DNA methylome biomarkers of rheumatoid arthritis-associated interstitial lung disease reflecting lung fibrosis pathways, an exploratory case-control study

Abstract

Rheumatoid Arthritis-associated Interstitial Lung Disease (RA-ILD) significantly reduces life quality and survival, necessitating improvements in its understanding and clinical management. We addressed these goals using DNA methylation analysis, which has not been done in RA-ILD samples, by comparing 32 RA patients with ILD diagnosed less than one year before (cases) and 32 matched RA patients without ILD (controls). This analysis identified 6679 differentially methylated positions (DMPs) with Δβ ≥ 2% and FDR < 0.05, and 576 differentially methylated regions in RA-ILD. Some DMPs were near mucin, collagen, and telomere maintenance genes. Also, the most notably enriched gene set (up to p_adj = 1.9 × 10^-38) included genes overexpressed in fibrosis by monocytes and alveolar macrophages. Other significantly enriched gene sets, known to be dysregulated in fibrosis, included the mitotic spindle and the Rho GTPases. Additionally, analysis of transcription factor binding sites around DMPs showed unique enrichment near the liver X receptor element (LXRE), which is associated with fibrosis in multiple tissues. These results were consistent and unaffected by stricter significance thresholds. They indicated that differential DNA methylation may serve as blood biomarkers for RA-ILD including some related to lung fibrosis pathways.

Keywords: Biomarkers; DNA methylation; Epigenetics; Interstitial lung disease; Lung fibrosis; Rheumatoid arthritis.

Fig.1

Differentially methylated features (DMPs and DMRs). (A) Circos plot representing the features as dots in their schematic position on chromosomes (alternating gray and black colors and numbered in the outermost circle) with the corresponding -log(p-value) in the y-axis toward the circle center (those over the red scattered lines are below FDR 0.05). The outer features are hypermethylated CpG, the middle circle represents hypomethylated CpG and the inner circle shows the DMR. (B) Violin plots of DNA methylation β values corresponding to DMP in selected genes. The x-axis shows the β values adjusted for sex, age, smoking, and anti-CCP in the logit scale. The right y-axis shows the CpG code and its localization in the promoter (P) or gene body (B) of the selected gene (indicated in the left y-axis). Red violin plots correspond to RA-ILD and gray plots to RA_controls, respectively. (C) Line plots of DNA methylation β values (y-axis) in selected DMR. The blue bars over the lines represent CpG included in the EPIC array. The upper part shows the gene-specific tracks corresponding to different splice isoforms.

Fig. 2

Enriched gene sets and pathways in differentially methylated features (DMPs and DMRs). This exploratory ORA was done with Enrichr. We show the most informative gene sets in increasing order of p values. The legend indicates with the circle color the library where the enriched gene set was found (none in the GO: Cellular Compartment library), with the line type the genome stratum (none in the promoters and CGI), and with the line color the DMP or DMR feature and the type of methylation change (none with the hypomethylated features). The complete results of the analysis are provided in Suppl. Table 7.

Fig. 3

Network representation of the enriched Reactome pathways in DMPs mapping to gene bodies. Each pathway and gene set corresponds to a node with a size proportional to the enrichment significance (-log(p_adj)) and color for the cluster. The edges represent shared genes between gene sets. We shortened the labels (detailed correspondence in Suppl. Table 9).

Fig. 4

Representation of the transcription factor binding sites (TFBS) enriched around the DMPs. We searched enriched motifs in the 500 bp (A,B) and 200 bp (C,D) centered on the DMPs and considering either all the DMPs (A,C) or only the DMPs annotated to gene promoters (B,D). The x-axis shows the -log(p) of enrichment, whereas the y-axis represents the fold enrichment of the motifs.