PONE-D-21-38918The impact of PNPLA3 allele dose on biliary bile acid composition and disease course in patients with primary sclerosing cholangitisPLOS ONE

Dear Dr. Färkkillä,

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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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: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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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: The title reflects inappropriately the content of the study, in which no impact of PNPLA3 allele dose was find neither on BA composition nor disease course in patients with PSC. Please rephrase.

The allele groups of patients are not balanced in numerosity: only 5.9% of patients were mutated.

Was there any difference between the wildtype group and the CG-GG group?

To the knowledge of this reviewer, the pathogenesis of PSC is still unknown: published evidences address the PSC development to defects in mechanisms protecting against bile acid toxicity. In addition, the strong relationship of PSC in patients with IBD support the hypothesis of pro-inflammatory microbial components to the portal circulation and the possibility of an antigenic trigger of microbial origin. In the discussion authors say that to date, no data is available on the PNPLA3 variants and the expression of FXR. Though, to the knowledge of this reviewer this field has been explored in NAFLD patients, also with the analysis of gut microbiome. (Chen F et al., Lean NAFLD: A Distinct Entity Shaped by Differential Metabolic Adaptation. Hepatology. 2020) It could be interesting to know whether a correlation between PNPLA3, FXR expression, BA composition and gut microbiome could be found in PSC patients.

The statement in the discussion should be amended.

Authors reported 73%, 81%, 73% of patients with associated IBD according to CC, CG, GG PNPLA3 haplotype. There is no description of the severity of the disease, nor the medical-surgical treatment. Could you present these data?

Authors presented in the results section that P-AST and P-GT were significantly increased with increasing allele dose. Could you give an interpretation of this finding in the discussion?

Reviewer #2: Dear authors, thanks for giving me the opportunity to review your manuscript.

In the manuscript, the authors aim at demonstrating how the presence of different alleles of PNPLA3 might influence clinical outcome and bile composition in patients with primary sclerosing cholangitis.

Despite the conflicting results of the other studies available in the literature and the numerous sample studied (not always available in case of rare diseases), in the reviewer's opinion, the rationale of the study is not convincing and, unsurprisingly the results confirm it. PNPLA3 has been implied in fibrogenesis and carcinogenesis and has been linked to hepatocarcinoma which is not a frequent tumor in PSC patients. Furthermore, PSC patients usually develop liver fibrosis as a consequence of cholestasis and in many cases, the fibrosis is confined to the bile ducts. The mechanisms by which PNPLA3 induces liver damage are not completely understood and, in the reviewer's opinion, the rationale for studying their alleles in PSC is weak. However, the evaluation of bile acid composition may be interesting in these patients but a lot of confounding factors might be involved in this analysis (i.e. microbiota composition and IBD activity) which are not taken into account.

Furthermore, there are many punctuation errors in the manuscript and imprecisions.

Introduction:

The introduction is not fluent and the rationale is not well explained.

I suggest rephrasing sentences in lines 76 and 81.

In line 88 I would add that the UDCA effect on LT-free survival and liver-related death has never been demonstrated.

Methods:

The part on statistical analysis is too long and full of detail with respect to the data shown in the results section.

You mention a "compound" outcome in the results but I suppose composite outcomes have never been mentioned in the methods section. I suggest specifying the endpoints in this section.

Results:

line 162, specify the unit of measure of median values in brackets.

line 175, time to the first dilation since when? diagnosis? ERC?

in table 2 I would have distinguished HCC from CCA, which is, in the reviewer's opinion, quite important to the aim of the manuscript.

An English editing is suggested.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-38918R1PNPLA3 allele frequency has no impact on biliary bile acid composition and disease course in patients with primary sclerosing cholangitisPLOS ONE

Dear Dr. Färkkilä,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Nov 18 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Antonio De Vincentis

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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

Reviewer #2: 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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: Yes

Reviewer #2: Yes

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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: Yes

Reviewer #2: Yes

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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: Thank to the authors who addressed properly to all the raised questions. No further comments from this reviewer

Reviewer #2: In the abstract:

I would change “clinical manifestations” with clinical outcomes.

In the conclusions “Conclusions: PNPLA3 I148M variants have no a significant impact on either on bile composition including UDCA content, clinical manifestations, progression of liver fibrosis, the risk for hepatobiliary cancers, transplantation, or overall survival”. Check the grammar. For example on both before and after either and a before significant.

In the introduction:

- The reviewer would shorten the very long introduction. For example the sentence “However, PSC is associated with markedly increased risk of cholangiocarcinoma (CCA) (2), with lifetime risk 10%-20% (3,4), or 398 - 1000-fold compared to the general population (4,5). Several risk factors for CCA in patients with PSC, such as inflammatory bowel disease (IBD), especially ulcerative colitis (6), and older age at PSC diagnosis have been identified (4,7).” Is not so important in the context of the manuscript, in fact in the following part of the introduction you mention in patients with PNPLA3 variants have an increased risk of HCC and you don’t mention anymore CCA.

Some of the points explained in the introduction should be moved to the discussion which is shorter with respect to the introduction.

Results:

- Wording error in line 192.

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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

Reviewer #2: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PNPLA3 allele frequency has no impact on biliary bile acid composition and disease course in patients with primary sclerosing cholangitis

PONE-D-21-38918R2

Dear Dr. Färkkilä,

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.

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Academic Editor

PLOS ONE