PONE-D-21-21760Prevalence of uncoupling protein one genetic polymorphisms and their relationship with cardiovascular and metabolic healthPLOS ONE

Dear Dr. Flouris,

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 Dec 20 2021 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,

Narasimha Reddy Parine, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

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

1. 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. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.

3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. 

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

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

The case-control study was supported by funding from the European Union 7th Framework Program (FP7-PEOPLE-2013-IRSES Grant No. 319010; U-GENE project). The case-control study also received funding by the Russian Foundation for Basic Research (grant 19-34-51003). The Polish research center has received additional funding for the case-control study from the Polish Ministry of Science and Higher Education 2016-2017 international project co-financed W15/7.PR/2016.

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: 

NO

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

5. Thank you for stating the following in your Competing Interests section:  

NO authors have competing interests

Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now 

 This information should be included in your cover letter; we will change the online submission form on your behalf.

6. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[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: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: No

Reviewer #2: N/A

**********

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: No

Reviewer #2: No

**********

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: No

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, Dinas and co-workers carried out a population-based genetic epidemiology study, aimed to document the frequency of four UCP1 gene polymorphisms and to examine its association with cardiovascular-metabolic health in 2283 multiethnic European individuals. Besides, the authors have carried out a meticulous meta-analysis cum systemic review to extend their findings. Overall, the manuscript is well written, and the attempt of the authors should be praised. However, there are some significant issues in the manuscript that need to be addressed.

Comments:

1. The authors are advised to revise the language of the manuscript, as some passages are inaccurate or contain semantic and syntactic mistakes. Further, there are several typos and spacing issues in the manuscript (for example: on line 86, the word ‘relat-ed’, on line 154 and 212, the word ‘be-tween’, on line 236, the word ‘statis-tical’, on line 360 ‘SNPs UCP1 SNPs’etc). This needs to be addressed.

2. The most important result of the study might have also been influenced by a selection bias. How were the probands chosen? How do the authors define an individual as Russian, Armenian ethnic, and healthy? This is a population-based genetic epidemiological study, and it is important to briefly describe the influence of ethnicity/race on inter/intra ethnic variation in the frequency distribution of germline variants.

3. I was wondering why there is no data on the clinical and biochemical features of the study subjects. And whether multivariable analysis to evaluate independent associations has been performed. Further, the paper is assessing several associations facing the risks of multiple testing. Correction is warranted.

4. Add a note in the introduction/methods stating the putative function of the studied SNPs (alters expression, alters binding site, etc), chromosome location, and position. Further, the author should comment on the possible molecular mechanism by which the studied polymorphisms influence the disease risk.

5. What is the time span for the study recruitment? Did they validate the genotyping methodology? If yes, please mention it. Also, the authors need to elaborate on whether a phenotype-blind genotyping process was pursued.

6. Several other demographic/environmental factors and social determinants were not explored in this study which could potentially affect the associations. Do the authors think that this study has any limitations? It is missing in the discussion section.

7. What about the association analysis of UCP1 gene polymorphisms with CMP risk, stratified by gender?

8. Potential ancestral differential effects should be discussed more as they present a considerable contribution to the study. Throughout the manuscript, the gene names should be in italics.

Reviewer #2: # General Comments

- The authors performed a genetic association study to test the influence of genetic variants of UCP1 on cardiometabolic pathologies (CMP) and their risk factors. They included Caucasian individuals from different populations. UCP1 is a key protein in the function of brown adipose tissue, which is implicated in the non-shivering thermogenesis. The authors found different allele frequencies between cases (CMP) and controls for some UCP1 SNPs in some populations. In addition, some alleles/genotypes were associated with CMP risk factors. I think this study can be relevant for improving our understanding of the genetic architecture of CMP and the role of UCP1. However, I have some comments and concerns.

# Specific comments

- Line 67: Missing comma before "which".

- Lines 76-78: The G allele of A-3826G associates with higher HDL? This would mean less cardiometabolic risk.

- Line 86: "related" instead of "relat-ed"

- Line 92: do you mean the frequency of the GENOTYPE AG among persons with CMP? Maybe add "AG genotype".

- Lines 102-107: I agree with the point that a better knowledge of the genetic architecture of CMP can help in their prevention and treatment, but this statement focused on UCP proteins is too strong. Cardiometabolic traits are complex, being influenced by many genes with small effect sizes. Improving our knowledge about the impact of UCP1 variants can contribute to precision medicine, but within the context of polygenic scores considering thousand of other variants. It is unlikely that these common variants can have practical relevance in isolation. Please, modify and soften this statement.

- There is any control for population structure? I understand that you performed analyses within each region, removing the influence of genetic clusters across Europe. However, structure could also exist within a population. You had evidence that all individuals in each group belonged to the same race/ethnicity and individuals were not related? If not, please clearly state this as limitation of the study.

- I do not fully understand table 1.1.3 in supplementary. The prevalence is the result of dividing cases with a given genotype by the total sample size. Then, the prevalence should be between 0 and 1. Maybe the prevalence is shown in brackets, while the actual number of genotype carriers is show above? Please, clarify.

- I am not very familiar with the methodology of meta-analyses, this could be also the case for other readers. Could you extend the explanations and rationale of the decisions made? Several choices were made based on previous guidelines. Maybe you could explain briefly the rationale in the supplementary.

- Figure 1: I would avoid the colors in Figure 1. It makes difficult the visualization of the patterns. I think it is enough with the country name at the bottom. I would also add a legend in the plot reminding that patterned bars are "healthy" and solid bars are "CMP".

- I have not seen any control for multiple comparison. When multiple tests are performed, the risk of false positives accumulates across the different P<0.05 tests performed, increasing the probability of type I error. I am aware that this study is using just a few SNPs and they show linkage between them, which decreases the number of independent tests. Under that scenario, a stringent control of false positives like the Bonferroni correction would be too stringent. However, I think the authors could use other approaches less stringent, like the False Discovery Rate. They are considering several populations and phenotypes, so the total number of tests is not low. At minimum, a mention to this aspect should be done in the Discussion, indicating that a control for increased false positive rate has not been done despite the multiple tests performed.

- In line with the previous comment about the risk of false positives, I think it would be good to add some additional evidence about the functional relevance of the variants. The impact of Ala64Thr is clear, but what about variants from non-coding regions? For example, A-112C seems to modify 4 transcription factor binding sites and its region has specific patterns of chromatin accessibility in several tissues (i.e., DNAse). However, a SNP in linkage disequilibrium (rs72941746) alters much more transcription motifs and more proteins binds in that position (from Haploref, see link below). This could be the causal variant behind the associations of A-112C. Indeed, RegulomeD gives a score of 0.81 to this variant for its support as functional, while A-112C gets 0.61 (the score ranges from 0 to 1). I think additional support coming from functional data can be relevant, specially considering the negative results in the meta-analysis.

- https://pubs.broadinstitute.org/mammals/haploreg/haploreg.php

- https://regulomedb.org/regulome-summary/?regions=rs10011540%0D%0Ars72941746&genome=GRCh37&maf=0.01

- line 260: "compared" instead of "com-pared". There are several typos like this through the manuscript. Please, check it.

- Individuals of the Greek population carrying the C allele of A-112C are 37% less likely to develop CMP. However, healthy individuals with the AC genotype have higher BMI than AA carriers for the entire sample. Do you have any potential explanation for this pattern? You mention in Discussion the existence of contrasting patterns between healthy and CMP groups, but I do not see a convincing explanation.

**********

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.

PONE-D-21-21760R1Prevalence of uncoupling protein one genetic polymorphisms and their relationship with cardiovascular and metabolic healthPLOS ONE

Dear Dr. Flouris,

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 31 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,

Narasimha Reddy Parine, Ph.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.

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

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: (No Response)

**********

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: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: (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

Reviewer #2: (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: Yes

Reviewer #2: (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: In this revised manuscript, the authors have satisfactorily responded to all the concerns from the previous review and made tremendous improvements in the revision. However, a few things need to be taken care of before any publication is warranted.

On line 246-247: the haplotype analysis revealed that CMP individuals 247 were 24% less likely to carry the GAC haplotype, what do you mean by?

On line 400: It should be written as ‘the A-3826G, A-1766G, Ala64Thr and A-112C SNPs of UCP1 gene’

Reviewer #2: # General Comments

- In general, the authors has addressed the comments well. I have just a few specific comments about the revision.

# Specific comments (lines refer to the manuscript version with track changes)

- Line 114: As a modification of my own previous comment, it would be better to say "Improving our knowledge about the impact of UCP1 variants can contribute to precision medicine, within the context of approaches that consider the polygenicity of cardio-metabolic traits (e.g., polygenic risk scores)."

- Line 176: I do not agree with the argumentation made by the authors. I do think that the risk of false positives and false negatives should be considered depending on the question and the goal. Despite this, I respect their decision of not doing it. This has to be stated in the manuscript, as they have already done, so the reader can take this aspect into account when interpreting the results.

- Line 330: Strange wording: "the A-3826G (AG) was the most prevalent of the four SNPs studied in persons with CMP was 40%,". I guess the prevalence was 40% overall. Please, modify.

**********

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

[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.

Prevalence of uncoupling protein one genetic polymorphisms and their relationship with cardiovascular and metabolic health

PONE-D-21-21760R2

Dear Dr. Flouris,

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,

Narasimha Reddy Parine, Ph.D

Academic Editor

PLOS ONE