PONE-D-21-01793

Is there a role for neuregulin 4 in human nonalcoholic fatty liver disease?

PLOS ONE

Dear Dr. Verbeek,

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an interesting well-conducted study. I have only a few specific points, all addressable with current data

Specific points:

The authors should provide further clarification of the microarray studies. They refer to their previous Gastroenterology patient, in which indeed, 15 patients had both adipose tissue and liver biopsies. I understand that these 15 patients were selected on the basis of availability of liver samples. If this was not the criteria, how were these 15 patients selected?

It is unclear from the report what is the starting point for the microarray analysis: previous frozen samples, previously isolated and preserved RNA, or was based on the analysis of the previous microarray readings (that have been made available in a public repository with the previous paper). If it is the latter, in the case of fat analysis (in which there were samples from 35 patients) is there any reason why the authors did not analyze all patients?

In the report of the microarrays, especially taking into account the small number of samples, the authors should provide additional heatmaps showing expression of individual patients (it is ok to show the means in main paper)

The authors point out that age was different between controls and NAFLD. The authors could show in a regression analysis that age does not explain the lack of differences in NRG4 levels between healthy controls and NAFLD

Since fibroscan provides a quantitative assessment of fibrosis (more or less imprecise), the association between NRG4 levels and fibrosis should be showed with the continues values of fibroscan, rather than categorizing the fibroscan values. Fig 2B should be like figure 2A, showing the scatterplot fibroscan vs plasma NRG4

The metrics of correlations shown in table 2 are not very informative for the reader. The authors should provide all the scatterplots as supplementary data.

Reviewer #2: The subject investigated by De Munck et al. in their manuscript is of interest. Despite evidence in mouse models that Nrg4 (likely to be originated in brown fat and perhaps in other adipose depots) may exert hepatoprotective effects for NAFLD, the relevance of these findings in humans is unclear. De Munck et al. show an associational study of NRG4 levels variations (plasma levels, gene expression) in patients with distinct conditions in relation to NAFLD. Despite the results were negative (in the sense that no clear correlations are found between NRG4/NRG4-mediated pathways and signs of pathology) data are worth to be reported in light of existing doubts on the role of NRG4 in human liver pathophysiology.

The study is reasonably performed and, despite the associational character of the study in relation to establishment of case-and-effect evidence (a limitation intrinsic to most human studies in this area), conclusions appear sound.

Suggestions for improvement are:

- In table 1, provision of standard values of metabolites, glucose/insulin parameters, hepatic enzymes, etc in the HC group (page 9) would had improved quality in the presentation of the cohort/s.

- I recommend that the authors perform a correlation study of TNFa and IL6 levels in relation to the NALFD-related parameters,..it would be interesting to see whether these inflammation-related parameters associate with NAFLD or parameters of metabolic disease in their cohort.

- Some statements provided in a clear-cut manner in Discussion: " microarray analysis did not show a difference of Nrg4 expression levels,..." (lines 293-4) or "none of the pro-inflammatroy cytokines correlated with plasma Nrg4 levels" (lines 309-310) are difficult to be identified as such in the Results section. Please include them explicitly in the Results section for clarity.

- Consider the possibility to validate at least a few key gene expression data (e.g. NRG4 gene expression itself) from microarray analysis using a qRT-PCR specific assay.

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Reviewer #1: No

Reviewer #2: No

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Is there a role for neuregulin 4 in human nonalcoholic fatty liver disease?

PONE-D-21-01793R1

Dear Dr. Verbeek,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Robin D Clugston, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No