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PONE-D-22-05551Leveraging large multi-center cohorts of Alzheimer Disease endophenotypes to understand the role of Klotho heterozygosity on disease riskPLOS ONE

Dear Dr. Cruchaga,

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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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Please respond to reviewers' comments.

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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: 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: No

Reviewer #2: 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: 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: Authors conducted a candidate-gene association study with five different AD endophenotypes, brain amyloidosis (PiB PET scan image; and n = 5,541) and CSF Ab42 (n = 5,093), Tau (n = 5,127), pTau (n = 4,778) and sTREM2 (n = 2,123). The target gene in this study was Klotho gene (KL) on chromosome 13, previously reported as a protective gene of AD. As described in the main body of the text (rows 353 – 356 on p19 in Discussion), the sample size is the largest in terms of evaluation of the genetic association of KL-VS^HET+ with the endophenotypic data of AD. The study is well designed, and the conclusion is very clear although the functional relationship between KL and Ab/tau metabolism is not yet well understood and is an important issue to be addressed in the future. Judging from the MAF of the two single nucleotide variants, rs9536314 (p.Phe352Val) and rs9527025 (p.Cys370Ser), such studies are indeed considered valid in Caucasians: actually, the MAF (0.1 - 0.2 in gnomAD) is common, for example, compared to East Asian’s MAF (0.001 in gnomAD), making it a bit easy to find variant carriers. The fact that these two variants have a protective role against APOE-e4 allele in cognitive normal subjects aged 60-80 y.o. is indeed a promising finding although it does not pass the multiple comparison correction. Overall, there are no major revisions, but there are several minor points that need to be corrected and are listed below.

Minor revision:

1) There is no uniformity in the way papers are cited: for example, references #8, 12, 17, 35, 36, and 52. Concerning reference 56, publication date is wrong: 2014 > 1997. Please carefully check the section of References again.

2) Please correct the word "KL-VSHET+" to superscript (KL-VS^HET+) in Table 2 (p15).

3) I wonder if a description of Figure S1 is not able to find.

4) It is helpful for readers to present representative brain imaging data (i.e., amyloid PET images) of KL-VS^HET+ to easily understand the protective impact of the variants in the main body of the manuscript or a supplementary figure.

Reviewer #2: Ali et al. examined the association between APOEε4 and five AD endophenotypes (Amyloid PET, CSF Aβ42, CSF Tau, CSF pTau, and CSF sTREM2) in a hetero group (KL-VSHET+) with one copy of the VS haplotype, a functional haplotype of the Klotho gene, using a total of 9,500 participants. The results showed that KL-VSHET+ was significantly associated with CSF Aβ42, CSF Tau, and CSF pTau, especially in the APOEε4+ group. As the authors mentioned in the introduction, previous studies on the association between KL-VS and AD pathology have reported conflicting results, but this study supports that KL-VS is associated with AD pathology by validating it with a larger sample than previous studies. Overall, the study is well written in detail and the use and interpretation of statistical methods are reasonable.

Please respond to the following comments and minor concerns.

1. The authors mentioned in the introduction that the hetero group with one copy of KL-VS is more protective of the brain than the homo group with two copies of KL-VS (KL-VSHET-). Intuitively, I thought that having more KL-VS haplotypes would be important for brain protection, but this does not seem to be the case. Although the authors examined only the group with 1 copy of KL-VS in this study, I am also interested in the association between APOEε4 and AD pathologies in the group with 0 copies of KL-VS and the group with 2 copies of KL-VS.

2. In Table 2, the authors found a significant decrease in CSF Tau and pTau in APOEε4 carriers compared to APOEε4 non-carriers. To show more clear statistical results, I recommend that the authors perform an association analysis including an interaction effect for the presence or absence of APOE4 and the status of each biomarker (positive or negative) and include the statistics in the paper.

3. Figure S1 is not included in the text.

4. The PCA plot in Figure S3 should have a smaller plot size and be color-coded by the study as in Figure S2.

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Reviewer #1: No

Reviewer #2: No

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Leveraging large multi-center cohorts of Alzheimer Disease endophenotypes to understand the role of Klotho heterozygosity on disease risk

PONE-D-22-05551R1

Dear Dr. Cruchaga,

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.

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Kind regards,

Kensaku Kasuga

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors fully responded to the reviewer's comments.

The manuscript deserves to be published.

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: No

Reviewer #2: 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 #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 have responded to all my comments/suggestions with precision.

Therefore, there are no further corrections.

Reviewer #2: The authors have addressed all of my concerns. The revised manuscript is ready for publication. I recommend publication of this manuscript in PLOS ONE.

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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