PONE-D-22-19738Whole exome sequencing study identifies candidate loss of function variants and locus heterogeneity in familial cholesteatomaPLOS ONE

Dear Dr. Cardenas,

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. Specific concerns:

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PP CP BJ Rosetrees Trust Grant number R203056 https://rosetreestrust.co.uk/project-grant-applications/  no

PP Modi pump priming grant Royal College of Surgeons https://www.rcseng.ac.uk/dental-faculties/fds/research/fds-pump-priming-grants/ no”

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DSB acknowledges support received for the UEA Cancer Genetics team from Prostate Cancer Research, Prostate Cancer UK, Big C, and the Bob Champion Cancer Trust. Some of the research presented in this paper was carried out on the High Performance Computing Cluster supported by the Research and Specialist Computing Support service at the University of East Anglia.”

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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: Partly

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: Yes

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: Recommend to be cautious when concluding anything from this very explorative study.

Recommend in line 403 and 403 to find an alternative and less strong word than 'Evidence'

The authors provide analyses within families, between families and between/across single participants. Which of these is the main priority of the study? Explain your motivation for- and discuss the implications of all three approaches (interpritation the results). Cholesteatoma mostly occurs sporadically - not inheritably. Everything that leads to chronic ear disease (e.g. mucosal or skin diseases) can indirectly increase the risk for cholesteatoma. This is a point that I recommend to add when discussing rare familial/inherited genetic variants with consequences for e.g. cilial function - and the link between this and cholesteatoma.

Emphasize, that this is an explorative study and discuss the false discovery rate/ likelyhood of incidental findings in these small groups.

Reviewer #2: The authors addressed familial cholesteatoma genetic architecture via whole exome sequencing (WES) for 21 individuals treated for cholesteatoma recruited from ten affected families. They searched for single nucleotide variants, insertions and deletions and performed a hard filtering to these variants to obtain high confidence variants. They fetched for variants that are shared between affected individuals within families. They performed gene-level mutational burden analysis and enrichment analyses. They detected the genes that showed rare loss of function (LOF) variants and identified six genes (DENND2C, DNAH7, NBEAL1, NEB, PRRC2C, and SHC2) which have LOF variants in two or more families. Functional enrichment analysis of the detected genes revealed 6 common pathways including calcium ion binding, extra-cellular matrix (ECM) organization, and GTPase activity. The missense variants were analyzed to predict the impact on protein functionality. All variants were classified as damaging/deleterious missense variants. One of DNAH7 variants (rs115474479) is classified as an indel (stop gained) mutation. Authors suggest that DNAH7 variants are of interest because they encode a protein component of human cilia, whose functional mutations have been associated with primary ciliary dyskinesia (PCD) which accompanies Cholesteatoma. The study used multiple bioinformatics tools in for analysis. There are some concerns for the improvement of the manuscript.

1. Please, mention the number of participants and number of corresponding families in the materials and methods section.

2. Would you please, mention the storage conditions of samples (line 160)?

3. Please, include fig S1 in the main figures as it shows the 6 common pathways of genes obtained by the two methods of analyses. Please, add a figure caption for it and unify the way of writing the labels (Upper case or lower case for the first letter).

4. Lines 321-323: ‘Calcium ion binding’ is repeated in the following part ‘’ These 6 included calcium ion binding, extra-cellular matrix (ECM) organization, GTPase activity and calcium ion binding, containing a larger number of genes for each term in the mutational burden analysis (Fig S1)'', so please correct it.

5. There are some typos and punctuation errors need to be corrected.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-22-19738R1Whole exome sequencing study identifies candidate loss of function variants and locus heterogeneity in familial cholesteatomaPLOS ONE

Dear Dr. Brewer,

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 Mar 09 2023 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,

Rafael da Costa Monsanto, M.D.

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.

Additional Editor Comments:

Please address the final comments made by the reviewers.

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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

**********

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

**********

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 further comments.

Again, the comparison of findings with findings from other studies is very interesting. It would be interesting to know more about the roles of COCH and S100 proteins i cholesteatoma pathogenesis.

Reviewer #2: Comments

The authors have addressed all mentioned comments in a satisfying manner.

However, I have a couple of questions:

1) Regarding figure 4 “Fig 4. Common pathways enriched. Common pathway and ontology terms found to be enriched for genes containing deleterious variants (p < 0.01; Hypergeometric test) in both the family overlap (red) and TRAPD (blue) analysis. The number of genes with deleterious variants in each pathway or ontology term are shown.”

Is there a significant difference between the number of genes of family overlap (red) and TRAPD (blue) in each pathway? Can you show this on the bar chart and the error bars ?

2) According to your findings do you have any future perspectives concerning the study of genetic variants in cholesteatoma within and between families?

3) Please, revise the typos.

Example:

Our study objectives were

1. to establish a database of multiply affected families; to record their family histories

(for otology and genetics); and to collect biological samples from participants for

DNA extraction and storage in a biobank.

Put it in uppercase please.

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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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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.

Whole exome sequencing study identifies candidate loss of function variants and locus heterogeneity in familial cholesteatoma

PONE-D-22-19738R2

Dear Dr. Brewer,

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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Kind regards,

Rafael da Costa Monsanto, M.D.

Academic Editor

PLOS ONE

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