A 6-CpG validated methylation risk score model for metabolic syndrome: The HyperGEN and GOLDN studies

Bertha A. Hidalgo; Bre Minniefield; Amit Patki; Rikki Tanner; Minoo Bagheri; Hemant K. Tiwari; Donna K. Arnett; Marguerite Ryan Irvin

doi:10.1371/journal.pone.0259836

Peer Review History

Original SubmissionApril 19, 2021
24 Jun 2021 Decision Letter - Kyle J Burghardt, Editor PONE-D-21-04138 A 6-CpG Validated Methylation Risk Score Model for Metabolic Syndrome: The HyperGEN and GOLDN Studies PLOS ONE Dear Dr. Hidalgo, 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 enhance your rationale and justification for your candidate CpG approach described in the methodology to ensure that one can understand how your selection process is robust and accounts for all the critical literature for your investigation. Please submit your revised manuscript by Aug 06 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. 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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. In your Methods section, please ensure that sufficient information to make the study reproducible are provided (for example, by describing the models and equations used, and describing parameters and assumptions applied). 3. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. 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Dr. Hidalgo is funded by 5K01HL13060904. Ms. Minniefield is funded by 3R01HL123782-02S1." We note that you have provided funding information that is not currently declared in your Funding Statement. However, 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: "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. 5. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. [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: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: 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 Reviewer #3: 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 Reviewer #3: 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 study Hidalgo, et al develop a risk score for metabolic syndrome, based on DNA methylation at 6 CpG sites identified through a prior literature review. Additionally, this study is novel for its focus on a population of primarily African ancestry in addition to one of European ancestry (in contrast to the many studies that primarily focus on the latter). The article is generally well-written but I believe that it does not go far enough into its analyses, and does not provide enough crucial details, to merit publication without substantial revisions. My primary concern is the lack of detail regarding the selection process for the six CpGs. Based on my own (admittedly, cursory) literature search, it seems like a lot more has been published than that for MetS alone (not to mention its individual components). Why did these six CpGs make the cut, and others did not? MetS and/or related traits is a very, very broad category of the literature. Surely there are more than 6 CpGs that have been associated with them, so how did the authors narrow their list further? The reader needs more information on this selection process in order to evaluate its rigor. Where did they search? How many articles did they review? How many CpGs did they decide against including? I find this particularly puzzling given the focus on African-ancestry populations in the introduction and discussion- four of the six CpGs used to construct the MRS were only validated in European- and Asian-ancestry individuals, and by the authors’ own arguments they shouldn’t be assumed to have a relationship in African-ancestry individuals. Additionally, a number of the single-CpG results are non-significant if you account for multiple testing. That isn’t necessarily a problem, but it does mean that the theoretical basis for selecting these six as candidates needs to be very well-justified. I also think that this discussion needs to be rewritten and expanded upon in more detail. There is no mention of the specific genes used to construct the MRS, their functions, their roles in MetS pathogenesis, or why there might be an association at all. Instead, the authors discuss other risk scores developed to measure related but different cardiovascular endpoints. There is very little presented here to relate the authors’ findings to the wider literature, beyond the fact that they all studied a methylation risk score for something. On a related point, I think the authors could improve the impact of this study (and find more to talk about in the discussion) by running a set of secondary analyses. Specifically, similar models as for Table 4, but instead of MetS as the dichotomous outcome use each of the individual components of MetS and model them separately. By comparing these associations (even without assessing statistical significance) the authors can potentially identify which specific components the MRS is capturing, which in turn could provide some insight into the relationship between DNA methylation biomarkers and the development of MetS. Some minor comments: - Consider adding the MRS risk score to Table 1, as some descriptive analysis will be a good way to assess its performance. - The methods section for HyperGEN should specify the source tissue for the DNA. It’s pretty clear elsewhere that this is blood, but it still should be specified in the methods. - On page 8 the authors refer to Table 1 when talking about the consistency between their results and the prior literature, in terms of direction of association. However, this information is not presented in Table 1- only the citations are. The authors should either a) add this information to Table 1, or b) cite specific studies and not Table 1 when making this point in the discussion. - Why was model fit in Table 5 compared in GOLDN but not HyperGEN? - The authors may want to consider de-emphasizing the model fit results in their concluding paragraph. I’m not sure a ~70-point difference in AIC/BIC is worth making it a primary conclusion, even if does represent an improvement in fit. Reviewer #2: The current study tries to create a methylation risk score (MRS) for metabolic syndrome (MetS) and performs an analysis to demonstrate the utility of the MRS across two different race groups. The study is well designed and executed and the topic is interesting for the scientific community and if validated in a longer cohort could have clinical utility. From my point of view there are no relevant concerns to be addressed, I have only minor comments: -The main objective should be stated more clearly in both abstract and introduction. -I suggest to include a paragraph in the introduction section regarding to the association between epigenetic mechanisms, mainly methylation marks, on the obesity and metabolic syndrome to highlight the rationale of this study more in depth. Some of these studies could be on help (PMID: 28211912, PMID: 27477082, PMID: 30887406). Reviewer #3: It is a very well written article and authors should be commended for this. Few comments 1. Since MetS parameters are known to show differential patterns in sex, and owing to the fact both HYPER AND GENO studies have higher female participants-i would like authors to discuss more about how sexual dimorphism could have impacted the MRS score. 2. The authors should also comment why only one cell type was used in the validation but not all or the vice versa, because one would assume that the gene expression would alter between different cell types. 3. The authors tend to ignore referencing the facts throughout the study, I would like the authors to please go back to the introduction and discussion and use appropriate references. for example-in the first paragraph in introduction " To date, despite impressive effect sizes, strong statistical significance, and successful external replication in the EWAS literature (even across race/ethnic groups), few studies have examined the polygenomic effects of CpG sites (e.g., methylation risk scores (MRS)) on complex diseases ". I don't see use of reference to literature ****** 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 Reviewer #3: Yes:** Farha Ramzan [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. https://doi.org/10.1371/journal.pone.0259836.r001
Revision 1
18 Oct 2021 Author Response We thank the reviewers for their thorough review of our paper and the astute feedback provided. We have addressed the reviewers’ comments as follows: Responses to Reviewer Comments Reviewer #1: Major comments 1. “…lack of detail regarding the selection process for the six CpGs.” We thank the reviewer for highlighting this. We have added additional details about our CpG selection process to the Methods section, specifically the subsection titled ‘CgG candidate selection’. (lines 170-188) 2. “…discussion needs to be rewritten and expanded upon in more detail.” We have added additional context to the discussion section accordingly. (lines 292-315 and 341-345) 3. “…running a set of secondary analyses. Specifically, similar models as for Table 4, but instead of MetS as the dichotomous outcome use each of the individual components of MetS and model them separately.” We performed additional analyses and have subsequently updated Table 2 and Table 4. In our prior work, we have shown the robustness of CpGs included in our score with each of the MetS components, as well as with MetS. We have included references to those studies in Table 1, including directions of effect and relevant references. Minor comments: 1. “Consider adding the MRS risk score to Table 1, as some descriptive analysis will be a good way to assess its performance.” We have included the MRS risk score to table 2, and have included all references of published methylation risk scores (MRS) to Table 1. 2. “The methods section for HyperGEN should specify the source tissue for the DNA. It’s pretty clear elsewhere that this is blood, but it still should be specified in the methods.” We have updated the methods section to make this detail clearer. (line 158) 3. “On page 8 the authors refer to Table 1 when talking about the consistency between their results and the prior literature, in terms of direction of association. However, this information is not presented in Table 1- only the citations are. The authors should either a) add this information to Table 1, or b) cite specific studies and not Table 1 when making this point in the discussion. We have added further details about each of the components and relevant literature to the text (lines 92-96), added results from analyses performed in each of the components, as well as results from our sex-stratified analyses. (Table 4) 4. “Why was model fit in Table 5 compared in GOLDN but not HyperGEN?” We have removed this information to avoid confusion about model fits, and have added results for additional analyses performed in tables 2 and 4. 5. “The authors may want to consider de-emphasizing the model fit results in their concluding paragraph. I’m not sure a ~70-point difference in AIC/BIC is worth making it a primary conclusion, even if does represent an improvement in fit.” We have made changes to the conclusion paragraph accordingly. (line 356) Reviewer #2: “…there are no relevant concerns to be addressed, I have only minor comments”: 1. “The main objective should be stated more clearly in both abstract and introduction.” We thank the reviewer for this recommendation. We have updated the text in the introduction and abstract to make this point clearer. (lines 35-37 and lines 74-75) 2. “I suggest to include a paragraph in the introduction section regarding to the association between epigenetic mechanisms, mainly methylation marks, on the obesity and metabolic syndrome to highlight the rationale of this study more in depth. Some of these studies could be on help (PMID: 28211912, PMID: 27477082, PMID: 30887406).” We thank the reviewer for the suggested references. We have integrated them into both the introduction section and Table 1 of our paper. (line 82 and Table 1) Reviewer #3: It is a very well written article and authors should be commended for this. Few comments 1. “Since MetS parameters are known to show differential patterns in sex, and owing to the fact both HYPER AND GENO studies have higher female participants-i would like authors to discuss more about how sexual dimorphism could have impacted the MRS score.” We performed these additional analyses and have added the results to the paper. (lines 218-219, 255-260, and 305-315) 2. “The authors should also comment why only one cell type was used in the validation but not all or the vice versa, because one would assume that the gene expression would alter between different cell types.” We agree with the reviewer that additional cell types would have been important to study, however, for this study we only had one cell type available in both HyperGEN and GOLDN. We have expanded on this point in the discussion section of the paper. (lines 341-345) 3. “The authors tend to ignore referencing the facts throughout the study, I would like the authors to please go back to the introduction and discussion and use appropriate references.” We regret that we did not include this information previously, and have added this literature and relevant references throughout, including Table 1 of the paper. Attachments Attachment Submitted filename: Reviews for MetS paper_clean.docx https://doi.org/10.1371/journal.pone.0259836.r002
28 Oct 2021 Decision Letter - Kyle J Burghardt, Editor A 6-CpG Validated Methylation Risk Score Model for Metabolic Syndrome: The HyperGEN and GOLDN Studies PONE-D-21-04138R1 Dear Dr. Hidalgo, 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, Kyle J Burghardt Academic Editor PLOS ONE Additional Editor Comments (optional): Dr. Hidalgo, Thank you for your revisions to your manuscript. You have answered all reviewer critiques in a clear and complete manner. Thank you for your patience as well. I did receive your earlier query regarding manuscript status but the manuscript was not in my control at that time. I received the revision this morning and processed immediately. Best. Kyle https://doi.org/10.1371/journal.pone.0259836.r003
Formally Accepted
4 Nov 2021 Acceptance Letter - Kyle J Burghardt, Editor PONE-D-21-04138R1 A 6-CpG Validated Methylation Risk Score Model for Metabolic Syndrome: The HyperGEN and GOLDN Studies Dear Dr. Hidalgo: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Kyle J Burghardt Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0259836.r004

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