Dear Dr. Ghasemi Semeskandeh,

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“The CHRIS study was funded by the Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol. This work was carried out within the TrainCKDis project, funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement H2020-MSCA-ITN-2019 ID:860977 (TrainCKDis).”

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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?

Reviewer #1: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: This is an interesting article which takes GWAS meta analysis data and undertakes a systematic mediation to look for quantitative traits that may underlie the SNPs leading to chronic kidney disease. Some additions are needed to maximise the utility of this analysis.

Page 4 - the rather convoluted text regarding replication needs to be revised / simplified. The authors say it is not a replication analysis but then refer to it as a replication analysis. I understand their meaning but it needs to be phrased more clearly.

The association of variants in SLC34A1 and aPTT - this needs to be dissected further, as the authors point out the snp is near to F12 which is more likely to be biologically relevant. Some eQTL or candidate cis-regulatory elements (cCREs) analysis for the SNPs of interest should be investigated (see Loeb GB et al Nature Genetics).

The PDILT snp is usually associated with UMOD - can this be mentioned clarified

The SHROOM3 findings related to Magnesium are interesting and perhaps a mechanistic diagram would help here to understand how SHROOM3 might be mediating magnesium regulation. Again for the SHROOM3 snp some QTL or candidate cis-regulatory elements (cCREs) analysis for the SNPs of interest should be investigated.

It would be useful to know if any of the SNPs are in coding regions.

The associations with abnormal thyroid function as modifiers are really interesting and that adds to the novelty of this study

Reviewer #2: Ghasemi-Semeskandeh et al., in this study of the CHRIS cohort (n=10,146), replicated associations for 11 kidney function loci, showing effect sizes up to 5.4 times larger than those observed in the CKDGen kidney function meta-GWAS involving 1 million individuals. Mediation analysis highlighted serum magnesium and activated partial thromboplastin time as partial mediators at SHROOM3 and SLC34A1, respectively. Additionally, thyroid function was identified as a genetic effect modifier, with significant SNP-by-TSH interactions at STC1. These findings underscore the value of individual population studies in elucidating genetic mechanisms underlying kidney function. However, I have several suggestions for the authors to consider:

1.Ethical Statement Placement: In the Methods section, I suggest beginning with the Ethical Statement, which is currently placed at the end of the section, for improved organization and clarity.

2.Selection of Loci: The authors selected 147 kidney function loci from over 264 independent loci reported in the CKDGen study. Please clarify how this number was reduced to 147 after excluding CHRIS samples from the GWAS summary statistics. Additionally, it is unclear how MetaSubtract was used to exclude CHRIS data from the summary statistics. Why not conduct the analysis solely on independent CHRIS subjects (n=~10,000), even if the sample size is smaller, to ensure cleaner results?

3.Correction for Multiple Testing: Did the authors account for multiple testing in the mediation analysis, given that 70 quantitative traits were assessed? While multiple testing correction for SNPs was mentioned, clarifying this point for the quantitative trait analysis would strengthen the study.

4.Evaluation of Collider Bias: Did the authors assess the potential for collider bias during mediation analysis, particularly for the quantitative traits? Addressing this would help validate the findings further.

5.Discrepancy in Variant Count: The authors mention testing 147 variants, but in the Results section (Page 9, Line 256), they state that 163 variants were tested at 11 loci. Are these independent SNPs? Please clarify this discrepancy and provide a detailed explanation.

6.Figure Legends: Some figures lack clear explanations in their legends, making it difficult to understand what tests were conducted. Providing detailed figure legends will significantly improve comprehension.

7.PCA Plot for Ancestry: To enhance clarity, consider including a PCA plot of the CHRIS cohort alongside the 1000 Genomes dataset to better illustrate ancestry composition.

Overall, addressing these points will enhance the clarity, organization, and rigor of the study.

Reviewer #3: The manuscript titled "Systematic mediation and interaction analyses of kidney function genetic loci in a general population study" explores the genetic factors influencing kidney function, specifically focusing on chronic kidney disease. The authors conducted a study using data from the Cooperative Health Research In South Tyrol (CHRIS) involving 10,146 participants to assess the association of 147 kidney function-related genetic loci with estimated glomerular filtration rate (eGFR) based on serum creatinine levels. The manuscript addresses a topic of great clinical importance, the relationship between genetic factors and kidney function, especially in a specific population context. The identification of genetic loci associated with glomerular filtration rate (GFR) may contribute to understanding the genetic basis of chronic kidney disease (CKD) and its implications for treatment and prevention.

Introduction

The introduction could be more robust in contextualizing the problem. It would be useful to include a literature review explaining the relevance of the genetic loci chosen and how they relate to kidney function and chronic kidney disease.

Statistical Issues:

The statistical analysis is adequate, but the choice to penalize the significance level for multiple tests must be clearly justified.

The use of a large and homogeneous sample, such as that of the CHRIS study, is a strength, as it allows for a more robust analysis of genetic associations. However, it is important to discuss the limitations of the methodology, such as the dependence on specific markers (such as creatinine) to estimate GFR and the need for validation in different populations.

Results:

It would be beneficial to include a section discussing the validation of the results in independent cohorts or the comparison with previous studies. This will help strengthen the credibility of the findings.

Discussion:

The discussion about the limitations of the study should be more comprehensive. Consider discussing the homogeneity of the sample and how this may affect the generalizability of the results.

Conclusion:

The conclusions are consistent with the results presented, but should emphasize the need for future investigations to explore the underlying mechanisms and applicability of the findings in clinical contexts.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Systematic mediation and interaction analyses of kidney function genetic loci in a general population study

PONE-D-24-44950R1

Dear Dr. Ghasemi-Semeskandeh,

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.

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Kind regards,

Andrew J. Mallett

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

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??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: The revised manuscript has answered my previous concerns. The updated draft allows a more complete understanding of the data and its interpretation.

Reviewer #2: All of my comments have been thoroughly addressed, and after reviewing the changes, I don’t have any further suggestions.

Reviewer #3: (No Response)

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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

Reviewer #3: No

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