PONE-D-22-28189

Study protocol for the epigenetic characterization of angor pectoris according to the affected coronary compartment: Global and comprehensive assessment of the relationship between invasive coronary physiology and microRNAs.

PLOS ONE

Dear Dr. Fernandez,

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.

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ACADEMIC EDITOR: All issues raised by expert reviewers are required. Please edit the manuscript point by point.

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Vincenzo Lionetti, M.D., PhD

Academic Editor

PLOS ONE

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“DdGC (Miguel Servet 2020: CP20/00041) has received financial support from Instituto de Salud Carlos III co-funded by the European Social Fund (ESF)/ “Investing in your future”.”

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6. We note that the original protocol that you have uploaded as a Supporting Information file contains an institutional logo. As this logo is likely copyrighted, we ask that you please remove it from this file and upload an updated version upon resubmission.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Partly

Reviewer #2: Yes

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript addresses an interesting topic. The research question is clearly stated. Data will be collected from multiple centers. Some comments follow.

1. The descritpion of the study design must be extended. It is rather unclear how the sample is obtained. Up to what I understand, it is not easy to collect a "reasonable" number of patients for each group (please, explain and justify the sample size was not calculated using standard procedures), but I do not really get why 25 subjects per group is a suited number of collected information. References to other studies should be better discussed.

2. I strongly believe that the difference with other approaches is currently missing. Some relevant references are included, but the novelty of the proposed study is a bit swept under the carpet.

3. I cast several doubts about the employed statistical methods.

a) Counfounders may play a role and statistical matching is rather not feasible with the proposed sample size. This aspects is completely overlooked. Please, remark that association/correlation does not imply causation.

b) The study is a multicenter one. Center-specific effects are neglected. Observations collected at a specific site are more similar than observations collected at a another site. Data have a clear hierarchical strucure that should be accounted for in any statistical analyses.

c) It is rather unclear why a two-step procedure (dimensionality reduction first and clustering then) is assumed. It is well known that this is a not efficient procedure. Clustering and dimensionality reduction can be jointly performed, see e.g. https://link.springer.com/article/10.1007/s11222-008-9056-0.

d) Hierarchical clustering is a too basic approach. Finite mixtures, and in general, model-based approaches should be preferred. PCA is valid only if linear associations can be depicted in the (standardized) data, but it is very likely that non-linear correlations arise.

e) It is rather unclear how the listed methods are related to the research questions.

f) Whataver approach, e.g. testing, regression, etc., you will use, please check that the underlying assumptions are met; otherwise, statistical inference is not valid.

Reviewer #2: The manuscript “Study protocol for the epigenetic characterization of angor pectoris according to the affected coronary compartment: Global and comprehensive assessment of the relationship between invasive coronary physiology and microRNAs.” by Lucía Matute-Blanco et al., described the methodological framework and the objectives of a pilot study aimed at evaluating the relationship between different patterns of coronary involvement and circulating miRNA levels in patients with chronic coronary syndrome. This manuscript is well designed and organized. The focus of this manuscript is new and original. However, tables are not included in the final PDF and some improvements are suggested.

1. Introduction. Please, add Ref. after the sentence “To date, the role of miRNAs in coronary function has been independently evaluated for macro and microcirculation”.

2. Material and Methods.

a. Study design: Please, add Ref. for FFR and IMR; rewrite the sentence “Among the secondary hypotheses to be tested are whether cardiovascular risk factors, medical conditions, medications, echocardiographic findings, angiographic findings, and other invasive physiological indexes are co-related with miRNAs expression.”.

b. Patient selection: “This study will include patients with suspected angina referred for coronary angiography and eventual angioplasty.” Which GL will be followed? Tables are not included in the final PDF.

c. Invasive physiological evaluation of the coronary circulation. Again, more references should be added in order to validate the methods suggested for this study (FFR and IMR), including for guidelines, and for suggested cut-off.

d. microRNA profiling. The methods described in this manuscript is not quantitative and allows to obtain just relative results. Thus, please remove “quantification” from the manuscript and replace with “evaluation”.

e. Please, add Ref. after the sentence “A panel of 14 circulating miRNAs has been selected after an extensive review of the literature.”

f. Sample size estimation. A better explanation should be added for the sample size evaluation. Which is the meaning of “Because studies evaluating the association between circulating miRNAs and coronary patterns are currently lacking, the sample size was not calculated using standard procedures.”?

3. Discussion.

a. Please, add Ref. after the sentence “To date, multiple miRNAs related to chronic coronary syndromes have been identified, being proposed as biomarkers with potential clinical applications in diagnosis and prognosis.”

b. The comment related to the methods for miRNAs evaluation should be modified. Again, the method described in this manuscript is the classical semiquantitative real-time PCR. A quantitative determination of miRNAs is possible only using digital PCR.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

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Study protocol for the epigenetic characterization of angor pectoris according to the affected coronary compartment: Global and comprehensive assessment of the relationship between invasive coronary physiology and microRNAs.

PONE-D-22-28189R1

Dear Dr. Fernandez,

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.

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Kind regards,

Vincenzo Lionetti, M.D., PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All my comments are included in this revised version. I feel that the work is publishable as it stands.

Reviewer #2: The manuscript improved following comments of Reviewers and it is now suitable for publication.

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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

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