PONE-D-21-30440

A de novo variant in the keratin 1 gene (KRT1) in a Shar Pei dog with severe congenital cornification disorder and non-epidermolytic ichthyosis

PLOS ONE

Dear Dr. Affolter,

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.

Editor and reviewers find the manuscript to be largely sound and worthy of publication, however numerous inconsistencies in language and clarity need to be addressed as detailed in the reviewer comments.

Please submit your revised manuscript by May 27 2022 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Brian W Davis

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 financial disclosure:

“This research was funded by the Swiss National Science Foundation, grant number 310030_200354 PI: Dr. Tosso Leeb, University Bern Switzerland”

Please state what role the funders took in the study.  If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.""

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

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.

[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

********** 

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

Reviewer #1: Yes

Reviewer #2: N/A

********** 

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

********** 

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

********** 

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 manuscript entitled “A de novo variant in the keratin 1 gene (KRT1) in a Shar Pei dog with severe congenital cornification disorder and non-epidermolytic ichthyosis” is a case study that describes the identification of a putative causal genetic variant in a single dog. While the identified variant is deemed to be de novo, and is not expected to have broad clinical relevance or importance in carrier identification within vulnerable populations, the discovery does expand the knowledge base of congenital epidermal disorders and gene function.

Minor corrections

- Throughout, a consistent scheme for capitalization of dog breed names needs to be followed. The generally accepted convention is to capitalize only the proper nouns in the name. Likewise, the official breed names should be used when relevant.

o Throughout: Shar Pei = Chinese shar pei

o Pg 3, line 49: Golden retriever = golden retriever

o Pg 3, line 54-55: German shepherd = German shepherd dog

o Pg 3, line 58: soft-coated Wheaten terriers = soft-coated wheaten terriers

o Pg 3, line 59: West Highland White terriers = West Highland white terriers

o Pg 3, line 59: English Springer spaniels = English springer spaniels

o Pg 4, line 78: Standard Poodle = standard poodle

o Pg 4, line 78: Terrier-mix = terrier mix

o Pg 4, line 78: Boxer = boxer

o Pg 10, line 244: German shepherd = German shepherd dog

o Pg 11, line 264: Norfolk Terriers = Norfolk terriers

- Pg 3, line 61: people = humans

- Pg 3, line 67: board-certified dermatologist = board certified veterinary dermatologist

- Pg 4, line 74: board-certified pathologist = board-certified veterinary pathologist

- Pg 4, line 77: “… three biopsy samples of the patient …” = “… three biopsy samples from the patient …”

- Pg 5, line 112: “… 793 control genomes (S1 Table).” Tell us roughly what the control genomes are. “… consisting of ### domestic dogs and ### wild canids.”

- Pg 6, ine 145: “… resented for scaly skin and reduced growth when …” Growth of what? Hair, skin, body size?

- Pg 7, lines 151 – 152: should read “At the time of presentation, the dog appeared …”

- Pg 7, line 159: “… for Demodex mites was negative.” Should be “… for Demodex mites were negative.”

- Pg 10, line 241: What is the envelope?

- Pg 10, line 241: Add a period after “desquamation”.

- Pg 10, line 257: “Electron microscopic revealed …”. Do you mean “electron microscopy” or is there a missing word after “microscopic”?

- Pg 11, line 259: Remove the “; it” at the end of the line.

- Pg 11, line 261: people = humans

- Pg 11, line 268: Should read “… reflecting the histologic features observed in the affected Chinese shar pei.”

- Pg 12, lines 286 – 288: The last sentence does not belong here. Move it up somewhere into the body of the Discussion.

Moderate to substantial corrections

- In the introduction, add 1 – 2 sentences describing “hereditary cornification disorders” and “ichthyosis”.

- Pg 3, lines 47 – 57: This is a single sentence that runs nearly 11 lines. It needs to be restructured.

- Pg 8, line 180 mentions that dilute hair coat color was considered incidental to the skin findings. None of the candidate genes investigated later were related to coat color or associated hair/skin conditions. Should any consideration be given to the possible overlap with color dilution alopecia, which is commonly seen with the “D locus” dilute coloration?

- Why were only known ichthyosis candidate genes considered? In the trio analysis, you identified 542 protein-changing variants, which is a very reasonable number of genes to investigate for possible functional implications. You may wind up with the same answer, I think you have sufficient evidence to suspect that the KRT1 variant is correct, but by not limiting yourself to a list of candidate genes, you are better able to make your case. What other genes were affected by the 542 protein-changing variants, and would any of them be potential functional candidates?

- Table 1: What does “Genotypes of parents compatible with a pathogenic effect” mean? It sounds like there is one variant found in the parents that may be pathogenic? What variant is that, what gene?

Reviewer #2: The authors have produced an interesting manuscript identifying a putatively associated variant in a Shar Pei with ichthyosis. The 3 base pair heterozygous deletion was uncovered by selecting private variants against a large set of public genomes, filtering with known candidate genes, and compatibility with parental genotypes. Immunohistochemistry indicated the deletion does not alter protein expression. Based on these observations and previous literature, the authors suggest the deletion may interfere with protein interaction of a known partner (or other function) and is the presumed cause of disease.

Comments

• While this does appear to be a good candidate variant, there are numerous instances where the language describing the potential role is not appropriate based on the analyses yet completed. For example lines 34-35 (“…strongly suggests…caused”), line 63 (“…due to a 3 base pair…”), and line 276 (“This strongly suggest…”). The authors should rephrase these (and any other) passages to reflect potential association as opposed to causation.

• Line 31 (and the discussion) speculate that the deletion “interferes with keratin dimerization”. While this certainly could be true based on the previous literature, this has not been thoroughly investigated here. The immunohistochemistry presented herein does not suggest this really. If the authors want to include a description of this potential mechanism the language should be toned down (as in the first comment) and likely removed from the abstract. Alternatively, and if possible, it would be really interesting to see the results from some type of in-vitro protein-protein interaction assay.

• How was the variant filtering accomplished with the 793 public control genomes? In addition, I noticed there were no Shar Pei’s in the control set. Were there none available or were they excluded? A reference (14) was used to cover how the mapping and variant calling were conducted but additional descriptions of how the variant filtering was conducted, why these 793 were selected, and why no other healthy Shar Pei’s were included is needed.

• Why was confirmation of parentage done with VCFtools as opposed to a more commonly used parentage/genetic marker test? In this section they are referred to as “presumed parents” but “unaffected parents” earlier suggesting not presumed. Similarly, lines 211-212 state parentage was “confirmed based on the genome sequence data.” Please include clarification as to the why and how here – this is important so that we have absolute confidence that the parents are in fact the parents.

• Line 122 the authors should include a description of the multiple sequence alignment method.

• The candidate gene set appears to be from three previous papers, one of which is for a dog with ichthyosis. Were there attempts to expand the search for candidate genes via databases such as OMIM or PubMed? Additional descriptions on why only these sources were used should be included. Perhaps a structured systematic review is not needed but a more complete explanation on how the candidate gene set was developed would be helpful in understanding why this severe filtering (92k to 500 variants) was needed.

• Table 1 lists 92k private variants, were there any attempts to examine this set for other potential variants compatible with an autosomal recessive inheritance pattern that could explain the phenotype? The definition of “protein-changing” should be probably be included somewhere as well. I would suggest including some type of analyses on the 92k private variants. For example, theses could be categorized by location, type, and predicted impact (I believe SnpEff outputs summary files containing this type of data?). Similar to the candidate gene comment above, the authors should examine at least the predicted high-impact variants for other potential genes that may be associated with keratinization disorder.

• Lines 231 and 235 state expression was “comparable” between the affected and normal dogs. It looks a bit a subjective and to the untrained eye, these appear rather different on paper. It would seem that in order to make statements about comparability, protein quantification should be conducted. Transcript quantification via RNA-seq or qPCR may be warranted as well to uncover any potential differences in expression. Baring any additional experiments, perhaps there is a pathologist associated with this study that could describe how these were identified as comparable?

• There should probably be some mention in the discussion of the uncertainty inherent in using the public annotation as there are known issues. For example, does this deletion appear in the same amino acid and domain using the Ensembl annotation which lists two isoforms?

Minor comments

• There is a discrepancy in author order between the cover sheet and manuscript. Please correct.

• Lines 46-57 is a single sentence and as a result a little difficult to follow. I would suggest breaking this up or restructuring it.

• Table 1 legend “genome resequencing”. Is this supposed to be “genome sequencing” or was this dog previously sequenced?

• Figure 3 legend has a reference error {Bray, 2015 #17}.

• Minor inconsistency in naming the protein dimer (line - 266 KRT1/10; line 273 - KRT1/KRT10). I am not sure which is correct but should be consistent I would think.

********** 

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: No

Reviewer #2: 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.

A de novo variant in the keratin 1 gene (KRT1) in a Chinese shar pei dog with severe congenital cornification disorder and non-epidermolytic ichthyosis

PONE-D-21-30440R1

Dear Dr. Affolter,

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,

Gerhard Wiche, 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

**********

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: N/A

**********

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: 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

**********

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 adequately addressed all of the points that I raised in the initial review. I am comfortable now recommending this manuscript for publication.

Reviewer #2: All comments/questions were addressed thoughtfully by the authors. The manuscript is technically sound and the underlying appears to be publicly available.

**********

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

**********