Genome-wide DNA methylation analysis of systemic lupus erythematosus reveals persistent hypomethylation of interferon genes and compositional changes to CD4+ T-cell populations

Abstract

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 Methylation 450 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.

Figure 1. Common and cell type-specific DNA methylation changes in SLE.

Differences in mean methylation between SLE and controls are plotted for each cell type at each probe near two genes. A. The IRF7 gene shows hypomethylation across all three cell-types at a CpG island, plus monocyte-specific hypomethylation further into the gene body. 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%.

Figure 2. SLE QQ-Plots and the ratio of hyper- and hypomethylation events.

A. QQ-Plots of the p-values from the SLE association analysis for each cell-type, with a unique inflation in CD4+ T-cells. B. Log2 ratios of hyper-to-hypomethylated CpGs within bins of the p-value distribution for each cell type, showing a unique enrichment for hypermethylation among the significant CpGs in CD4+ T-cells.

Figure 3. Disease activity QQ-Plots and the persistence of hypomethylation in quiescent patients.

A. 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. 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.

Figure 4. Comparison of the SLE-control p-values in the sorted T-cells from the validation cohort.

(A) Distribution of the −log10 of the p-values from association tests in the sorted T-cells cells from the validation cohort at CpGs that were highly significant (p<1×10⁻⁸) in the initial cohort. (B) Percentage of CpGs that reported p<0.01 in the sorted T-cells cells from the validation cohort. Values in the left set of bars are from all 1,031 CpGs tested. Values in the right set of bars are from the subset of 62 CpGs near interferon-regulated genes.

Figure 5. Comparison of the SLE-control methylation differences in sorted T-cell populations.

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 (A) total CD4+, (B) CD4+Memory, (C) CD4+Naïve, or (D) 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).