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PONE-D-23-37920Rare Genomic Copy Number Variants Implicate New Candidate Genes for Bicuspid Aortic ValvePLOS ONE

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Additional Editor Comments:

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The study is relevant and of interest. The reviewers are in agreement that the mauscript at a number of points needs clarification and generally needs some word processing. Plieas pay attention to reviewer comments and adhere to these.

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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: Partly

Reviewer #3: Yes

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

Reviewer #1: No

Reviewer #2: I Don't Know

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 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: Yes

Reviewer #3: 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

Reviewer #3: 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: The paper by Carlisle et al describes rare copy number variants in two sets of patients with bicuspid aortic valves. While this type of investigation has been described before, the paper is of interest as it has two different cohorts, one early onset and one late onset, and describes the differences and overlap between these findings. However, because of the two patient cohorts, multiple control cohorts and the differences between these cohorts, the paper could do with some textual cleaning up, as at times it is confusing.

The use of control cohorts to compare against needs more detail. WLS and HRS and Illumina Genotypes are used to compare CNVs against. What are these cohorts? Basic information on this is needed. Why are certain control cohorts used to compare against certain patient cohorts? Why not use one cohort, etc etc. How sure are the authors that these cohorts do not also contain patients with BAV (as BAV is a rather frequent phenotype), and would this be a problem?

The authors do three comparisons, EBAV alone, BAVGWAS alone and overlap between the two. However this is not quite clear from the results and the way it is written. I would like a clear separation between these comparisons in both text and tables, as that makes it much easier to follow. Is there a table in which the overlap between the two sets is described?

Table S9 is unclear. How can there be segregation when none of the family have the CNV? Eg BAV787. I would suggest including pedigrees, as that makes this type of information much easier to parse.

On line 191/193 prevalence is discussed, but I don’t see any statistics here to compare between the two groups.

On line 98 references are missing to the various papers that investigated the genes mentioned.

In the discussion, before line 335 there is a paragraph on GJA5 deletions , after this there is just a summary genes, each on their own sentence. This reads rather jarring in comparison to the previous part. I would put a connecting or explanatory sentence here, to prevent it reading like a summary.

Reviewer #2: The manuscript reports the association of copy number variants with bicuspid aortic valve disease. The authors report that CNVs could explain a notable number of cases. This is a very interesting study and advances genetic discovery efforts for BAV. I did however find some areas of the manuscript difficult to interpret. I believe the manuscript could be improved with a clearer description of findings. I list several points below.

Cohort numbers are confusingly presented. For EBAV 272 probands are mentioned in the abstract, 293 families on Page 6, a cohort size of 544 (comprising both affected and unaffected samples) presented in Table 1 and 279 probands in Table 2. For BAVGWAS the text refers to 5040, but Table 2 summarises just 3141. It is therefore unclear how many samples have contributed to the analyses. Can the authors please explain and address this so that numbers analysed are clearly presented.

I don’t see the need for Table 1 with the information included.

Page 9, paragraph 2. Presentation of Tables 3 and S2 in relation to the text could be improved. Is the “prevalence of large and rare CNVs”, referring to the PE for number of CNVs per individual?

Table S3 could include a column listing affected genes.

Where is the data supporting the page 5 statement “Seven of these genic CNVs were

205 enriched in EBAV cases compared to WLS controls with a genome-wide adjusted empiric P < 0.05.”

Which BAV genes are in the CNVs assessed in Table 4?

Page 10. “Large duplications involving the 208  Velocardiofacial (VCFS) region in 22q11.2 and 1q21.1 microduplications were also enriched in 209  EBAV cases (Table S4)”.

The table does not present evidence for enrichment.

Page 10. Which CHD genes were scrutinized? A supplementary table listing genes assessed would be helpful. I think important to show CHD associated genes where no CNV was identified.

Page 10, Table 5 and table S6. I believe “subtle” should be properly defined to clarify criteria of the CNVs analysed in this section. Relationship of the two tables is unclear – for some genes (e.g., MYH11) the CNVs described in Table S6 appear to correspond to the numbers presented in Table 5, however, this is not true for others (e.g., GATA4). Please explain.

Burden testing P-values should be presented to the data in Table 5 and Table 6.

Burden testing method is not properly described.

Abstract and Page 13, opening paragraph of discussion. The authors report potentially pathogenic variants for 8% of BAV cases in their EBAV cohort. It is not immediately clear to me which CNVs were considered as likely pathogenic. A table summarising the cases where CNVs have been interpreted in this way, with a clinical description of proband/family could be included.

The methods should include a section clearly describing the statistical tests performed.

Reviewer #3: The authors analyzed the frequency and genetic relevance of large and rare CNVs in a cohort of early onset bicuspid aortic valve disease. BAV is the most common congenital heart defect and a major cause of severe cardiac complications (early) or later in life. As the genetic causes of the disease are still incompletely understood and especially risk stratification is unclear the study is of great clinical interest. The genetic methods are appropriate, and the manuscript is well written.

However, I have some comments:

1. Did the authors check whether large and rare CNVs tend to occur more often in patients with BAV developing an aortic aneurysm or is the distribution equal?

2. Where the CNVs more common in families with more than 1 affected individual?

3. Interestingly, CNVs only affected the TGFB family in late onset BAV. How do the authors explain this finding?

3, The average clinical reader may not be used to CNVs. I therefore recommend including a simple graphical abstract.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Rare Genomic Copy Number Variants Implicate New Candidate Genes for Bicuspid Aortic Valve

PONE-D-23-37920R1

Dear Dr. Prakash,

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,

Nejat Mahdieh

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 #3: 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 #3: Yes

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

Reviewer #1: Yes

Reviewer #3: 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 #3: 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 #3: Yes

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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: All my comments have been addressed, the manuscript has improved and is of interest. I have no further suggestions.

Reviewer #3: The reviewers comments have been fully addressed. Therefore, I am happy to recommend the manuscript for publication.

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Reviewer #1: No

Reviewer #3: No

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