Background Epigenetic dysregulation is implicated in the pathogenesis of lupus. We performed a longitudinal analysis of DNA methylation in lupus patients and assessed epigenetic changes over time and across disease activity status. Combining genetic and epigenetic analyses, we also examined ancestry-specific DNA methylation and DNA methylation changes influenced by genetic variants across the genome.
Methods A total of 54 female lupus patients, including 32 European-American and 22 African-American, were followed for up to 4 years. Blood samples were obtained at routine follow up visits and during disease flares, with a total of 229 samples collected. Disease activity at each blood draw was determined by SLEDAI. Granulocytes were isolated and DNA extracted. Genotyping was performed using the Infinium Global Screening Array v2.0, and genome-wide DNA methylation was assessed at each time-point using the Infinium MethylationEPIC array. Ancestry-specific DNA methylation changes and methylation quantitative trait loci (meQTL) were identified. A linear mixed effects model was implemented to identify DNA methylation alterations that vary with disease activity and the development of lupus nephritis during follow up.
Results We identified 487 hypomethylated and 420 hypermethylated CpG sites in African-American compared to European-American lupus patients, annotated to 391 and 316 unique genes, respectively. Differentially methylated genes include type I interferon-response genes such as IRF7 and IFI44, and genes related to the NFkB pathway. After adjusting for age, medications, and genetic background, DNA methylation levels in 142 (15.7%) differentially methylated sites were found to be allele-specific and influenced by at least one genetic variant located within 1kb. TREML4, which plays a vital role in toll-like receptor signaling, was hypomethylated in African-American patients and demonstrated a strong cis-meQTL association (R 2=0.91). The associated genetic variant (rs9369265) significantly differs in allele frequencies between African-American and European-Americans, and is located within an active enhancer region in neutrophils and modifies TREML4 expression. In vitro patch methylation experiments confirmed the regulatory effects of TREML4 methylation upon gene expression. Experiments to assess the functional effects of TREML4 overexpression in human neutrophils are underway in our laboratory. Interestingly, the DNA methylome was highly stable across disease activity levels and over time. Two sites cg26104306 (SNX18; FDR-adjusted P-value = 3.38 x 10−2) and cg06708913 (FDR-adjusted P-value = 3.43 x 10−2) were associated with changing disease activity levels in African-American patients. Demethylation of a CpG site located within GALNT18 was associated with the development of active lupus nephritis.
Conclusion Lupus granulocytes demonstrate significant differences in DNA methylation patterns between African-American and European-American patients. DNA methylation profiles in lupus patients are influenced by ancestry-specific genetic variants and are highly stable over time independent of disease activity levels. Progressive demethylation in SNX18 was observed with increasing disease activity in granulocytes from African-American lupus patients, and demethylation in GALNT18 was associated with the development of lupus nephritis in our cohort during follow up.
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