Hypomethylation of miR-17-92 cluster in lupus T cells and no significant role for genetic factors in the lupus-associated DNA methylation signature

Abstract

Objectives: Lupus T cells demonstrate aberrant DNA methylation patterns dominated by hypomethylation of interferon-regulated genes. The objective of this study was to identify additional lupus-associated DNA methylation changes and determine the genetic contribution to epigenetic changes characteristic of lupus.

Methods: Genome-wide DNA methylation was assessed in naïve CD4⁺ T cells from 74 patients with lupus and 74 age-matched, sex-matched and race-matched healthy controls. We applied a trend deviation analysis approach, comparing methylation data in our cohort with over 16 500 samples. Methylation quantitative trait loci (meQTL) analysis was performed by integrating methylation profiles with genome-wide genotyping data.

Results: In addition to the previously reported epigenetic signature in interferon-regulated genes, we observed hypomethylation in the promoter region of the miR-17-92 cluster in patients with lupus. Members of this microRNA cluster play an important role in regulating T cell proliferation and differentiation. Expression of two microRNAs in this cluster, miR-19b1 and miR-18a, showed a significant positive correlation with lupus disease activity. Among miR-18a target genes, TNFAIP3, which encodes a negative regulator of nuclear factor kappa B, was downregulated in lupus CD4⁺ T cells. MeQTL identified in lupus patients showed overlap with genetic risk loci for lupus, including CFB and IRF7. The lupus risk allele in IRF7 (rs1131665) was associated with significant IRF7 hypomethylation. However, <1% of differentially methylated CpG sites in patients with lupus were associated with an meQTL, suggesting minimal genetic contribution to lupus-associated epigenotypes.

Conclusion: The lupus defining epigenetic signature, characterised by robust hypomethylation of interferon-regulated genes, does not appear to be determined by genetic factors. Hypomethylation of the miR-17-92 cluster that plays an important role in T cell activation is a novel epigenetic locus for lupus.

Keywords: autoimmunity; lupus erythematosus, systemic; polymorphism, genetic.

Figure 1.

Distribution of average CpG methylation levels within 1500bp of the TSS for the respective genes differentially methylated in naïve CD4+ T cells of lupus patients compared to healthy controls.

Figure 2.

Heatmap of hierarchical clustering of pairwise Pearson correlation coefficient values of 51 differentially methylated gene promoters (TSS1500) in global tissue signature derived from 16,541 samples. Range from +1 (red) to −1 (blue), represent a greater to lower correlation in global tissue, respectively. KEGG pathways are significantly enriched (FDR-adjusted P-value < 0.05) in a block of 21 genes (green bars).

Figure 3.

(A) Violin plots of the seven CG probes in lupus patients and healthy controls used to calculate the average promoter methylation (TSS1500) for the miR-17-92 cluster. The solid black bar represents the median value and the dashed lines the first and third quartiles. Genomic visualization and annotation are from WashU Epigenome Browser using AuxillaryHMM tracks from peripheral naïve CD4+ T cell tissues (E038 and E039, top and bottom tracks, respectively). For P-values: n.s. = not significant, * = P < 0.05, ** = P < 0.01. (B) Correlation of median microRNA expression in naïve CD4+ T cells of a subset (n = 16) of lupus patients with SLEDAI score. Hsa-miR-18a-5p and hsa-miR-19b1-5p had a Pearson correlation (r) of 0.52 (P-value = 0.038) and 0.51 (P-value = 0.042), respectively.

Figure 4.

Proportion of differentially methylated CpG sites in naïve CD4+ T cells of lupus patients compared to healthy controls associated with an meQTL in (A) lupus patients, (B) healthy controls, and (C) the subset of meQTL shared between lupus patients and healthy controls.

Figure 5.

(A) Gene structure diagram of IRF7 depicting the location of rs1131665 and cg16486109. (B) The presence of the lupus risk allele at rs1131665 shows a significant negative correlation with DNA methylation of cg16486109 located in IRF7.