PONE-D-20-18740

Biallelic loss-of-function in NRAP is a common cause of recessive dilated cardiomyopathy

PLOS ONE

Dear Dr. Koskenvuo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

1. Address reviewer 1's concern on the lack of detail on the clinical phenotyping of patients and include more details on these.

2. Provider more details for the pipeline used in the bioinformatics analysis.

3  Address reviewer's comments on improvements for Tables 1 and 2.

4. Fix discrepancies in numbers identified by reviewer 2.

5. Respond to reviewer 4's comments related to exome sequencing and lack of detection of other DCM genes.

6. Consider adding in the reference suggested by Reviewer 4.

7.  Provide greater detail on the inclusion and exclusion criteria for variants.  See comments from reviewers.  Also address how these variants meet ACMG criteria.

8.  Address the other points raised by the reviewers.

Please submit your revised manuscript by Sep 14 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Amanda Ewart Toland, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Competing Interests section:

"Drs. Koskenvuo, Saarinen, Ahonen, Tommiska, Seppälä, Tuupanen, Kangas-Kontio, Schleit, Hathaway, Kytölä, Muona, Sistonen, Salmenperä, Gentile, Paananen, Myllykangas, Alastalo are full-time employees of Blueprint Genetics, which offers genetic diagnostics for cardiomyopathies. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose."

We note that one or more of the authors are employed by a commercial company: Blueprint Genetics, a Quest Diagnostics Company.

2.1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2.2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. 

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

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

Reviewer #3: Partly

Reviewer #4: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

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: No

Reviewer #4: Yes

**********

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: In this manuscript by Koskenvuo and colleagues utilize a large genetic testing database to identify a number of rare variants in NRAP associated with DCM. Overall, this is an appropriately concise report that is well written. It attempts to tackle a common, yet challenging problem – what is the cause of genotype negative DCM?

The size of the cohort analyzed, and the limited co-segregation present are strengths of this paper. However, there are a number of points which should be expanded or clarified in my opinion.

In the introduction, a brief explanation of the physiologic role of NRAP would be helpful. Is it expressed in the heart? Solely in the heart? Would its expression pattern lend itself to a cardiac myocardium only disease?

A major limitation of this manuscript is the lack of clinical phenotyping of the patients with DCM. This should be addressed. What is the evidence that the cohort that was screened had non-ischemic, congenital DCM? This is even more of any issue for the NRAP-positive individuals.

The bioinformatic pipeline used to identify these variants is not well delineated. How were other variants that localized to genes which are known to cause DCM excluded as possibilities?

Were copy number variants identified? This is important to address as there are CNVs associated with DCM (1p36 deletion syndrome) which may confound this data.

The variant inclusion and exclusion data is unclear. For example, having an exclusionary criteria which appears to be <100 individuals is not appropriate as coverage in gnomAD is variable. Thus, a poorly covered area with fewer individuals sequenced may thus have fewer minor allele positive individuals that are nonetheless a high minor allele frequency. Minor allele frequency should be used.

Inclusion criteria for missense variants are particularly unclear. What criteria is used to determine whether a missense variant is included?

Table 1 seems to be extraneous and seems odd. I think it should be removed and potentially referenced in the Discussion

What was the pathogenicity assessment of the NRAP variants by 2015 ACMG guidelines?

Reviewer #2: Koskenvuo et al. presented interesting novel data on NRAP gene’s implication in the pathogenesis of autosomal recessive dilated cardiomyopathy (DCM).

Authors reviewed genetic results of 577 patients either with a confirmed or suspected diagnosis of DCM and compared them with data of control cohort of 31062 individuals, including both non-cardiac controls (n>25000) and non DCM cardiac controls (n>5000).

They identified two rare variants in NRAP gene [(four homozygous truncating (PTV), or compound heterozygous (one patient had two different PTV and 6 had PTV/missense)] in 11 unrelated probands with DCM (1.9%) but none in the controls, with OR of 1052 in comparison to controls. They concluded that NRAP variants could explain 0.25%-2.46% of all DCM cases.

Also, Authors stress the fact that NRAP patients seem to have an earlier onset of major cardiac end-points compared to cardiac laminopathy or DCM, and higher requirement for cardiac transplantation/LVAD.

This is a very important, well designed and rigorously performed study, rather nicely presented.

However, page 10:

Authors state:

“In these 11 individuals four had a homozygous PTV, one had two heterozygous PTVs and two were compound heterozygous for a PTV/missense variant.” This adds up to 7 , and not to 11.

Although later we can learn that the same variants were present in other patients, and the Table 2 lists them, it is unclear and should be corrected.

Page 19

Authors state:

"In the first study suggesting association of NRAP with cardiomyopathy, the proband’s 35-year-old brother who was compound heterozygous for two PTVs in NRAP”. This is untrue. As Authors correctly state in the Table 1, the first identified variant was homozygous nonsense NRAP variant (p.Arg1502*).

Reviewer #3: Koskenvuo et al report a study where they identified 4 cases with biallelic NRAP protein truncating variants (PTV), 2 cases with NRAP compound heterozygous PTV and a missense variant, 1 case with two PTVs (phase unknown) and 4 cases with a PTV and a missense variant (phase unknown), in a large dilated cardiomyopathy cohort (n=577). The aim was to assess the association of bi-allelic loss of function (LoF) variants in NRAP with autosomal recessive cardiomyopathy, since previously, only 4 such cases have been reported. Large disease cohorts are a useful resource to assess for the presence of any gene’s variants and to study their association with the disease. This study provides supports the role of NRAP biallelic loss of function variants and their association with autosomal recessive cardiomyopathy, though with reduced penetrance. The data presented in the study does not support for a causal role of NRAP LoF variants in autosomal dominant dilated cardiomyopathy. However, this manuscript in its current form needs substantial revision before it is suitable for publication in PLos One. This report can benefit from addition of phenotype details of the affected individuals and further discussion on incomplete penetrance or age related penetrance etc.

Major comments:

In this study the authors found 6 cases with biallelic NRAP variants (4 homozygous & 2 with PTV and a missense variant in trans/compound heterozygous). This needs to be clearly stated in the text as the other 5 sets of NRAP variants may or may not be in trans.

Were the variants Sanger confirmed in the 11 cases ? If so, please mention that in the methods.

Table 1

• It is best to list the variants in the “chr-genomic coordinate- Ref allele- Alt allele” format. This format is most useful along with the HGVS nomenclature for the cDNA , protein and the coding exon to which the LoF variants maps to (for example 3/20).

• Please add the units and reference ranges for the CK & ProBNP levels listed

• Addition of the gender of the affected individual will also be helpful

• In the footnotes, it needs to be mentioned that homozygotes for these variants in Gnomad were not seen.

• Please mention the NRAP NM_ID used by the published reports listed in this table.

Table 2

• Table 2 contains information on the cases reported in the current study; however, the organization of this table is not reader friendly.

• Please reword the legend for clarity and make it concise

Suggested Legend “NRAP variants identified in individual with confirmed or suspected dilated cardiomyopathy”

• It is best to have one row each for one affected individual

• Please add available phenotype information for each individual including gender, age of onset, CK levels, available cardiac biopsy

• It is best to list the variants in the “chr-genomic coordinate- Ref allele- Alt allele” format

• List the variants in Hgvs nomenclature (cDNA and protein)

• Add a column to clarify whether it is compound heterozygous or phase unknown

• The cases where unambiguous biallelic NRAP variants were seen (6/11) should be listed first

• the number of coding exon where the variant is located will be a useful addition especially, for the C terminal LoF variants, to assess whether they are predicted to undergo NMD or will result in truncation

• The “type of variant” column is redundant and may be removed

• SIFT and conservation data can be listed in the footnotes

• In the footnotes, it needs to be mentioned that homozygotes for these variants in Gnomad were not seen

For calculation of the population based prevalence using the frequency of NRAP LoF variants in Gnomad v.2.1, were the NRAP LoF variants seen in the last coding exon also included? The LOF variants in the last coding exon of NRAP may not undergo NMD and may result in a truncated product. Hence if these were included, the prevalence estimate derived for NRAP associated recessive cardiomyopathy might be a slight overestimate. Further, there were two high quality homozygous LoFs seen for NRAP in gnomad v.2.1.1 – were they included/removed for the prevalence calculation?

Discussion

If the age of onset data is available for the 6 individuals, along with the 4 published reports the heterogeneity seen should be discussed. If a large variability is seen, it might be supportive of age related penetrance. In addition, the age of onset or its variability, if seen for other AR non syndromic dilated cardiomyopathy genes, is also worth a mention.

Since NRAP is predominantly expressed in the heart & skeletal muscle, in the 6 cases with bialleleic variants were there any muscle issues seen in the affected individuals? Were any muscle issues noted in the 4 previously published cases? If so, this needs to be mentioned in the discussion

The authors list a handful of genes, which cause AR non-syndromic cardiomyopathy. Is age related penetrance or incomplete penetrance a feature seen for these genes as well ? in individuals who carry biallelic variants in these genes. Or this is uniquely seen for NRAP?

This will be insightful; Further, Gnomad V.2.1.1 data has two high quality homozygous LoF variants in NRAP. The authors should attempt to provide a potential explanation for this observation.

It is interesting that a single missense variant (p.Gln24His) which could potentially affect splicing is seen in 4 affected individuals.

Minor comments

Several sentences lack clarity and I urge the authors to read through the manuscript carefully and edit the manuscript for clarity and typos.

For example,

Title

“ Biallelic loss-of-function in NRAP is a common cause of recessive dilated cardiomyopathy”

Instead the following title is clearer

“ Biallelic loss-of-function variants in NRAP are a common cause of recessive dilated cardiomyopathy”

Abstract

“Confirmed or potentially biallelic NRAP variants were enriched in DCM”

the usage of the word confirmed is confusing ; instead it is suggested to use “Biallelic or potential bi-allelic”

“Biallelic (n=6) or two (n=5) NRAP variants (two PTVs or PTV+missense) were”

(two PTVs or PTV+missense)

Instead the following addition will make it more clear.

“Biallelic (n=6) or two (n=5) NRAP variants (two PTVs or PTV+missense) were”

(two PTVs or PTV+missense, phase unknown )

Methods: Patients

Please clearly mention whether the HQSA also included NRAP.

Page 5

estimated int the range of 1:500 to 1:3,000 [2–4].

Page 10:

“Visual Splicing software v2.11 (Interactive Biosoftware, France) predicts that this variant either leads to either loss of the native splice donor”

Instead

“Visual Splicing software v2.11 (Interactive Biosoftware, France) predicts that this variant either leads to loss of the native splice donor”

“Enrichment of NRAP variants in DCM

We identified two rare NRAP variants, of which at least one was a PTV in 11 out”

Instead

We identified cases, which carried two rare NRAP variants, of which atleast one was a PTV

Gene names should be italicized in the manuscript text.

Reviewer #4: The authors identified biallelic loss-of-function in NARP as a common cause of recessive dilated cardiomyopathy with next generation sequencing. However, there are some issues to address.

Major issues:

1. The study didn’t detect the variants of well-established causal genes of DCM. Is the DCM phenotype caused by biallelic loss-of-function in NARP or variants of the known causal genes, such as TTN, RBM20, et, al? It would better to perform whole exome sequencing or targeted next generation sequencing with causal genes of DCM on the cohort.

2. It would be better to confirm the variants in NARP by Sanger sequencing, especially in family segregation analysis due to the false positive rate of next generation sequencing.

Minor issues:

1. Vasilescu et, al reported NRAP underlying childhood dilated cardiomyopathy. It would be better to mention their work in discussion section. (J Am Coll Cardiol. 2018 Nov 6;72(19):2324-2338.) in NARP.

**********

6. 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: Yes: Andrew Landstrom

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-18740R1

Biallelic loss-of-function in NRAP is a common cause of recessive dilated cardiomyopathy

PLOS ONE

Dear Dr. Koskenvuo,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

1.  Soften language suggesting that biallelic NRAP loss is a common cause of AR DCM throughout manuscript and in title.

2.  Expand description of bioinformatics analysis of NGS data in the methods.

3. Include details of inclusion/exclusion criteria for study population.  See reviewer's comments.

4. Consider doing Sanger to confirm the 11 variants found.  If Sanger is not done, list lack of confirmation of these variants as a study limitation in the Discussion.

5.  Check for spelling errors.  See reviewer's comments.

Please submit your revised manuscript by Jan 30 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Amanda Ewart Toland, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #4: (No Response)

**********

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

Reviewer #4: Yes

**********

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #4: 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 Response)

Reviewer #2: Yes

Reviewer #4: Yes

**********

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

Reviewer #4: 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 revisions to this manuscript appear reasonable and the manuscript is greatly improved. I have one lingering concern.

Overall, I think the language associating biallelic loss of NRAP is too strong. For example, the title stating that it is the most common cause of AR DCM, the conclusion in the abstract and discussed in the paper is outside the scope of the data. Given the heterogeneity of the genetic substrate of DCM, additional, independent validation studies are needed to corroborate this finding. This should be highlighted in the manuscript, including in the title, and the language appropriately softened.

Reviewer #2: The manuscript is sound, concise and interesting. Although clinical data are not robust, the report nicely underlies severe clinical endpoints related to the biallelic loss-of-function NRAP variants causing autosomal recessive DCM.

Spelling mistakes:

fatigability

wildtype

Reviewer #4: The authors found that rare NRAP variants were associated with the pathogenesis of autosome recessive dilated cardiomyopathy through genetic testing performed on a large cohort. Moreover, co-segregation analysis provided a strong supporting evidence for this conclusion.

However, there are some issues to address.

1. Have the authors performed Sanger sequencing to validate the 11 variants as NGS possess relatively high error rate?

2. Please described the bioinformatics analysis in Materials and methods section in details.

3. It is better to list out the inclusion and exclusion criteria of the recruited subjects. How to define a DCM patient and what kind of individuals can be classified as suspected DCM instead of non-DCM cardiac group?

**********

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: Yes: Andrew Landstrom

Reviewer #2: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Biallelic loss-of-function in NRAP is a cause of recessive dilated cardiomyopathy

PONE-D-20-18740R2

Dear Dr. Koskenvuo,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Amanda Ewart Toland, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: