Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex illness characterized by disabling fatigue, disturbed sleep patterns, pain, and flu-like symptoms. Patients report a high degree of physical disability, and a decreased quality of life with 24% being homebound (1), causing a US economic loss ranging from of $9.1 to $51 billion (2). Currently, there are three main sources of diagnosis criteria, the Center for Disease Control (CDC) Empiric (3), Fukuda (4), and Canadian Consensus (5), showing 2.54, 1.0, and 0.10% of the population affected, respectively. This variation highlights the lack of a concrete illness definition. Although research studies have identified various aspects such as immune abnormalities and exposure to toxins relevant to the pathogenesis of ME/CFS (6), ME/CFS is still not yet fully understood. The genetic and environmental pathogenesis of ME/CFS remains unclear. Currently, treatment of ME/CFS is dependent on management of symptomology and improvement on quality of life (6). An improved understanding of the molecular mechanisms affected and dysfunction in the regulatory systems will translate into better diagnostic methods and more targeted approaches to treatment. There are numerous studies suggesting that genes and single nucleotide polymorphisms (SNPs) within those genes might play a role in the development and progression of ME/CFS (7–9). Results of these studies are very interesting and useful, however, one of these studies was focused on mitochondrial DNA (7), and the other two were limited by 80 study subjects (8, 9). The aim of the current study is to increase the size of the ME/CFS cohort and identify the most harmful variants associated with ME/CFS.

Functional analysis of SNPs identified three main clusters of pathways as sharing at least 30% SNP related genes (Figure 1). The first is dominated in size via the pathway Cytokine Signaling in Immune System and includes other immune-related pathways such as interferon signaling, autoimmune responses, and T-cell receptor signaling. This cluster highlights a module of immune-related SNPs.

The second cluster is dominated in size via the Nuclear-Receptors Meta-Pathway and includes hormone related pathways such as steroid hormone, estrogen, and androgen biosynthesis, glucuronidation, and the pregnane x receptor pathway. This cluster highlights modules of hormone-related SNPs.

The final cluster is dominated in size by Pathways in Cancer, however, closer inspection shows many metabolic processes such as enzyme reactions (protein kinase A, calcium and calmodulin signaling), and G proteins signaling which regulate metabolic enzymes, which are all involved in the regulation of glycogen, sugar and lipid metabolism. This cluster highlights a module of metabolism-related SNPs.

While there is an overlap between the metabolic and immune modules, the hormone module remains isolated with main connections only formed via Ovarian Steroidogenesis and the Wnt signaling pathway. Finally, there is a group of loosely connected pathways involved in an extracellular matrix organization.

While this organization highlights the interplay between immune, hormone and metabolic activity underlying ME/CFS, overlay of the location of CADD scores illustrates where the most deleterious effects occur (Figure 1; lower panel).

Of the 11,485 SNPs that passed prefiltering according to the annotations (see Methods), 8,593 SNPs had frequency more than 10% in either reference or ME/CFS cohort. Out of them, 5,693 SNPs had a two-fold difference between ME/CFS and the reference cohorts in either direction (Supplementary Table 1).

SNPs that prevailed in ME/CFS cohort were scored using the CADD algorithm (12). According to the CADD algorithm, C-scores above 10 indicate that these SNPs are predicted to be among the 10% most harmful, and C-scores above 20 indicate the 1% most deleterious substitutions (12). Table 1 shows 50 SNPs that are the most frequent in the ME/CFS cohort and have C-scores above 10.

Of the 50 most frequent deleterious SNPs found in our ME/CFS cohort compared to the reference database (Table 1), 10 were found to have a frequency of 70% or more in the ME/CFS group. This includes CYP2D6, PRRT4, and PRSS56 at a frequency over 90%, C14orf37, ANKDD1B, at over 80%, and GPBAR1, LHB, ADAMTS19, VARS2, and CPLX2 at over 70%.

CYP2D6 (Cytochrome P450 2D6) is primarily expressed in the liver, but also highly expressed in areas of the central nervous system, including the substantia nigra, and is one of the most important enzymes involved in the metabolism of xenobiotics in the body. A significantly higher frequency of polymorphisms CYP2D6 was found in ME/CFS study subjects with Fibromyalgia than in controls and could differentiate these study subjects s from study subjects with multiple chemical sensitivity (27). CYP2D6 was found in the xenobiotics metabolism, androgen and estrogen biosynthesis and metabolism, tyrosine metabolism, codeine and morphine metabolism, oxidation by cytochrome P450, metapathway biotransformation phase I and II, and cytochrome P450—arranged by substrate type pathways all of which belong to the hormone related cluster.

PRSS56 (putative serine protease 56) is a serine protease that has been implicated in human eye development (28) and in the regulation of cerebellum activity of mice in exercise (29). It was not found to be a member of any of the annotated pathways.

GPBAR1 (G Protein-Coupled Bile Acid Receptor) functions as a cell surface receptor for bile acids and participates in the production of intracellular cAMP and activation of a MAP kinase signaling pathway. This receptor plays a big role in the suppression of macrophage functions and regulation of energy homeostasis by bile acids (30). Finding of the deleterious SNP in GPBAR1 (Table 1) is in agreement with the results of the recent study that showed disturbances in bile acid metabolism in ME/CFS study subjects (31). GPBAR1 was not among any of the pathways annotated.

LHB (luteinizing hormone beta polypeptide) is expressed in the pituitary gland and is essential for spermatogenesis and ovulation by stimulating the testes and ovaries to synthesize steroids (32, 33). LHB was found among the GnRH signaling pathway and ovarian steroidogenesis pathway.

ADAMTS19 is a member of the large ADAMTS (a desintegrin-like and metalloprotease with thrombospondin type 1 motif) family of metalloproteases (metal binding enzymes). ADAM proteins are responsible for the proteolytic cleavage of many transmembrane proteins and the release of their extracellular domain. ADAMTS19 is considered as a possible candidate for premature ovarian failure (34). Only the O-linked glycosylation pathway was found to contain ADAMTS19.

VARS2 (valyl-tRNA synthetase 2, mitochondrial) is important for the mitochondrial protein synthesis. Mutations in this gene are associated with cardiomyopathy (35), microcephaly and epilepsy (36), deficiency of the mitochondrial respiratory chain complex I and oxidative phosphorylation deficiency (37). VARS2 was not found among any of the annotated pathways.

CPLX2 gene encodes the complexin 2 protein that participates in neurotransmitter release by directly interacting with the neuronal SNARE complex (38). CPLX2 is known to be overexpressed in aging and downregulated by sleep deprivation (39), and this shows a connection of CPLX2 expression to fatigue. CPLX2 was also not found among any of the annotated pathways.

The remaining genes PRRT4, C14orf37, and ANKDD1B are obscure without much literature to support their function and not found among any of the annotated pathways. It was determined that PRRT4 (proline-rich transmembrane protein 4) showed biased expression in adult ovary, lung, adrenals, CNS and whole brain, while C14orf37 showed bias in brain, kidney, and ovary (ncbi.nlm.nih.gov). Little information was found for ANKDD1B (ankyrin repeat and death domain containing 1B).

Although SNPs in MYBPC3 and HLA genes have lower frequencies in ME/CFS cohort (0.19 for MYBPC3 and 0.13-0.44 for various HLA isoforms, respectively), these SNPs could be used for subgrouping of ME/CFS study subjects in larger studies because of their possible association with ME/CFS and fatigue. Multiple deleterious SNPs in HLA genes are in agreement with known impairment of the immune system in ME/CFS (40). Increased frequency of HLA-DQA1 alleles and decreased expression of HLA-DRB1 was found to be associated with ME/CFS (41). MYBPC3 (myosin binding protein C, cardiac) dysfunction is also associated with hypertrophic cardiomyopathy and corresponding fatigue (42).

These results contrast with previous SNP studies in ME/CFS (9, 43–45) which have found statistically significant associations in multiple loci including in neuroendocrine effector and receptor genes (43), TRP ion channels and AChRs (44, 45), and genes regulating the HPA axis (9, 46). This difference is most likely due to a combination of factors such as, (i) differences in array types used between studies, (ii) difference in the methods of analysis, (iii) differences between cohorts and the general heterogeneity of ME/CFS, and (iv) small-effect variants due to the relatively small sample sizes in each of these previous studies, compared to our relatively large cohort.

To date, this is the largest study known using SNP data and its affected pathways in combination with study subjects' self-reported symptoms. The results generated from our study will enhance the current understanding of ME/CFS and will generate new studies, all of which will lead to a better method for diagnosis and targeted genetic therapy. Replicative larger studies are warranted to improve the reliability of the results.

While these results are novel there are some limitations to the current analysis that are worth noting. First, there is no control over the chip version used by 23andMe for genotyping. This can result in loss of precision in the determination of study subject genotyping signature. Second, this initial pilot analysis was only conducted on SNPs in protein coding regions, miRNA regions, and regions close to splice junctions. SNPs in non-coding regions may be important in the cause of the illness. Finally, this analysis does not include rare variants. Moving forward, future studies based on this on-going collection of study subject information will address these limitations, will increase sample size, and provide more detailed statistical analyses. Building on this dataset we also aim to correlate these findings with our on-going research on gene expression (47), miRNA expression and DNA methylation (48).

All subjects signed an informed consent approved by the Institutional Review Board of Nova Southeastern University. Ethics review and approval for data analysis was also obtained by the IRB of Nova Southeastern University.

NK, LN, and TC conceived the study. MP, RJ, TC, and LN analyzed data. NK consulted on ME/CFS symptoms. AD, KH, KG, and MP led the recruitment of study participants.