Differential DNA methylation patterns in whole blood from ACPA-positive patients with DMARD-naïve rheumatoid arthritis at clinical disease onset

Abstract

Objective: Epigenetic DNA imprints are increasingly being recognized as co-drivers of disease in complex conditions. In this exploratory and hypothesis-generating epigenome-wide association study (EWAS), we investigated differential methylation patterns in peripheral blood leucocytes from patients with early untreated ACPA-positive rheumatoid arthritis (RA) versus controls.

Methods: Whole blood DNA was isolated from 101 disease-modifying anti-rheumatic drug (DMARD)-naïve patients with recent clinical onset of ACPA-positive RA and 200 controls. DNA methylation was studied using the Illumina MethylationEPIC BeadChips (Illumina). We assessed our findings against previously reported differentially methylated DNA positions associated with RA including an EWAS on peripheral blood leucocytes from a similar Drop Nordic cohort.

Results: We identified 16,583 CpG sites and 14 differentially methylated regions (DMRs) associated with RA. The most robust DMRs were in the gene body of LAMP1 and the TNSF14 GENE known as LIGHT. We identified three novel Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, the taste transduction pathway, the olfactory pathway, and the viral carcinogenesis pathway, which have not previously been associated with RA. We replicated 2,248 CpG sites reported earlier in an EWAS on peripheral blood leukocytes from RA patients of Scandinavian ancestry with incipient untreated ACPA-positive disease.

Conclusion: We have detected a considerable number of epigenetic marks with potential relevance to the pathogenesis of RA. These findings may pave the way for the development of narrowly targeted new drugs and possibly assist to retrieve persons at particular risk of acquiring RA.

Keywords: DNA-methylation; anti-CCP antibodies; epigenetics; incidence; rheumatoid arthritis.

Figure 1

Manhattan plot of the linear mixed model of DNA methylation and rheumatoid arthritis. The vertical axis represents the –log10(P value of the mixed effect model) versus genomic position for RA-associated CpGs. The solid horizontal line represents genome-wide significance of unadjusted p value = 5.0 × 10 −8 (612 sites) and the dash-dotted line represent suggestive significance of unadjusted p value = 1.0 × 10 −5.

Figure 2

Volcano plot. Volcano plot representing differentially methylated probes. The negative dots on the horizontal axis represent hypomethylated probes in RA and positive dots hypermethylated probes in RA. The vertical axis represents the -log10 p value. The figure indicates a major methylation deficiency in the RA methylome.

Figure 3

GO enrichment analysis. Dot plots of GSEA results illustrating GO biological processes associated with RA. On the left is the 15 GO terms significantly enriched after correcting for multiple testing. The 15 GO processes with the largest gene ratios are plotted in order of gene ratio. Gene ratio is the fraction of differentially expressed genes found in the gene set. The size of the dots represents the number of genes in the significant methylation gene list associated with the GO term and the color of the dots represent the P-adjusted values.

Figure 4

Enrichment plot of top two enrichment KEGG terms (ranked in descending order of normalized enrichment score). P-values are significant (p < 0.001).