Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This narrative review article by Elremaly et al., explores nuclear and mitochondrial DNA contributions to the development and biological mechanisms of ME/CFS. It covers genetic risk factors, findings from genome-wide and multi-omics studies, and the impact of mtDNA variants on energy metabolism and symptoms. Epigenetic changes, such as DNA methylation and transposable elements, are discussed as links between genes and environmental triggers. The review also highlights the potential of DNA-based biomarkers and therapies to improve diagnosis and treatment of ME/CFS.

Strengths of the manuscript include its comprehensive coverage of molecular aspects of ME/CFS, presented in a well-organized format that makes it a valuable resource for researchers new to the field. The main weakness lies in the occasionally overstated enthusiasm when presenting experimental findings, which may affect the objectivity of the interpretation. Additionally, some key references seem to be missing, as noted in the recommendations for improvement provided below.

CONTENTS

Page 2: It is not clear how the information from Citation 19-“over 95% of symptoms shared and nearly half of Long COVID patients meeting ME diagnostic criteria,..” was extracted. Readers may wonder which/how many are the symptoms included in the 95% considered as “shared” and which/how many are the 5% not “shared”.

Similarly, the statement: “nearly half of Long COVID patients…” needs detail on how was this calculated. In this sense, other authors may need to be cited as well. For example: Jaon and Dorri, 2022 (https://pubmed.ncbi.nlm.nih.gov/36648965/)

The sentence: “..examining specific gene variants thought to be involved in the disease.”  and followings need citations to support the arguments provided for the timeline.

Page 3: “Multiple studies consistently support…” need more than 1 citation (cite 20). As per style, void subjective expressions such as “strongly, consistently support.. unless the attributes are backed up by objective information/findings. When multiple studies explore a common possibility (hypothesis) recompilation of summary data (number of individuals, methods applied, main findings, and other relevant info) on tables is recommendable.

Section 2.1: The term “significant” may mislead readers if not accompanied by statistic data. Please, review the sentences including: “..significantly increased risk” and “..a significant portion of the susceptibility to ME is indeed heritable.”

The twin studies referred include 2 experimental and 1 review/opinion study. In addition, the 2 experimental studies cited were performed by a single research group. On one side, supporting data should be strictly experimental. On another the number of cases (independent observations) must be detailed to show the level of evidence.

GWAS studies (Section 2.3) need updating to include the recent preprint including results for the DECODE ME study (https://www.medrxiv.org/content/10.1101/2025.08.06.25333109v1 ), and additional information, such as a publication by the Garder group focused on ME (https://pubmed.ncbi.nlm.nih.gov/36517845/) instead, or in addition to, reference 32. Zhang et al., 2025 (https://pubmed.ncbi.nlm.nih.gov/40321247/ ) and other.

Section 2.4, please define “strong associations of HP genotypes with severe PEM and cognitive dysfunction ” referred for the study of Moezzi et al.,

Page 5: Section 3.2 Please indicate in what cell types were “swollen or fragmented mitochondria, suggesting structural damage [48-49]” found. Also, cell type for: “WASF3 knockdown in patient-derived cells..”

Page 6: In the sentence: “This figure illustrates the key differences in energy metabolism between a healthy mitochondrion and an ME mitochondrion” revisit the term “the”. Is this indicating “some” or “reported/shown/informed”¿? Include citations used to illustrate Figure 2. The whole paragraph following Fig 2 lacks citations.

Table 2, the text: “Baseline for comparison” is not clear.

Table 3, could be more complete. Very few studies are included.

Page 8: The following sentence needs a citation: “Notably, altered methylation of D-loop regions and mitochon drial tRNA genes has been linked to reduced respiratory efficiency and increased oxidative stress.”

Page 11: HERV activation in ME and Long COVID-19, is also supported at the protein level, as shown in the following articles: https://pubmed.ncbi.nlm.nih.gov/40726775/

And https://pubmed.ncbi.nlm.nih.gov/36389746/

Page 12: Although the sentence “Recent large-scale whole-genome sequencing studies..” refers “studies” in plural, only one article is cited. If this is a review article, authors should consider to mention “as reviewed by…”

Page 13: in the sentence: “The top panel shows how methylation alteration..” “how” should be replaced by “that” . And the term “can” should be replaced by “could” or “may”. Likewise, this revision should be extended in the sentences that follow these.

 

FORMAT/STYLE

Page 2: “Early investigations…” replace by “Initial investigations..or similar, since the timeline starts in early 2000, so to avoiding repeating the term “Early”

Page 3: The text “Figure created using BioRender(SG28PQS95K)” should appear at the end of the Figure legend. This applies to the additional figures created with this tool.

Please review the sentence including: “increase with increasingly..”

In the sentence: “Early investigations into the genetic basis of ME often focused on candidate gene studies.. ”the word “often” seems dispensable as all the evidence refers to focused studies.

Page 5: “post-exertional malaise” should be replaced by PEM, as the full term was already introduced in the text. Please review this throughout the document for all abbreviations.

On Fig 4. It seems that “Diets and Drugs” should appear on the left side. Similarly, the lower panel background should appear in pink tone for consistency of “disease” status susceptible of treatment.

 

 

Author Response

Comment 1:  Page 2: It is not clear how the information from Citation 19-“over 95% of symptoms shared and nearly half of Long COVID patients meeting ME diagnostic criteria,..” was extracted. Readers may wonder which/how many are the symptoms included in the 95% considered as “shared” and which/how many are the 5% not “shared”.

Response 1:  Thank you for this insightful comment regarding the clarity of our statement on the symptom overlap between Long COVID and ME. We fully agree that the original phrasing “over 95% of symptoms shared” lacked sufficient detail and could leave readers uncertain about which symptoms are included in the shared set and which fall outside it.

To address this, we have revised the text on page 2 to provide greater specificity. The updated passage now reads: “Over 95% of symptoms are shared between Long COVID and ME, with a significant proportion of Long COVID patients meeting the diagnostic criteria for ME. One study found that 58% qualified under the Canadian Consensus Criteria (CCC) [19,20]. This extensive clinical and pathophysiological overlap includes elevated oxidative stress and common epigenetic signatures. Core symptoms shared by both conditions include fatigue, PEM, cognitive impairment, sleep disturbances, and orthostatic intolerance. In contrast, anosmia, dysgeusia, rash, and hair loss are more frequently observed in Long COVID, whereas painful lymph nodes, chemical sensitivities, and tinnitus are more characteristic of ME [19].”

Comment 2: Similarly, the statement: “nearly half of Long COVID patients…” needs detail on how was this calculated. In this sense, other authors may need to be cited as well. For example: Jaon and Dorri, 2022 (https://pubmed.ncbi.nlm.nih.gov/36648965/)

Response 2:  Thank you for the precise comment regarding the statement, “nearly half of Long COVID patients meeting ME diagnostic criteria,” and the request for clarification of its calculation and supporting citations. We also greatly appreciate your suggestion of the Jaon and Dorri (2022) reference, which proved highly valuable. Upon review, this study directly confirms that a substantial proportion of individuals with persisting COVID-19 symptoms fulfill ME diagnostic criteria, specifically reporting that 58% of their Long COVID cohort met the Canadian Consensus Criteria (CCC) for ME. We have now cited this reference in the manuscript to support our statement. Revision made on page 2 of the manuscript, as quoted in Comment 1.

Comment 3: The sentence: “..examining specific gene variants thought to be involved in the disease.”  and followings need citations to support the arguments provided for the timeline.

Response 3:  Thank you for this essential comment. We fully agree that the statements outlining the historical progression of genetic and epigenetic research in ME, particularly those associated with Figure 1's timeline, require robust citation to support the arguments provided.

In response to your valuable feedback, we have carefully reviewed the paragraph describing the timeline and have integrated specific citations to substantiate each phase of research. These references now support the claims regarding candidate gene studies, twin studies/heritability, initial epigenetic research, and the emergence of large-scale and multi-omics approaches. Furthermore, to enhance the logical flow and ensure Figure 1 (the timeline) appears in the most appropriate context, we have repositioned the figure in the manuscript to follow the detailed discussion and supporting studies that describe these different research phases. This placement should improve readability and reinforce the connections between the text and the visual representation.

The revised text on page 6 now reads as follows: “This timeline illustrates the evolving research into the genetic and epigenetic underpinnings of ME. In the early 2000s, studies primarily focused on candidate gene, examining specific gene variants thought to play a role in the disease [26-30]. The 2010s marked a shift toward broader approaches, with twin studies providing strong evidence for heritability [24-26] and the start of initial epigenetic research exploring how environmental factors influence gene expression without altering DNA sequence [39]. In the 2020s, the field entered a new phase with the launch of large-scale projects, such as the DecodeME project [31,32], and the rise of multi-omics approaches that analyze multiple biological layers to understand ME complexity [31,40]. This period also saw significant discoveries regarding the genetic overlap with Long COVID, highlighting shared molecular pathways between the two conditions [36-38]. Figure created using BioRender(SG28PQS95K).”

Comment 4: Page 3: “Multiple studies consistently support…” need more than 1 citation (cite 20). As per style, void subjective expressions such as “strongly, consistently support.. unless the attributes are backed up by objective information/findings. When multiple studies explore a common possibility (hypothesis) recompilation of summary data (number of individuals, methods applied, main findings, and other relevant info) on tables is recommendable.

Response 4:  Thank you for this highly valuable and insightful comment. We appreciate the guidance on citation backing, the use of objective language, and particularly the excellent suggestion to include a summary table for clarity.

In response, we  have created  a new Table 1 (Summary of Studies on Familial Clustering and Heritability in Myalgic Encephalomyelitis) in the manuscript on Page 3. This table directly addresses the reviewer's recommendation by compiling key data, methods applied, main findings, and other relevant information from multiple studies exploring the genetic component and familial clustering in ME.

Revised text to add new references on page 2: “Evidence supports a genetic component and heritability in ME. Familial clustering, indicating an elevated risk among relatives of affected individuals compared to the general population, has been consistently reported across multiple studies (Table 1) [21-23, 17] ”

Comment 5: Section 2.1: The term “significant” may mislead readers if not accompanied by statistic data. Please, review the sentences including: “..significantly increased risk” and “..a significant portion of the susceptibility to ME is indeed heritable.”

Response 5:  Thank you for your thoughtful comment regarding the use of the term “significant.” We agree that it may be assumed to mean statistical significance even whennot supported by appropriate data. To address this concern, we have revised the relevant sentences in Section 2.1 and rephrased the language to avoid unintended implications of statistical significance.

Revised text on page 2: “Evidence supports a genetic component and heritability in ME. Familial clustering, indicating an elevated risk among relatives of affected individuals compared to the general population, has been consistently reported across multiple studies (Table 1) [21-23, 17]”

Comment 6: The twin studies referred include 2 experimental and 1 review/opinion study. In addition, the 2 experimental studies cited were performed by a single research group. On one side, supporting data should be strictly experimental. On another the number of cases (independent observations) must be detailed to show the level of evidence.

Response 6:  We thank the reviewer for bringing this to our attention. We agree that the strength of evidence needs to be made more transparent and that sample sizes should be explicitly stated. In our revision, we now distinguish between review/opinion pieces and empirical twin studies, and we provide the number of twin pairs and concordance rates where available. This clarifies both the experimental basis and the level of evidence supporting heritability in ME.

Revised Text for Manuscript on pages 2 and 3: “To further discern the contributions of genetic versus environmental factors to familial aggregation in ME, investigators have used classic twin methodology. Three independent twin studies provide empirical data in this regard. In one study of 124 twin pairs (79 monozygotic [MZ], 45 dizygotic [DZ]), the cross-twin correlation for prolonged fatigue was substantially higher in MZ twins (r = 0.49) than in DZ twins (r = 0.16), indicating strong genetic influence on familial aggregation of fatigue. Interestingly, while immune activation markers were more strongly shaped by shared environmental factors, fatigue itself showed a major genetic component [24]. A larger study of 1,004 adult twin pairs (533 MZ, 471 DZ, all >50 years) simillarly found higher concordance for fatigue in MZ twins compared to DZ twins. Multivariate modeling revealed that a common genetic factor contributed to fatigue alongside distress, anxiety, and depression, but also that fatigue was influenced by an additional independent genetic factor, distinct from psychiatric traits. Notably, 44% of the genetic variance for fatigue was unique and not shared with other forms of psychological distress [25]. A third study of 146 female-female twin pairs, in which at least one twin reported ≥6 months of fatigue, found higher concordance rates for fatigue in MZ than in DZ twins across increasingly stringent diagnostic definitions. For idiopathic chronic fatigue (aligned with ME case criteria), concordance was 55% in MZ twins vs. 19% in DZ twins (p = .042), with heritability estimated at 51% (95% CI 7–96%) [26]. Together, these studies provide consistent experimental evidence for a heritable contribution to ME susceptibility, particularly when case definitions are more stringent. While shared environmental factors such as infections or stress are also relevant, the magnitude of the twin data indicates that genetic factors play an essential role in disease risk.”

Comment 7: GWAS studies (Section 2.3) need updating to include the recent preprint including results for the DECODE ME study (https://www.medrxiv.org/content/10.1101/2025.08.06.25333109v1 ), and additional information, such as a publication by the Garder group focused on ME (https://pubmed.ncbi.nlm.nih.gov/36517845/) instead, or in addition to, reference 32. Zhang et al., 2025 (https://pubmed.ncbi.nlm.nih.gov/40321247/ ) and other.

Response 7: We thank the reviewer for this helpful suggestion. We have carefully considered the DecodeME preprint. However, in line with journal policy and to ensure the use of peer-reviewed sources, we are unable to formally integrate preprint data into the manuscript. Instead, we have updated Section 2.3 to incorporate the most relevant published references, including the recent work by the Gardner group, alongside the Zhang et al. (2025) HEAL2 study, which complements GWAS findings through deep learning–based whole-genome analysis. The revised section now clarifies methodological distinctions, sample sizes, and major findings of each study, providing a comprehensive and up-to-date overview of the genomic landscape of ME.

Revised text on page 4: “The advent of genome-wide association studies (GWAS) has revolutionized the search for genetic associations by systematically scanning the entire genome for common variants linked to disease [31]. A recent study, the DecodeME project, recruited over 17,000 individuals with ME in its initial launch phase and provided important insights into disease heterogeneity [32]. Analysis of detailed questionnaire data revealed a clear female predominance (83.5% of participants), with females also reporting more comorbidities than males. Greater illness severity was significantly associated with female sex, older age, and illness duration beyond 10 years. Stratification by onset type further highlighted clinical subgroups, including cases following infectious mononucleosis, COVID-19, other infections, non-infectious onset, or onset of unknown infectious status, with the latter notably associated with comorbid fibromyalgia. These findings demonstrate how large-scale, population-based cohorts can identify phenotype–severity relationships that were not previously evident. Complementing this, Das et al. applied GWAS and a combinatorial analytics approach to UK Biobank-derived ME cohorts, identifying 199 SNPs across 14 genes, which stratified into 15 genetic clusters [33]. Many of these genes were linked to mitochondrial dysfunction, immune dysregulation, sleep disturbance, and autoimmunity, with replication across post-viral fatigue and fibromyalgia cohorts. In parallel, novel computational approaches have advanced genomic discovery. Zhang et al. employed whole-genome sequencing combined with the HEAL2 deep learning framework, identifying 115 candidate ME risk genes with intolerance to loss-of-function mutations [34]. Functional analyses implicated central nervous system and immune cell pathways, particularly cytotoxic CD4 T cells, with reduced expression of risk genes in patient-derived multi-omics data. This study supports the notion that both rare variants and gene expression changes contribute to disease risk, complementing common-variant GWAS results. Together, these advances demonstrate that both common and rare variants contribute to ME susceptibility, with converging evidence for roles of immune dysregulation, infection response, mitochondrial function, and neurological pathways.”

Comment 8:Section 2.4, please define “strong associations of HP genotypes with severe PEM and cognitive dysfunction ” referred for the study of Moezzi et al.,

Response 8: We thank the reviewer for this comment. We have clarified that the “strong associations” reported by Moezzi et al. refer to the correlation between Hp genotype distribution and symptom severity (PEM and cognitive dysfunction).

Revised Section 2.4 (with clarifications) on page 4: “Recent work by Moezzi et al. has highlighted the importance of structural variants in ME genetics, particularly their role in genotype–phenotype correlations underlying symptom heterogeneity [35]. Using high-resolution genomic mapping coupled with detailed clinical phenotyping, the study found that the distribution of haptoglobin (HP) genotypes, most notably Hp2-1, was correlated with more severe PEM and cognitive dysfunction. These findings provide a molecular explanation for some of the clinical variability seen in ME and support genotype-informed stratification as a strategy for biomarker discovery and therapeutic development. Incorporating structural variant analysis represents a pivotal step toward precision phenotyping, complementing SNP-based approaches and offering deeper insight into the genomic architecture driving PEM, cognitive impairment, and other hallmark symptoms.”

Comment 9: Page 5: Section 3.2 Please indicate in what cell types were “swollen or fragmented mitochondria, suggesting structural damage [48-49]” found. Also, cell type for: “WASF3 knockdown in patient-derived cells..”

Response 9: We appreciate the reviewer's helpful comment. We have clarified in Section 3.2, the specific cell types in which mitochondrial alterations and the effects of WASF3 knockdown were observed. The revised text now specifies that structural mitochondrial abnormalities were described in skeletal muscle fibers and in PBMC-derived T cells, while WASF3-related findings were derived from patient skeletal muscle cells and validated in transgenic mouse skeletal muscle.

Revised Section 3.2 on page 7: “Additionally, electron microscopy studies have revealed altered mitochondrial morphology in ME patients, with swollen or fragmented mitochondria observed in skeletal muscle fibers (subsarcolemmal and intermyofibrillar compartments) [56] and in stimulated peripheral blood mononuclear cells (particularly T cells) [57], suggesting structural damage. Changes in the expression of genes involved in mitochondrial biogenesis have also been reported [58–60]. A 2023 study by Wang et al. identified WASF3 as a key regulator of mitochondrial respiration and exercise intolerance in ME, based on analyses of skeletal muscle cells from patient biopsies. Overexpression of WASF3 disrupted respiratory supercomplex formation and reduced complex IV activity, whereas WASF3 knockdown in patient-derived muscle cells restored mitochondrial membrane potential and improved ATP output. These findings were further validated in transgenic mice, linking cytoskeletal signaling to mitochondrial inefficiency and highlighting WASF3 as a potential therapeutic target [59]”

Comment 10: Page 6: In the sentence: “This figure illustrates the key differences in energy metabolism between a healthy mitochondrion and an ME mitochondrion” revisit the term “the”. Is this indicating “some” or “reported/shown/informed”¿? Include citations used to illustrate Figure 2. The whole paragraph following

Response 10: We appreciate the reviewer’s comment. We have revised the sentence to clarify that the figure illustrates reported and proposed differences in mitochondrial energy metabolism between healthy controls and ME patients, rather than implying an absolute or exhaustive description. We have also included key citations in the paragraph to indicate the sources that informed the schematic representation in Figure 2.

Revised Text (for Figure 2 paragraph) on pages 7and 8: “This figure illustrates reported key differences in energy metabolism between a healthy mitochondrion and an ME mitochondrion, highlighting the proposed mechanisms contributing to the profound fatigue and post-exertional malaise (PEM) characteristic of the illness. Left Panel: Healthy Mitochondrion. Glucose is metabolized to pyruvate, which is then converted to acetyl-CoA to enter the Tricarboxylic Acid (TCA) cycle. The electron transport chain (Complexes I, II, III, IV, and Cytochrome C) efficiently drives oxidative phosphorylation, leading to robust ATP synthesis [52,61]. The Translocator Protein (TSPO, also known as ADP/ATP translocase or ANT) typically functions to facilitate the import of ADP into the mitochondrial matrix for ATP synthesis and the export of newly synthesized ATP to the cytosol for cellular energy utilization [62]. Right Panel: ME Mitochondrion. In contrast, ME mitochondria exhibit reported dysfunction. Glucose metabolism is often shunted towards anaerobic glycolysis, leading to increased lactate production even with minimal exertion [51,52]. ATP synthesis is markedly reduced, contributing to cellular energy deficits [53,54]. A major factor in this dysfunction is the proposed partial blocking of Translocator Protein (TSPO) sites, which impairs both ADP import and ATP export [62]. Additionally, ME is characterized by elevated oxidative stress, leading to an increased production of Reactive Oxygen Species (ROS), which can damage mitochondrial components and further impair function [45,63]. Deficiencies in essential cofactors like intracellular magnesium [64] and Coenzyme Q10 (CoQ10) [65] are also observed, further compromising ATP production. These combined metabolic impairments contribute to the inability of ME patients’ cells to meet energy demands, particularly under exertional stress, providing a mechanistic explanation for hallmark symptoms such as fatigue and PEM. Figure created using BioRender (WU28PQTCCB).”

Comment 11: Fig 2 lacks citations.

Response 11 :Thank you for this essential comment. We appreciate you pointing out  the omission of citations for Figure 2. We fully agree that all figures must be appropriately sourced. In response, we have reviewed the content of Figure 2 and have integrated relevant citations into its figure legend on pages 7 and 8.

Comment 12: Table 2, the text: “Baseline for comparison” is not clear.

Response 12: Thank you for this valuable comment regarding the clarity of the text 'Baseline for comparison' in Table 2 (now Table 3 in the revised manuscript). We fully agree that this phrasing was ambiguous and required greater precision. For Haplogroup H, the term was originally intended to convey that, as the most common haplogroup in Europe, it often serves as a reference group in genetic studies of ME due to its typical metabolic characteristics, in contrast to other haplogroups that carry more specific hypothesized effects. However, to completely avoid any potential confusion or misinterpretation, we have excluded the phrase 'Baseline for comparison' and revised the entry to be more descriptive and neutral, focusing solely on the established general characteristics of Haplogroup H.

Comment 13: Table 3, could be more complete. Very few studies are included.

Response 13: We appreciate the reviewer's suggestion. We have expanded Table 3 (revised as Table 4) on page 10 to include additional epigenetic mechanisms reported in ME, along with representative studies and examples of their potential impact. This provides a more comprehensive overview of the field.

Comment 14: Page 8: The following sentence needs a citation: “Notably, altered methylation of D-loop regions and mitochon drial tRNA genes has been linked to reduced respiratory efficiency and increased oxidative stress.”

Response 14: We thank the reviewer for pointing this out. We have added an appropriate citation to support the statement regarding altered methylation of mitochondrial D-loop and tRNA genes and their impact on respiratory efficiency and oxidative stress on page 10.

Comment 15: Page 11: HERV activation in ME and Long COVID-19, is also supported at the protein level, as shown in the following articles: https://pubmed.ncbi.nlm.nih.gov/40726775And https://pubmed.ncbi.nlm.nih.gov/36389746/

Response 15: We appreciate the reviewer's important suggestion. We have revised the section on HERV activation to clarify that evidence extends beyond transcriptomic data and is now supported at the protein level, as shown by recent studies detecting HERV-W ENV expression in both ME and post-COVID-19 condition patients. The references suggested by the reviewer have been added.

Text added on page 12: “Importantly, this activation is also supported at the protein level, as the toxic human endogenous retrovirus W (HERV-W) ENV protein has been found actively expressed (antigenemia) in post-COVID-19 condition patients long after infection and in pre-pandemic cases of ME and Fibromyalgia [109,110]”

Comment 16: Page 12: Although the sentence “Recent large-scale whole-genome sequencing studies..” refers “studies” in plural, only one article is cited. If this is a review article, authors should consider to mention “as reviewed by…”

Response 16: Thank you for pointing out this inconsistency regarding the plural use of 'studies' with only a single citation. We appreciate this attention to detail. In response, we have now located and integrated additional large-scale whole-genome sequencing studies into the discussion. This allows the use of the plural 'studies' to be accurately supported by multiple references page 13.

Comment 17: Page 13: in the sentence: “The top panel shows how methylation alteration..” “how” should be replaced by “that” . And the term “can” should be replaced by “could” or “may”. Likewise, this revision should be extended in the sentences that follow these.

Response 17: Thank you for this valuable comment regarding the precision and tonality of the language used in the Figure 4 legend. We fully agree that because these strategies represent potential therapeutic avenues, the language should reflect possibility rather than certainty.

We have reviewed the entire Figure 4 legend to implement the suggested revisions. The word 'how' has been replaced with 'that' to improve the grammatical flow of the descriptive sentences. The term 'can' has been replaced with modal verbs such as 'may' or 'could' throughout the figure legend and surrounding text to accurately convey the potential or hypothesized nature of these therapeutic approaches.

The revised Figure 4 legend on page 14 now reads as follows: “The top panel shows that methylation alteration, influenced by environmental factors such as diet and drugs, may be modulated to restore normal gene expression. The bottom-left panel illustrates that mitochondrial dysfunction, a key feature of ME, could be addressed with supplements such as Coenzyme Q10 and L-carnitine to enhance ATP production and mitigate energy deficits. This section also shows that antioxidants such as N-acetylcysteine may reduce cell damage by neutralizing reactive oxygen species (ROS), which contribute to oxidative stress. The bottom-right panel illustrates the potential for gene editing technologies to modulate the gene expression of specific ME targets, providing a precision medicine approach to correct underlying genetic dysfunctions. Figure created using BioRender(TU28PQT2GO).”

Comment 18: Comment Page 2: “Early investigations…” replace by “Initial investigations..or similar, since the timeline starts in early 2000, so to avoiding repeating the term “Early”

Response 18:  Thank you for this valuable suggestion. We agree that 'Early investigations' was repetitive. We have replaced it with 'Initial investigations’ on page 5 to improve clarity and avoid repetition.

Comment 19: Page 3: The text “Figure created using BioRender(SG28PQS95K)” should appear at the end of the Figure legend. This applies to the additional figures created with this tool.

Response 19:  Thank you for pointing this out. We apologize for the oversight. The BioRender attribution (SG28PQS95K) was indeed intended to be part of the figure legend for all relevant figures. We have now ensured that it is integrated directly at the end of each figure legend, correcting the formatting/spacing issue, as shown in the revised manuscript.

Commnt 20: Please review the sentence including: “increase with increasingly..”

Response 20:  Thank you for this valuable suggestion regarding the redundant phrasing. We have addressed this by revising the sentence to eliminate the repetition and improve clarity. This specific revision was implemented simultaneously with the comprehensive changes made to the twin studies section (Comment 6, page 2).

Comment 21: In the sentence: “Early investigations into the genetic basis of ME often focused on candidate gene studies.. ”the word “often” seems dispensable as all the evidence refers to focused studies.

Response 21: Thank you for this precise suggestion regarding the use of the word 'often.' We agree that, given the context, 'often' is dispensable and creates a subtle redundancy. We have removed it to make the sentence more concise and direct.

Revised sentence on page 3: “Initial investigations into the genetic basis of ME focused on candidate gene studies, examining specific gene variants or single-nucleotide polymorphisms (SNPs) that are thought to be involved in relevant biological pathways.”

Comment 22: Page 5: “post-exertional malaise” should be replaced by PEM, as the full term was already introduced in the text. Please review this throughout the document for all abbreviations.

Response 22: Thank you for this valuable comment. We agree that 'post-exertional malaise' should be abbreviated to 'PEM' after its initial introduction. We have thoroughly reviewed the entire manuscript and replaced all subsequent occurrences of 'post-exertional malaise' with 'PEM' to ensure consistency and improve readability throughout the document.

Comment 23: On Fig 4. It seems that “Diets and Drugs” should appear on the left side. Similarly, the lower panel background should appear in pink tone for consistency of “disease” status susceptible of treatment.

Response 23: Thank you for these insightful suggestions regarding Figure 4. We agree that these changes will significantly improve the figure's clarity, logical flow, and visual consistency with the proposed concepts. The revised Figure 4, incorporating these changes, is included on page 14 of the updated manuscript.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

I like this review, but I believe that with a few adjustments, the manuscript could be further improved:

1) Notably, recent mechanistic hypotheses implicate dysregulation of mitochondrial dynamics. For example, viral proteins (e.g., herpesvirus dUTPases) have been shown in experimental settings to induce hyperfusion and clumping of mitochondria (Franco A., Life, 2022), which resist mitophagy and recycling, thereby disrupting mitochondrial turnover and energy homeostasis. Could the authors please add a few sentences addressing this point?

2) Moreover, mitochondrial fusion and mtDNA exchange are closely correlated (Dang X., JMC, 2021). It would be helpful if the authors could also include a brief discussion on this relationship.

Author Response

Comment 1: 1) Notably, recent mechanistic hypotheses implicate dysregulation of mitochondrial dynamics. For example, viral proteins (e.g., herpesvirus dUTPases) have been shown in experimental settings to induce hyperfusion and clumping of mitochondria (Franco A., Life, 2022), which resist mitophagy and recycling, thereby disrupting mitochondrial turnover and energy homeostasis. Could the authors please add a few sentences addressing this point?

Response 1: We sincerely appreciate the reviewer’s insightful comment, which highlights the emerging hypothesis of dysregulated mitochondrial dynamics in ME. We agree that this is a critical aspect, particularly regarding the potential role of viral proteins in impairing mitochondrial turnover. To address this, we have revised Section 3.2 (Evidence of Mitochondrial Abnormalities in ME) to include discussion of mitochondrial dynamics, hyperfusion, and their potential links to viral agents and energy homeostasis. The suggested reference has also been incorporated into the text.

Added text in the manuscript on page 6: “Notably, recent mechanistic hypotheses also implicate dysregulation of mitochondrial dynamics (the balance between fusion and fission), which is essential for quality control. For example, in experimental settings, specific viral proteins such as herpesvirus dUTPases have been shown to induce hyperfusion and clumping of mitochondria [48]. These hyperfused mitochondria resist degradation and recycling via mitophagy, thereby disrupting overall mitochondrial turnover and energy homeostasis, which could directly contribute to the observed structural damage and functional deficits in ME [48].”

Comment 2:  2) Moreover, mitochondrial fusion and mtDNA exchange are closely correlated (Dang X., JMC, 2021). It would be helpful if the authors could also include a brief discussion on this relationship

Response 2: Thank you for this highly pertinent comment, which highlights the crucial biological link between mitochondrial fusion and mtDNA exchange. We agree that this relationship is foundational to mitochondrial quality control and should be included to complete the discussion on mitochondrial dynamics in Section 3. We integrated a clarifying sentence on this relationship immediately following the discussion of viral protein-induced mitochondrial hyperfusion in Section 3.2 (Evidence of Mitochondrial Abnormalities in ME). The suggested reference has also been incorporated into the text.

Added text in the manuscript on pages 6 and 7: “This process of mitochondrial fusion is crucial as it facilitates the exchange and complementation of mitochondrial contents, including mtDNA and other factors essential for maintaining respiratory function, further emphasizing the link between disrupted dynamics and energy deficits in ME [49].”