target of osteoarthritis genetic risk at this locus. Genotype at rs11732213 impacts DNA methylation at the enhancer, which, in turn, modulates TMEM129 expression. TMEM129 encodes an enzyme involved in protein degradation within the endoplasmic reticulum, a process previously implicated in osteoarthritis. TMEM129 is a compelling osteoarthritis susceptibility target.

Prioritization of PLEC and GRINA as Osteoarthritis Risk Genes Through the Identification and Characterization of Novel Methylation Quantitative Trait Loci.

OBJECTIVE: To identify methylation quantitative trait loci (mQTLs) correlating with osteoarthritis (OA) risk alleles and to undertake mechanistic characterization as a means of target gene prioritization. METHODS: We used genome-wide genotyping and cartilage DNA methylation array data in a discovery screen of novel OA risk loci. This was followed by methylation, gene expression analysis, and genotyping studies in additional cartilage samples, accompanied by in silico analyses. RESULTS: We identified 4 novel OA mQTLs. The most significant mQTL contained 9 CpG sites where methylation correlated with OA risk genotype, with 5 of the CpG sites having P values <1 × 10-10 . The 9 CpG sites reside in an interval of only 7.7 kb within the PLEC gene and form 2 distinct clusters. We were able to prioritize PLEC and the adjacent gene GRINA as independent targets of the OA risk. We identified PLEC and GRINA expression QTLs operating in cartilage, as well as methylation-expression QTLs operating on the 2 genes. GRINA and PLEC also demonstrated differential expression between OA hip and non-OA hip cartilage. CONCLUSION: PLEC encodes plectin, a cytoskeletal protein that maintains tissue integrity by regulating intracellular signaling in response to mechanical stimuli. GRINA encodes the ionotropic glutamate receptor TMBIM3 (transmembrane BAX inhibitor 1 motif-containing protein family member 3), which regulates cell survival. Based on our results, we hypothesize that in a joint predisposed to OA, expression of these genes alters in order to combat aberrant biomechanics, and that this is epigenetically regulated. However, carriage of the OA risk-conferring allele at this locus hinders this response and contributes to disease development.

Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk.

Osteoarthritis (OA) is a common, multifactorial and polygenic skeletal disease that, in its severest form, requires joint replacement surgery to restore mobility and to relieve chronic pain. Using tissues from the articulating joints of 260 patients with OA and a range of in vitro experiments, including CRISPR-Cas9, we have characterized an intergenic regulatory element. Here, genotype at an OA risk locus correlates with differential DNA methylation, with altered gene expression of both a transcriptional regulator (RUNX2), and a chromatin remodelling protein (SUPT3H). RUNX2 is a strong candidate for OA susceptibility, with its encoded protein being essential for skeletogenesis and healthy joint function. The OA risk locus includes single nucleotide polymorphisms (SNPs) located within and flanking the differentially methylated region (DMR). The OA association SNP, rs10948172, demonstrates particularly strong correlation with methylation, and two intergenic SNPs falling within the DMR (rs62435998 and rs62435999) demonstrate genetic and epigenetic effects on the regulatory activity of this region. We therefore posit that the OA signal mediates its effect by modulating the methylation of the regulatory element, which then impacts on gene expression, with RUNX2 being the principal target. Our study highlights the interplay between DNA methylation, OA genetic risk and the downstream regulation of genes critical to normal joint function.

Methylation quantitative trait locus analysis of osteoarthritis links epigenetics with genetic risk.

Osteoarthritis (OA) is a common, painful and debilitating disease of articulating joints resulting from the age-associated loss of cartilage. Well-powered genetic studies have identified a number of DNA polymorphisms that are associated with OA susceptibility. Like most complex trait loci, these OA loci are thought to influence disease susceptibility through the regulation of gene expression, so-called expression quantitative loci, or eQTLs. One mechanism through which eQTLs act is epigenetic, by modulating DNA methylation. In such cases, there are quantitative differences in DNA methylation between the two alleles of the causal polymorphism, with the association signal referred to as a methylation quantitative trait locus, or meQTL. In this study, we aimed to investigate whether the OA susceptibility loci identified to date are functioning as meQTLs by integrating genotype data with whole genome methylation data of cartilage DNA. We investigated potential genotype-methylation correlations within a 1.0-1.5 Mb region surrounding each of 16 OA-associated single-nucleotide polymorphisms (SNPs) in 99 cartilage samples and identified four that function as meQTLs. Three of these replicated in an additional cohort of up to 62 OA patients. These observations suggest that OA susceptibility loci regulate the level of DNA methylation in cis and provide a mechanistic explanation as to how these loci impact upon OA susceptibility, further increasing our understanding of the role of genetics and epigenetics in this common disease.