PONE-D-21-37584Early onset adult deafness in the Rhodesian Ridgeback dog is associated with an in-frame deletion in the EPS8L2 genePLOS ONE

Dear Dr. Kawakami,

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PONE-D-21-37584

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(I have read the journal’s policy and the authors of this manuscript have the following competing interests: TK, VR, ALR, MKJ, AM, TCN, and ARB are employees of Embark Veterinary, a canine DNA testing company which offers commercial testing for the variant described in this study. ARB is co-founder and part owner of Embark.)

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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: Yes

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: The data is interesting, and I agree with the conclusion made by the authors; that they could not definitely conclude a causal relationship between EOAD and the 12-bp deletion in EPS8L2 gene. I also believe that this is the most interesting finding of all variants presented.

The authors only sequenced the 12-bp deletion in eight adult Pembroke Welsh Corgis that were homozygous for the core haplotype. Would it be possible to increase that number? Where these the only ones that were homozygous for the haplotype?

In-frame deletions can be more difficult to predict in terms of pathogenicity. I think it would be interesting to further explore how the deletion affects the protein, in particularly the alpha-helix structure, and down stream signaling if possible. If the 12-bp deletion would be classified from “likely/possibly pathogenic” to “pathogenic” it could be included is breeding programs for Rhodesian Ridgebacks.

You state that the owners provided informed consent to participate in the study. Is that enough or should the study be approved by an ethics committee?

Line 291, I believe it should be “deletion”.

Figure 2 is upside down in the version I received.

Reviewer #2: Manuscript Summary

This is a well-written and relatively straightforward manuscript describing the identification of an associated variant in the EPS8L2 gene with early onset adult deafness in Rhodesian Ridgeback dogs. The paper is clear, the scientific conclusions are sound, and the manuscript overall is worthy of publication as it will make a strong contribution to the literature on hearing loss in dogs and humans. My comments are all relatively minor (except one major point, but easily addressable), however there quite a few minor points that merit addressing.

Major

1. Please indicate somewhere in the manuscript where the genotyping and WGS data is available publicly. This is mentioned in the cover page from PLoS ONE but I didn’t see it anywhere in the manuscript itself, so not sure whether/how this information will be conveyed to readers.

Minor

1. Line 24: “disorder” not “disorders” – should be singular

2. Line 55: after [8] should be a comma rather than a period

3. Line 58: “an” accurate diagnosis

4. Line 59: “the” relative contribution

5. Line 88: strike the word “Since”

6. Line 94: should be: “translational medical value for better understanding childhood…” rather than as written.

7. Lines 109-111: can you clarify how you identified these RR owners? Through the breed club? Through the Embark database?

8. Line 117: Can you clarify how/where the BAER test was done? Did you have owners bring their dogs to a veterinary neurologist and did he/she interpret the test?

9. Line 121: Awkwardly worded with “of Rhodesian Ridgebacks” – consider: “Three additional EOAD-affected and 12 control Rhodesian Ridgebacks were recruited…”

10. Line 124: Again, please clarify how the BAER tests were performed and analyzed.

11. Line 128: “are” listed

12. Lines 136-139: This really should be part of the results section rather than the methods – consider moving these sentences to the beginning of the section labeled “A novel association on chromosome 18 with EOAD”

13. Line 145: “A Wald test” rather than “Wald test”

14. Line 147: The beginning of this sentence is incomplete: “To evaluate whether marginally significant associations identified in the main GEMMA analysis,…” – either remove the word “whether” or add something like “were relevant” or “remained significant”

15. Line 150-151: I would clarify this by saying something like “We only considered regions showing consistent association….EAOD-associated region in downstream analyses.” Also – can you clarify what you mean by “consistent association”? Did it have to remain associated in 100/100 randomization experiments?

16. Line 165: Please state the VEP version

17. Line 175: should be “these breeds are” rather than “these breeds were”

18. Line 180: please add the word “region” so that it reads “to amplify regions in and around this gene”

19. Line 187: “genotype and phenotype” and should be “were used”; change “nor” to “and”

20. Line 189: I don’t think “Personal Information” should be capitalized?

21. Line 195: Can you clarify that the 23 EOAD dogs you are reporting here do NOT include the 3 “additional” EOAD dogs that you report in the methods? Same for the 162 control dogs – do these exclude the 12 additional controls (line 197)?

22. Line 202: “both had” rather than “were both”

23. Line 212: “were” rather than “are”

24. Lines 214-215: Should be “, where we repeated the GWAS analysis 100 times”

25. Line 216: please change to “of 23 case and 162 control dogs” rather than “with”

26. Lines 213-218: Can you clarify the results of these repeated experiments? What does “not detected as significantly associated regions in replicated runs” mean? In some fraction of the 100 runs? In none of them? Same with “consistently significantly associated” – was this in 100% of the replicated runs? I get the big picture here but I think some more details are warranted.

27. Lines 226-227: should be “an EOAD-associated haplotype” rather than “the EOAD-associated haplotypes.”

28. Liner 230: should be “an EOAD-associated haplotype” rather than “the”

29. Line 232: should be “was a carrier of an EOAD-associated haplotype,”

30. Line 234: should be “have any EOAD-associated haplotype.”

31. Line 243: EOAD not EAOD

32. Line 248: Kill the word “A” at the beginning of the sentence.

33. Line 253: should be “were homozygous for the non-reference alleles”

34. Line 254: consider changing “hearing conditions” to “auditory phenotypes”

35. Line 259: get rid of “the” in “the Plassais et al”

36. Lines 259-260: You can delete the sentence “The 50 bp deletion…” b/c this is repetitive from the previous sentence, and then start the next sentence: “Because the 50 bp deletion was not in LD…”

37. Line 281: should be “11 EOAD affected” not “11 of EOAD affected”

38. Line 283: “intron 13” not “the intron 13”

39. Lines 281-294: I think this paragraph could be replaced, or at least complemented by, a table showing the results for the 3 SNPs and 2 indels. You might have columns that show the position, whether SNP or indel, the location within the gene (intron vs. exon), and for both cases and controls – numbers of homozygous reference, het, and homozygous variant. It would make the point more clearly for readers. I wouldn’t require this, but just something for you to consider.

40. Line 298: I would say “likely causes” rather than “likely resulted in”

41. Line 300: “consists of” and not “consisted of”

42. Line 311: “has” not “had”

43. Line 313: “are”, not “were”

44. Line 318: I would say “three additional EOAD-affected dogs and 12 additional control dogs”

45. Line 321: delete “chromosomes with”

46. Line 331: please say “deletion and EOAD in Rhodesian Ridgebacks” for clarification.

47. Line 339: I would say “..was not seen by short-read whole genome sequencing” rather than “characterized by” as characterized implies a different type of analysis.

48. Discussion: I would consider adding a sentence or two (max) about why one of the mutation prediction programs did not classify the deletion as being pathogenic (MutPred) but the other one did (PROVEAN).

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Reviewer #1: No

Reviewer #2: No

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Early onset adult deafness in the Rhodesian Ridgeback dog is associated with an in-frame deletion in the EPS8L2 gene

PONE-D-21-37584R1

Dear Dr. Kawakami,

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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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: 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: (No Response)

Reviewer #2: Thank you for addressing my concerns and for preparing a well-written manuscript. This should be well-received by the community.

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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