HERV activation segregates ME/CFS from fibromyalgia while defining a novel nosologic entity
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eLife Assessment
This important study investigates the implications of human endogenous retrovirus (HERV) activity in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and fibromyalgia (FM). These findings indicate significant associations that coincide with previous literature, which has suggested roles for differential HERV activity in degenerative, inflammatory, and aging-related pathologies of the central nervous system (CNS), as well as neurotropic infections. These seminal studies can be strengthened with minor improvements to the methodologies of characterizing differential HERV activity, further characterizing downstream mechanisms by which HERV activity impacts disease and by an expansion of the datasets utilized to include additional cohorts. These compelling findings are of immediate importance to clinicians, policymakers, and researchers interested in the underlying etiology of human health and disease.
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Abstract
Research of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Fibromyalgia (FM), two acquired chronic illnesses affecting mainly females, has failed to ascertain their frequent co-appearance and etiology. Despite prior detection of human endogenous retrovirus (HERV) activation in these diseases, the potential biomarker value of HERV expression profiles for their diagnosis, and the relationship of HERV expression profiles with patient immune systems and symptoms had remained unexplored. By using HERV-V3 high-density microarrays (including over 350k HERV elements and more than 1500 immune-related genes) to interrogate the transcriptomes of peripheral blood mononuclear cells from female patients diagnosed with ME/CFS, FM or both, and matched healthy controls (n=43), this study fills this gap of knowledge. Hierarchical clustering of HERV expression profiles strikingly allowed perfect participant assignment into four distinct groups: ME/CFS, FM, co-diagnosed, or healthy, pointing at a potent biomarker value of HERV expression profiles to differentiate between these hard-to-diagnose chronic syndromes. Differentially expressed HERV-immune-gene modules revealed unique profiles for each of the four study groups and highlighting decreased γδ T cells, and increased plasma and resting CD4 memory T cells, correlating with patient symptom severity in ME/CFS. Moreover, activation of HERV sequences coincided with enrichment of binding sequences targeted by transcription factors which recruit SETDB1 and TRIM28, two known epigenetic silencers of HERV, in ME/CFS, offering a mechanistic explanation for the findings. Unexpectedly, HERV expression profiles appeared minimally affected in co-diagnosed patients denoting a new nosological entity with low epigenetic impact, a seemingly relevant aspect for the diagnosis and treatment of this prevalent group of patients.
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eLife Assessment
This important study investigates the implications of human endogenous retrovirus (HERV) activity in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and fibromyalgia (FM). These findings indicate significant associations that coincide with previous literature, which has suggested roles for differential HERV activity in degenerative, inflammatory, and aging-related pathologies of the central nervous system (CNS), as well as neurotropic infections. These seminal studies can be strengthened with minor improvements to the methodologies of characterizing differential HERV activity, further characterizing downstream mechanisms by which HERV activity impacts disease and by an expansion of the datasets utilized to include additional cohorts. These compelling findings are of immediate importance to clinicians, …
eLife Assessment
This important study investigates the implications of human endogenous retrovirus (HERV) activity in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and fibromyalgia (FM). These findings indicate significant associations that coincide with previous literature, which has suggested roles for differential HERV activity in degenerative, inflammatory, and aging-related pathologies of the central nervous system (CNS), as well as neurotropic infections. These seminal studies can be strengthened with minor improvements to the methodologies of characterizing differential HERV activity, further characterizing downstream mechanisms by which HERV activity impacts disease and by an expansion of the datasets utilized to include additional cohorts. These compelling findings are of immediate importance to clinicians, policymakers, and researchers interested in the underlying etiology of human health and disease.
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Reviewer #1 (Public review):
Summary:
Giménez-Orenga et al. investigate the origin and pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and fibromyalgia (FM). Using RNA microarrays, the authors compare the expression profiles and evaluate the biomarker potential of human endogenous retroviruses (HERV) in these two conditions. Altogether, the authors show that HERV expression is distinct between ME/CFS and FM patients, and HERV dysregulation is associated with higher symptom intensity in ME/CFS. HERV expression in ME/CFS patients is associated with impaired immune function and higher estimated levels of plasma cells and resting CD4 memory T cells. This work provides interesting insights into the pathophysiology of ME/CFS and FM, creating opportunities for several follow-up studies.
Strengths:
(1) Overall, …
Reviewer #1 (Public review):
Summary:
Giménez-Orenga et al. investigate the origin and pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and fibromyalgia (FM). Using RNA microarrays, the authors compare the expression profiles and evaluate the biomarker potential of human endogenous retroviruses (HERV) in these two conditions. Altogether, the authors show that HERV expression is distinct between ME/CFS and FM patients, and HERV dysregulation is associated with higher symptom intensity in ME/CFS. HERV expression in ME/CFS patients is associated with impaired immune function and higher estimated levels of plasma cells and resting CD4 memory T cells. This work provides interesting insights into the pathophysiology of ME/CFS and FM, creating opportunities for several follow-up studies.
Strengths:
(1) Overall, the data is convincing and supports the authors' claims. The manuscript is clear and easy to understand, and the methods are generally well-detailed. It was quite enjoyable to read.
(2) The authors combined several unbiased approaches to analyse HERV expression in ME/CFS and FM. The tools, thresholds, and statistical models used all seem appropriate to answer their biological questions.
(3) The authors propose an interesting alternative to diagnosing these two conditions. Transcriptomic analysis of blood samples using an RNA microarray could allow a minimally invasive and reproducible way of diagnosing ME/CFS and FM.
Weaknesses:
(1) The cohort analysed in this study was phenotyped by a single clinician. As ME/CFS and FM are diagnosed based on unspecific symptoms and are frequently misdiagnosed, this raises the question of whether the results can be generalised to external cohorts.
(2) The analyses performed to unravel the causes and effects of HERV expression in ME/CFS and FM are solely based on sequencing data. Experimental approaches could be used to validate some of the transcriptomic observations.
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Reviewer #2 (Public review):
Summary:
Giménez-Orenga carried out this study to assess whether human endogenous retroviruses (HERVs) could be used to improve the diagnosis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Fibromyalgia (FM). To this end, they used the HERV-V3 array developed previously, to characterize the genome-wide changes in the expression of HERVs in patients suffering from ME/CFS, FM, or both, compared to controls. In turn, they present a useful repertoire of HERVs that might characterize ME/CFS and FM. For the most part, the paper is written in a manner that allows a natural understanding of the workflow and analyses carried out, making it compelling. The figures and additional tables present solid support for the findings. However, some statements made by the authors seem incomplete and would …
Reviewer #2 (Public review):
Summary:
Giménez-Orenga carried out this study to assess whether human endogenous retroviruses (HERVs) could be used to improve the diagnosis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Fibromyalgia (FM). To this end, they used the HERV-V3 array developed previously, to characterize the genome-wide changes in the expression of HERVs in patients suffering from ME/CFS, FM, or both, compared to controls. In turn, they present a useful repertoire of HERVs that might characterize ME/CFS and FM. For the most part, the paper is written in a manner that allows a natural understanding of the workflow and analyses carried out, making it compelling. The figures and additional tables present solid support for the findings. However, some statements made by the authors seem incomplete and would benefit from a more thorough literature review. Overall, this work will be of interest to the medical community seeking in better understanding of the co-occurrence of these pathologies, hinting at a novel angle by integrating HERVs, which are often overlooked, into their assessment.
Strengths:
(1) The work is well-presented, allowing the reader to understand the overall workflow and how the specific aims contribute to filling the knowledge gap in the field.
(2) The analyses carried out to understand the potential impact on gene expression mediated by HERVs are in line with previous works, making it solid and robust in the context of this study.
Weaknesses:
(1) The authors claim to obtain genome-wide HERV expression profiles. However, the array used was developed using hg19, while the genomic analysis of this work are carried out using a liftover to hg38. It would improve the statement and findings to include a comparison of the differences in HERVs available in hg38, and how this could impact the "genome-wide" findings.
(2) The authors in some points are not thorough with the cited literature. Two examples are:
a) Lines 396-397 the authors say "the MLT1, usually found enriched near DE genes (Bogdan et al., 2020)". I checked the work by Bogdan, and they studied bacterial infection. A single work in a specific topic is not sufficient to support the statement that MLT1 is "usually" in close vicinity to differentially expressed genes. More works are needed to support this.
b) After the previous statement, the authors go on to mention "contributing to the coding of conserved lncRNAs (Ramsay et al., 2017)". First, lnc = long non-coding, so this doesn't make sense. Second, in the work by Ramsay they mention "that contributed a significant amount of sequence to primate lncRNAs whose expression was conserved", which is different from what the authors in this study are trying to convey. Again, additional work and a rephrasing might help to support this idea.(3) When presenting the clusters, the authors overlook the fact that cluster 4 is clearly control-specific, and fail to discuss what this means. Could this subset of HERV be used as bona fide markers of healthy individuals in the context of these diseases? Are they associated with DE genes? What could be the impact of such associations?
Appraisals on aims:
The authors set specific questions and presented the results to successfully answer them. The evidence is solid, with some weaknesses discussed above that will methodologically strengthen the work.
Likely impact of work on the field:
This work will be of interest to the medical community looking for novel ways to improve clinical diagnosis. Although future works with a greater population size, and more robust techniques such as RNA-Seq, are needed, this is the first step in presenting a novel way to distinguish these pathologies.
It would be of great benefit to the community to provide a table/spreadsheet indicating the specific genomic locations of the HERVs specific to each condition. This will allow proper provenance for future researchers interested in expanding on this knowledge, as these genomic coordinates will be independent of the technique used (as was the array used here).
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Reviewer #3 (Public review):
The authors find that HERV expression patterns can be used as new criteria for differential diagnosis of FM and ME/CFS and patient subtyping. The data are based on transcriptome analysis by microarray for HERVs using patient blood samples, followed by differential expression of ERVs and bioinformatic analyses. This is a standard and solid data processing pipeline, and the results are well presented and support the authors' claim.
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Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
Giménez-Orenga et al. investigate the origin and pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and fibromyalgia (FM). Using RNA microarrays, the authors compare the expression profiles and evaluate the biomarker potential of human endogenous retroviruses (HERV) in these two conditions. Altogether, the authors show that HERV expression is distinct between ME/CFS and FM patients, and HERV dysregulation is associated with higher symptom intensity in ME/CFS. HERV expression in ME/CFS patients is associated with impaired immune function and higher estimated levels of plasma cells and resting CD4 memory T cells. This work provides interesting insights into the pathophysiology of ME/CFS and FM, creating opportunities for several follow-up …
Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
Giménez-Orenga et al. investigate the origin and pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and fibromyalgia (FM). Using RNA microarrays, the authors compare the expression profiles and evaluate the biomarker potential of human endogenous retroviruses (HERV) in these two conditions. Altogether, the authors show that HERV expression is distinct between ME/CFS and FM patients, and HERV dysregulation is associated with higher symptom intensity in ME/CFS. HERV expression in ME/CFS patients is associated with impaired immune function and higher estimated levels of plasma cells and resting CD4 memory T cells. This work provides interesting insights into the pathophysiology of ME/CFS and FM, creating opportunities for several follow-up studies.
Strengths:
(1) Overall, the data is convincing and supports the authors' claims. The manuscript is clear and easy to understand, and the methods are generally well-detailed. It was quite enjoyable to read.
(2) The authors combined several unbiased approaches to analyse HERV expression in ME/CFS and FM. The tools, thresholds, and statistical models used all seem appropriate to answer their biological questions.
(3) The authors propose an interesting alternative to diagnosing these two conditions. Transcriptomic analysis of blood samples using an RNA microarray could allow a minimally invasive and reproducible way of diagnosing ME/CFS and FM.
Weaknesses:
(1) The cohort analysed in this study was phenotyped by a single clinician. As ME/CFS and FM are diagnosed based on unspecific symptoms and are frequently misdiagnosed, this raises the question of whether the results can be generalised to external cohorts.
Thank you for your comment. Surely the study of larger cohorts will determine the external validity of these results in a clinical scenario. However, this pilot study, first of its kind, was designed to maximize homogeneity across participants which seemed primarily ensured by inclusion of females only diagnosed by a single experienced observer.
(2) The analyses performed to unravel the causes and effects of HERV expression in ME/CFS and FM are solely based on sequencing data. Experimental approaches could be used to validate some of the transcriptomic observations.
Certainly, experimental approaches may add robustness to our findings. We in fact consider taking this avenue to deepen in the observations presented here. However, the limited knowledge of HERV-mediated physiological functions may hinder the task of revealing causes and effects of HERV expression in ME/CFS and FM in the short term.
Reviewer #2 (Public review):
Summary:
Giménez-Orenga carried out this study to assess whether human endogenous retroviruses (HERVs) could be used to improve the diagnosis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Fibromyalgia (FM). To this end, they used the HERV-V3 array developed previously, to characterize the genome-wide changes in the expression of HERVs in patients suffering from ME/CFS, FM, or both, compared to controls. In turn, they present a useful repertoire of HERVs that might characterize ME/CFS and FM. For the most part, the paper is written in a manner that allows a natural understanding of the workflow and analyses carried out, making it compelling. The figures and additional tables present solid support for the findings. However, some statements made by the authors seem incomplete and would benefit from a more thorough literature review. Overall, this work will be of interest to the medical community seeking in better understanding of the co-occurrence of these pathologies, hinting at a novel angle by integrating HERVs, which are often overlooked, into their assessment.
Strengths:
(1) The work is well-presented, allowing the reader to understand the overall workflow and how the specific aims contribute to filling the knowledge gap in the field.
(2) The analyses carried out to understand the potential impact on gene expression mediated by HERVs are in line with previous works, making it solid and robust in the context of this study.
Weaknesses:
(1) The authors claim to obtain genome-wide HERV expression profiles. However, the array used was developed using hg19, while the genomic analysis of this work are carried out using a liftover to hg38. It would improve the statement and findings to include a comparison of the differences in HERVs available in hg38, and how this could impact the "genome-wide" findings.
This is an important point. However, the low number of probes that were excluded from our analysis by lack of correspondence with hg38, less than 100 among the 1,290,800 probesets, was interpreted as insignificant for "genome-wide" claims. An aspect that will be detailed in the revised version of this manuscript.
(2) The authors in some points are not thorough with the cited literature. Two examples are:
a) Lines 396-397 the authors say "the MLT1, usually found enriched near DE genes (Bogdan et al., 2020)". I checked the work by Bogdan, and they studied bacterial infection. A single work in a specific topic is not sufficient to support the statement that MLT1 is "usually" in close vicinity to differentially expressed genes. More works are needed to support this.
b) After the previous statement, the authors go on to mention "contributing to the coding of conserved lncRNAs (Ramsay et al., 2017)". First, lnc = long non-coding, so this doesn't make sense. Second, in the work by Ramsay they mention "that contributed a significant amount of sequence to primate lncRNAs whose expression was conserved", which is different from what the authors in this study are trying to convey. Again, additional work and a rephrasing might help to support this idea.
Certainly, these two sentences need rephrasing to better adjust statements to current evidence and will be replaced in the revised version of this manuscript.
(3) When presenting the clusters, the authors overlook the fact that cluster 4 is clearly control-specific, and fail to discuss what this means. Could this subset of HERV be used as bona fide markers of healthy individuals in the context of these diseases? Are they associated with DE genes? What could be the impact of such associations?
Using control DE HERV as bona fide markers of healthy individuals seems like an interesting possibility worth exploring. Control DE HERVs (cluster 4) are indeed associated with DE genes involved in apoptosis, T cell activation and cell-cell adhesion (modules 1 and 6) (Figure 3A). The impact of which deserves further study.
Appraisals on aims:
The authors set specific questions and presented the results to successfully answer them. The evidence is solid, with some weaknesses discussed above that will methodologically strengthen the work.
Likely impact of work on the field:
This work will be of interest to the medical community looking for novel ways to improve clinical diagnosis. Although future works with a greater population size, and more robust techniques such as RNA-Seq, are needed, this is the first step in presenting a novel way to distinguish these pathologies.
It would be of great benefit to the community to provide a table/spreadsheet indicating the specific genomic locations of the HERVs specific to each condition. This will allow proper provenance for future researchers interested in expanding on this knowledge, as these genomic coordinates will be independent of the technique used (as was the array used here).
We agree with the reviewer that sharing genomic locations of DE HERVs in these pathologies would contribute to further development of our findings. Unfortunately, we do not hold the rights to share probe coordinates from this custom HERV-V3 microarray which we used under MTA agreement with its developer.
Reviewer #3 (Public review):
The authors find that HERV expression patterns can be used as new criteria for differential diagnosis of FM and ME/CFS and patient subtyping. The data are based on transcriptome analysis by microarray for HERVs using patient blood samples, followed by differential expression of ERVs and bioinformatic analyses. This is a standard and solid data processing pipeline, and the results are well presented and support the authors' claim.
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