PGENETICS-D-25-00725

Genomic profiling of active vitamin D colonic responses in African- and European-Americans identifies an ancestry-related regulatory variant of POLB

PLOS Genetics

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Heather J Cordell

Academic Editor

PLOS Genetics

Hua Tang

Section Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

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Reviewer #1:

Reviewer #2:

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At this stage, the following Authors/Authors require contributions: Bharathi Laxman, Hina Usman, Margaret Bielski, Kristi Lawrence, and Sonia Kupfer. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: This is an interesting and thorough study of the effect of 1,25D on color organoids, with emphasis on the POLB findings. In particular, this reviewer appreciated the multi-omic approach and thoughtful consideration of ancestry. Below are concerns about the overall approach, as well as some comments related to interpretation.

1) I am a bit confused by the information presented in Figures 1D and 1F the 1D, the authors state ~4000 peaks were significantly associated with 1,25D treatment. But in 1F, the number by ancestry (one alone or both) is only ~500, as the legend states.

2) For eQTL analysis, the author states that they used BRIdGE and MatrixQTL to identify eQTLs; however, there is no mention of multiple testing correction commonly done in eQTL analysis. An FDR (Or posterior probability cut-off) is not sufficient for the number of SNPs tested, of the number of genes assessed. More up-to-date methods have now been created to assess Context-specific eQTLs (Meta-Tissue, MashR).

3) In the eQTL analysis, no covariate (age, sex, etc) seems to have been added to the analysis. Were both ancestors mapped together? This may pose a real issue if no PC were added. No SVA or PEER was included to correct for hidden variables in gene expression.

4) The authors focus on POLB because of the difference in transcriptional response with treatment. In the LFC on ATAC seq peak near this locus, only 9 to 10 points are shown. Why are there so few samples in this analysis? The methods are stated 12-13 in each group.

5) 3 models of DE analysis are described in the methods, but only one is shown in the results (Model 1). The other. Models are very interesting, and the results of those should also be presented.

6) It would be interesting to know if the eQTL identified after treatment are located in DA genomics regions, with the idea that some SNPs may not exert a regulatory effect until they are located within open chromatin.

7) Missing from the discussion is a clear delineation of limitations, such as the use of organoid cultures, which may differ from colonic tissue in vivo, the relatively small sample size, and the power to detect association inherent in the study, especially once divided by ancestry.

8) People of African Ancestry are known to have lower levels of Vitamin D as well as higher levels of VDR. How would these differences relate to your findings? While genetic regulation is interesting, would the long-noted difference long noted blunt the findings?

Minor Comments

1) As a result, the authors state that they controlled for Ancestry (line 102) in the DE analysis (Model 1). I believe they mean population as Ancestry is controlled for in Model 3.

Reviewer #2: review will be uploaded as attachment

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Reviewer #1: None

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes: Syed Munim Husain

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Attachments

Attachment

Submitted filename: plosgenetics_vitD_review.docx

Dear Dr Kupfer,

We are pleased to inform you that your manuscript entitled "Genomic profiling of active vitamin D colonic responses in African- and European-Americans identifies an ancestry-related regulatory variant of POLB" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Yours sincerely,

Heather J Cordell

Academic Editor

PLOS Genetics

Hua Tang

Section Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have addressed all comments fully.

Reviewer #2: The authors have fully addressed my previous comments. Well done on the substantial and excellent work.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy , and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes: Syed Munim Husain

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