Genome-wide DNA methylation analysis in dermal fibroblasts from patients with diffuse and limited systemic sclerosis reveals common and subset-specific DNA methylation aberrancies

Abstract

Background: The aetiology of systemic sclerosis (SSc) is not clear, but there is an emerging evidence of gene-specific epigenetic dysregulation in the pathogenesis of SSc.

Methods: We performed a genome-wide DNA methylation study in dermal fibroblasts in six diffuse cutaneous SSc (dSSc) patients, six limited cutaneous SSc (lSSc) patients compared with 12 age-matched, sex-matched and ethnicity-matched healthy controls. Cytosine methylation was quantified in more than 485 000 methylation sites across the genome. Differentially methylated CpG sites between patients and controls with a fold difference ≥1.2 were identified. Quantitative real-time RT-PCR was performed to assess correlation between DNA methylation changes and gene expression levels.

Results: We identified 2710 and 1021 differentially methylated CpG sites in dSSc and lSSc, respectively. Of the differentially methylated sites, 61% in dSSc and 90% in lSSc were hypomethylated. There were only 203 CpG sites differentially methylated in both dSSc and lSSc, representing 118 hypomethylated and 6 hypermethylated genes. Common hypomethylated genes include ITGA9, encoding an α integrin. Other relevant genes such as ADAM12, COL23A1, COL4A2 and MYO1E, and transcription factors genes RUNX1, RUNX2 and RUNX3 were also hypomethylated in both dSSc and lSSc. Pathway analysis of differentially methylated genes in both dSSc and lSSc revealed enrichment of genes involved in extracellular matrix-receptor interaction and focal adhesion. We demonstrate significant correlation between DNA methylation status and gene expression in the majority of genes evaluated.

Conclusions: Our data highlight common and subset-specific aberrancies in dSSc and lSSc fibroblasts at the epigenomic levels and identify novel candidate genes in SSc.

Keywords: Autoimmunity; Fibroblasts; Systemic Sclerosis.

Figure 1

Correlation of gene expression and methylation status. To evaluate the functional significance of methylation status on gene expression, we performed RT-qPCR for eight selected genes that were hypomethylated in both diffuse and limited SSc fibroblasts. Overall most of the hypomethylated genes were overexpressed in SSc fibroblasts. The columns in the graph show the average individual gene expression level in limited and diffuse SSc in reference to control fibroblasts, error bars represent the SD. t test was used to compare the mean of individual gene expression levels in each SSc subset, as well as all SSc samples. *p value ≤0.05.