An imprinted rheumatoid arthritis methylome signature reflects pathogenic phenotype

Abstract

Background: A DNA methylation signature has been characterized that distinguishes rheumatoid arthritis (RA) fibroblast like synoviocytes (FLS) from osteoarthritis (OA) FLS. The presence of epigenetic changes in long-term cultured cells suggest that rheumatoid FLS imprinting might contribute to pathogenic behavior. To understand how differentially methylated genes (DMGs) might participate in the pathogenesis of RA, we evaluated the stability of the RA signature and whether DMGs are enriched in specific pathways and ontology categories.

Methods: To assess the RA methylation signatures the Illumina HumanMethylation450 chip was used to compare methylation levels in RA, OA, and normal (NL) FLS at passage 3, 5, and 7. Then methylation frequencies at CpGs within the signature were compared between passages. To assess the enrichment of DMGs in specific pathways, DMGs were identified as genes that possess significantly differential methylated loci within their promoter regions. These sets of DMGs were then compared to pathway and ontology databases to establish enrichment in specific categories.

Results: Initial studies compared passage 3, 5, and 7 FLS from RA, OA, and NL. The patterns of differential methylation of each individual FLS line were very similar regardless of passage number. Using the most robust analysis, 20 out of 272 KEGG pathways and 43 out of 34,400 GO pathways were significantly altered for RA compared with OA and NL FLS. Most interestingly, we found that the KEGG 'Rheumatoid Arthritis' pathway was consistently the most significantly enriched with differentially methylated loci. Additional pathways involved with innate immunity (Complement and Coagulation, Toll-like Receptors, NOD-like Receptors, and Cytosolic DNA-sensing), cell adhesion (Focal Adhesion, Cell Adhesion Molecule), and cytokines (Cytokine-cytokine Receptor). Taken together, KEGG and GO pathway analysis demonstrates non-random epigenetic imprinting of RA FLS.

Conclusions: The DNA methylation patterns include anomalies in key genes implicated in the pathogenesis of RA and are stable for multiple cell passages. Persistent epigenetic alterations could contribute to the aggressive phenotype of RA synoviocytes and identify potential therapeutic targets that could modulate the pathogenic behavior.

Figure 1

The RA methylome signature is stable over passages. (A) Hierarchical clustering RA, OA, and NL samples at P3, P5, and P7. Each column shows the methylation level at one of the 1,859 DML. Additionally, samples from P5 of Nakano et al. 2012 are shown as reference and are labeled as rep. (B) The distribution of the difference in beta values between passages for RA sample.

Figure 2

DMGs in the KEGG RA pathway. The methylation status at the promoters of genes within the pathway is shown. The coloring scheme is as follows: yellow are hyper-methylated in RA, blue are hypo-methylated in RA, pink contain both hyper and hypo loci in their promoters, and green contain no significantly DML in their promoters.

Figure 3

DMGs in the KEGG 'Complement and coagulation cascades' pathway. The methylation status at the promoters of genes within the pathway is shown. The coloring scheme is as follows: yellow are hyper-methylated in RA, blue are hypo-methylated in RA, pink contain both hyper and hypo loci in their promoters, and green contain no significantly DML in their promoters.

Figure 4

DMGs in the KEGG 'Focal Adhesion' pathway. The methylation status at the promoters of genes within the pathway is shown. The coloring scheme is as follows: yellow are hyper-methylated in RA, blue are hypo-methylated in RA, and green contain no significantly DML in their promoters. Note that the following extracellular matrix (ECM) genes not specified in the figure are hypo-methylated: COL1A1, COL1A2, COL2A1, COMP, LAMA2, LAMB3, and VWF.

Figure 5

DMGs in the KEGG 'Toll-like receptor signaling' pathway. The methylation status at the promoters of genes within the pathway is shown. The coloring scheme is as follows: yellow are hyper-methylated in RA, blue are hypo-methylated in RA and green contain no significantly DML in their promoters.