Formerly known as chronic fatigue syndrome, myalgic encephalomyelitis/chronic fatigue syndrome has long been neglected by physicians, researchers, and funding agencies, not least due to its mysterious causes. It’s often hard for patients to find doctors who can diagnose ME/CFS, a widespread condition characterized by debilitating post-exertion fatigue and other symptoms.

A new study appears to make headway toward solving those difficulties. A recent analysis from more than 40 ME/CFS patients reports that a disease-specific stress test leaves a distinct signature of 11 microRNAs in their blood that change in abundance compared with blood drawn before the test. Most of these microRNAs are involved in regulating immunity, supporting the idea that immune dysfunction plays a key role in the disease’s pathology. The findings lay the groundwork for developing a molecular diagnostic test for the disease, the authors write in their study, which was published on...

Although the findings need to be replicated in larger cohorts, “I think there are many strengths here,” remarks Mady Hornig, an immunologist at the Columbia University Mailman School of Public Health who wasn’t involved in the research. “There’s a lot here, in terms of the findings, that are really intriguing and important clues.”

MicroRNAs are short nucleotide snippets that act inside cells to regulate gene expression and have garnered much research interest over the past decade as potential diagnostic tools for several conditions in which they’re dysregulated. To Alain Moreau, who specializes in the molecular genetics of musculoskeletal diseases at the University of Montreal and the Sainte-Justine University Hospital, they were an attractive focus for exploring ME/CFS, in part because microRNAs also circulate in the blood, providing an easily accessible source of gene-regulatory information. 

Previous studies have flagged distinctive circulating or cellular microRNAs in ME/CFS patients, but some had very small sample sizes or didn’t compare patients with appropriate controls, making the data often hard to interpret, Moreau says. He and his colleagues wanted to overcome these issues, while also looking for microRNAs tied to specific symptoms in the condition, which he says he hoped would yield robust biomarkers. 

The hallmark symptom of ME/CFS is post-exertional malaise (PEM), a worsening of fatigue and other symptoms following physical or mental exercise that can leave patients bedridden for weeks. The researchers sought to probe microRNAs associated with this symptom, but to spare patients a full-blown bout of PEM in the clinic, Moreau’s team figured out that they could use a therapeutic massager—an inflatable arm cuff that exerts gentle pulsating compressions—to induce a milder form of PEM, as evidenced by headaches, muscle pain, and profound fatigue that patients reported in later questionnaires.

Starting with 11 severely affected, housebound ME/CFS patients, the team drew plasma samples before and 90 minutes after this challenge and screened for differences in levels of microRNAs. Computational analysis revealed 17 microRNAs whose levels had changed significantly after the test; their response also differed from that of eight age- and sex-matched healthy individuals who had been subjected to the massager but didn’t report any PEM symptoms.

Repeating this analysis in a larger cohort of 32 ME/CFS patients and 17 matched controls, the team discovered the same response patterns for 11 microRNAs. In fact, a machine learning algorithm the researchers trained could correctly diagnose someone with ME/CFS based solely on the change in concentration of these microRNAs after the massage intervention. “We were unable to misdiagnose a [healthy] control as ME/CFS, or inversely, ME/CFS as a control,” Moreau says.

The need for diagnostics and drugs for ME/CFS

There’s an overwhelming need for definitive diagnostic tests for ME/CFS, says Frances Williams, a genomic epidemiologist at King’s College London who wasn’t involved in the research. Diagnosis is currently made by excluding other conditions, which is difficult, time-consuming, and a frustration for patients. The fact that Moreau’s team could replicate their results in a larger cohort is encouraging, but she says she doubts that microRNAs will form a standalone test for ME/CFS, simply because of the genetic and epigenetic complexity of the disease. However, microRNAs “could be helpful in combination with other things,” she says.

Hornig says the test has many strengths in terms of translating it for use in a clinical setting. For one, its focus on free-floating sequences in the blood makes it technologically simpler than a technique used in a previous study that requires extracting microRNAs from inside blood cells. She also praises the team’s PEM-inducing stressor, which reflects the sensitivity of severely affected ME/CFS patients to their sensory environment, but is less taxing than physical exertion “and respectful of the patients’ experience and fears,” she says.

We were unable to misdiagnose a [healthy] control as ME/CFS, or inversely, ME/CFS as a control.

—Alain Moreau, University of Montreal and Sainte-Justine University Hospital

Moreau encourages other institutions to replicate the results. The findings will require validation in larger cohorts to ensure the test can diagnose patients with all subtypes of ME/CFS, different stages of the disease, and those from a variety of regions and ethnicities. Hornig adds it’s also important to investigate differences with the results of other studies—for instance, previous work on cellular microRNAs in ME/CFS suggested disparities in how men and women responded to exercise, whereas Moreau’s results found no sex-based differences. She’s also curious about where the microRNAs come from, suspecting immune cells or muscle tissue as possible origins. “This opens a lot of questions.” 

Using a different algorithm, Moreau’s team found that they could cluster the patients into four discrete groups based on the precise pattern of their microRNA responses to the PEM challenge. Interestingly, these groups happened to also share clinical features—one group, for instance, had markedly severe symptoms. To Moreau, this indicates that different mechanisms operate in different subsets of the disease, which could help explain why drug trials for ME/CFS patients have so far been largely inconclusive. But he says he hopes to use microRNA data soon to match patients with drugs they’re more likely to respond to. His team suspects, for instance, that the immunomodulating drug rintatolimod, which activates Toll-like receptor 3, might not be effective in patients with high levels of microRNAs that impede the transcription of that receptor. “I [hope] the test will open the door to more . . . precision medicine in the field of ME/CFS,” Moreau says.

To Williams, the major value of the study lies in an analysis Moreau’s team conducted with the microRNA data that untangles the molecular pathways the 11 sequences are involved in.

This revealed that 7 out of the 11 microRNAs were involved in regulating immune functions, which “certainly fits with one arm of the research that suggests that immune activation is very important in leading to chronic fatigue,” she says.

An additional network analysis flagged the key genes each microRNA is associated with and other diseases they’ve been linked to, which included viral infection, sleep disorders, and cognitive impairments. “Using a network approach, you can start to shed light on which cellular processes are important. And then if that ties in with what we know already about the cellular processes in ME/CFS, then that all begins to paint a bit of a picture.”

The findings are relevant in light of the COVID-19 pandemic and the increasing numbers of “long-hauler” patients left with enduring fatigue symptoms, a phenomenon that has many experts concerned that the coronavirus could trigger ME/CFS in a subset of infected people. The pandemic “will bring a huge number of new [ME/CFS] patients, and some of them are relatively young. That will create some devastating impacts for themselves and their families,” Moreau says. “That’s why we need to hurry and hope that we will convince governments and funding agencies to [put more funding into the field].” 

E. Nepotchatykh et al., “Profile of circulating microRNAs in myalgic encephalomyelitis and their relation to symptom severity, and disease pathophysiology,” Sci Rep, doi:10.1038/s41598-020-76438-y, 2020. 

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