PONE-D-21-25624Genetic Variations of CYP2R1 (rs10741657) in Bangladeshi adults with low serum 25(OH)D levelPLOS ONE

Dear Dr. Rahman,

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. Besides the important reviewers' comments, authors should address the following points:

• Please, follow appropriate reporting guidelines, such as STROBE
• Please, better specify the objectives of the study; it would be better to add the word to determine “frequency” of variants…)
• The small sample size is a major limitation. Authors stated that this is a pilot study (this should be better also included in the title, discussion, and conclusion).
• Regarding Data availability, authors stated “Yes - all data are fully available without restriction” and data are “within the manuscript and its as supporting information files”. However, the submission doesn’t contain unidentified patients’ data. According to PLOS ONE publication criteria, authors are required to make all data underlying the findings described fully available, without restriction, and from the time of publication. PLOS allows rare exceptions to address legal and ethical concerns, that should be clearly explained.
• Authors should clearly describe participants’ inclusion and exclusion criteria, time of recruitment, how participants were selected (convenience sample?)
• Authors should provide definitions/diagnostic criteria for described conditions/outcomes, including vitamin D deficiency.
•  The authors didn’t study other genetic determinants (other vitamin D associated genes), which could be possible confounders.
• In statistical analysis, were data tested for normality (some data are reported as mean (SD) but appear not normally distributed). What about confounders?
• Study results and tables should be reported in the results section.
• Tables require better organization with comparison of variables between the study and control groups.
• In figure 1, it would be better to annotate the polymorphisms.
• Figures 2 & 3 seem to be of little value as its data can be incorporated in tables or text.
• Discussion requires major revision, critically discussing main findings of study (without repeating results) and study implications.
• In light of being a pilot, the current conclusion seems to overestimate study findings; authors should reduce the strength of their statement, confirming that this is a pilot study, and recommend further well-powered studies.
• Please, revise the references to follow PLOS ONE style.
• Authors should revise the manuscript for several errors in language, sentence structure, grammar, and punctuation.

Please submit your revised manuscript by Nov 04 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,

Elsayed Abdelkreem, MD, PhD

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

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2. Please ensure that you refer to Figure 3 in your text as, if accepted, production will need this reference to link the reader to the figure.

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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

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: I Don't Know

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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

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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: No

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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: I have a major concern regarding the sample size of the study. With the current small sample size and the used study design, the study probably is underpowered. Was the sample size and power of the study was calculated prior starting the study?

There are other points to consider as follows:

1. In line 73 (in the introduction), Please remove DBP as GC is already mentioned and It is the gene encoding vitamin D binding protein (DBP). In addition, please include CYP27B1 gene with the other major common vitamin D genes as it is one of the significant genes involved in vitamin D metabolism (it is the gene encoding 1α-hydroxylase, the enzyme responsible for the 2nd step of activation of vitamin D in the kidney).

2. Please mention the exclusion and inclusion criteria in the methods and the inter and intra-assay CV for the performed tests.

3. Please move Table 1 and 2 to the Results section.

4. Information in line 186-187 (in the results) is already mentioned in the methods, please remove.

Reviewer #2: First of all, I would like to congratulate the researchers for this very good work. The "CONCLUSION" section is also finished with a very good conclusion. However, the deficiency that I see in the study and other similar studies is that it is in the form of statistically researching the negativities associated with vitamin D deficiency and making a connection between them. This should provide an advantage if a selective mutation is dominant in geographic regions where this mutation is present. What advantage does vitamin d provide at lower levels? What protection does it provide for people in those areas? The answer to this question should also be explored in the comments. Natural selection never happens without a reason. When I look at it from the point of view of the country I live in, kidney stones come to mind. But I think this should be investigated. If researchers can add an answer and comment to this question in terms of their own country, I think that their work will be more beneficial than other studies. Beautiful original work worth publishing. The journal meets all publication criteria. I wish the researchers success in their new work.

Reviewer #3: Thank you for your work. Some comments

1. Grammar and sentence construction i nthe entire manuscript have to be revised/ improved

2. What is the significance of yorus tidu if this has been reported in the literature already?

3.inclusion and exclusion criteria not stated

4. how di you come up with the final nuber of subjects and how did you choose the initial of 54. please state clearly how you got yoru subjects

5. what was the significantce of all the other biochemical parameters that were taken apart form vitamin D levels

6. for tables 1 and 2 make them reader friendly-remove too many lines and make it cleaner. yo ulaso did not specify the statistical significance betwenn the two groups of all the parameters

7. for table 3 you did not compare the results between groups. you only compared them within the groyp. it wuld be ebtter if you compared for exampe the genoype GG of affected and controls and the corresponding statistical significance

8. i nthe discussion, do not repeat anymore your results but instead interpret and explain them. your tables hsould be self explanatory and in the discussion you will only give its importance

9. what is the impact of the study?

10. what is the implciation i nterms of treatment?

11. make the discussion mroe concise

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Reviewer #1: Yes: Shatha Alharazy

Reviewer #2: No

Reviewer #3: 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-21-25624R1Genetic Variations of CYP2R1 (rs10741657) in Bangladeshi adults with low serum 25(OH)D level- A Pilot StudyPLOS ONE

Dear Dr. Rahman,

Thank you for submitting your manuscript to PLOS ONE. The revised manuscript is much better. However, some minor, but important, comments have to be addressed.   You are kindly requested to respond to some concerns raised by Reviewer #3. Moreover, authors should briefly emphasize, in the results section, most important findings in the table (not just saying data are shown in tables). Last, the limitations should be moved to appear before the conclusion (could be integrated with the last paragraph in the discussion)  

Please submit your revised manuscript by Nov 26 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,

Elsayed Abdelkreem, MD, PhD

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.

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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 #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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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 #2: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: Yes

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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 #2: (No Response)

Reviewer #3: 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 #2: (No Response)

Reviewer #3: Yes

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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 #2: Since I think that this and similar studies, which have been done despite limited technical possibilities, should be supported, I would like to be approved for publication. Good luck to the researchers.

Reviewer #3: Thank you for your revisions. The manuscript has improved a lot i nterms of grammar, sentence construction, tables, and explanation of results

I still have some minor comments though. (I would like to apologize for all the typo errors during my initial comments).

1. You did not discuss the relevance of the significant differences in the biochemical parameters like sgpt, calcium, PT between subjects and controls. What were the reference values for these parameters? Or they were both in the normal ranges for both groups thats why they were not discussed anymore?

2. Clarification: were your subjects all healthy with incidental findings of low vitamin D levels? pls state that they were all healthy "meaning no other medical problem?"

3. I also wanted to know what is the implication in terms of management. Will you recommend vitamin D supplement to those who have low levels? If you will not give supplement, again what is the significance of the study if you will not be able to translate it to clinical practice? Likewise, did you consider checking if the subjects had concomittant disorders associated with low vitamin D levels? In your introduction you said that low vitamin D is related to several disorders. How can you correlate it with your results-- did you ask the medical histories of your subjects and check if these could be related to low vitamin D? (or are they all healthy?)

4. Will you also recommend to test for this polymorphism routinely among the population? If yes, what is the significance. Will this be harmful for their health?

5. For a better organization, I suggest created subheadings in your methodology. First discuss about the "study subjects" then the " biochemical analysis" and lastly "molecular analysis". Do not lump them altogether.

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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 #2: No

Reviewer #3: 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.

Genetic Variations of CYP2R1 (rs10741657) in Bangladeshi adults with low serum 25(OH)D level- A Pilot Study

PONE-D-21-25624R2

Dear Dr. Rahman,

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,

Elsayed Abdelkreem, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #3: All comments have been addressed

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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 #3: Yes

**********

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

Reviewer #3: I Don't Know

**********

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 #3: 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 #3: No

**********

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 #3: (No Response)

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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 #3: No