Background/Purpose: A DNA methylation signature has been characterized that distinguishes RA FLS from osteoarthritis (OA) and normal (NL) FLS. The presence of epigenetic changes in these cells suggest that rheumatoid FLS imprinting might contribute to pathogenic behaviour. Differentially methylated loci (DML) RA FLS are located in the promoters of numerous genes implicated in RA, including validated therapeutic targets like TNF, CXCL10, and IL-1Ra. To understand how methylated genes might participate in the pathogenesis of RA, we evaluated how DML in RA FLS cluster in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.  

Methods: Genomic DNA was isolated from fifth passage RA (n=11), OA (n=11) and normal (NL) (n=6) FLS lines obtained at the time of joint replacement (OA, RA) or from the tissue bank (NL). Methylation was evaluated using the Illumina HumanMethylation450 chip. A Welch’s t-test identified RA vs. OA and RA vs. NL DML using a false discovery rate, q-value, cut off of 0.05. Genes containing DML within their promoter regions ([-2.5 kb, 500 bp] of TSS) were labeled as differentially methylated genes (DMG). The significance of DMG in KEGG human pathways was determined and resulting p-values represented the fraction of randomly selected background gene sets that were at least as enriched in genes found in the tested pathway as the DMG set. A q-value threshold of 0.01 determined significance.  

Results: 2109 DMGs in RA FLS were identified for KEGG analysis. 20 out of 252 KEGG pathways were significantly altered with q <0.01 for RA compared with OA or NL FLS. The greatest differences were in the Focal Adhesion pathway (q<10e-4), with 34 DMG, including key matrix genes (COLA1A2), signaling genes (MAPK10, PIK3CG and AKT2), and integrins (ITGA4, ITGA7, ITGA10). The Cell Adhesion pathway (26 DMG; q<10e-4) was also differentially methylated, suggesting that RA FLS imprinted abnormalities could affect adhesion and migration. Additional critical pro-inflammatory pathways implicated in RA were also differentially methylated, including Toll-like Receptors and Complement Cascade. The former encompassed 17 DMG, including differential methylation of TLR1, TLR4, and TLR5 as well as RIPK1 and MAP kinases (q<10e-3). The latter included C1QB, C3, C3AR1, C4A, C4A, C4B, C4B, C4BPA, and C8A(18 DMG; q=10e-4). Perhaps most intriguing, the pre-defined KEGG  “Rheumatoid Arthritis” pathway was significantly different in RA FLS compared to the controls (20 DMG; q<10e-4), while the “Systemic Lupus Erythematosus” pathway was not differentially methylated.  

Conclusion: KEGG pathway analysis demonstrates non-random imprinting of RA FLS. The patterns include anomalies in key cell adhesion genes (integrins, focal adhesion) and inflammation genes (signalling and innate immunity). These persistent epigenetic alterations could contribute to the aggressive phenotype of RA synoviocytes and identify potential therapeutic targets that could modulate the pathogenic behaviour.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-dna-methylation-signature-in-fibroblast-like-synoviocytes-fls-defines-critical-pathogenic-pathways-in-rheumatoid-arthritis-ra/