PONE-D-23-09055Seven naturally variant loci serve as genetic modifiers of Lamc2jeb induced non-Herlitz Junctional Epidermolysis Bullosa in micePLOS ONE

Dear Dr. Sproule,

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 manuscript is in very good shape and the reviewers only had minor concerns with writing and asked for a few clarifications.

Please respond to those carefully, and review the writing and formatting (particularly of supplements and figure legends) to tidy up any potential typos or errors.

Please submit your revised manuscript by Jun 23 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,

Arnar Palsson, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

2. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. [Data from inbred strains (B6, 129, MRL, FVB) homozygous Lamc2jeb/jeb used as controls in this manuscript for congenics and F2 crosses (also Lamc2jeb/jeb) was mostly previously published as new findings in PMID 24550734. Inclusion of that information is necessary to make comparisons.] Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This work was supported by grants from DEBRA Austria, DEBRA UK, and The Jackson Laboratory.”

We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“DCR received awards 'Roopenian 1' from Debra UK (https://www.debra.org.uk/) and 'Roopenian 2' from Debra Austria (https://www.debra-austria.org/). The Jackson Laboratory (jax.org) provided supplemental support for these studies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. 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 (if provided):

The manuscript is in very good shape and the reviewers only had minor concerns with writing and asked for a few clarifications.

Please respond to those carefully, and review the writing and formatting (particularly of supplements and figure legends) to tidy up any potential typos or errors.

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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

**********

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: In this manuscript, the authors present a highly rigorous analysis of modifier QTL in a mouse model of Epidermolysis Bullosa (EB). Expanding on their 2014 PLoS Genetics publication that identified Col17a1 as the first definitive genetic modifier of EB, the authors present six additional QTL that, while apparently inconsequential in wild-type mice, significantly affect EB phenotypic severity in the context of homozygous, hypomorphic Lamc2 mutations. While causal variants are not definitively identified in the current study, likely candidates are presented based on several convincing lines of evidence. The manuscript is well written, and conclusions are justified as stated, though some sections may be challenging for non-experts to understand due to the complexity of genetic analyses. Overall, the study warrants publication in PLoS One. A few minor modifications/clarifications, as detailed below, may improve overall clarity of the manuscript.

General comments:

Some aspects of this study are quite complex, involving multiple distinct strains with important though not always straightforward genetic differences. It may be helpful to include a summary Table or Figure that details predicted (or known) genotypes for each QTL along with predicted effector gene(s).

In the final two sentences of the Discussion section, the authors provide solid rationale for how the data obtained in this study could be applied translationally to improve patient outcomes. It would be beneficial to include this rationale in the Introduction and/or Abstract as well to highlight its importance.

Specific comments:

Page 4, Line 75: The abbreviation "LTA" is not previously defined and is not obvious.

Page 9, Lines 166-167: It would be helpful to clarify that the short congenic strain was generated as part of the current study.

Page 11, Lines 192-194: Inclusion of body weight as a covariant may be appropriate, but it is not immediately clear how this variable would influence tail tension test results or why it was considered as a potential covariant, aside from the substantial variation observed. If inclusion as a covariant affects QTL significance for this cohort, is it appropriate to apply it to others as well (even though effects may be less)? Perhaps a bit more explanation would clarify matters for readers.

Page 14, Line 230: The authors refer to a specific dystonin isoform (Dst-e) as a modifier candidate. More background should be provided on dystonin isoforms and why this one was specifically mentioned (presumably due to expression patterns?).

Figure 3A: It is interesting that several, perhaps the majority, of F2 mice exhibit a tail tension phenotype indicative of lower disease severity (higher tension) than either of the parental strains. What is the interpretation of this result?

Figure 3B: The extremely high LOD score for the chr1 QTL makes it somewhat difficult to appreciate the significance of other QTL. It may be beneficial to include a sub-panel with a y-axis similar to that of Fig. 4B-C or use a staggered y-axis.

Reviewer #2: Epidermolysis Bullosa (EB) is a group of skin disorders caused by genetic mutations, several of which have been described in literature. Published genetic mutations were able to account for all subtypes of EB, but there still remains clinical variations within each subtypes that were not explained by the primary mutations. These phenotypic variations within the same primary mutations may be caused by enviromental factors or other genetic mutations. This manuscript dissects genetic modifiers that may contribute to the severity and clinical variations found in EB by using multiple F2 crosses of strains that are homozygotes for Lamc2jeb/jeb, which produces insufficient healthy laminin 332.

The present study reports six novel QTLs that were proposed to play a role in the clinical outcome of EB. For each QTL, the authors investigate the genomic area identified by the 95% confidence interval for missense SNPs/Indels. The authors used genetic variants in related additional crosses to reduce genomic regions of interest. Overall, the study was performed using appropriate experimental and statistical methods, and conclusions were supported based on the presented data.

Minor issues that authors should consider to address are:

1. Please provide the percentage of phenotypic variations (Tensions) explained by each QTL reported, which will help readers put into prespective the impact of each QTL on the outcome of EB.

2. The third effect plots in Fig 3C showed that mouse carrying allele BB has the highest tension, unlike the other three effect plots in the same figure. Could you please provide explanations.

3. This study identifies potential DNA variants (SNPs/Indels) that may regulate EB outcomes, which will be useful for other researchers. Please provide a list of SNPs/Indels of interest as in Fig. 5J that were identified for the rest of QTLs.

**********

6. 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

Reviewer #2: No

**********

[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.

Seven naturally variant loci serve as genetic modifiers of Lamc2jeb induced non-Herlitz Junctional Epidermolysis Bullosa in mice

PONE-D-23-09055R1

Dear Dr. Sproule,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Arnar Palsson, 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

Reviewer #2: All comments have been addressed

**********

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)

Reviewer #2: Yes

**********

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

Reviewer #1: (No Response)

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: (No Response)

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: (No Response)

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 Response)

Reviewer #2: (No Response)

**********

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

Reviewer #2: No

**********