Background/Purpose: Recent studies revealed epigenetic changes in DNA methylation associated with rheumatoid arthritis (RA) synovial fibroblasts (SF). In addition, we have shown that SF exhibit large anatomic differences in their epigenome, transcriptome and function. Here, we investigated the role of DNA methylation in the regulation of joint specific gene expression.

Methods: RNA and DNA were isolated from cultured SF obtained from hands (n= 1 OA/4 RA), shoulders (n = 3 OA/4 RA) and knees (n = 1 OA/4 RA) of RA and OA patients. RNA sequencing (Illumina HiSeq2000) of SF was used to identify differentially expressed genes (n=3 for each joint and condition). DNA from the same samples was subjected to the Illumina HumanMethylation 450 array. After quality control, we calculated differentially methylated CpG sites and islands using the COHCAP Bioconductor package (version 3.3) in R statistical program.

Results: We compared the methylation profile of the following joints shoulder-hand, knee-hand and knee-shoulder. The analysis identified 66, 32 and 64 differentially methylated CpG sites, respectively (methylated beta > 0.7 and unmethylated beta <0.3, p<0.05, FDR <0.05). Principal component analysis showed clustering of samples in relation to the joint localization. In detail, we found 40 CpG sites to be hypomethylated and 26 hypermethylated in hand compared to shoulder SF. Of these, 13 and 6 CpG sites were hypomethylated on the CpG island of lncRNAs HOXA11AS and LOC145845. 8 hypermethylated CpG sites were found on the CpG island of MEIS1 in hand. In hand versus knee SF 28 CpG sites were hypomethylated and 4 hypermethylated. Of these, 5 CpG sites were hypomethylated on the gene body of the lncRNA HOTAIR and 4 CpG sites along the region of HOXC6, C5, C4 genes in hand. Last, we compared SF of shoulders versus knees and identified 51 hypomethylated and 13 hypermethylated CpG sites. Of these, 4 CpG sites were hypomethylated on the CpG island of HAND2 and 5 hypomethylated CpG sites were spread along the MEIS1 gene body in shoulders. Functional annotation analysis revealed that the majority of differential methylated genes are associated with limb development. The grade of DNA methylation matched the expression of most but not all differentially methylated genes, pointing to other regulating mechanisms of joint specific transcription. HOXA11-AS was higher (log2 ratio 2.89, p=1.18×10^-23) and MEIS1 lower expressed in hand than in shoulder SF (log2 ratio -1.63, p=2.77×10^-21). Expression of LOC145845 was not detectable. HOTAIR and HOXC6, C5, C4 were however overexpressed in knee compared to hand SF (log2 ratios 5.85, 3.04, 3.23, 3.08, p<5.6×10^-21). Expression of HAND2 (log2 ratio 3.13, p=3.53×10^-8) and also MEIS1 (log2 ratio 0.94, p=1.52×10^-5) was increased in shoulder compared to knee SF.

Conclusion: We show that DNA methylation regulates the expression of genes in a joint specific manner. These results point towards a fundamental role of epigenetics in maintaining location specific identities in joint stromal cells. We suggest that these joint specific differences in gene expression mediate the occurrence of distinct patterns of joint involvement in the development of arthritides.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dna-methylation-defines-joint-specific-differences-in-synovial-fibroblasts-from-oa-and-ra-patients/