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PONE-D-21-10394

HIGH RISK APOL1 GENOTYPES AND KIDNEY DISEASE AMONG TREATMENT NAÏVE HIV PATIENTS AT KANO, NIGERIA

PLOS ONE

Dear Dr. Aliyu Abdu, 

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 all the points raised  by 3 experts in the fields, which I agreed. Please respond in a point-by-point manor, and please pay special attention to the concern raised by reviewer #1 and #2. Please revise your manuscript carefully, as this revision suggestion may not necessarily lead to publication.

Please submit your revised manuscript by 10/7/2021. 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.

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Weijing He, M.D.

Academic Editor

PLOS ONE

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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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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: No

Reviewer #2: Yes

Reviewer #3: Yes

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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: General Comments:

The study was performed to determine the frequency of APOL1 gene frequencies in HIV-1 infected- ART naïve patients at KANO, the northern part of Nigeria. 142 samples having HIV negative and HIV positive (with or without chronic kidney disease)) were analyzed for risk alleles by PCR-RFLP assay. The authors found an association between risk alleles (G1 and G2) frequencies and CKD. While these observations in part confirm previous studies, the APOL1 gene frequencies at the KANO region

remain to be determined. Simultaneously, the obtained results with respect to published results do not give any clear picture of the association between the tested variables. Consider increasing the sample size, which might help draw definitive conclusions, especially when a broad spectrum of frequency associations is reported. Overall, the data appear to be essential to the complex area of HIV-1 therapy.

Specific Comments:

1. I would ask the authors to include line and page numbers in all future submissions as it makes reviewing difficult without these.

2. Consider building a hypothesis in the introduction

3. Table 2 was mentioned two times and did not come complete on paper when printed.

4. Consider describing Table legends.

Reviewer #2: The authors have presented data on a limited sample size on the prevalence of APOL-1 genotypes in HIV positive patients (with and without kidney disease) in Northern Nigeria, West Africa. The describe the prevalence of high-risk and low-risk genotypes on kidney disease in the region.

I have a number of concerns about the study:

Major concerns:

1. The aim of the study was to “investigate the prevalence of high-risk variants and their effects on kidney disease among HIV infected patients in Northern Nigeria with hitherto limited information despite earlier reports of high population frequencies of these alleles from the Southern part of the country.” There are a number of prevalence studies from the same country region including this one from the same centre with a much larger sample size:

(Apolipoprotein-1 risk variants and associated kidney phenotypes in an adult HIV cohort in Nigeria. Wudil UJ, Aliyu MH, Prigmore HL, Ingles DJ, Ahonkhai AA, Musa BM, Muhammad H, Sani MU, Nalado AM, Abdu A, Abdussalam K, Shepherd BE, Dankishiya FS, Burgner AM, Ikizler TA, Wyatt CM, Kopp JB, Kimmel PL, Winkler CA, Wester CW.Kidney Int. 2021 Apr 23:S0085-2538(21)00430-0. doi: 10.1016/j.kint.2021.03.038. Online ahead of print.).

So, it is unclear what added value this study has provided. Rather, I think their results are conflicting with recent results from the same centre.

2. Leading on to the study rationale was this statement “Genetic predilection to HIVAN has been described among African-Americans but has not been widely studied among native Africans except for a few studies such as the study among the South African black population by Kasembeli et al and another study among children in the DRC.” This is not correct as there have been several other studies e.g.:

- Association of Genetic Polymorphisms of TGF-β1, HMOX1, and APOL1 With CKD in Nigerian Patients With and Without HIV. Ekrikpo UE, Mnika K, Effa EE, Ajayi SO, Okwuonu C, Waziri B, Bello A, Dandara C, Kengne AP, Wonkam A, Okpechi I.Am J Kidney Dis. 2020 Jul;76(1):100-108. doi: 10.1053/j.ajkd.2020.01.006. Epub 2020 Apr 27.

- APOL1 risk alleles among individuals with CKD in Northern Tanzania: A pilot study. Stanifer JW, Karia F, Maro V, Kilonzo K, Qin X, Patel UD, Hauser ER.PLoS One. 2017 Jul 21;12(7):e0181811. doi: 10.1371/journal.pone.0181811.

- APOL1 genetic variants, chronic kidney diseases and hypertension in mixed ancestry South Africans. Matsha TE, Kengne AP, Masconi KL, Yako YY, Erasmus RT.BMC Genet. 2015 Jun 26;16:69. doi: 10.1186/s12863-015-0228-6.

3. The method section is not very detailed. For instance, where were the HIV negative patients recruited from and how?, little information is provided about the methods of the kidney biopsy and how it was reported, statistical methods are not sufficiently reported, etc The methods section needs to have more details to ensure clarity.

4. The results section is also lacking in detail:

- The tables do not have a legend to describe abbreviations within each Table

- Some variables do not have units (e.g. what is the unit of measurement of CD4 count?)

- Why were there two Tables labelled as “Table 3”?

- What do the empty cells in the Tables mean – “no data”, “zero”, or “not reported”?

- Why are there inconsistencies with the decimals – most of the blood pressure reported have no decimals?

- In reporting the histologies, this was stated in the results section: “…and the commonest histological diagnosis was FSGS seen in 20 (37.7%) patients followed by HIVAN seen in 19 (35.9%) …” Is this primary FSGS? How did you differentiate primary FSGS in HIV positive patients from HIVAN which also has an FSGS pattern on histology?

5. The discussion needs to probe more into the implications of your study findings, especially relative to other recent studies in the region and in Nigeria as well as implications related to kidney disease prevalence and outcomes in Nigeria.

Minor comment:

I am not sure why most of the references are very old despite there being more recent citations from the region and country.

Reviewer #3: The study investigates the frequency of high risk alleles of the APOL1 gene in HIV infected patients of Northern Nigeria and establishes their effects on kidney disease. A High Risk Genotype (HRG) is associated with an increased risk to develope HIVAN and FSGS in HIV positive group respect to the control group. In particular, carriers of 2 vs 0 risk alleles have the highest odds.

The manuscript is interesting and, despite limitations on sample size and study design that can be improved with further investigations, provides new findings on the frequencies of APOL1 genotypes in a region with limited information so far. I have some comments to clarify and better explain some matters. The PDF file doesn’t include the number of lines or the number of pages, so it was difficult to report my comments, I hope I was clear.

In the ABSTRACT, please explain the acronyms FSGS and HIVAN

In the INTRODUCTION:

1. In the phrase “these variants include the APOL1 G1 allele…..and the G2 allele” the verb “include” is not the most appropriate, the allele includes the variants and not vice-versa: please explain better the concept

2. the correct nomenclature for a protein variation is p.Ser342Gly and p.Ile384Met: please correct

3. in the phrase “such as the study among the South African black population by Kasembeli et al and another study among children in the DRC” please explain the acronym DRC

In the METHODS:

1. Which is the treatment of HIV patients (both HIV positive no CKD and HIV positive CKD)? Is there any difference between the two groups? Please explain better this subject

2. eGFR is Estimated Glomerular Filtration Rate: please correct

3. It is not specified if it has been tested the Hardy-Weinberg equilibrium: please clarify

In the RESULTS:

1. In the table 1 “Characteristics of the study participants” the unit of measure of eGFR is ml/min/1.73m2 and the unit of measure of CD4 is missing, please correct. Furthermore in my opinion it would be important to have other information such as the possible presence of coinfection (hepatitis B/C), the type of ART and the HIV viral load: please indicate whether or not you have these data, and eventually why you do not have it.

2. In the table 2 please explain what haplotypes G0, G1GM, G1G+, G1+M, G2 mean

3. In the text after table 3 you state “these associations however were not significant after adjusting for age and gender”, but the association with 2 vs 1 or 0 risk alleles is already not significant (p=0.07): please explain better this concept.

4. Why do you adjust only for age and gender? There are other risk factors that could be included as covariates in the model, such as CD4 count, viral load, ART type and blood pressure

5. There are two table 3

6. It is not specified how you determined that G1 and G2 are on two different chromosomes, therefore in compound heterozygosity (G1/G2), and not on the same: is it based on literature data or have you tested it? Please clarify this concept.

In the DISCUSSION:

1. In the phrase “variations in the frequencies of these alleles among diverse African ethnic populations…” was should be were, please correct and add the number of reference of the cited study by Tzur et al.

2. Please change the sentence “the association was also strong with HIVAN…..with a less strong association in the recessive form”, a p=0.07 is not a less strong association, is not an association at all.

3. What does it mean the expression “from this environment” in the sentence “this study provides the first information on the high frequencies of the APOL1 genotypes….”? Please explain better.

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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: Yes: Himanshu Batra

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-21-10394R1HIGH RISK APOL1 GENOTYPES AND KIDNEY DISEASE AMONG TREATMENT NAÏVE HIV PATIENTS AT KANO, NIGERIAPLOS ONE

Dear Dr. Abdu,

Sorry for the long process, thank you for submitting your revised 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. Specifically, if you could carefully response reivewer#3's comments, 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 May 23, 2022. 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,

Weijing He, M.D.

Academic Editor

PLOS ONE

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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 #1: All comments have been addressed

Reviewer #3: (No Response)

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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 #1: Yes

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #3: N/A

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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 #1: Yes

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 #1: Yes

Reviewer #3: Yes

**********

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 #1: I thank authors for addressing all the concerns which I raised. Therefore, I recommend the article to be published.

Reviewer #3: Reviewing again the manuscript, I realized that there is a serious inaccuracy in the calculation of allelic frequencies, that I have not considered before.

The 142 subjects participating in the study have 284 chromosomes and consequently alleles, so the G0 alleles in each group are not 35, 50 and 49, but 55, 88 and 76 respectively (20 individuals with the G0/G0 genotype have 40 G0 alleles; 38 G0/G0 have 76 G0 alleles; 27 G0/G0 have 54 G0 alleles); the G1 alleles are 38 and the G2 alleles are 27; in total there are 284 alleles and not 198 alleles resulting from the sum of the haplotypes listed in table 2 "Frequencies of the APOL1 risk alleles". Therefore, the sentence "The frequency of the individual risk allele among all study participants was 26.1% (37/142) for G1 and 19.0% (27/142) for G2" on page 9 is incorrect, the exact G1 prevalence is 38/284=13.4% and the G2 prevalence is 27/284=9.5%. It would be correct to consider the 142 subjects, if you are talking about the frequency of the genotype or of the individuals with a number of risk alleles of 0,1,2. If you are talking about allelic frequencies, you must consider all 284 alleles. In my opinion in a study dealing with genetics, this concept needs to be revised.

Another important issue, already included in my revision, concerns the concept on page 10, where you state "five had the compound heterozygote G1/G2 form". I would like to know how you say that, are you based on literature data or have you tested it and know that G1 and G2 are on two different chromosomes? You didn't answer to my previous question.

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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: Yes: Himanshu Batra

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.

HIGH RISK APOL1 GENOTYPES AND KIDNEY DISEASE AMONG TREATMENT NAÏVE HIV PATIENTS AT KANO, NIGERIA

PONE-D-21-10394R2

Dear Dr. Aliyu Abdu,

My apologies for the long review process, 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,

Weijing He, M.D.

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 #1: 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 #1: Yes

**********

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

Reviewer #1: Yes

**********

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 #1: 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

**********

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 #1: I thank the authors for making the necessary changes.

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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: Yes: Himanshu Batra

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