Genome-Wide DNA Methylation Analysis of Systemic Lupus Erythematosus Reveals Persistent Hypomethylation of Interferon Genes and Compositional Changes to CD4+ T-cell Populations

<div><p>Systemic lupus erythematosus (SLE) is an autoimmune disease with known genetic, epigenetic, and environmental risk factors. To assess the role of DNA methylation in SLE, we collected CD4+ T-cells, CD19+ B-cells, and CD14+ monocytes from 49 SLE patients and 58 controls, and performed genome-wide DNA methylation analysis with Illumina Methylation450 microarrays. We identified 166 CpGs in B-cells, 97 CpGs in monocytes, and 1,033 CpGs in T-cells with highly significant changes in DNA methylation levels (p<1×10⁻⁸) among SLE patients. Common to all three cell-types were widespread and severe hypomethylation events near genes involved in interferon signaling (type I). These interferon-related changes were apparent in patients collected during active and quiescent stages of the disease, suggesting that epigenetically-mediated hypersensitivity to interferon persists beyond acute stages of the disease and is independent of circulating interferon levels. This interferon hypersensitivity was apparent in memory, naïve and regulatory T-cells, suggesting that this epigenetic state in lupus patients is established in progenitor cell populations. We also identified a widespread, but lower amplitude shift in methylation in CD4+ T-cells (>16,000 CpGs at FDR<1%) near genes involved in cell division and MAPK signaling. These cell type-specific effects are consistent with disease-specific changes in the composition of the CD4+ population and suggest that shifts in the proportion of CD4+ subtypes can be monitored at CpGs with subtype-specific DNA methylation patterns.</p></div

Common and cell type-specific DNA methylation changes in SLE.

<p>Differences in mean methylation between SLE and controls are plotted for each cell type at each probe near two genes. <b>A.</b> The IRF7 gene shows hypomethylation across all three cell-types at a CpG island, plus monocyte-specific hypomethylation further into the gene body. <b>B.</b> The IKZF4 gene shows T-cell-specific hypomethylation at the 5′ end of the gene. Red dots indicate p<1×10⁻⁸. Yellow dots indicate FDR<1%.</p

Disease activity QQ-Plots and the persistence of hypomethylation in quiescent patients.

<p><b>A.</b> QQ-Plots of the p-values from the flare versus quiescent association analysis for each cell type, illustrating the lack of activity-dependent DNA methylation. <b>B.</b> Boxplots of the methylation difference between each individual and the mean of all controls at CpGs in IFN-regulated genes among those that were highly significant in the SLE-control tests. The groups are labeled C, Control, F, SLE collected during a flare, and Q, SLE collected during quiescence.</p

Comparison of the SLE-control methylation differences in sorted T-cell populations.

<p>Each scatter plot represents 1,031 CpGs that had p<1×10⁻⁸ in CD4+ T-cells in our SLE-control association tests. The Y-axis for all plots is the mean SLE-control methylation delta at these CpGs in the initial cohort. The X-axis for each plot is the mean SLE-control methylation delta at the same CpGs in our validation cohort, using (<b>A</b>) total CD4+, (<b>B</b>) CD4+Memory, (<b>C</b>) CD4+Naïve, or (<b>D</b>) CD4+Regulatory cells. The red dots represent CpGs near IFN-regulated genes and the squared correlation coefficients (R²) represent the values for all plotted CpGs (upper left) or IFN CpGs only (lower right).</p

Functional analysis of significant CpGs in three cell types.

<p><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003678#s2" target="_blank">Results</a> from DAVID/Panther GO term analysis for the highly significant CpGs in each cell type and the mildly significant CpGs in T-cells.</p

Distributions of heritability values by CpG island proximity and gene region.

<p>Histogram of heritability values (A) for all 174,445 tested CpGs with overlaid density estimate (dotted-line) and boxplots of heritability values for CpGs annotated by CpG-island proximity (B), and gene region (C).</p

Distributions of heritability values across heritable CpGs by SNP-association group, CpG island proximity, and gene region.

<p>Boxplots show heritability values for heritable CpGs under our threshold within their respective SNP-association groups (A). Boxplots of heritability values were also plotted for each SNP-association group subdivided by CpG island proximity (B). Barplots depict relative percentages of heritable CpGs across SNP-association groups that were positioned within CpG island proximity (C) or gene region (D) annotations.</p

Boxplots of twin data for strong cell-difference related GICs and non-cell difference related GICs.

<p>Boxplots of H2/h2 values across our cell-difference related (cell delta) or non-cell type difference related (cell delta depleted) with respect to our H² values (lmekin) and twin data (h2 twin) (A). Boxplots depicting twin data values within cell delta or cell delta depleted GICs: h2 values associated with genotype (h2 SNPs; B), common environmental values (c2 twinACE;C), non additive genetic effects (d2 twin ADE;D), unique environmental values (1-rMZ; E), and additive genetic effects (2*rMZ-rDz; F).</p

Trans-ethnic fine-mapping narrowed the association signals.

a<p><i>P</i> meta: <i>P</i> values from meta-analysis combining samples of African American, East Asian and European ancestries.</p>b<p>Direction: effect direction of each individual studies in the order of ARIC, MEC, WHI batch1, WHI batch2, HyperGEN, CLHNS, TAICHI, Finnish T2D, Finnish unaffected, Norwegian T2D and Norwegian unaffected.</p>c<p><i>P</i> het: <i>P</i> values for heterogeneity, indicating whether observed effect sizes are homogeneous across ancestry samples.</p>d<p><i>I</i>²: index of the degree of heterogeneity.</p

LDL-C locus <i>PCSK9</i> exhibited seven signals in African Americans.

<p>Initial association in the main analysis (A). Residual association in sequential conditional analysis by sequentially adding the lead SNPs into the regression model (B–G). Each SNP was colored according to its LD (<i>r²</i>) in the PAGE consortium, with the strongest SNP colored in purple and symbols designating genomic annotation defined in the ‘annotation key’. Genomic coordinates refer to build 36 (hg18).</p