Abstract
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a prevalent but poorly understood illness characterised by severe debilitating fatigue, affecting approximately 1 % of the global population and disproportionately females. There are no agreed diagnostic markers or definitive clinical tests, and the causative agent and disease pathophysiology are ill defined. However, immune dysfunction, chronic viral infection and, recently, metabolic and mitochondrial dysregulation are proposed causes of ME/CFS. Abnormal upregulation and activation of an innate antiviral immune response protein, Protein Kinase R (PKR), has also been observed. This PhD project hypothesised that a precision medicine approach, studying a NZ pilot study group, could identify ME/CFS pathology in agreement with recent larger studies. A further hypothesis was that an increased ratio of phosphorylated PKR to non-phosphorylated inactive PKR could be detected in the NZ pilot study group, to ascertain the protein's utility as a diagnostic biomarker.
This project aimed at identifying biological pathway dysfunctions by comprehensive analysis of different classes of molecules, including micro-RNA, expressed genes (transcriptome), and cellular proteomes in an study group of ten ME/CFS patients and ten age-gender matched controls. For a potential diagnostic test, antibodies against (i) a non-phosphorylated PKR fragment and (ii) a phosphorylated PKR peptide were raised and purified to investigate the ratio of active: inactive PKR in ME/CFS patients. The regulation of PKR by cellular RNAs was investigated, using the known PKR regulator nc886 as a control.
Principal component analysis and t-tests were used to identify significant changes in the molecular data. Regulators of metabolic, immune and oxidative pathways were significantly increased. An example was Interleukin 8 (Fold-change = 5.57, P = 0.02). Several key mitochondrial proteins were decreased in the ME/CFS transcriptome and proteome analyses, for example NADH dehydrogenase 1 alpha subcomplex, 5 and NADH dehydrogenase flavoprotein 1 (Fold-change < 0.78, P < 0.01). Possible disease biomarkers were identified including plasma microRNAs hsa-miR-142-5p (P = 0.036), hsa-let-7g (P = 0.02), hsa-miR-1825 (P = 0.02), and a gene transcript TNFAIP3 (P = 3.23x10-21). Two antibodies against (i) a non-phosphorylated PKR fragment and (ii) a Thr-446 phosphorylated PKR peptide were raised and purified by negative and positive selection using antigen affinity chromatography. Western immunoblots of lymphocyte (PBMC) and neutrophil protein extracts detected an increased ratio of active: inactive PKR in ME/CFS patients. An RNA binding protocol was developed using an N-terminal fragment of PKR containing double-stranded RNA binding motifs, and some miRNA and small RNAs were identified that could bind to PKR, including hsa-miR-769-5p and the messenger RNA encoding PKR itself (EIF2AK2).
This study of a range of biologically important molecules within a well-characterised ME/CFS patient group identified significant dysregulation in immune inflammatory, apoptosis, oxidative stress, and metabolic pathways, and in mitochondrial functioning. A changed ratio of active: inactive PKR was identified, suggesting this ratio may be suitable for further development as a diagnostic test for ME/CFS.