PONE-D-21-24235Additive genetic effect of GCKR, G6PC2, and SLC30A8 variants on fasting glucose levels and risk of type 2 diabetesPLOS ONE

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Reviewers' comments:

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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: No

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Reviewer #1: Yes

Reviewer #2: 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: Chen et al conducted GWAS with 23,535 individuals, either nondiabetics or

298 untreated diabetics, and identified and replicated three independent SNPs in GCKR, G6PC2, and SLC30A8 associated with fasting glucose levels.

Here are my comments:

1. In Abstract line24, authors should clarify whether the they have multiple outcomes in the regression model. If it is only one outcome in the model, the 'multivariate' should be 'multivariable'.

2. In the association analysis part, The fasting glucose is log-transformed. It seems the density plot still have tail after transformation. I suggest authors check the normal test such as Shapiro–Wilk test before and after transformation.

3. If the log-transformation for FG works, the residual should follow normal distribution. If this is the case, author should provide the reason why they also need the inverse normal transformation for the residuals.

4. How was the distribution of FG in the replication?

5. Authors should describe the statistical method for T2D.

6. In line 203, author should include the backward selection criteria.

Reviewer #2: The Chen et al performed a GWAS of fasting glucose in non-diabetic and untreated diabetic individuals of European ancestry. 3 loci displayed significant associations (SNPs with P<5x10-8). The association of the 3 lead SNPs (after fine mapping) was replicated in trans-ethnic meta-analysis of European, Chinese, Hispanic and African ancestry. The authors further test the possible consequences of non-synonymous SNPs using various in silico tools. Finally, a genetic score combining the FPG reducing alleles was derived. The score was associated with FPG and T2D.

The manuscript is interesting and easy to read. I have the following comments/suggestions:

Methods :

- in replications studies that are family studies (eg, FHS and HUFS), did you only keep unrelated individuals as in discovery ?

- I suppose all the alleles shown in the manuscript tables/text are on the + strand ? please specify.

- Replication studies : please clearly state the genome build used in methods and tables (same as discovery study so, hg19 ? ).

- All R packages: provide version.

- Reference the R software and provide version.

Row 82: Is there a missing "." here ? if not, the sentence is not really clear.

Row 83: "Among Europeans" : please specify how the EUR ancestry was determined (self report, genetic data ? or both ?).

Row 88: use "race and ethnicity" instead or "race" only.

Row 89: T2D definitions in row 79 and 91 are slightly different, please clarify.

Row 96: GWAS QC: no sex check control performed? no ethnicity check? Also maybe provide some references for the QC of replication studies?

Row 107: Can the author provide a brief justification as to why they used a two-stage mixed model? (i.e. first regressing on Age sex BMI on log FPG, then use ranked inverse normalized residuals for stage 2? Also, does this mean that all FPG related results (Beta, SE, etc) we see in the tables are back transformed and are in mmol/l? if yes, this should be mentioned.

Row 111: How many PCs adjusted for?

Row 118: What was the random factor in the mixed models?

Row 118-119: Please provide a full dbGAP accession number with the version (phsxxxxxx.vx.px format).

Row 121: Add citation to the dbGAP database itself.

Row 126: Consider giving the number of case controls by ethnicity form each study in a supplementary table.

Row 131: For each study (both stage 1 and replication studies) and for each ethnic group, provide a supplementary table with genotyping array, imputation panel used, filtering criteria (e.g. info score and MAF) and the resulting number of SNPs used (in the analysis in addition to the paragraph lines 131 to 138).

Row 164: Add ref to R package Meta.

Row 152: How do you define the presence of "LD between SNP".

Results:

Row 188: "The associations at all three lead SNPs were replicated", please specify that this is for the overall Meta-A, and briefly describe the replication results by ethnic groups.

Row 188: "Of the two suggestive loci": Please specify what you mean by suggestive locus ( 5*10-7< P <5*10-8 ?)

Row 192 + 198: SIFT, Polyphen-2 and CADD not mentioned at all in the methods. Please correct. also Ensembl is referenced instead of the programs and original papers themselves, if SIFT, Polyphen-2 and CADD results were extracted from Ensembl, then this should be specified in the methods, and references should include the original SIFT, Polyphen-2 and CADD papers in addition to Ensembl.

row 193 + Figure S5: "The model protein structure of GCKR with L446P predicted an altered interaction between GCKR and GCK Figure S5)": not very clear from the figure S5 how the "interaction is altered", do you have a metric you use? a prediction P-value? also, maybe you could add arrows that point to where we're supposed to look on Figure S5 + specify what the purple color means?

Row 230: eQTL and sQTL analysis not described in methods and results.

Row 209: Please correct "nomarl".

Row 209: "compared with normal values are less ...". Please consider changing the phrasing.

Row 210: Please correct "mmol/".

Discussion:

Row 236: it is a bit confusing that authors describe rs560887 as lead SNP after fine mapping, having a low functional impact (row 195), but then discuss eQTL and sQTL effects in the discussion, and later suggest that rs573225 might be a better candidate. Was rs573225 included in fine mapping? what was its posterior probability? has the fine mapping performed in the different ethnic groups or restricted to EUR?

Row 258-261: " based on the sequence to structure... for predicted protein function": should not be included in discussion, it reads more like a methods paragraph.

Introduction:

Row 37: Please replace ".." by "."

Abstract:

Row 34: The authors should not just mention the results of 0 vs 6 T alleles and emphasize the gradual change of outcome based on the number of risk alleles (linear trend) instead.

Row 37: "Since non of the individuals homozygous for the ... identify individuals at low vs. high risk of developing type 2 diabetes": Not sure about this statement... I think if you derive a genetic score using more than 3 SNPs it will have a far better predictive power than a score with 3 SNPs.

All Tables, Figures and Supplementary Figures: Please add abbreviations and units when applicable.

Table S3:

-Add an Ethnicity column.

-Show meta-analysis heterogeneity results.

-Show meta-A results by ethnic group (Europeans, Africans + African descent, Chinese, Latinos)

Figure S3: Figure Resolution too low.

Figure S6: The figure title does not reflect the fact that you are testing the number of protective alleles.

Figure S7: The figure title does not reflect the fact that you are re testing the number of protective alleles.

References: I believe I found a couple of references that do not match what is being said in the main text, please double check the references.

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Reviewer #1: No

Reviewer #2: Yes: Amel Lamri

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Additive genetic effect of GCKR, G6PC2, and SLC30A8 variants on fasting glucose levels and risk of type 2 diabetes

PONE-D-21-24235R1

Dear Dr. Chen,

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,

Nicholette D. Palmer, Ph.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

Reviewer #2: 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

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: 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

Reviewer #2: (No Response)

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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 #2: 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: The authors addressed all my concerns. I have no further comments.

Reviewer #2: (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 #1: No

Reviewer #2: No