DNA Methylation and Systemic Sclerosis

Background/Purpose:

Epigenetic modifications are stable and heritable alterations in gene expression and cellular function that do not involve changes to the original DNA sequence.  They are now known to be essential regulators of multiple key biological processes.   Epigenetic mechanisms are also vital for the development and function of the immune system.

DNA methylation is one of them and is mostly achieved by methylation of CpG dinucleotides of the mammalian genome. When methylated they cause a stable, heritable repression of transcription of the affected gene.  DNA methylation was shown to play a role in the pathophysiology of systemic lupus erythematosus (SLE).   A few studies have also shown important DNA methylation changes in systemic sclerosis (SSc).

The objective of this study was to establish the DNA methylation pattern differences between systemic sclerosis (SSc) and control fibroblasts as well as differences based on antibody profile.

Methods:

DNA methylation profiling of fibroblasts from 16 SSc patients compared to 10 healthy controls was performed.  Seven patients were anti-topoisomerase I (topo-1) positive and 9 were anti-centromere antibody (ACA) positive.

Roche NimbleGen 385K RefSeq promoter array was used. Candidate genes that were significantly hypomethylated in SSc fibroblasts compared to controls and are part of fibrotic or inflammatory pathways were selected for confirmation studies. Bisulfite conversion and sequencing methods for methylation detection was performed. We designed two sets of PCR primers (one for DNA without bisulfite conversion, and one for post-conversion). Bisulfite conversion of sample DNA was performed using EZ DNA methylation kit (Zymo Research, Irvine, CA). The samples were PCR-sequenced and examined for methylation using QUAMA software.

Results:

Methylation changes of CpG islands of multiple genes showed significant difference between SSc and control fibroblasts by initial global profiling.

Ten candidate genes that were significantly hypomethylated in SSc fibroblasts compared to controls were chosen for confirmation studies (table 1).

Bisulfite sequencing analyses showed hypomethylation of DNA topoisomerase I (TOP1) and mitochondrial DNA topoisomerase I (TOP1-MT) genes in ACA positive SSc patients compared to controls with a statistically significant difference in percentage methylation of these genes (p=0.02 for TOP1-MT and p=0.005 for TOP1). No difference was found for any of the studied genes between topo-1 positive patients and controls.

Conclusion:

DNA methylation is an important epigenetic mechanism implicated in the pathophysiology of SSc. There are several candidate hypomethylated genes involved in collagen production and inflammatory pathways. Bisulfite sequencing showed significant hypomethylation of TOP1 and TOP1-MT in ACA positive SSc patient fibroblasts.

Table 1. Hypomethylated genes selected for confirmatory studies