Objective. To perform a genome-wide DNA methylation study to identify DNA methylation changes in osteoarthritic (OA) cartilage tissue.Methods. The contribution of differentially methylated genes to OA pathogenesis was assessed by bioinformatic analysis, gene expression analysis, and histopathologic severity correlation. Genome-wide DNA methylation profiling of >485,000 methylation sites was performed on eroded and intact cartilage from within the same joint of 24 patients undergoing hip arthroplasty for OA. Genes with differentially methylated CpG sites were analyzed to identify overrepresented gene ontologies, pathways, and upstream regulators. The messenger RNA expression of a subset of differentially methylated genes was analyzed by reverse transcription-polymerase chain reaction. Histopathology was graded by modified Mankin score and correlated with DNA methylation.Results. We identified 550 differentially methylated sites in OA. Most (69%) were hypomethylated and enriched among gene enhancers. We found differential methylation in genes with prior links to OA, including RUNX1, RUNX2, DLX5, FURIN, HTRA1, FGFR2, NFATC1, SNCAIP, and COL11A2. Among these, RUNX1, HTRA1, FGFR2, and COL11A2 were also differentially expressed. Furthermore, we found differential methylation in approximately one-third of known OA susceptibility genes. Among differentially methylated genes, upstream regulator analysis showed enrichment of TGFB1 (P ‫؍‬ 4.40 ؋ 10 ؊5 ) and several microRNAs including miR-128 (P ‫؍‬ 4.48 ؋ 10 ؊13 ), miR-27a (P ‫؍‬ 4.15 ؋ 10 ؊12 ), and miR-9 (P ‫؍‬ 9.20 ؋ 10 ؊10 ). Finally, we identified strong correlations between 20 CpG sites and the histologic Mankin score in OA.Conclusion. Our data implicate epigenetic dysregulation of a host of genes and pathways in OA, including a number of OA susceptibility genes. Furthermore, we identified correlations between CpG methylation and histologic severity in OA.

Objective To perform a genome-wide DNA methylation study to identify differential DNA methylation patterns in subchondral bone underlying eroded and intact cartilage from patients with hip osteoarthritis (OA) and to compare these with DNA methylation patterns in overlying cartilage. Methods Genome-wide DNA methylation profiling using Illumina HumanMethylation 450 arrays was performed on eroded and intact cartilage and subchondral bone from within the same joint of 12 patients undergoing hip arthroplasty. Genes with differentially methylated CpG sites were analyzed to identify shared pathways, upstream regulators, and overrepresented gene ontologies, and these patterns were compared with those of the overlying cartilage. Histopathology was graded by modified Mankin score and assessed for correlation with DNA methylation. Results We identified 7,316 differentially methylated CpG sites in subchondral bone underlying eroded cartilage, most of which (~75%) were hypomethylated, and 1,397 sites in overlying eroded cartilage, 126 of which were shared. Samples clustered into 3 groups with distinct histopathologic scores. We observed differential DNA methylation of genes including the RNA interference–processing gene AGO2, the growth factor TGFB3, the OA suppressor NFATC1, and the epigenetic effector HDAC4. Among known susceptibility genes in OA, 32 were differentially methylated in subchondral bone, 8 were differentially methylated in cartilage, and 5 were shared. Upstream regulator analysis using differentially methylated genes in OA subchondral bone showed a strong transforming growth factor β1 signature (P = 1 × 10−40) and a tumor necrosis factor family signature (P = 3.2 × 10−28), among others. Conclusion Our data suggest the presence of an epigenetic phenotype associated with eroded OA subchondral bone that is similar to that of overlying eroded OA cartilage.

Background/Purpose Osteoarthritis (OA) is the leading cause of chronic disability affecting 40% of individuals over the age of 70 and costing $128 billion annually in the US alone. Little is known regarding changes in gene expression that occur regionally within these affected joints. Herein, we perform RNA-seq analysis of eroded and intact cartilage from human OA, and correlate transcript levels with histopathologic disease severity. Methods Six femoral heads were obtained at the time of hip arthroplasty for primary OA. Articular cartilage tissue was dissected from grossly affected and grossly normal areas from the same joints, flash frozen in liquid nitrogen, and RNA was extracted. Tissue samples from grossly affected and normal joint regions were histologically examined for OA severity using Mankin scoring. Following confirmation of RNA quality (RIN value ≥6), samples were sequenced with the Illumina TruSeq system on a MiSeq sequencer. Raw data were analysed and mapped using the GeneSifter software package. Genes with GeneSifter quality score <1.0 were excluded. For categorical analysis, EdgeR p-values <0.05 with Benjamini-Hochberg q ≤ 0.1 and expression ratios ≤0.83 or ≥ 1.2 between affected and normal tissues were considered significant. For correlations with histologic score, Pearson’s r > 0.75 or <-0.75 with p ≤ 0.05 were considered significant. Gene ontology and pathway analysis was performed using Ingenuity IPA and DAVID. Results Categorical analysis identified 43 overexpressed and 313 underexpressed genes in eroded compared to intact cartilage. Both over- and underexpressed genes were overrepresented in the fibroblastic growth factor (FGF) signalling pathway (p = 0.004). FGFR2 demonstrated aneroded to intact cartilage expression ratio = 0.46, was highly inversely correlated with OA histologic score severity (r = 0.92), and is hypermethylated in eroded OA cartilage. The WNT pathway genes, WNT11 (ratio 0.27) and WNT9A (ratio 0.45), and STAT3 pathway was also overrepresented, including both over- and underexpressed genes (p = 0.001). A top predicted upstream regulator in differentially expressed genes was mir-9 (p = 0.005), known to be associated with metalloproteinase production. Further, we identified 1576 genes positively correlated with histopathologic score. Among these, the NFAT pathway was highly overrepresented including both positively and inversely correlated genes (24, p = 0.002). The RIG-I innate immunity pathway was also overrepresented among inversely correlated genes (p = 0.008), as were several genes related to chromatin remodelling (overall p = 0.009: HDAC1, r = -0.89, MECP2 r = -0.78, RBBP4 r = -0.82, SAP130 r = -0.81, SIN3A r = -0.80). Conclusions Using RNA-seq we detected significant changes in gene expression in eroded compared to intact OA cartilage, as well as expression changes correlated with histologic disease progression in OA. Our data strongly suggest involvement of several signalling pathways, many of which are potential therapeutic targets for OA. This work reinforces ...

Results: Participants were young (median age 25), mostly male, largely Caucasian and had substantial impairment and pain by KOOS at baseline (within 8 weeks of their injury, median time to baseline 17 days). For rs143383 genotype of 131 participants, 37 (28%) were TT, 81 (62%) were heterozygotes and 13 (10%) CC. There was no significant difference between the KOOS 4 of individuals with TT, TC or CC genotypes at baseline. There were similar increases in KOOS 4 in TT and TC individuals over the 3 month period (P<0.0001 for each). In contrast, KOOS 4 did not increase significantly over 3 months in CC homozygotes (P ¼ 0.35). A very similar pattern was seen for the rs143384 genotype, in terms of genotype frequency and change in KOOS over time. In a linear regression model of change in KOOS 4 over the 3 month period, CC at rs143383 was significantly associated with a smaller improvement in KOOS 4 compared with TC/TT (unadjusted coeff. À12.58 (À24.15, À1.0), P ¼ 0.033. When adjusted for other pre-defined explanatory variables (Age, Gender, extent of injury) a significant effect remained for the CC genotype (coeff. À11.89 (À23.48, À0.32), P ¼ 0.044. Conclusions: Possession of the CC genotype at SNP rs143383 of GDF-5 would appear to be an adverse prognostic factor for early clinical outcome after knee injury in this cohort.This finding should be tested in other joint injury cohorts, in larger numbers of individuals and in other populations. What effect this polymorphism has on subsequent incidence of post-traumatic osteoarthritis remains to be established.