Abstract
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating and often life-long condition affecting over 20,000 New Zealanders. Symptoms include muscle and joint pain, severe fatigue, unrefreshing sleep, hypersensitivity to light and sound, and cognitive dysfunction. However, the condition lacks a reliable diagnostic biomarker, impairing patient diagnosis and treatment development. Previous research has identified several key physiological areas of promise from which markers might come, including mitochondrial dysfunction, HPA-axis impairment, immune alterations, increased oxidative stress and epigenome modifications.
This project focuses on post-exertional malaise (PEM), a cardinal symptom of ME/CFS. Post-exertional malaise is defined as an exacerbation of ME/CFS symptoms after physical, mental or emotional exertion. As PEM distinguishes ME/CFS from other fatigue-related conditions, it may aid in identifying the molecular basis of ME/CFS.
To determine the molecular changes leading to this malaise, five ME/CFS-affected individuals and two healthy controls performed an exercise paradigm where they were made to cycle on an ergometer until their peak work rate was reached. 20 mL of blood was taken from each individual before performing the exercise, and after two exercise episodes that were 24 hours apart.
Peripheral blood mononuclear cells (PBMCs) were purified from the blood by centrifuging on Ficol gradients. The mitochondrial function was determined on live cells using the Seahorse XF Cell Mito Stress Test Kit. The genomic DNA was extracted from the PBMCs and 8-hydroxy 2 deoxyguanosine (a marker of oxidative stress) was determined using an 8-hydroxy 2 deoxyguanosine ELISA Kit. Methylome changes were detected in the DNA by digestion of the DNA and preparation of 40-220bp fragment libraries before performing Reduced Representation Bisulfite sequencing.
A decrease in the mitochondrial function after the first exercise was observed in all ME/CFS individuals and one control. Contrary to existing literature, the ME/CFS group had, on average, a lower level of 8-hydroxy 2 deoxyguanosine compared to healthy controls.
An average of 1.25% of DNA fragments were differentially methylated between the baseline 24-hour and baseline and 48-hour samples. Results of a STRING protein network analysis on the differentially methylated fragments present in the promoter show interaction between upregulated mitochondrial, nervous system, immune function, and HPA-axis -associated genes. Additionally, hypermethylation and potentially decreased expression of POU3F4, a transcription factor with high expression levels in the basal ganglia (which regulates motor activity and motivation) provides evidence of how the PEM symptoms of increased fatigue and perceived exertion may arise.
Whilst the molecular changes during PEM are varied and complex, these results contribute to the knowledge of the processes underlying the symptoms, and by proxy, the overall pathophysiology of ME/CFS.