Genetic evidence for amlodipine’s protective role in gastroesophageal reflux disease: A focus on CACNB2

Liuzhao Zhang; Quanwang Chu; Shuyue Jiang; Bo Shao

doi:10.1371/journal.pone.0309805

Peer Review History

Original SubmissionJune 7, 2024
26 Jul 2024 Decision Letter - Asif Jan, Editor PONE-D-24-22095Genetic Evidence for Amlodipine's Protective Role in Gastroesophageal Reflux Disease: A Focus on CACNB2PLOS ONE Dear Dr. Shao, 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 Sep 09 2024 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, Asif Jan 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. Please remove your figures from within your manuscript file, leaving only the individual TIFF/EPS image files, uploaded separately. These will be automatically included in the reviewers’ PDF. 3. We notice that your supplementary figures are uploaded with the file type 'Other'. Please amend the file type to 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list. 4. 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. 5. 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: Please incorporate reviewers comments and re-submit. Reviewer 1. Overall the “Genetic Evidence for Amlodipine's Protective Role in Gastroesophageal Reflux Disease: A Focus on CACNB2” is a very interesting research manuscript. Very much informative and well written, however needs some minor corrections. 1) The overall English is easy and understandable, however needs improvement. 2) Introduction chapter is relevant, however needs some latest inclusion of some references relevant to the context. 3) The methodology is relevant and very well written. 4) The results chapter is also written in a very good way. 5) Remove double spacing in Study Design line 3. 6) Check the last paragraph in Study Design for grammatical mistake. 7) Correct the spacing errors in Data sources and processing section. 8) Correct the grammatical mistake and spacing errors in section Instrumental variable selection and data harmonization. 9) No need to write Version as capital in section Construction of ceRNA network of Drug Target Genes and Identification of Core Targets. 10) Too many spacing errors. Kindly correct all the spacing errors in the overall research manuscript. I accept the manuscript after the minor correction. Reviewer 2: This is a well written mansucript which investigates the relationship between genetically predicted amlodipine use and the risk of gastroesophageal reflux disease (GERD) using a bidirectional Mendelian Randomization approach, finding a significant protective effect of amlodipine against GERD, supported by genetic and molecular analyses. I would siggest some minor corrections before considering it for publication. In mAterial and methods; Materials and Methods, improve clarity and conciseness of the following statement. "The data utilized in our analysis were openly accessible and had been authorized by the institutional review board in the relevant studies." Suggested revision: "The data used in our analysis were publicly available and had been authorized by the institutional review board in the relevant studies." Overall, after making this minor change, this study can be published in its current form. Reviewer 3: Major Concerns 1. Methodological Clarity and Justification: o Instrumental Variable Selection: The paper describes the selection and clustering of SNPs but lacks detail on the criteria used to validate these SNPs as strong instruments. While the linkage disequilibrium and significance thresholds are mentioned, the justification for excluding certain SNPs is not clearly explained. o Data Harmonization: The harmonization process, especially handling palindromic and ambiguous SNPs, needs more clarity. The rationale behind proxy SNP usage and its impact on the results should be more thoroughly discussed. 2. Results Interpretation: o Association vs. Causation: The paper concludes a protective effect of amlodipine on GERD risk. However, the distinction between association and causation needs stronger emphasis, especially given the nature of MR studies. 3. Functional Analysis and Biological Pathways: o Enrichment Analysis: The connection between identified pathways and GERD is inferred but not deeply explored. How calcium ion transport and related pathways specifically influence GERD pathophysiology should be more explicitly discussed so that readers may better understand it. 4. General o Comparison with Other Medications: While amlodipine is highlighted, comparisons with other calcium channel blockers or antihypertensive medications are not well developed. This comparison could strengthen the argument for amlodipine's unique role. Minor Concerns 1. Writing and Presentation: o Abstract and Introduction: The abstract is comprehensive, but the introduction could benefit from a clearer statement of the study's novelty and specific hypotheses. o Figures and Tables: The figures (e.g., funnel plots, enrichment analyses) are informative, but the legends should provide more contexts to aid interpretation. 2. Statistical Methods: o Software and Packages: While the use of R and specific MR packages is mentioned, a more detailed description of the statistical methods and assumptions would help readers replicate the study. o Sensitivity Analyses: The results of sensitivity analyses are presented, but the implications of findings like the MR-Egger intercept and Cochran’s Q-test are not explained. 3. Technical Accuracy: o Genetic Terms and Definitions: Some genetic terms (e.g., linkage disequilibrium, pleiotropy) are used without definitions. Including brief explanations would benefit readers who are not geneticists. o Gene Lists and Databases: The sources of gene lists and databases used for functional analysis are cited, but the process of selecting and validating these resources should be clearer. Decision: Minor revision [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 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: REVIEW REPORT OF PONE-D-24-22095 - [EMID: dce6b8afba788c30] Overall the “Genetic Evidence for Amlodipine's Protective Role in Gastroesophageal Reflux Disease: A Focus on CACNB2” is a very interesting research manuscript. Very much informative and well written, however needs some minor corrections. 1) The overall English is easy and understandable, however needs improvement. 2) Introduction chapter is relevant, however needs some latest inclusion of some references relevant to the context. 3) The methodology is relevant and very well written. 4) The results chapter is also written in a very good way. 5) Remove double spacing in Study Design line 3. 6) Check the last paragraph in Study Design for grammatical mistake. 7) Correct the spacing errors in Data sources and processing section. 8) Correct the grammatical mistake and spacing errors in section Instrumental variable selection and data harmonization. 9) No need to write Version as capital in section Construction of ceRNA network of Drug Target Genes and Identification of Core Targets. 10) Too many spacing errors. Kindly correct all the spacing errors in the overall research manuscript. I accept the manuscript after the minor correction. Reviewer #2: This is a well written mansucript which investigates the relationship between genetically predicted amlodipine use and the risk of gastroesophageal reflux disease (GERD) using a bidirectional Mendelian Randomization approach, finding a significant protective effect of amlodipine against GERD, supported by genetic and molecular analyses. I would siggest some minor corrections before considering it for publication. In mAterial and methods; Materials and Methods, improve clarity and conciseness of the following statement. "The data utilized in our analysis were openly accessible and had been authorized by the institutional review board in the relevant studies." Suggested revision: "The data used in our analysis were publicly available and had been authorized by the institutional review board in the relevant studies." Overall, after making this minor change, this study can be published in its current form. Reviewer #3: Major Concerns 1. Methodological Clarity and Justification: o Instrumental Variable Selection: The paper describes the selection and clustering of SNPs but lacks detail on the criteria used to validate these SNPs as strong instruments. While the linkage disequilibrium and significance thresholds are mentioned, the justification for excluding certain SNPs is not clearly explained. o Data Harmonization: The harmonization process, especially handling palindromic and ambiguous SNPs, needs more clarity. The rationale behind proxy SNP usage and its impact on the results should be more thoroughly discussed. 2. Results Interpretation: o Association vs. Causation: The paper concludes a protective effect of amlodipine on GERD risk. However, the distinction between association and causation needs stronger emphasis, especially given the nature of MR studies. 3. Functional Analysis and Biological Pathways: o Enrichment Analysis: The connection between identified pathways and GERD is inferred but not deeply explored. How calcium ion transport and related pathways specifically influence GERD pathophysiology should be more explicitly discussed so that readers may better understand it. 4. General o Comparison with Other Medications: While amlodipine is highlighted, comparisons with other calcium channel blockers or antihypertensive medications are not well developed. This comparison could strengthen the argument for amlodipine's unique role. Minor Concerns 1. Writing and Presentation: o Abstract and Introduction: The abstract is comprehensive, but the introduction could benefit from a clearer statement of the study's novelty and specific hypotheses. o Figures and Tables: The figures (e.g., funnel plots, enrichment analyses) are informative, but the legends should provide more contexts to aid interpretation. 2. Statistical Methods: o Software and Packages: While the use of R and specific MR packages is mentioned, a more detailed description of the statistical methods and assumptions would help readers replicate the study. o Sensitivity Analyses: The results of sensitivity analyses are presented, but the implications of findings like the MR-Egger intercept and Cochran’s Q-test are not explained. 3. Technical Accuracy: o Genetic Terms and Definitions: Some genetic terms (e.g., linkage disequilibrium, pleiotropy) are used without definitions. Including brief explanations would benefit readers who are not geneticists. o Gene Lists and Databases: The sources of gene lists and databases used for functional analysis are cited, but the process of selecting and validating these resources should be clearer. Decision: Minor revision ****** 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: Yes: Dr. Naveed Rahman Reviewer #2: Yes: Tahir Muhammad Reviewer #3: Yes: Dr. Waheed Ali Shah ******** [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. Attachments Attachment Submitted filename: Review report of PONE-D-24-22095 - [EMIDdce6b8afba788c30].docx Attachment Submitted filename: comments for Plos one .docx https://doi.org/10.1371/journal.pone.0309805.r001
Revision 1
9 Aug 2024 Author Response Major Concerns 1. Methodological Clarity and Justification: o Instrumental Variable Selection: The paper describes the selection and clustering of SNPs but lacks detail on the criteria used to validate these SNPs as strong instruments. While the linkage disequilibrium and significance thresholds are mentioned, the justification for excluding certain SNPs is not clearly explained. o Response: Thank you for your insightful comment regarding the selection and clustering of SNPs. We agree that further clarification is needed. In our study, we selected SNPs based on genome-wide significance (P < 5 × 10−8) and ensured independence by clustering them with a linkage disequilibrium threshold of r² < 0.001 within a 10,000 kb window. This approach was chosen to minimize confounding due to linkage disequilibrium. SNPs were excluded if they did not meet these criteria or if they were identified as palindromic and ambiguous, as these can introduce biases in Mendelian Randomization (MR) analysis. We will revise the manuscript to provide a more detailed explanation of these criteria and their implementation. For example, we applied a PhenoScanner search to assess all known phenotypes related to the considered genetic instruments in our analyses. o Data Harmonization: The harmonization process, especially handling palindromic and ambiguous SNPs, needs more clarity. The rationale behind proxy SNP usage and its impact on the results should be more thoroughly discussed. o Response: We appreciate your concern regarding data harmonization. In our study, palindromic SNPs were carefully managed to avoid strand ambiguity, and ambiguous SNPs were excluded from the analysis to maintain data integrity. When necessary, proxy SNPs were used, selected based on high linkage disequilibrium (r² > 0.8) with the original SNPs to preserve the instrument's validity. We acknowledge that the impact of using proxy SNPs on the results can be significant and will include a discussion on the potential biases and limitations associated with this approach in the revised manuscript. 2. Results Interpretation: o Association vs. Causation: The paper concludes a protective effect of amlodipine on GERD risk. However, the distinction between association and causation needs stronger emphasis, especially given the nature of MR studies. o Response: We appreciate the reviewer's feedback on this critical point. While our Mendelian Randomization study suggests a potential causal relationship between amlodipine use and reduced GERD risk, we recognize that MR studies have inherent limitations and cannot establish causation definitively. We will revise the discussion section to emphasize the distinction between association and causation more clearly, acknowledging the assumptions and limitations of the MR approach, such as potential pleiotropy and unmeasured confounding. 3. Functional Analysis and Biological Pathways: o Enrichment Analysis: The connection between identified pathways and GERD is inferred but not deeply explored. How calcium ion transport and related pathways specifically influence GERD pathophysiology should be more explicitly discussed so that readers may better understand it. o Response: Thank you for pointing out the need for a deeper exploration of the identified pathways. Our enrichment analysis highlighted calcium ion transport and signalling pathways as key biological processes involved. These pathways are crucial in regulating smooth muscle tone, including the lower oesophageal sphincter (LES), which plays a significant role in GERD pathophysiology. We will expand the discussion to provide a more detailed explanation of how these pathways influence GERD development and the potential mechanisms through which amlodipine exerts its protective effects. 4. General o Comparison with Other Medications: While amlodipine is highlighted, comparisons with other calcium channel blockers or antihypertensive medications are not well developed. This comparison could strengthen the argument for amlodipine's unique role. o Response: We appreciate the reviewer’s suggestion regarding the comparison with other calcium channel blockers or antihypertensive medications. However, the scope of our study was specifically designed to focus on the potential protective effect of amlodipine against GERD. Due to the study’s design and the available data, we were not equipped to conduct a comprehensive comparison with other medications within this framework. Our study aimed to elucidate the genetic relationship between amlodipine use and GERD risk, utilizing a Mendelian Randomization approach. Expanding the analysis to include other medications would require additional data sets and potentially different methodological approaches, which were beyond the scope of the current study. We acknowledge that such comparisons could provide valuable insights and strengthen the argument for amlodipine's unique role. Therefore, we suggest that this could be an important direction for future research. Future studies that include direct comparisons between different classes of calcium channel blockers or antihypertensive drugs may further clarify the unique effects of amlodipine on GERD risk. Such research could help identify specific pharmacological properties of amlodipine that contribute to its protective effects, providing a more comprehensive understanding of its role in GERD management. Minor Concerns 1. Writing and Presentation: o Abstract and Introduction: The abstract is comprehensive, but the introduction could benefit from a clearer statement of the study's novelty and specific hypotheses. o Response: Thank you for your constructive feedback. We agree that the introduction could better highlight the study's novelty and hypotheses. We will revise the introduction to explicitly state our research question and the novel aspects of our study, such as the application of a bidirectional MR approach and the construction of a ceRNA network to explore the genetic relationship between amlodipine use and GERD. o Figures and Tables: The figures (e.g., funnel plots, enrichment analyses) are informative, but the legends should provide more contexts to aid interpretation. o Response: We appreciate the feedback on the figures and tables. We will revise the figure legends to include more detailed explanations, ensuring that they provide sufficient context for readers to interpret the data accurately. This will include descriptions of the methods used, the significance of the results, and any relevant biological implications. 2. Statistical Methods: o Software and Packages: While the use of R and specific MR packages is mentioned, a more detailed description of the statistical methods and assumptions would help readers replicate the study. o Response: Thank you for highlighting the need for a more detailed description of our statistical methods. We will expand the methods section to include a comprehensive description of the statistical software, and packages used, as well as the assumptions underlying our MR analyses. This will help ensure that the study can be replicated by other researchers. o Sensitivity Analyses: The results of sensitivity analyses are presented, but the implications of findings like the MR-Egger intercept and Cochran’s Q-test are not explained. o Response: We acknowledge that the implications of our sensitivity analyses require further elaboration. The MR-Egger intercept and Cochran’s Q-test are crucial in assessing pleiotropy and heterogeneity, respectively. We will revise the manuscript to explain the significance of these tests and how they support the robustness and validity of our findings. 3. Technical Accuracy: o Genetic Terms and Definitions: Some genetic terms (e.g., linkage disequilibrium, pleiotropy) are used without definitions. Including brief explanations would benefit readers who are not geneticists. o Response: We appreciate the suggestion to provide definitions for genetic terms. We will include brief explanations of key genetic terms, such as linkage disequilibrium and pleiotropy, to enhance the manuscript's accessibility for readers from diverse backgrounds. o Gene Lists and Databases: The sources of gene lists and databases used for functional analysis are cited, but the process of selecting and validating these resources should be clearer. o Response: Thank you for pointing out the need for clarity in our selection and validation process for gene lists and databases. We will revise the manuscript to provide a detailed description of the criteria and methods used to select and validate these resources, ensuring transparency and reproducibility of our analysis. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0309805.r002
20 Aug 2024 Decision Letter - Asif Jan, Editor Genetic Evidence for Amlodipine's Protective Role in Gastroesophageal Reflux Disease: A Focus on CACNB2 PONE-D-24-22095R1 Dear Dr. Shao 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Asif Jan, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0309805.r003
Formally Accepted
18 Sep 2024 Acceptance Letter - Asif Jan, Editor PONE-D-24-22095R1 PLOS ONE Dear Dr. Shao, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Asif Jan Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0309805.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .