PONE-D-22-07467APOL1 Genotype Associated Risk for Preeclampsia in African populations: Rationale and protocol design for studies in women of African ancestry in resource limited settingsPLOS ONE

Dear Dr. OSAFO,

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.

==============================

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: No

Reviewer #2: Yes

**********

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

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

Reviewer #1: No

Reviewer #2: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: No

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

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

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

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

Reviewer #1: General comments:

This is a well-powered study to assess the role of fetal and maternal APOL1 renal risk variants in the development of preeclampsia in a West African setting. The authors also will be measuring a number of biomarkers but make no mention on how these will be used in the analyses. The authors mention, but do not elaborate on a longitudinal component to assess maternal events in mothers experiencing preeclampsia but this is not developed in the manuscript. Critical details on biomarkers, placental histology, and definitions of outcomes and events are not provided. Any information about statistical tests would be helpful.

Abstract:

Methods: The study will not recruit all pregnant – there are exclusion criteria. Change all to only or just delete all/

Introduction:

Common variants in the apolipoprotein L-1 gene (APOL1) only found on African

chromosomes termed G1 and G2, are potent risk factors for a spectrum of kidney diseases (14) but very rare in European ancestry individuals. This sentence needs to be re-written, the haplotypes are termed G1 and G2—not the chromosomes.

There is a strong relationship between kidney function and hypertension (15, 16) and looking at the uniqueness of the APOL1 gene to people of African descent, an in-depth role of this gene in preeclampsia in indigenous African women will be key to understanding preeclampsia genetics and biomarker innovations. Uniqueness seems like the wrong word here and sentence construction is awkward—try rewriting. I would suggest replacing with “and considering the importance of the APOL1 gene to people of African descent,…” and adding …and advance biomarker innovations.

Define high-risk genotype at first usage.

Among populations from Nigeria and Ghana, the APOL1 high-risk genotype frequency approaches 13% among those with kidney disease. What is the overall frequency of high-risk genotypes?

It is known that preeclampsia results in part from microangiopathy in the glomerulus of the kidney suggesting a potentially important role of APOL1in preeclampsia (19, 20). This statement should be supported by briefly stating the evidence that APOL1 is associated with (micro)angiopathy. There is other evidence suggesting a role for APOL1 in preeclampsia: mouse model with APOL1 placental expression with preclampsia and fetal wasting, higher circulating antibodies against APOL1 in preclampsia mothers.

The studies did not confer a 2-fold risk for preclampsia, APOL1 did. Please revise sentence.

Most studies have been African Americans and sampling has been mostly underpowered.

This statement should be toned down. Strengths of the two studies is that one had a replication cohort, which showed similar strong effect sizes and was powered at nearly 80% to detect OR>1.8 and the second was well powered. In aggregate these studies provide convincing evidence that APOL1 risk alleles increase risk of preeclampsia in African Americans. This study should allow a more precise OR and give some idea of the penetrance in African populations experiencing different demographics and environmental stressors than African descendants living in the USA.

Study Details

Aims and objectives:

Aim 2 is to determine perinatal outcomes, but elsewhere it is hinted that there will be 3 yrs follow-up of mothers to assess longitudinal outcomes—but this not mentioned. What is the aim and hypothesis and outcomes/events that will be measured for the follow-up?

Study design/population

The authors state that the main recruiting facility is in Ghana—are there others and have they been IRB approved? Or do they mean the only facility is at the KMTH?

Inclusion and exclusion criteria (Table 1): what is the justification for excluding women with a prior history—most of the participants are multiparious. Excluding mothers with prior history might under-estimate the true effect size of APOL1 risk genotypes.

Ethnicity:

5 ethnicities are listed, all with the potential of having different APOL1 allele frequencies. Have the investigators ascertained the allele frequencies for these different populations? How will population stratification be handled in the analyses? For example the high risk allele frequency among Hausa is ~7% , Akan 42%, Ga 21% and Ewe 28%.

Sample handling:

This section is less about sample handling and more about technology for documentation. Are the study participants the same as enrolled in an earlier study? Why given identifiers from an earlier study, presumably with the same investigators. It would make more sense to give each participant a unique identifier which identified both the study and the participant to avoid mix-ups in freezers or over time.

Will samples be barcoded—essential to reduce sample id errors. There is considerable detail about pitfalls and solutions to recruitment and sample collection, but not much detail of how samples will be stored, QC measures, etc.

Biomarkers:

More details about biomarkers—what are the specific biomarkers that will be quantified? At what time points. This protocol design will be used as a reference for the study design and protocol for ancillary studies in the future—more detail is needed. Details of study could go into supplementary data. Where will testing for these biomarkers be done?

Quality control:

Will creatinine and albumin be assessed at multiple time points? How will QC be implemented; how will consistency between labs and with labs over time be ascertained? How will CKD or other outcomes be defined for mother follow-up studies? Will two uACR and creatinine levels be collected at least 3 months apart? Will random duplicate samples be collected to validate laboratory consistency over time?

The section on pitfalls on recruitment and how they were overcome is useful, but could be shortened. More information on laboratory and histology would be welcome. For example, what section of placenta will be collected, how will this be standardized across time, and is there a pathologist specializing in placental pathology on the team.

Expand on the longitudinal arm of the study—what incident outcomes/events will be captured? Will all mothers be followed? Is this a stated aim of the study? What analyses will be performed?

What platform will be used to call APOL1 alleles. Will the genotyping be performed in Ghana or the USA? What is the capacity building plan for this study in Ghana?

Overall: This is a compelling and important study, but this reviewer finds the rationale and protocol design to be overly descriptive and short on technical details, particularly for details about timing of sample collection for biomarkers, laboratory protocols, and QC protocols, which would be helpful to investigators interested in ancillary studies or citing this study. A table listing the biomarkers for preeclampsia, kidney function, inflammation and time points for collection would be extremely helpful.

Reviewer #2: 1. Page 11 (numbers indicate overall page number in the pdf): Please clarify whether the study included early onset vs late onset preeclampsia – were both groups approached for enrollment?

2. Page 11: c/c study design of 700 cases and 700 controls. this should be clarified as the targe number, and an presentation of the interim enrollment number. It should be clarified that this was 1400 is the goal and that they only enrolled sl more than half of each intended group at the current time

Pages 12/13- refer to figures which are not included

Page 13: in what % of deliveries wase cord blood unavailable/not obtained? Similarly for placental tissue- in what percent of their current sample was it available.

Page 13: what are ‘endothelial markers for mother and child’?

Table 2: clarify what is meant by the titles: @20 and @30 weeks. Is this time of enrollment, time of onset of dx, time of delivery? this is unclear

similarly in table 2: please clarify what is meant by booking vs diagnosis (table 2)

Table2: lots of missing data – explain why this is the case?

there are some patients with a prior hx of PE when first onset was the criteria? What do the authors plan to do with them?

What birth defects were included?

Page 18: what do they mean by 1033 ‘data collection tools; have been obtained? They probably mean values, not tools.

Page 19: at the beginning of recruitment patients were ..recruited early in their pregnancy – but then they modified the recruitment strategy to make sure the time between recruitment and delivery did not take too long.The authors should discuss the implications of this approach, as I believe it will weight the cohort more towards late-onset preeclampsia

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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

Reviewer #1: No

Reviewer #2: No

==============================

Please submit your revised manuscript by Aug 04 2022 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,

Vicente Sperb Antonello, MD, MSc, 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 

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 update your submission to use the PLOS LaTeX template. The template and more information on our requirements for LaTeX submissions can be found at http://journals.plos.org/plosone/s/latex.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

This project was supported by the Fogarty International Center of the National Institutes of Health

under Award Number K43TW011160. 

However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

CO- Fogarty International Center of the National Institutes of Health under Award Number K43TW011160.

The funders had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

5.  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-22-07467R1APOL1 Genotype Associated Risk for Preeclampsia in African populations: Rationale and protocol design for studies in women of African ancestry in resource limited settingsPLOS ONE

Dear Dr. OSAFO,

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 Dec 01 2022 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,

Vicente Sperb Antonello, MD, MSc, 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.

Additional Editor Comments:

APOL1 Genotype Associated Risk for Preeclampsia in African populations: Rationale and protocol design for studies in women of African ancestry in resource limited settings

After careful evaluation of the article and data from by reviewers, I understand that the article should pass through a minor revision to be accepted into Plos One. Please check the reviewers´ recommendations and adjust in the present article.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

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

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

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

Reviewer #1: The authors have addressed this reviewer's comments. I have only two minor edits: APOL1 when referring to the gene is always italicized; when referring to the protein it is not. Please correct in abstract and throughout text

Preeclampsia is a disease and is therefore not capitalized in abstract and elsewhere.

Reviewer #2: I believe the authors have addressed the reviewer's initial comments satisfactorily. There are two areas that could use some additional clarification (but not re-review). First, limiting the sample to women without a history of preeclampsia removes from the population women who might have recurrent preeclampsia due to their (or their fetus's) APOL1 status. This would result in an underestimate of the relationship between apol1 and preeclampsia generally, and is worthy of a statement to this regard in the discussion.

Second, there is still a little confusion in the manuscript about when the maternal blood draw occurs. According to table 2, biomarkers and blood samples are taken at recruitment, but I believe that they are actually taken at delivery (as stated in the section above entitled 'Sample collection and laboratory assays'. it would be good to go thru the manuscript one last time and clarify exactly when these samples are obtained.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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

Reviewer #1: No

Reviewer #2: No

**********

[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.

APOL1 Genotype Associated Risk for Preeclampsia in African populations: Rationale and protocol design for studies in women of African ancestry in resource limited settings

PONE-D-22-07467R2

Dear Dr. OSAFO,

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,

Vicente Sperb Antonello, MD, MSc, Phd

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

APOL1 Genotype Associated Risk for Preeclampsia in African populations: Rationale and protocol design for studies in women of African ancestry in resource limited settings

Regarding the present manuscript, all reviewers' notes have been clarified and I believe that this article should be accepted for publication in Plos One.