PONE-D-21-16348

Clinical and genetic spectra in patients with dystrophinopathy in Korea: A single-center study

PLOS ONE

Dear Dr. Park,

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.

Both reviewers ask for a number of additional pieces of information as well as some editorial changes.  Please address these, either by making changes, or by providing an explanation.

Please submit your revised manuscript by Jul 26 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. 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,

Michael Kyba

Academic Editor

PLOS ONE

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.

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:

"NO authors have competing interests"

We note that one or more of the authors are employed by a commercial company: 3bilion, Inc..

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. 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

**********

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

Reviewer #1: Yes

Reviewer #2: 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: 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: This is a solid, well-written manuscript describing the clinical and genetic characteristics of a large single center cohort of dystrophinopathy patients from Korea. This information will be valuable if the authorities in Korea are considering regulatory and access issues for the new molecular therapies for this disease. I have the following recommendations to improve the manuscript.

1. I recommend alternate references to replace #1 and #2. The authors are correct in citing Louis Kunkel, as his laboratory was responsible for the initial cloning of DMD and discovering the protein dystrophin, but the seminal articles are: (1) Monaco AP et al, Nature 1986;323:646-650 (initial cloning of DMD); (2) Koenig M et al, Cell 1987;50:509-517 (cloning of entire DMD gene); and Hoffman EP et al, Cell 1987;51:919-928 (initial discovery of the protein dystrophin). Some or all of these articles should be cited instead.

2. Regarding the life expectancy into the 30s and 40s for DMD, that is correct but only in the context of good supportive care. Without such care, the life expectancy is much shorter and thus this caveat should be noted.

3. The Introduction notes 2 antisense oligonucleotide drugs but the Discussion mentions 4. There are 4 such compounds approved in the US to date so the Introduction should be corrected.

4. I am a little confused about the composition of the UD cohort. The Methods section suggests that they are presymptomatic patients, and in the Results it suggests that the UD cohort has incomplete medical records. One or both of these passages should be clarified for consistency.

5. It is best to avoid the term "wheelchair-bound", it is better to say "non-ambulatory" instead.

6. In the methods, it is best not to say "We also assessed the serum creatine kinase..." and "Echocardiography was performed..." as the wording suggests that these data were collected prospectively. As this is a retrospective study, it is better to say that results of prior CK and echocardiography studies were extracted from the medical records.

7. In the Genetic spectrum section, the first sentence should end with ", respectively."

8. The description of the 4 exonic duplications affecting two regions. I assume these are large duplications rather than two duplications for each patient but this should be clarified, perhaps even by indicating the start and end exons for the duplications.

9. The genotype-phenotype correlation data is in line with the findings of other studies so I believe this information is largely correct. However, given that steroids prolong the ambulatory phase of the disease, the lack of steroid information potentially introduces errors as a patient who would ordinarily be classified as DMD may walk a bit past the age of 16 with steroid therapy. If possible, it would help to obtain steroid information. If this is not possible, at the very least this limitation should be expanded in the Discussion and also information should be provided on how widespread steroid use is in the DMD population in Korea.

10. In Figure 2, it is difficult to interpret the exact CK levels at the later age ranges. It might be more informative to show two separate graphs, one for ages 0-12 or so and the other for ages 12 and above. Thus the second graph would be capped at 10,000 and it will be more clear what the later numbers are.

11. Figure 3 would be more informative if bars or lines were shown indicating the actual rearranged DNA segments.

Reviewer #2: The Authors report on 227 Korean patients from 218 unrelated families with dystrophinopathy; they describe retrospectively the clinical course of the disease. In all the patients they performed manual muscle testing (MMT) scores and CK dosage, only some patients underwent Echocardiography and muscle biopsy.

In all the patients was identified a pathogenic mutation in the dystrophin gene.

Clinical findings are compatible with those of previous reports; the Authors include an additional phenotype named undetermined phenotype (UD) for the patients who were not wheelchair-bound and too young to be defined as DMD or BMD. Regarding this group, in the supplementary table 1 18 patients are reported as “clinical phenotype pending”. Is the UD phenotype?

I think the group UD is confounding, if the muscle biopsy has been performed I would suggest to define the phenotype as BMD or DMD based on the immunohystochemical analysis ; if not performed I would classify these patients referring to the phenotype expected based on the genotype. All the 18 mutations are reported in the LOVD pages and only the phenotype associated to the duplication of exon 14-45 it could be difficult to predict ( transcript analysis is necessary)

A part form this group, it would be interesting to add some information on the muscle biopsy performed in 43 patients and to correlate it with the genotype.

Regarding the genetic data the frequencies of exonic deletions/duplications and small mutations are similar to the ones previously reported in other countries. Do the Authors have cases of DMD confirmed at the biopsy ( absence of dystrophin) and negative to molecular analysis?

This paper has some limitations as described by the Authors but it is important because is the first report on dystrophinopathies in Korean patients and give data for developing therapeutic drugs and designing clinical trials.

I have few comments :

Lane 37 “dystrophinopathies is a genetic muscle disease” Is not correct, is a group of inherited phenotypes all due to dystrophin mutations.

ABSTRACT and Lane 58 instead of “sequence variants” I’d rather say “mutations”

Lane 62 1% of deep intronic mutations is missing

Lane 65 is the most common type of muscular dystrophy I would add “IN CHILDREN”

Lane 105 “undetermined phenotype (UD) (for the patients who were not wheelchair-bound and too young to be defined as DMD or BMD) “ it is not specificed the age considered as cut off

Lane 219 “Nine unrelated probands did not follow the reading frame rule” A part from the patients with the del 3-7 for which the explanation is known, in the other patients it would be worth to study the transcript if the RNA from muscle biopsy is available.

**********

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: Peter B. Kang

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.

Clinical and genetic spectra in patients with dystrophinopathy in Korea: A single-center study

PONE-D-21-16348R1

Dear Dr. Park,

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,

Michael Kyba

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank your for addressing the reviewers' comments. Paper is good to go.

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: 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: The authors have responded constructively to the prior comments. The manuscript has been improved significantly. No further concerns.

Reviewer #2: (No Response)

**********

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