Epigenetic modifications and glucocorticoid sensitivity in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

Abstract

Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating idiopathic disease characterized by unexplained fatigue that fails to resolve with sufficient rest. Diagnosis is based on a list of symptoms and exclusion of other fatigue-related health conditions. Despite a heterogeneous patient population, immune and hypothalamic-pituitary-adrenal (HPA) axis function differences, such as enhanced negative feedback to glucocorticoids, are recurring findings in ME/CFS studies. Epigenetic modifications, such as CpG methylation, are known to regulate long-term phenotypic differences and previous work by our group found DNA methylome differences in ME/CFS, however the relationship between DNA methylome modifications, clinical and functional characteristics associated with ME/CFS has not been examined.

Methods: We examined the DNA methylome in peripheral blood mononuclear cells (PBMCs) of a larger cohort of female ME/CFS patients using the Illumina HumanMethylation450 BeadChip Array. In parallel to the DNA methylome analysis, we investigated in vitro glucocorticoid sensitivity differences by stimulating PBMCs with phytohaemagglutinin and suppressed growth with dexamethasone. We explored DNA methylation differences using bisulfite pyrosequencing and statistical permutation. Linear regression was implemented to discover epigenomic regions associated with self-reported quality of life and network analysis of gene ontology terms to biologically contextualize results.

Results: We detected 12,608 differentially methylated sites between ME/CFS patients and healthy controls predominantly localized to cellular metabolism genes, some of which were also related to self-reported quality of life health scores. Among ME/CFS patients, glucocorticoid sensitivity was associated with differential methylation at 13 loci.

Conclusions: Our results indicate DNA methylation modifications in cellular metabolism in ME/CFS despite a heterogeneous patient population, implicating these processes in immune and HPA axis dysfunction in ME/CFS. Modifications to epigenetic loci associated with differences in glucocorticoid sensitivity may be important as biomarkers for future clinical testing. Overall, these findings align with recent ME/CFS work that point towards impairment in cellular energy production in this patient population.

Keywords: Chronic fatigue syndrome; Dna methylation; Epigenetics; Glucocorticoid; Hpa axis; Immune cells; Myalgic encephalomyelitis.

Fig. 1

Dexamethasone suppression assay results. Dexamethasone suppression results from PBMCs of ME/CFS patients (n = 33) and healthy controls (n = 24) after stimulation with PHA. Inhibition % represents the amount of suppressed cell replication with 10^-6 M dexamethasone compared to stimulated and unstimulated conditions. Each point represents the mean % inhibition of a single subject. The mean is represented along with standard error bars. Glucocorticoid sensitivity was greater among ME/CFS patients overall compared to healthy controls and between ME/CFS GC-Hypersensitive (red circle) compared to ME/CFS GC-Typical (blue circle; p’s ≤0.05)

Fig. 2

Bisulfite pyrosequencing of nominally significant sites. Pyrosequencing (white) results of (a) cg24634471, (b) cg10596483, and (c) cg20592995, sites that showed nominal significance on the 450 K array (black) when comparing means of ME/CFS GC-Hypersensitive (n = 14), ME/CFS GC-Typical (n = 19), and healthy controls (n = 33) assessed using an in vitro dexamethasone suppression assay. Error bars represent standard error. Lines indicate the group comparisons, where the first symbol indicates the result when compared using the 450 K array while the second statistic indicates the result compared by pyrosequencing. * = p ≤0.05, # = p ≤0.10, t-test

Fig. 3

Venn diagram comparing the number of differentially methylated sites across three comparisons. Venn diagram depicting the overlap between the ME/CFS GC-Hypersensitive vs. ME/CFS GC-Typical (green), ME/CFS GC-Hypersensitive vs. Control (blue), and ME/CFS GC-Typical vs. Control (red) comparisons. Numbers within each circle and overlap correspond to the number of differentially methylated CpG sites

Fig. 4

Top 3 glucocorticoid sensitive sites, based on magnitude of methylation difference. Increased methylation was observed at GpG sites of (a) GSTM1, (b) MYO3B, and (c) GSTM5 in the ME/CFS GC-Hypersensitive (black) compared to ME/CFS GC-Typical (grey) and Controls (white). * = p ≤0.05, Wilcoxon rank-sum test

Fig. 5

Enriched biological themes in differentially methylated sites that are likely associated with ME/CFS. A network map of 4,699 CpG sites showing significantly different methylation associated with ME/CFS, grouped according to GO terms and summary annotations (boxes). The size of the nodes (red circles) is proportional to the number of genes within each GO term and the thickness of the edges (green lines) represents the number of genes in common between GO terms