Analysis of early changes in DNA methylation in synovial fibroblasts of RA patients before diagnosis

Abstract

DNA methylation is an important epigenetic modification that is known to be altered in rheumatoid arthritis synovial fibroblasts (RASF). Here, we compared the status of promoter DNA methylation of SF from patients with very early RA with SF from patients with resolving arthritis, fully established RA and from non-arthritic patients. DNA was hybridized to Infinium Human methylation 450k and 850k arrays and differential methylated genes and pathways were identified. We could identify a significant number of CpG sites that differed between the SF of different disease stages, showing that epigenetic changes in SF occur early in RA development. Principal component analysis confirmed that the different groups of SF were separated according to their DNA methylation state. Furthermore, pathway analysis showed that important functional pathways were altered in both very early and late RASF. By focusing our analysis on CpG sites in CpG islands within promoters, we identified genes that have significant hypermethylated promoters in very early RASF. Our data show that changes in DNA methylation differ in RASF compared to other forms of arthritis and occur at a very early, clinically yet unspecific stage of disease. The identified differential methylated genes might become valuable prognostic biomarkers for RA development.

Figure 1

Differentially methylated CpGs at promoter sites (DMPs) differ between diseases and disease stage. DNA methylation at CpGs of promoter regions were compared between SF isolated from healthy individuals (nSF) and patients with resolving, acute arthritis (rSF), very early rheumatoid arthritis (veRA) and patients fulfilling the criteria for the diagnosis of RA (estRA), respectively. (A) Principal component analysis (PCA) showed a clear separation of SF based on DMPs between the different conditions. (B) Heatmap of beta values of DMPs across synovial fibroblasts of the different groups. Every row represents the beta value of a DMP CpG and every column is one patient. The colour refers to the level of DNA methylation as shown in the color key.

Figure 2

Hypermethylated genes and pathways between different disease stages. (A) An increase of the average beta values of PM20D1, MFAP2 and SHROOM1 occurred between nSF, rSF, veRASF and estRASF. The box plots show the media, minimum, maximum and quartiles of beta values. (B) Hypermethylation was observed in the CpG Island within the PM20D1 promoter of rSF, veRASF and estRASF. The graph represents DNA methylation beta values for each sample aligned to the genomic reference and CpG Island of hg19 UCSC genomic assembly by IGV viewer software. (C) The top enriched pathways in significantly differential methylated sites (DMS) in very early RASF (veRASF), established RASF (estRASF) and resolving arthritis (rSF) respectively are depicted.