Genome-wide association studies (GWAS) have identified in total six independent SNPs within the 5′ region of the RUNX2 gene to be robustly associated with five different cartilage and bone related phenotypes. We aim to elucidate the effect of the identified SNPs on the regulation and expression of RUNX2 and how these confer susceptibility to cartilage and bone related disorders, such as osteoarthritis and osteoporosis. Independent GWAS signals and SNPs in LD with the GWAS loci were identified with GCTA conditional joint analysis, the SNAP tool, and HaploReg (V2.2, Broad Institute). GWAS SNPs and SNPs in high LD were analysed for enrichment in genomic regulatory regions, and co-location with DNA binding proteins using data from the ENCODE-Project, Roadmap epigenetics project, and the FANTOM5 database. In human cartilage explants we measured RUNX2 expression by RNA sequencing, CTCF-DNA binding by ChIP-qPCR and preformed eQTL analysis to determine the effect of the SNPs on gene expression. We found six genetically independent GWAS signals to co-localise to regions with enrichment of active enhancer markers, H3K4me1, H3K27ac, DNase1 hypersensitivity enrichment and bi-directional CAGE reads, in osteoblast and chondrogenic cells. The BMD associated SNP located ~700 kb away from the RUNX2 promoter, had a significant effect (P<0.05) on RUNX2 gene expression in human cartilage. In addition, we observed that when we stimulated RUNX2 expression in human chondrocytes by TGFβ stimulation, there is an increase in binding of the chromatin-loop mediating protein, CTCF, near the RUNX2 promoter. We have found that variants in the SUPT3H-RUNX2 locus associated to cartilage and bone phenotypes are located in gene regulatory regions, and affect RUNX2 gene expression. We hypothesise that the SNPs are localised in long-range enhancers which, mediated by a CTCF chromatin-loop to the RUNX2 promoters, regulate RUNX2 gene expression in bone and cartilage development.
Disclosure: The authors declared no competing interests. This work was supported by the Netherlands organisation for scientific research (NWO) VIDI-scheme.