Dear Dr Fegraeus,

Thank you very much for submitting your Research Article entitled 'An endothelial regulatory module links blood pressure regulation with elite athletic performance' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

If you decide to revise the manuscript for further consideration at PLOS Genetics, please aim to resubmit within the next 60 days, unless it will take extra time to address the concerns of the reviewers, in which case we would appreciate an expected resubmission date by email to plosgenetics@plos.org.

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Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool.  PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. 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 us at figures@plos.org.

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To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Carrie J Finno, DVM, PhD

Guest Editor

PLOS Genetics

Gregory Barsh

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Fegraeus et al. report a follow-up study on earlier work regarding the genetics of athletic performance in horses. Starting from a previously identified and published sweep region of 19.6 kb downstream of the EDN3 gene, the authors were able to refine the associated region to 5.5 kb. This region harbors 14 single nucleotide variants (SNVs) that are in strong linkage equilibrium with each other. The authors determined that only two major haplotypes for these 14 SNVs segregate in the general horse population, which they termed elite performing haplotype (EPH) and subelite performing haplotype (SPH). Extrapolating from existing human data, Fegraeus et al. identify potential regulatory networks that provide new plausible and testable hypotheses how the variants might ultimately affect endothelin signalling and blood pressure regulation. The proposed phenotypic effect on endothelin serum levels and blood pressure was validated in horses with the different genotypes. Overall, this is a comprehensive and well presented study that enables a significantly improved understanding of the complex traits under investigation.

Specific comments:

(1)

The data availability statement is inacceptable. The raw data must be made publicly available prior to the publication of this manuscript and accession numbers must be given in the manuscript. Individual phenotypes and marker genotype data of the studied horses must be given in one or more supplementary tables to allow interested readers to repeat the presented analyses.

(2)

Figure 1: The figure would be much easier to grasp, if the displayed species were indicated in each of the four panels A-D (horse or human). The authors may want to consider to redesign panel D in order to better display the correspondence between horse and human. Alternatively, another supplementary figure may be added to display more details of the horse-human correspondence (e.g. a dot plot and/or a full sequence alignment).

(3)

Can you add some additional text with more information why the human sequence is twice as long as the horse sequence (11 kb vs 5.5 kb)? What is the repeat content in the two species in this region?

(4)

Figure S2: Please expand the figure legend and give more explanations. The legend should contain an easy to grasp list/explanation of all transcription factors, for which binding site consensus sequences are shown. Is a score or any other kind of measure available, how likely it is that these transcription factor will bind to the reference and alternate alleles? A supplementary table with all TF binding scores for all analyzed variable sites should be added.

(5)

Consider to replace SNP with SNV.

(6)

I assume that all horse genomic coordinates in this study refer to the EquCab3.0 assembly. If true, please state this explicitly at the beginning of the methods section.

(7)

Line 266: "Data not shown" is not allowed in PLoS Genetics publications. All underlying raw data must be made publicly available.

(8)

Line 389: Myostation -> myostatin

(9)

Line 664: Suit --> Suite (?)

Reviewer #2: The authors describe the identification of a causative genomic region (5.5 kb/14 SNPs, regulatory element acting on EDN3 transcription) for blood pressure regulation and athletic performance in horses based on fine-mapping and comparative analyses. Then, the authors defined and identified an elite performing haplotype (EPH) and a sub-elite performing haplotype (SPH). EPH is associated with higher plasma levels of EDN3 and lower levels of EDN1, and lower exercise-related blood pressure.

This research is well organized, but the following revision or comment are necessary.

I have read this manuscript in a previous round of peer review. In the previous manuscript, it was unclear whether the 5.5 kb site truly functioned as an enhancer site, as there were no results from gene expression analysis. The new Human HiCap analysis performed in this study may have compensated for previous weaknesses.

However, this reviewer has some concerns about Human HiCap analysis. While the author's main research is conducted on horses, HiCap analysis is conducted on humans. Is it appropriate to conduct an analysis using cells from different species in this way? Has this approach been used in other studies?

So, the authors should discuss discrepancies with horse FAANG and human HiCap analyses.

The author conducted WGS to comprehensively identify variants, but the number of animals analyzed seems to be small. Is there a possibility that a causative variant exists at a site distant from the 5.5 kb site?

Reviewer #3: Please see attachment.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: No: Please see my comments ot the authors.

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #3: No

Attachments

Attachment

Submitted filename: CBTreview.pdf

Dear Dr Fegraeus,

Thank you very much for submitting your Research Article entitled 'An endothelial regulatory module links blood pressure regulation with elite athletic performance' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address in a revised manuscript.

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. 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 us at figures@plos.org.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

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.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Carrie J Finno, DVM, PhD

Guest Editor

PLOS Genetics

Gregory Barsh

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have adressed my comments satisfactorily.

The data availability statement is now acceptable to me. I find it regrettable that the ethics permit for this study was issued by a Human Research Ethics Committee rather than a committee related to animal welfare (an IACUC). This study does not relate to human patients, it relates to animals. Furthermore, it is my understanding that this study is about variation of physiological blood pressure parameters, not a disease. I therefore think that the term "living patient" in the data availability statement is inappropriate. I do not understand why sequencing data of animals cannot be made publicly available. This sets a very problematic precedence for future research involving animals. However, I assume that it is not in the power of the authors to change legal requirements in Sweden.

Reviewer #2: This study comprehensively identified about 5.5 kb-region as endothelial regulatory module involved in blood pressure control based on racehorse exercise performance and blood pressure measurements, and the human HiCap data and transcription factor analysis.

This manuscript has been revised multiple times, with revisions noted by this reviewer. Therefore, this reviewer considers the current manuscript to be suitable for publication.

However, this reviewer requests comments or revisions on the following:

Comments:

In this study, elite haplotypes showed a high frequency in CBT and SB, but a low frequency of 0.07 in TB. Since TB is a racehorse, it may be expected that the frequency of this haplotype would be higher as the identified region is associated with racing ability. Why isn't that the case?

Is it due to the difference in thoroughbred and trotter racing? Or does this allele not function significantly in thoroughbreds? This reviewer was concerned because the title stated "with elite athletic performance."

Line 389: myostatin?

Reviewer #3: please see attached

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #3: No

Attachments

Attachment

Submitted filename: EDN_revision_review.docx

Dear Dr Fegraeus,

Thank you very much for submitting your Research Article entitled 'An endothelial regulatory module links blood pressure regulation with elite athletic performance' to PLOS Genetics.

 

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address in a revised manuscript.

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. 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 us at figures@plos.org.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

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.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Carrie J Finno, DVM, PhD

Guest Editor

PLOS Genetics

Gregory Barsh

Section Editor

PLOS Genetics

Dear Dr Fegraeus,

We are pleased to inform you that your manuscript entitled "An endothelial regulatory module links blood pressure regulation with elite athletic performance" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Carrie J Finno, DVM, PhD

Guest Editor

PLOS Genetics

Gregory Barsh

Section Editor

PLOS Genetics

www.plosgenetics.org

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----------------------------------------------------

Comments from the reviewers (if applicable):

Thank you for revising this manuscript based on the reviewers' comments.

----------------------------------------------------

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