Novel insights into systemic sclerosis using a sensitive computational method to analyze whole-genome bisulfite sequencing data

Abstract

Abstract Background Abnormal DNA methylation is thought to contribute to the onset and progression of systemic sclerosis. Currently, the most comprehensive assay for profiling DNA methylation is whole-genome bisulfite sequencing (WGBS), but its precision depends on read depth and it may be subject to sequencing errors. SOMNiBUS, a method for regional analysis, attempts to overcome some of these limitations. Using SOMNiBUS, we re-analyzed WGBS data previously analyzed using bumphunter, an approach that initially fits single CpG associations, to contrast DNA methylation estimates by both methods. Methods Purified CD4+ T lymphocytes of 9 SSc and 4 control females were sequenced using WGBS. We separated the resulting sequencing data into regions with dense CpG data, and differentially methylated regions (DMRs) were inferred with the SOMNiBUS region-level test, adjusted for age. Pathway enrichment analysis was performed with ingenuity pathway analysis (IPA). We compared the results obtained by SOMNiBUS and bumphunter. Results Of 8268 CpG regions of ≥ 60 CpGs eligible for analysis with SOMNiBUS, we identified 131 DMRs and 125 differentially methylated genes (DMGs; p-values less than Bonferroni-corrected threshold of 6.05–06 controlling family-wise error rate at 0.05; 1.6% of the regions). In comparison, bumphunter identified 821,929 CpG regions, 599 DMRs (of which none had ≥ 60 CpGs) and 340 DMGs (q-value of 0.05; 0.04% of all regions). The top ranked gene identified by SOMNiBUS was FLT4, a lymphangiogenic orchestrator, and the top ranked gene on chromosome X was CHST7, known to catalyze the sulfation of glycosaminoglycans in the extracellular matrix. The top networks identified by IPA included connective tissue disorders. Conclusions SOMNiBUS is a complementary method of analyzing WGBS data that enhances biological insights into SSc and provides novel avenues of investigation into its pathogenesis.