Association between NF-kB polymorphism and age-related macular degeneration in a high-altitude population

Yan Xin; Kang Zefeng; Li Ling; Guan Ruijuan

doi:10.1371/journal.pone.0251931

Peer Review History

Original SubmissionJanuary 4, 2021
4 Mar 2021 Decision Letter - Ravirajsinh Jadeja, Editor PONE-D-20-40186 Association between NF-kB polymorphism and Age-related macular degeneration in high altitude populationAssociation between NF-kB polymorphism and A ge-related macular degeneration in high altitude population PLOS ONE Dear Dr. Guan, 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. Authors are required to address all the issues raised by both reviewers. Specifically, the authors need to clarify the use of age 40 for AMD and ethical committee number. Additionally, language needs to be edited and spelling and grammar to improve the clarity for the reader. Please submit your revised manuscript by Apr 12 2021 11:59PM. 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Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests 4. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files 5. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 2 in your text; if accepted, production will need this reference to link the reader to the Table. [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: No Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: 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 ****** 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: Yin et al examined 55 patients and 57 controls from the Qinghai Provicial People’s Hospital and genotyped them for four SNPs in NFK-B. The paper claims a risk association with NFK-B for risk of AMD in a population that had lived more than 20 years in a high altitude climate (>2000m). The study is interesting but there are some major issues. 1) The usual age of diagnosis of AMD is >60. However, here >age 40 was used. Could the authors please clarify? 2) The sample size is extremely small (slightly over 100 people), and therefore conclusions regarding age, gender, or other differentiation is extremely hard to ascertain, including association of NFK-B to AMD. Could the authors provide please how they were able to have the power to draw the conclusions they did? 3) No comparison to non-high altitude patients was obtained, even a larger study to evaluate the NFK-B polymorphism to see how common the alleles are in the regular population- which means that this study cannot come to the conclusion as to whether or not the alternate allele is associated with AMD in the high-altitude population alone. The reader does not even know how common the alternate alleles are in the general population through examining databases like GnomeAD or ExAC. Could the authors please clarify? 4) The p-values are extremely high (0.04) to declare an association. 5) The paper language needs to be edited along with spelling and grammar, to improve the clarity for the reader. Reviewer #2: Association between NF-kB polymorphism and Age-related macular degeneration in high altitude population The authors have intended to investigate the association between NF-kB gene polymorphism and age-related macular degeneration in high altitude population. The study concludes that AA genotype at rs3774932 and CC genotype at rs3774937 in NF-kB gene could be the risk genotypes for AMD. However, this reviewer has few major concerns to be addressed by the authors in order to enhance the quality of the manuscript. • The ethical committee numbers are different on the ethic statement and in the manuscript. Introduction: Overall, this section needs to be revised. • The details in this section is not sufficient enough to substantiate the primary goal. I suggest including more information and citations relevant to Nf-KB gene and its association with AMD. • The subheading “Subjects” could be under the methods section. Methods: Overall, the method section needs a revision and more references could be added to enable the readers to follow without confusion. • 1.2.1 - Provide sample size with more details. • 1.2.2 - Provide reference and add more details. • 1.2.3 - Provide the primers and probe details in the table format. • 1.2.4 - The representative gel images could be added. • 1.2.5 - very less details • Did the author further confirm the genotypes/SNP of these four variants of NF-KB by PCR-RFLP? • Besides, either the ChIP assay to identify the binding activity of this genotypes or luciferase reporter assay to test the function of these polymorphisms, especially the NF-κB binding site activity in AA and CC alleles, could have been attempted. Results: Overall, the results are not presented well and need thorough revision. • 2.3 - grou-----group Discussion: The current data obtained from this study was not sufficient enough to conclude. Also, the findings are not discussed well. • The results should be discussed with respect to how these genetic variations (rs3774932 and rs3774937) might influence gene transcriptional regulation and expression, consequently affecting NF-κB pathway activation and/or the susceptibility to AMD pathology. Figures &Tables: Some of the details on table-3 and supplementary materials are provided in different language. Please consider. • The punctuations/ spacings throughout the manuscript should be rectified. ****** 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. https://doi.org/10.1371/journal.pone.0251931.r001
Revision 1
9 Apr 2021 Author Response 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 Response: We have revised our manuscript to meet PLOS ONE's style requirements. 2. PLOS ONE has specific criteria requiring that experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail (https://journals.plos.org/plosone/s/criteria-for-publication#loc-3). To that effect, please ensure that you have described who performed the methods and procedures and a more thorough discussion of how they were carried out, as well as further details regarding participant recruitment. If materials, methods, and protocols are well established, authors may cite articles where those protocols are described in detail, but your submission should include sufficient information to be understood independent of these references (https://journals.plos.org/plosone/s/submission-guidelines#loc-materials-and-methods). Please note that PLOS ONE does not copy edit accepted manuscripts (https://journals.plos.org/plosone/s/criteria-for-publication#loc-5). To that effect, please ensure that your submission is free of typos and grammatical errors. Response: We have revised the Methods according to PLOS ONE's requirements. 3. Thank you for stating the following in your Competing Interests section: "I have read the journal's policy and the authors of this manuscript have the following competing interests: [insert competing interests here]" 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. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests Response: We have added the statement "The authors have declared that no competing interests exist" to the manuscript. 4. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files Response: We have included all tables as part of the main manuscript and have removed the individual files. 5. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table Response: All tables have been referred to within the text of the revised manuscript. Responses to Reviewer #1: Reviewer #1: Yin et al examined 55 patients and 57 controls from the Qinghai Provincial People’s Hospital and genotyped them for four SNPs in NFK-B. The paper claims a risk association with NFK-B for risk of AMD in a population that had lived more than 20 years in a high-altitude climate (> 2000 m). The study is interesting but there are some major issues. Response: Thank you for the opportunity to improve our manuscript. 1) The usual age of diagnosis of AMD is > 60. However, here > age 40 was used. Could the authors please clarify? Response: We agree with the Reviewer that the use of ≥ 40 years of age as an inclusion criterion for AMD cases and controls may have compromised the representativeness of the samples in this study. Although AMD can be diagnosed as early as 35 years, most AMD cases are diagnosed at ≥ 60 years [Song P, Du Y, Chan KY, Theodoratou E, Rudan I. The national and subnational prevalence and burden of age-related macular degeneration in China. J Glob Health. 2017 Dec;7(2):020703.]. In addition, since control participants younger than 60 years may develop AMD later in life, our inclusion criterion of patients ≥ 40 years may have introduced selection bias in this study, resulting in lower power to detect real associations. We have added these statements to the Discussion. 2) The sample size is extremely small (slightly over 100 people), and therefore conclusions regarding age, gender, or other differentiation are extremely hard to ascertain, including association of NFK-B to AMD. Could the authors provide please how they were able to have the power to draw the conclusions they did? Response: We agree with the Reviewer that the power of our study to detect the association between the NF-kB gene polymorphisms and AMD was limited due to the small sample size. We estimated that this study had the power of 78% to detect the association at OR = 2.0 and the prevalence at 7.11% for people aged 65–69 years [Song P, Du Y, Chan KY, Theodoratou E, Rudan I. The national and subnational prevalence and burden of age-related macular degeneration in China. J Glob Health. 2017 Dec;7(2):020703.]. Therefore, the negative associations we observed after correcting for multiple tests in this study did not disprove potential associations between the NF-kB gene polymorphisms and AMD. We have added these statements to the Discussion. 3) No comparison to non-high-altitude patients was obtained, even a larger study to evaluate the NFK-B polymorphism to see how common the alleles are in the regular population- which means that this study cannot come to the conclusion as to whether or not the alternate allele is associated with AMD in the high-altitude population alone. The reader does not even know how common the alternate alleles are in the general population through examining databases like GnomeAD or ExAC. Could the authors please clarify? Response: One of the strengths of this study is that all patients with AMD were long-term (> 20 years) residents at a high altitude (> 2,000 m), which is a unique population that is under-represented in genetic studies of AMD. However, we agree that the lack of comparison to non-high-altitude patients in this study made it impossible to conclude that the risk alleles are associated with AMD in the high-altitude population alone. The allele frequencies of the four SNPs analyzed in this study are comparable to the corresponding allele frequencies in East Asians reported in the public gnomAD database (Table 3). We have added these statements to the Discussion. 4) The p-values are extremely high (0.04) to declare an association. Response: We agree that the associations we observed in this study were nominally significant and did not survive the Bonferroni correction (corrected P > 0.05). We have clarified this in the Results. 5) The paper language needs to be edited along with spelling and grammar, to improve the clarity for the reader. Response: Thank you for the comments. We have improved our language by correcting spelling and grammar. Responses to Reviewer #2: Reviewer #2: Association between NF-kB polymorphism and age-related macular degeneration in a high-altitude population. The authors have intended to investigate the association between NF-kB gene polymorphism and age-related macular degeneration in a high-altitude population. The study concludes that the AA genotype at rs3774932 and CC genotype at rs3774937 in NF-kB gene could be the risk genotypes for AMD. However, this reviewer has few major concerns to be addressed by the authors in order to enhance the quality of the manuscript. • The ethical committee numbers are different on the ethics statement and in the manuscript. Response: Thank you for pointing out this error. We have corrected it in the manuscript. Introduction: Overall, this section needs to be revised. Response: We have revised the Introduction. • The details in this section are not sufficient enough to substantiate the primary goal. I suggest including more information and citations relevant to the Nf-KB gene and its association with AMD. Response: We have added more information and citations relevant to the NF-kB gene and its association with AMD. • The subheading “Subjects” could be under the methods section. Response: We have revised the subheadings across the entire manuscript. Methods: Overall, the methods section needs a revision and more references could be added to enable the readers to follow without confusion. • 1.2.1 - Provide sample size with more details. • 1.2.2 - Provide reference and add more details. • 1.2.3 - Provide the primers and probe details in the table format. • 1.2.4 - The representative gel images could be added. • 1.2.5 - Very less details Response: We have revised the Methods according to the Reviewer’s comments. • Did the author further confirm the genotypes/SNP of these four variants of NF-KB by PCR-RFLP? Response: We did not further confirm the genotypes of these four SNPs in NF-kB by PCR-RFLP. However, we used the well-known MassARRAY system to genotype the SNPs. • Besides, either the ChIP assay to identify the binding activity of these genotypes or luciferase reporter assay to test the function of these polymorphisms, especially the NF-κB binding site activity in AA and CC alleles, could have been attempted. Response: Thank you for the comments on functional studies. Those will be one of the major aims in our future studies. Results: Overall, the results are not presented well and need thorough revision. Response: We have thoroughly revised the Results section. • 2.3 - grou-----group Response: We have corrected this typo. Discussion: The current data obtained from this study was not sufficient enough to conclude. Also, the findings are not discussed well. • The results should be discussed with respect to how these genetic variations (rs3774932 and rs3774937) might influence gene transcriptional regulation and expression, consequently affecting NF-κB pathway activation and/or the susceptibility to AMD pathology. Response: We have revised the Discussion. Figures &Tables: Some of the details on table-3 and supplementary materials are provided in a different language. Response: We have removed all non-English text from the manuscript. Please consider. • The punctuations/ spacings throughout the manuscript should be rectified. Response: We have reformatted the entire manuscript. Attachments Attachment Submitted filename: Response to reviewers.docx* https://doi.org/10.1371/journal.pone.0251931.r002
6 May 2021 Decision Letter - Ravirajsinh Jadeja, Editor Association between NF-kB polymorphism and age-related macular degeneration in a high-altitude population PONE-D-20-40186R1 Dear Dr. Guan, 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, Ravirajsinh Jadeja, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0251931.r003
Formally Accepted
14 May 2021 Acceptance Letter - Ravirajsinh Jadeja, Editor PONE-D-20-40186R1 Association between NF-kB polymorphism and age-related macular degeneration in a high-altitude population Dear Dr. Ruijuan: 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. Ravirajsinh Jadeja Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0251931.r004

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