Understanding the molecular changes of Post Exertional Malaise in ME/CFS, by Jemma Elley (Thesis, Bachelor of Biomedical Sciences with Honours). University of Otago, 2021
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.
Supervisor: Prof Warren Tate
In summary, changes were observed in the oxygen consumption rate of ME/CFS subjects, as well as evidence of a difference between ME/CFS patients and healthy controls. The similarity in OCR profiles between ME/CFS-affected individuals and an over-exerted control should be further explored, as it potentially links the “increased perceived exertion” hypothesis with a physiological output.
The statistically significant decrease of ATP production after exercise should also be explored, potentially explaining the fatigue ME/CFS sufferers experience.
8-OHdG levels, as a measure of oxidative stress, did not appear to have drastic changes during the exercise paradigm, nor between ME/CFS individuals and controls. However, the spread of 8-OHdG levels in ME/CFS and healthy individuals should be assessed, as we cannot conclude that this minor difference is not, in fact, significant enough to act as a biomarker for ME/CFS.
Finally, significant differences in the DNA methylation of one individual’s genome was observed throughout the exercise paradigm, especially in genes related to the HPA-axis and immune system, metabolism, and circadian rhythm.
These findings buttress those reported by many other research groups. Whilst many researchers have investigated ME/CFS by comparing it to controls, the investigation of its key symptom, PEM, is underdeveloped. Therefore, this thesis contributes to filling a major gap in the field.
Additionally, this study employs a “precision medicine” approach, using patients as their own controls. Because of the fatigue-induced limitations of those with ME/CFS in partaking in studies, genome-wide investigations employing thousands of participants are not possible. This project provides an alternative, which, with increased sharing of data and machine learning, may provide a better way to approach ME/CFS research and the study of chronic illnesses in general.