Gow JW, Hagan S, Herzyk P, Cannon C, Behan PO, Chaudhuri A
Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, UK
At present, there are no clinically reliable disease markers for chronic fatigue syndrome. DNA chip microarray technology provides a method for examining the differential expression of mRNA from a large number of genes. Our hypothesis was that a gene expression signature, generated by microarray assays, could help identify genes which are dysregulated in patients with post-infectious CFS and so help identify biomarkers for the condition.
Human genome-wide Affymetrix GeneChip arrays (39,000 transcripts derived from 33,000 gene sequences) were used to compare the levels of gene expression in the peripheral blood mononuclear cells of male patients with post-infectious chronic fatigue (n=8) and male healthy control subjects (n=7).
Patients and healthy subjects differed significantly in the level of expression of 366 genes. Analysis of the differentially expressed genes indicated functional implications in immune modulation, oxidative stress and apoptosis. Prototype biomarkers were identified on the basis of differential levels of gene expression and possible biological significance.
Differential expression of key genes identified in this study offer an insight into the possible mechanism of chronic fatigue following infection. The representative biomarkers identified in this research appear promising as potential biomarkers for diagnosis and treatment.
This manuscript is dedicated to the memory of Professor WMH Behan (1939–2005). The authors wish to thank Drs G Riboldi-Tunnicliffe and S Keir for expert assistance and would like to acknowledge the financial support of The Cunningham Trust, The Barclay Foundation, Scottish Enterprise, the ME Association and ME Research UK during the course of this work.
A major problem in the diagnosis of ME/CFS is that there are no reliable markers for the disease; that is, there are no clinical or laboratory measurements that can be used to determine whether an individual has ME/CFS or not. However, genetics is one field in which there is the potential to discover a signature for the disease that can be used in diagnosis.
We used DNA chip microarray technology to measure the expression of 39,000 genes across the whole human genome. This provides information on which genes (information inherited from our parents) are active and therefore involved in the creation of specific proteins which can be used in the body.
We identified 366 genes with altered levels of expression in patients with post-infectious chronic fatigue, compared with healthy controls. These genes are involved mainly in the immune system, oxidative stress and apoptosis (all of which have been reported to be abnormal in ME/CFS). It may be possible to use these genes as a signature that could aid in the diagnosis of ME/CFS.
Comment by ME Research UK
The work by Dr John Gow and colleagues, to whom ME Research UK was one of the contributors of interim funding, is one of a number of research projects seeking a ‘biomarker’ for the illness ME/CFS using novel microarray technology. In this experimental technique, a sample of blood or tissue is taken, applied to a glass slide (microarray) containing more than 20,000 gene identifiers, and examined to determine which genes in the sample are being expressed.
As was reported in a series of articles some time ago — in the Scotsman, the Evening Times, and on the BBC website — the initial pilot data obtained by Dr Gow’s team suggested alterations to genes controlling the metabolism of prostaglandin and those regulation-specific immune cells. In the work presented in the current paper published in 2009 in BMC Medical Genomics, the team has identified 366 genes with different levels of expression in CFS patients and control subjects. These genes are involved in the immune system, oxidative stress and apoptosis, and it is interesting to note that functional abnormalities in all three of these areas have previously been reported in ME/CFS patients.
Contemporaneously, microarray investigations using samples from ME/CFS patients are being undertaken by other research groups. One team, led by Dr Jonathan Kerr at St Mary’s Campus, Imperial College London, supported by the CFS Research Foundation, published a paper containing early results in the Journal of Clinical Pathology (pdf 175 KB) (1) in August 2005. An article on this work in New Scientist (2) reported that using real-time PCR 15 of the genes were up to four times as active in people with ME/CFS, while one gene was less active; and in late 2007, a paper was published (Journal of Clinical Pathology) outlining the identification of a putative ‘gene signature’ for the illness consisting of 88 human genes. As these 88 genes have been linked directly to the pathogenesis of ME/CFS, the logical next step is to begin the study of inherited determinants of susceptibility by examining single nucleotide polymorphisms (SNPs), and with funding from ME Research UK, the St George’s group will shortly begin the next phase of the work: identifying the key SNPs for each of these 88 genes.
Another group, led by Suzanne Vernon of the Centers for Disease Control and Prevention’s molecular epidemiology programme in Atlanta is investigating gene expression profiles in the large Wichita clinical data set. The preliminary findings of these groups suggest dysregulation of genes involved in immune pathways, supporting the many reports in the literature of immune dysregulation in the pathogenesis of ME/CFS.
These developments, including this new 2009 paper from Prof. Gow’s group in Glasgow are welcome: few areas of biomedical research into ME/CFS can boast more than two separate research groups simultaneously engaged on a common quest. It is likely to be a long search, however. Experience from the use of genome-wide scanning technologies for cancer screening has shown that discovery and validation of biomarkers requires multiple phases of research. In a lucid commentary, Sullivan Pepe et al (3) identified issues to be addressed for the design of biomarker studies, and outlined a five-phase structure for investigation — from phase 1 pre-clinical exploratory studies to identify leads for potentially useful biomarkers and prioritise identified leads, through phase 2 involving the development of a clinical biomarker assay and assessment of its ability to distinguish patients and controls, to subsequent phases of increasingly stringent validation involving longitudinal, prospective, and large, expensive control studies. The publicly available preliminary information on the status of genome-wide scanning technologies in ME/CFS suggests that most work is presently in or around the phase 1 stage, and that much progress in bioassay development and validation studies will be required before an indubitable and validated ‘gene signature’ can be unveiled.
Of course, the same problems that confront all researchers in ME/CFS also apply to research groups using microarray technology. One is that ‘diagnosis’ of the illness is most often based on a ragbag of common non-specific symptoms, resulting in a diverse group of patients. As Jasonet al (4) have pointed out in an excellent recent review, “
Subgrouping is the key to understanding how CFS begins, how it is maintained… and in the best case, how it can be prevented, treated and cured.” It is unlikely, therefore, that a single biomarker or cluster will be found able to detect all cases as currently defined, although microarray technology does have the potential to make diagnosis more precise in the long term. Another problem is that obtaining and maintaining funding haunts the efforts of all biomedical researchers in ME/CFS, and it is particularly acute in these gene biomarker studies which will require million of dollars to come to a definitive conclusion. At ME Research UK, we echo the comment of Alex Fergusson MSP in the Parliamentary members’ business debate (Thursday 9 June 2005, motion S2M-2852) on the subject of a cure for myalgic encephalomyelitis, that it is entirely unacceptable that major funding bodies seem prepared to see novel gene research grind to a halt — particularly when large traunches of money have been allocated to research on non-curative psychosocial strategies designed to ‘manage’ symptoms.
Illnesses are most easily accepted when they have a specific clinical or scientific ‘signature’ — a biochemical test, a cluster of specific symptoms or signs, etc. — that confers legitimacy in the eyes of healthcare professionals. Until then, patients are in a no-man’s land between the living and the well, subject to a variety of quasi-therapeutic interventions and the ministrations of charlatans. ME/CFS has been called the “
disease of a thousand names“, but it has also been the disease of a thousand false dawns and a thousand broken promises. Yet, the discovery of a clinical or scientific ‘signature’ for ME/CFS, indicative of the physical terrain, would transform this situation at a single sharp stroke. In the longer term, work using genome-wide scanning technologies has the potential to reveal such a ‘signature’. To quote Steinau et al (5):
Biomarkers characteristic of CFS could contribute to precision in case ascertainment, identify heterogeneity in the CFS population to clarify contributing pathways to disease, suggest novel therapeutic targets, and provide indicators of disease progression and prognosis.
- Kaushik N, Fear D, Richards SCM et al. Gene expression in peripheral blood mononuclear cells from patients with chronic fatigue syndrome. J Clin Pathol 2005; 58: 826–32.
- Hooper R. Chronic fatigue is not all in the mind. New Scientist 2005 July 21; issue 2509, page 9.
- Sullivan Pepe M et al. Phases of biomarker development for early detection of cancer. J Natl Cancer Inst 2001; 93: 1054–61.
- Jason LA et al. Chronic fatigue syndrome: the need for subtypes. Neuropsychology Review 2005; 15: 29–58.
- Steinau M et al. Differential-display PCR of peripheral blood for biomarker discovery in chronic fatigue syndrome. J Mol Med 2004; 82: 750–5.