December 22, 2009

NEW YORK (GenomeWeb News) – Epigenetic changes, specifically differences in DNA methylation, may contribute to environmental factors involved in systemic lupus erythematosus risk, according to an online study in Genome Research today.A Spanish, German, and American research team used bead arrays and targeted bisulfite sequencing to compare DNA methylation patterns in the genomes of more than a dozen sets of identical twins who were discordant for SLE or two other autoimmune diseases. While they did not detect methylation differences for two of the conditions, the team did detect intriguing epigenetic changes when one twin had SLE and the other did not.

"Our study suggests that the effect of the environment or differences in lifestyle may leave a molecular mark in key genes for immune function that contributes to the differential onset of the disease in twins," Esteban Ballestar, a researcher at the Bellvitge Biomedical Research Institute in Barcelona, said in a statement.

Lupus is an autoimmune disease in which an individual's immune system begins attacking his or her own tissues. The precise cause is still unknown. Previous genome-wide association studies in North American, European, and Asian populations have turned up several immune-related genes, but environmental factors — mediated in part by epigenetic changes — also appear to play a role in determining who's affected by SLE.

"Many complementary lines of evidence suggest that epigenetic factors may play a key role in the pathogenesis of autoimmune disease," Ballestar and his team wrote. "Interactions between environmental and genetic factors have been proposed to explain why certain individuals develop autoimmune disorders while others do not, and provide an explanation for the high discordance rates for autoimmune diseases in [monozygotic] twins."

To begin exploring the epigenetics of lupus, the researchers used Illumina GoldenGate Methylation Panel bead arrays to assess DNA methylation at more than 800 CpG promoter regions in DNA extracted from white blood cells from 15 pairs of monozygotic twins. Five sets of twins were discordant for SLE, and the other ten twin pairs were discordant for either rheumatoid arthritis or dermatomyositis, conditions which share symptoms with SLE. They also evaluated samples from 30 unrelated, matched control individuals.

While they did not find any methylation differences when comparing twins with and without rheumatoid arthritis or dermatomyositis, the team did detect significant changes in methylation at 49 genes in individuals with SLE compared with their unaffected twins and matched controls.

The team's gene ontology analyses suggest these methylation differences frequently occurred in immune-related genes.

To verify the methylation differences detected within SLE discordant twin pairs, the researchers did targeted bisulfite sequencing on eight genes and nearby CpG regions in two of the twin pairs. They also did similar experiments in healthy controls and twin sets in which both individuals had SLE.

Again, the researchers found methylation differences between individuals with SLE and their healthy twins. They also noted that methylation levels for these genes were similar in the affected twin as it was in twin pairs in which both twins had the condition.

The team found similar results when sequencing genes from a dozen more SLE-discordant sibling pairs, including one set of identical and four sets of non-identical twins.

Their subsequent experiments suggested individuals with SLE tend to have lower overall levels of 5-methylcytosine — and lower levels of methylation in 18S and 28S ribosomal genes — than unaffected twins or other healthy controls.

When they used quantitative RT-PCR to measure the expression of seven of the genes in five SLE-discordant identical twin pairs, the researchers found that the individuals with SLE had lower methylation and higher expression for the majority of genes tested.

Together, the findings suggest epigenetics contribute to the differences in SLE phenotypes that exist within some identical twins. And while the current study does not provide information about when the observed methylation changes began, the team speculated that they might coincide with the onset of SLE in affected individuals.

"Our findings not only identify potentially relevant DNA methylation markers for the clinical characterization of SLE patients but also support the notion that epigenetic changes may be critical in the clinical manifestations of autoimmune disease," the researchers wrote.