Abstract
BackgroundMyalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a lifelong debilitating disease with a complex pathology not yet clearly defined. Susceptibility to ME/CFS involves genetic predisposition and exposure to environmental factors, suggesting an epigenetic association. Epigenetic studies with other ME/CFS cohorts have used array-based technology to identify differentially methylated individual sites. Changes in RNA quantities and protein abundance have been documented in our previous investigations with the same ME/CFS cohort used for this study.ResultsDNA from a well-characterised New Zealand cohort of 10 ME/CFS patients and 10 age-/sex-matched healthy controls was isolated from peripheral blood mononuclear (PBMC) cells, and used to generate reduced genome-scale DNA methylation maps using reduced representation bisulphite sequencing (RRBS). The sequencing data were analysed utilising the DMAP analysis pipeline to identify differentially methylated fragments, and the MethylKit pipeline was used to quantify methylation differences at individual CpG sites. DMAP identified 76 differentially methylated fragments and Methylkit identified 394 differentially methylated cytosines that included both hyper- and hypo-methylation. Four clusters were identified where differentially methylated DNA fragments overlapped with or were within close proximity to multiple differentially methylated individual cytosines. These clusters identified regulatory regions for 17 protein encoding genes related to metabolic and immune activity. Analysis of differentially methylated gene bodies (exons/introns) identified 122 unique genes. Comparison with other studies on PBMCs from ME/CFS patients and controls with array technology showed 59% of the genes identified in this study were also found in one or more of these studies. Functional pathway enrichment analysis identified 30 associated pathways. These included immune, metabolic and neurological-related functions differentially regulated in ME/CFS patients compared to the matched healthy controls.ConclusionsMajor differences were identified in the DNA methylation patterns of ME/CFS patients that clearly distinguished them from the healthy controls. Over half found in gene bodies with RRBS in this study had been identified in other ME/CFS studies using the same cells but with array technology. Within the enriched functional immune, metabolic and neurological pathways, a number of enriched neurotransmitter and neuropeptide reactome pathways highlighted a disturbed neurological pathophysiology within the patient group.
Highlights
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a poorly understood disease estimated to affect an average of 0.89% [1] of the global population, though estimates have been higher such as 7%, as has Helliwell et al Clin Epigenet (2020) 12:167 been observed in Iceland [2]
peripheral blood mononuclear (PBMC) were purified from each subject and genomic DNA isolated for library preparation for reduced representation bisulphite sequencing (RRBS)
The first was the utilisation of Differential methylation analysis program (DMAP) [16, 17] with an ANOVA F test comparison that identified changes in sequence data on 40–220 bp DNA fragments in order to capture the DNA methylation patterns across fragment lengths produced in the process of creating the RRBS libraries
Summary
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a poorly understood disease estimated to affect an average of 0.89% [1] of the global population, though estimates have been higher such as 7%, as has Helliwell et al Clin Epigenet (2020) 12:167 been observed in Iceland [2]. The disease onset itself often follows a major ‘stress’ event, with a viral infection commonly reported by patients. This has established the hypothesis that onset of ME/CFS requires a stressor event coupled with a normally ‘silent’ component of genetic risk [3]. Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a lifelong debilitating disease with a complex pathology not yet clearly defined. Changes in RNA quantities and protein abundance have been documented in our previous investigations with the same ME/CFS cohort used for this study
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