PONE-D-24-11417Bidirectional mendelian randomization analyses explore the relationship between various cathepsins and uterine leiomyoma: Cathepsin B is the risk factor of uterine leiomyomaPLOS ONE

Dear Dr. Liu,

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.

the editorial office also wants to add the following:= albeit a large number of SNPs analysed, please ensure to use a relatively high genome wide significance threshold ie p < 10^-8 in order to produce an enough significant SNPs

= to include details on multiple testing correction to justify for an accurate data analysis

= it is necessary to use literature or a tool such as phenoscanner to validate whether the identified SNPs are associated with any other outcome or exposure

= interpretation and conclusions drawn must be supported by a robust methodology in order to fulfill our publication criteria 3 (https://journals.plos.org/plosone/s/criteria-for-publication)

Please submit your revised manuscript by Aug 07 2024 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.

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Academic Editor

PLOS ONE

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2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

- https://doi.org/10.1017/thg.2023.48

(among others)

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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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: No

Reviewer #2: Yes

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

Reviewer #1: No

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: Yes

Reviewer #2: Yes

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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: Summary:

Liu et al. present a well-motivated analysis on using Mendelian Randomisation (MR) to assess the genetically predicted causal effect of cathepsins on the risk of developing uterine leiomyoma UL). They used publicly available summary statistics to conduct two-sample MR with widely utilized software. They found that cathepsin B (CTSB) is associated with increased risk of uterine leiomyoma in sub-group analyses excluding cancer.

Cause for Major Concern:

Line 175: “Further MR analysis was performed on the subgroup of UL (excluding all cancers)” – please specify how this subgroup was identified. This is a major concern as I do not see a phenotype for UL (excluding all cancers) in the public FinnGen GWAS data release. I am only able to see GWAS results for three uterine cancers – carcinoma in situ of cervix uteri, malignant neoplasm of corpus uteri, and malignant neoplasm of cervix uteri, where the controls exclude all cancers. So please provide: 1. The FinnGen endpoint code (for example for UL it is: CD2_BENIGN_LEIOMYOMA_UTERI), 2. Number of cases and controls, and 3. An explanation from the FinnGen database of how this subgroup of UL (excluding all cancers) was selected. As this is the analysis with significant results, it is not possible to interpret the results presented without this information.

Other Major Comments:

1. The Introduction requires more references, for example: line 74, line 80, line 84, etc. that explain the prevalence, aetiology, and risk factors for UL.

2. Line 110: Please implement Steiger filtering, which is available in the TwoSampleMR package, to orient the causal relationship between cathepsins and UL. This can be in addition to the reciprocal MR results presented, but is necessary to ensure that instrument SNPs have stronger effects on the exposure than on the outcome. Ref: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1007081

3. Line 131: Please explain why such a high genome-wide significance threshold (P < 5E-6) was selected, when the standard in the field is P < 5E-8. Such a high threshold increases your chance of having spurious SNPs in the instrument, and also may lead to a weak instrument. Ref: https://www.nature.com/articles/s43586-021-00056-9

4. Line 132: Please explain the identification of independent SNPs in more detail. Did you use the LD-clumping procedure in the TwoSampleMR package? Please specify if so, and also move the distance for clumping (currently on line 131, which says 10,000 kb) to line 132.

5. Line 136: What are the “confounding traits” you tested? Please explain list which SNPs were removed due to association with confounders.

6. Line 138: Please briefly outline how MR-PRESSO identifies outliers, i.e. what criteria make a SNP an outlier?

7. Line 140: It is crucial to provide the F-statistics for all the instruments in all the various analyses.

8. Line 142: It is not correct that “IVW method is the gold standard of MR”, it depends on what assumptions are violated. If none are violated, then IVW is the most sensitive method. Please modify. Ref: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5506233/

9. Line 153: Since you are performing 9 MR tests (for all 9 cathepsins), you should adjust your P-value for multiple testing. Moreover, since you also perform reciprocal MR (another 9 tests), I would recommend Bonferroni correction (significance at P < 0.05/18) or for less stringency as the cathepsins are all somewhat correlated, FDR correction. If you apply Bonferroni, then none of the results will be significant.

10. Line 157: Please specify which SNPs were removed by sensitivity analysis, and which of the various sensitivity analyses you carried out (leave-one-out, heterogeneity tests, MR-PRESSO, etc.) caused this.

11. Lines 158-160: The leave-one-out analysis should produce 11 plots, as you have 11 SNPs in the instrument and each forest/scatter plot will display results from repeating the MR without one of the SNPs. You have only provided one forest/scatter plot – please check.

12. Lines 167-172: To my understanding, this is just re-stating the same things already reported in lines 158-161 (but the P-value reported is slightly different, 0.878 vs 0.876 – typo?) Remove this section or explain.

13. Line 178: “…remaining cathepsins showed no statistical significance.” Why is the protective association of Cathepsin O on UL excluding cancers (Figure 3) not discussed? The strength and significance of this association are comparable to CTSB (OR=0.93, P=0.019).

14. Figure 1:

a. Could the authors name some specific confounders they evaluated (as they mention in line 136)?

b. The “Exposure” box should cite the study from which genetic instruments were derived.

c. Minor comments: what does the symbol represent in the “Instrumental variables” box? In the “Mendelian randomization analysis” box, MR-PRESSO is mis-spelled.

15. Figures 2, 3, and 4:

a. Include instrument F-statistics in these plots

b. Adjust the P-value for multiple-testing correction

16. Figure 4: Please extend the axes to capture the full 95% CIs as these are not so large as to make the plots meaningless. In the top plot, include at least [0.95, 1.25] and in the bottom plot, [0.75, 1.30].

17. Supp. Table 1: It seems only one SNP for CTSB would pass a genome-wide significant threshold of P<5E-8, so I’m worried that the results are driven by a single SNP. The leave-one-out plots are also missing. You must also provide F-statistics so we can evaluate the strength of the instrument.

Minor Comments:

1. Please split the Introduction into three separate paragraphs – one on the aetiology of UL, one on cathepsins, and one on Mendelian Randomisation.

2. Line 107 & Figure 1 caption: Please correct that you use multiple GWASs as this is two-sample MR, not “a genome-wide association study (GWAS)” which means one single study.

3. Line 121: Please specify that you construct instruments for cathepsins for forward MR, and instruments for UL for reciprocal MR – it currently reads like you only did the latter.

4. Line 122: Which data release of FinnGen did you use?

Reviewer #2: The authors used bidirectional Mendelian Randomization to explore the causality of UL and found that Cathepsin B SNP is related to UL incidences. The authors used sound logic and methodology in this study. The data analysis was performed rigorously. However, I'd like to raise several questions for the authors:

1> In the abstract session, the authors stated that there are "Emerging evidence suggests a tentative association between cathepsins and uterine leiomyoma.", but could the authors give some citations to support this statement?

2>Can the authors specify the standard for eliminating the SNPs for the sensitivity analysis and quote studies using similar methodology?

3> Can the authors verify the findings using another independent cohort?

4> There's a grammar error in line 157-158.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-11417R1Association between various cathepsins and uterine leiomyoma: A Mendelian randomization analysisPLOS ONE

Dear Dr. Liu,

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 Sep 30 2024 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,

David Chau

Academic Editor

PLOS ONE

Journal Requirements:

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: (No Response)

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

**********

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

**********

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

**********

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: Summary:

Congratulations to Liu et al. for thoroughly addressing all my comments. My remaining concerns are mostly about the interpretation of results rather than the methodology.

Comments:

1. The association of cathepsin B with UL does not remain after:

a. Adjustment for multiple testing, or

b. Steiger filtering

Both of these give me cause for concern, as they indicate that this is likely a spurious association. While the authors mention this in the Limitations, these two major limitations should be presented in the Abstract, which currently has no indication that the result might not be real. Additionally, please make it clear that the presented finding is for cathepsin B on UL “excluding all cancers”.

2. The authors say they conducted independent validation of their findings using external datasets (in their response to reviewer #2). Why are these results not presented in the manuscript? I understand that they don’t have replication of the findings for UL-excluding cancer, but as the UL (not excluding cancer) result is also presented in the manuscript, it is worth showing the replication of this finding.

Reviewer #2: (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.

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Reviewer #1: No

Reviewer #2: No

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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.

Association between various cathepsins and uterine leiomyoma: A Mendelian randomization analysis

PONE-D-24-11417R2

Dear Dr. Liu,

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,

David Chau

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)

**********

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

Reviewer #1: (No Response)

**********

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)

**********

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)

**********

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

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