Radiographic endophenotyping in hip osteoarthritis improves the precision of genetic association analysis.

OBJECTIVE: Osteoarthritis (OA) has a strong genetic component but the success of previous genome-wide association studies (GWAS) has been restricted due to insufficient sample sizes and phenotype heterogeneity. Our aim was to examine the effect of clinically relevant endophenotyping according to site of maximal joint space narrowing (maxJSN) and bone remodelling response on GWAS signal detection in hip OA. METHODS: A stratified GWAS meta-analysis was conducted in 2118 radiographically defined hip OA cases and 6500 population-based controls. Signals were followed up by analysing differential expression of proximal genes for bone remodelling endophenotypes in 33 pairs of macroscopically intact and OA-affected cartilage. RESULTS: We report suggestive evidence (p<5×10-6) of association at 6 variants with OA endophenotypes that would have been missed by using presence of hip OA as the disease end point. For example, in the analysis of hip OA cases with superior maxJSN versus cases with non-superior maxJSN we detected association with a variant in the LRCH1 gene (rs754106, p=1.49×10-7, OR (95% CIs) 0.70 (0.61 to 0.80)). In the comparison of hypertrophic with non-hypertrophic OA the most significant variant was located between STT3B and GADL1 (rs6766414, p=3.13×10-6, OR (95% CIs) 1.45 (1.24 to 1.69)). Both of these associations were fully attenuated in non-stratified analyses of all hip OA cases versus population controls (p>0.05). STT3B was significantly upregulated in OA-affected versus intact cartilage, particularly in the analysis of hypertrophic and normotrophic compared with atrophic bone remodelling pattern (p=4.2×10-4). CONCLUSIONS: Our findings demonstrate that stratification of OA cases into more homogeneous endophenotypes can identify genes of potential functional importance otherwise obscured by disease heterogeneity.

A Damaging COL6A3 Variant Alters the MIR31HG-Regulated Response of Chondrocytes in Neocartilage Organoids to Hyperphysiologic Mechanical Loading.

The pericellular matrix (PCM), with its hallmark proteins collagen type VI (COLVI) and fibronectin (FN), surrounds chondrocytes and is critical in transducing the biomechanical cues. To identify genetic variants that change protein function, exome sequencing is performed in a patient with symptomatic OA at multiple joint sites. A predicted damaging variant in COL6A3 is identified and introduced by CRISPR-Cas9 genome engineering in two established human induced pluripotent stem cell-derived in-vitro neocartilage organoid models. The downstream effects of the COL6A3 variant on the chondrocyte phenotypic state are studied by a multi-omics (mRNA and lncRNA) approach in interaction with hyper-physiological mechanical loading conditions. The damaging variant in COL6A3 results in significantly lower binding between the PCM proteins COLVI and FN and provokes an osteoarthritic chondrocyte state. By subsequently exposing the neocartilage organoids to hyperphysiological mechanical stress, it is demonstrated that the COL6A3 variant in chondrocytes abolishes the characteristic inflammatory signaling response after mechanical loading with PTGS2, PECAM1, and ADAMTS5, as central genes. Finally, by integrating epigenetic regulation, the lncRNA MIR31HG is identified as key regulator of the characteristic inflammatory signaling response to mechanical loading.

Hyper-physiologic mechanical cues, as an osteoarthritis disease relevant environmental perturbation, cause a critical shift in set-points of methylation at transcriptionally active CpG sites in neo-cartilage organoids.

Background: Osteoarthritis (OA) is a complex, age-related multifactorial degenerative disease of diarthrodial joints marked by impaired mobility, joint stiffness, pain, and a significant decrease in quality of life. Among other risk factors, such as genetics and age, hyper-physiological mechanical cues are known to play a critical role in the onset and progression of the disease (1). It has been shown that post-mitotic cells, such as articular chondrocytes, heavily rely on methylation at CpG sites to adapt to environmental cues and maintain phenotypic plasticity. However, these long-lasting adaptations may eventually have a negative impact on cellular performance. We hypothesize that hyper-physiologic mechanical loading leads to the accumulation of altered epigenetic markers in articular chondrocytes, resulting in a loss of the tightly regulated balance of gene expression that leads to a dysregulated state characteristic of the OA disease state. Results: We showed that hyper-physiological loading evokes consistent changes in ML-tCpGs associated with expression changes in ITGA5, CAV1, and CD44, among other genes, which together act in pathways such as anatomical structure morphogenesis (GO:0009653) and response to wound healing (GO:0042060). Moreover, by comparing the ML-tCpGs and their associated pathways to tCpGs in OA pathophysiology, we observed a modest but particular interconnected overlap with notable genes such as CD44 and ITGA5. These genes could indeed represent lasting detrimental changes to the phenotypic state of chondrocytes due to mechanical perturbations that occurred earlier in life. The latter is further suggested by the association between methylation levels of ML-tCpGs mapped to CD44 and OA severity. Conclusion: Our findings confirm that hyper-physiological mechanical cues evoke changes to the methylome-wide landscape of chondrocytes, concomitant with detrimental changes in positional gene expression levels (ML-tCpGs). Since CAV1, ITGA5, and CD44 are subject to such changes and are central and overlapping with OA-tCPGs of primary chondrocytes, we propose that accumulation of hyper-physiological mechanical cues can evoke long-lasting, detrimental changes in set points of gene expression that influence the phenotypic healthy state of chondrocytes. Future studies are necessary to confirm this hypothesis.