Genome-Wide DNA Methylation Analysis of Chinese Patients with Systemic Lupus Erythematosus Identified Hypomethylation in Genes Related to the Type I Interferon Pathway

<div><p>Background</p><p>Epigenetic variants have been shown in recent studies to be important contributors to the pathogenesis of systemic lupus erythematosus (SLE). Here, we report a 2-step study of discovery followed by replication to identify DNA methylation alterations associated with SLE in a Chinese population. Using a genome-wide DNA methylation microarray, the Illumina Infinium HumanMethylation450 BeadChip, we compared the methylation levels of CpG sites in DNA extracted from white blood cells from 12 female Chinese SLE patients and 10 healthy female controls.</p><p>Results</p><p>We identified 36 CpG sites with differential loss of DNA methylation and 8 CpG sites with differential gain of DNA methylation, representing 25 genes and 7 genes, respectively. Surprisingly, 42% of the hypomethylated CpG sites were located in CpG shores, which indicated the functional importance of the loss of DNA methylation. Microarray results were replicated in another cohort of 100 SLE patients and 100 healthy controls by performing bisulfite pyrosequencing of four hypomethylated genes, <i>MX1</i>, <i>IFI44L</i>, <i>NLRC5</i> and <i>PLSCR1</i>. In addition, loss of DNA methylation in these genes was associated with an increase in mRNA expression. Gene ontology analysis revealed that the hypomethylated genes identified in the microarray study were overrepresented in the type I interferon pathway, which has long been implicated in the pathogenesis of SLE.</p><p>Conclusion</p><p>Our epigenetic findings further support the importance of the type I interferon pathway in SLE pathogenesis. Moreover, we showed that the DNA methylation signatures of SLE can be defined in unfractionated white blood cells.</p></div

Co-expression analysis of differentially hypomethylated genes.

<p>The differentially hypomethylated genes found in the 450k microarray study were input into GeneMANIA for co-expression analysis, which revealed that they are related to type I interferon pathways, including “response to type I interferon” (FDR = 1.45e-34), “type I interferon-mediated signaling pathway” (FDR = 1.45e-34) and “cellular response to type I interferon” (FDR = 1.45e-34). Genes identified in these three co-expression pathways are overlapped with each other and are indicated in blue circles. Black circles indicate the differentially hypomethylated genes found in the 450k microarray, whereas circles with alternating black and blue stripes indicate hypomethylated genes that are overrepresented in type 1 interferon pathways.</p

Comparison of the methylation level of four hypomethylated genes between control and SLE patients.

<p>Bisulfite pyrosequencing was carried out on four of the genes that were found to be differentially methylated in the 450k microarray and in a larger cohort of 100 SLE patients and 100 healthy controls. These genes are <i>MX1</i>, <i>IFI44L</i>, <i>NLRC5</i> and <i>PLSCR1</i>. Bisulfite pyrosequencing of all four genes confirmed the microarray findings, showing that SLE patients have a significant loss of methylation when compared to healthy controls.</p

<i>LINE-1</i> methylation level measurement in control and SLE patients.

<p>Bisulfite pyrosequencing was performed for 10 controls and 12 SLE patients on three different CpG sites in <i>LINE-1</i> to determine their average methylation level. There is no significant difference between the average methylation levels of <i>LINE-1</i> in controls and SLE patients.</p

Heat map visualization of differentially methylated CpG sites.

<p>Forty-four differentially methylated CpG sites of 10 controls and 12 SLE patients are shown. CpG sites are clustered using Manhattan clustering, and the corresponding gene name of the CpG site is also shown on the left (if any). A scale is shown on the right, in which red and green correspond to a higher and a lower methylation status, respectively.</p

Volcano plot showing DNA methylation data according to genomic distribution.

<p>Blue spots represent CpG sites that are considered to be differentially methylated, fulfilling the requirement of a beta difference > |0.1| and log transformed adjusted p-value > 1.3, whereas red spots represent CpG sites that are not differentially methylated. (A) CpG sites are classified in relation to CpG islands. Most of the differentially methylated probes are located in shores and non-islands. (B) CpG sites are classified in relation to the gene structure, and most of the differentially methylated CpG sites are located in the 5’UTR, 1500 bp upstream of the TSS and in the gene body.</p

Confirmation of association of <i>HLA</i> locus, and <i>TNFSF4</i>, <i>STAT4</i>, <i>TNFAIP3</i>, <i>IRF5</i>, <i>BLK</i> with SLE.

<p>Shown are association results comparing SLE patients with controls collected in Hong Kong analyzed by Plink (−log₁₀(<i>P</i>-value) of SNPs). The best SNP in Chromosome 11 is around <i>ETS1</i> gene (rs6590330), which is in high LD with rs1128334. And the best SNP in Chromosome 10 is in <i>WDFY4</i> (rs877819).</p

Linkage disequilibrium and sequence conservation of SNPs in <i>ETS1</i>.

<p>Shown are LD for significant SNPs in and around <i>ETS1</i> detected by GWAS (A), and replicated SNPs in the 3′-UTR and downstream of the gene (B), and sequence conservation for sequences around SNP rs1128334 among different species (C).</p

Involvement of <i>ETS1</i> in Th17 cell and B lymphocyte development and autoimmunity.

<p>Involvement of <i>ETS1</i> in Th17 cell and B lymphocyte development and autoimmunity.</p

Allelic expression of <i>ETS1</i> on SNP rs1128334 in PBMC of healthy individuals.

<p>PBMC cDNA processed from 33 healthy individuals heterozygous on rs1128334 were used for allelic expression detection of <i>ETS1</i> by pyrosequencing. (A) A case example of detection on the “A” allele and the “G” allele from both DNA and cDNA samples from the same individual. (B) The ratio of G/A allelic detection for both DNA and cDNA samples. The median G/A ratio for DNA is 1.09 (95% CI: 1.08–1.11) and the median G/A ratio for cDNA expression is 1.32 (95% CI: 1.21–1.43), <i>P</i><0.0001 by paired student's <i>t</i> test.</p