Changes in the pattern of DNA methylation associate with twin discordance in systemic lupus erythematosus

Abstract

Monozygotic (MZ) twins are partially concordant for most complex diseases, including autoimmune disorders. Whereas phenotypic concordance can be used to study heritability, discordance suggests the role of non-genetic factors. In autoimmune diseases, environmentally driven epigenetic changes are thought to contribute to their etiology. Here we report the first high-throughput and candidate sequence analyses of DNA methylation to investigate discordance for autoimmune disease in twins. We used a cohort of MZ twins discordant for three diseases whose clinical signs often overlap: systemic lupus erythematosus (SLE), rheumatoid arthritis, and dermatomyositis. Only MZ twins discordant for SLE featured widespread changes in the DNA methylation status of a significant number of genes. Gene ontology analysis revealed enrichment in categories associated with immune function. Individual analysis confirmed the existence of DNA methylation and expression changes in genes relevant to SLE pathogenesis. These changes occurred in parallel with a global decrease in the 5-methylcytosine content that was concomitantly accompanied with changes in DNA methylation and expression levels of ribosomal RNA genes, although no changes in repetitive sequences were found. 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.

Figure 1.

Representative scatterplots showing methylation profiles of twins with the three autoimmune diseases studied (SLE, RA, and DM) compared with their respective healthy twins. (A) Scatterplots corresponding to the average data from five MZ twin pairs discordant for RA (left) and DM (right). (Red) Genes with significant differences in averaged results from five SLE-discordant MZ twin pairs (see below). (B) (Left) Scatterplot corresponding to the averaged results from five SLE-discordant MZ twin pairs. (Right) Comparison of the averaged data of five pairs of unrelated controls matched by race, age, and gender with samples from the left panel. (Red) Significant genes (β > 0.10, P < 0.05). (C) (Left) A comparison of a single SLE-discordant MZ twin pair. (Red) Genes with significant differences in averaged results from five MZ twin pairs (see above). (D) Heat map including the data for the 49 genes (54 probes) showing differential methylation between SLE affected and healthy MZ twin pairs (five samples were included). Five additional age-, gender-, and race-healthy controls for each of the five MZ twin pairs are also included. Data from the Illumina array have been normalized as follows: xi = (xi − row.mean[i])/(0.333 × row.sd[i]). A scale is shown at the bottom, whereby positive (red) and negative (blue) values correspond, respectively, to a higher and a lower methylation status than average.

Figure 2.

A comparison of the DNA methylation levels of eight genes in paired samples discordant for SLE. Bisulfite pyrosequencing analysis was performed on the genes selected from experiments with methylation arrays. Methylation levels were normalized by dividing the percentage of methylation for each particular gene by the average percentage of methylation in the entire population (17 SLE discordant sibling pairs: six MZ twins, four DZ twins, and seven disease-discordant sibling pairs). Control samples are ordered right to left by decreasing level of methylation. SLE samples are on the left side of each graph, and the same order is maintained for their respective healthy twins or siblings. Each bar graph is accompanied by a scatterplot representing the normalized DNA methylation level of each gene for the control samples relative to their equivalent SLE sample.

Figure 3.

(A) Scatterplot comparing total 5mC content in SLE siblings and their matching healthy siblings. Data are normalized with respect to the average 5mC methylation content in the entire population. (B) Band patterning corresponding to the analysis of unmethylated/methylated Alu repeats. Two SLE discordant MZ twin pairs, two SLE concordant MZ twin pairs, and two concordant pairs of healthy MZ twins are shown. To show the sensitivity toward DNA methylation changes, a WBC sample is compared with the same sample following limited treatment with SssI DNA methyltransferase. (C) Individual bisulfite sequencing of consensus Alu repeat. Fifteen clones are shown. (Black squares) Methylated CpG sites; (white squares) non-methylated CpG sites. One pair of SLE discordant MZ twins and one pair of SLE concordant MZ twins are shown.

Figure 4.

DNA methylation changes in ribosomal genes in paired samples discordant for SLE. (A) Schematic representation of the rRNA gene depicting three regions in the gene repeat: the proximal promoter, including the transcription start site, and the initial sections of the 18S and 28S regions, were subjected to bisulfite genomic sequencing. Fifteen clones are shown. (Black squares) Methylated CpG sites; (white squares) non-methylated CpG sites. One SLE discordant pair of MZ twins, one SLE concordant pair of MZ twins, and one concordant pair of healthy MZ twins are shown. (B) Heat map including the normalized methylation data for 17 pairs of SLE-discordant samples and seven pairs of SLE-concordant samples. Data are normalized with respect to the average value of healthy siblings from discordant twin pairs, and a color scale is established between no-methylation (blue) and methylation (red) values near the average of normal samples. Three comparisons are made: SLE siblings from discordant pairs versus SLE twins from concordant pairs (upper P-value); healthy siblings from discordant pairs versus SLE twins from concordant pairs (lower P-value); SLE siblings from discordant pairs versus their matching healthy twins (P-value at left). (C) Bar graph showing the average percentage of DNA methylation for the 18S and 28S sequences of SLE siblings from discordant twin pairs, healthy siblings from discordant twin pairs, and SLE individuals from concordant twin pairs.