Source: University of Otago
Date: December 20, 2019
URL:
https://ourarchive.otago.ac.nz/handle/10523/10114
https://ourarchive.otago.ac.nz/bitstream/handle/10523/10114/HelliwellAmberAH2019GeneticsMasters.pdf
(210 Mb !)
An epigenetic analysis of Myalgic Encephalomyelitis/Chronic Fatigue
Syndrome
-------------------------------------------------------------------
Amber Mikaela Helliwell
- Department of Genetics, University of Otago, New Zealand
Abstract
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a
debilitating disease affecting approximately 20,000 New Zealanders.
Patients experience lifelong persistence of the disease with symptoms
including a characteristic post exertional malaise, and dysfunctions in
cognition, sleep and the autonomic nervous system. Symptoms are severe
enough to prevent normal function, leaving a significant proportion of
patients house or bed bound greatly reducing quality of life. Prior
research and disease presentation indicates a multi-systemic
pathophysiology primarily involving metabolic, immune and neurological
dysfunctions. Susceptibility is believed to be a combination of a
genetic predisposition in combination with environmental stressors.
Recent investigative efforts have turned to epigenetics in order to
further understand the disease. DNA methylation is a well-characterized
epigenetic modification that is linked to changes in gene expression,
especially when it is found within the regulatory regions of the genome
such as promoters. Additionally, DNA methylation is a malleable,
environmentally affected regulatory modification with the potential to
provide insights into systematic changes linked to the disease. This
investigation aimed to characterize, in depth, the DNA methylation
patterns of ME/CFS patients compared with age and gender matched healthy
controls.
In order to determine the DNA methylation variation in ME/CFS a cohort
of 10 patients and 10 matched controls were investigated in the study.
DNA was extracted from peripheral blood mononuclear cell fractions
purified from whole blood. Following quality assessment of isolated DNA,
DNA fragment libraries were prepared for Reduced Representation
Bisulfite Sequencing (RRBS). The RRBS libraries were sequenced using
high throughput next generation sequencing. The subsequent data were
analysed using multiple bioinformatic platforms in order to determine
patterns of differential methylation between the patient and control
groups. RRBS designed MethylKit and DMAP analysis pipelines were
utilised in order to investigate changes at each individual cytosine and
at clustered cytosines within DNA 40-220bp fragments respectively.
Additional methylation variation was investigated across genomic
features of interest including promoters, enhancers and gene bodies.
Genes identified associated with gene body differential methylation were
then utilised for further pathway enrichment analyses.
With appropriate statistical significance thresholds, Methylkit
identified 394 differentially methylated cytosines and DMAP identified
76 differentially methylated fragments. Manual inspection of the data
identified four clusters of MethylKit cytosines overlapping or in close
proximity to DMAP fragments. These clusters identified regions of
regulatory importance for 16 protein-coding genes. Further independent
regulatory feature analysis identified 22 promoter regions associated
with 45 differentially methylated cytosines (utilising MethylKit data)
and 11 promoters associated with 12 fragments (utilising DMAP data).
Analysis of gene body differential methylation identified 91 genes
associated with 121 individual differentially methylated cytosines and
31 genes associated with 31 fragments containing methylated cytosines.
Functional pathway enrichment analysis with a FDR <0.05 identified 7
functional pathways through analysis of MethylKit identified genes, and
23 functional pathways through analysis of DMAP identified.
The 16 genes associated with the regulatory regions identified by
clusters of differential methylation largely falling into either immune
or metabolic/mitochondrial related functions. Further gene body analysis
identified a number of enriched functional pathways including a number
of immune, metabolic and particularly neurological related functions.
Together the pattern of DNA methylation in patients implicated
components of a number of systems that may be differentially methylated
and therefore potentially differentially regulated compared to a healthy
population. Particularly the large number of enriched neurotransmitters
and neuropeptide reactome pathways identified by DMAP genes implicate an
irregular stimulation of the HPA axis through the 'Stress Centre'
(Paraventricular nucleus), a link between an irregular neuroendocrine
response to stress and the stress sensitivity of ME/CFS patients.
Overall this work shows specific changes in ME/CFS methylation with
compelling links to the pathophysiology of the disease.
Keywords: DNA methylation; Myalgic Encephalomyelitis/Chronic Fatigue
Syndrome; MethylKit; DMAP; Reduced representation bisulfite sequencing
--------
