Investigation of dermal collagen nanostructures in Ehlers-Danlos Syndrome (EDS) patients

Mehrnoosh Neshatian; Nimish Mittal; Sophia Huang; Aiman Ali; Emilie Khattignavong; Laurent Bozec

doi:10.1371/journal.pone.0307442

Peer Review History

Original SubmissionApril 11, 2024
5 Jun 2024 Decision Letter - Richard G. Haverkamp, Editor PONE-D-24-12071Investigation of dermal collagen nanostructures in Ehlers-Danlos Syndrome (EDS) patients.PLOS ONE Dear Dr. Bozec, 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. Please address each of the comments below from the reviewer. I strongly recommend that you make appropriate changes as suggested by the reviewer. Please submit your revised manuscript by Jul 20 2024 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. 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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. Please note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, all author-generated code must be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse. 3. Thank you for stating the following financial disclosure: “1.GoodHope Ehlers-Danlos Syndrome Foundation 2.Rosenstadt Fund, University of Toronto.” 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. 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: “The authors would like to acknowledge the participation of the EDS patients from the UHN GoodHope EDS clinic (University Health Network, Toronto, Canada) and its Director, Dr. Hance Clarke, for supporting the project. The authors would also like to acknowledge Dr. Grahame, who suggested this study in 2010. Partial funding was received from the UHN GoodHope EDS clinic and the Faculty of Dentistry Bridge Grant program to support the study. The authors would finally like to thank the CAMiLoD Facility at the Faculty of Dentistry, University of Toronto, for the extensive access to their facility. The authors declare no conflict of interest in the work presented.” We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “1.GoodHope Ehlers-Danlos Syndrome Foundation 2.Rosenstadt Fund, University of Toronto.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. Please remove your figures from within your manuscript file, leaving only the individual TIFF/EPS image files, uploaded separately. These will be automatically included in the reviewers’ PDF. Additional Editor Comments: I am sorry we have only one reviewer for your manuscript, but in the interests of time I have decided to proceed with a decision. I also have read the manuscript and feel the manuscript is suitable for publication after the points described are addressed. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: It is a well written paper addressing the possibility to use atomic force microscopy as an adjunct for clinical diagnosis of Ehlers Danlos. A have a number of comments: 1. The authors describe the use of genetic testing, including EDS related gene panel, in the studied individuals. Please provide the list of genes included in the aforementioned panel and include the molecular details and ACMG classification of the COL1A1 variant identified in the cEDS case. 2. The authors show skin biopsy of the hEDS-scleroderma patient to display patchy abnormalities in terms of collagen fiber density with three different regions that could be identified by light microscopy,, while the one with the highest fiber density was ascribed to scleroderma related changes. It seems prudent to analyze the AFM findings including collagen fiber elasticity measurements separately for each one of these three regions of the hEDS-scleroderma patient. This seems important since previous paper utilizing AFM including Young’s Modulus measurements in the setup of scleroderma showed increased collagen fiber stiffness (PMID 28138238). This point should be discussed. 3. The details of full clinical and laboratory findings of the hEDS-scleroderma patient should be included, with the emphasis on the clinical scleroderma subtype, autoantibodies present and disease duration. 4. The existing literature addressing the previous use of AFM for connective tissue disorders should be included in introduction. 5. minor comments: page 5 line 79 - "genetic variants of EDS" while implying additional genes causative of EDS. Please rephrase page 5 line 85 - typo, "relayed" should be "related" ****** 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. 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Revision 1
3 Jul 2024 Author Response To the Editor and the Reviewer, The research team would like to acknowledge and thank the two reviewers for reviewing our manuscript and providing us with constructive feedback, which has been addressed below: 1. The authors describe the use of genetic testing, including EDS related gene panel, in the studied individuals. Please provide the list of genes included in the aforementioned panel and include the molecular details and ACMG classification of the COL1A1 variant identified in the cEDS case. Thank you for your comment. The standard EDS related comprehensive gene panel was done at our institute employs Next generation Sequencing (NGS) with duplication and deletion analysis by exon targeted microarray. This test includes 22 genes. Details can be found online on the website (https://www.sickkids.ca/siteassets/care--services/for-health-care-providers/lab-information-sheets/connective-tissue-disorders-ehlers-danlos-syndrome.pdf). In the cEDS case, patient was found to have a pathogenic variant (c.1053delG in exon7) of the COL5A1 gene. The body of the manuscript has been edited accordingly (as follow). Section: Clinical cases: Next generation Sequencing (NGS) with duplication and deletion analysis by exon targeted microarray was performed at SickKids hospital for diagnosis. This test includes 22 genes. Details can be found online on the SickKids website. Study ID No 5 (labeled as cEDS throughout the paper) Genetic testing revealed a COL1A1 mutation (c.1053delG in exon7), confirming the diagnosis of classical EDS. 2. The authors show skin biopsy of the hEDS-scleroderma patient to display patchy abnormalities in terms of collagen fiber density with three different regions that could be identified by light microscopy, while the one with the highest fiber density was ascribed to scleroderma related changes. It seems prudent to analyze the AFM findings including collagen fiber elasticity measurements separately for each one of these three regions of the hEDS-scleroderma patient. This seems important since previous paper utilizing AFM including Young’s Modulus measurements in the setup of scleroderma showed increased collagen fiber stiffness (PMID 28138238). This point should be discussed. AFM images (5x5 µm) were acquired from three normal and three disrupted areas (guided by PS image) of the histological section of the skin biopsy. Subsequently, a total of 300 force curves were recorded for each of the normal and disrupted areas. A unimodal Yong’s moduli distribution was observed for both normal and disrupted collagen in all the three regions similar to hEDS-Scleroderma curves presented in Figure 3A. Suggesting that in each of these regions, a single population of collagen fibrils is present. These mechanical data must be interpreted carefully, as we only had one case with both EDS and scleroderma. Further association of the mechanical findings and H&E staining requires more skin tissue from similar patients These findings have been added as supporting information to the manuscript. And the body of the manuscript on the section “Collagen fibrils elasticity to determine EDS phenotype? “, has been edited as follow: “Additionally we have plotted the Yoong’s moduli distribution for all the three regions denoted as (, , ) and found similar distribution in all the three regions as shown in S1Fig” Figure S1 Shows the distributions of Young's moduli of collagen fibrils recorded in the , , and regions of the hEDS-Scleroderma case. A unimodal distribution of Young's moduli was observed for both normal and disrupted collagen across all three regions. The red dotted line represents the distribution of healthy collagen in the control group 3. The details of full clinical and laboratory findings of the hEDS-scleroderma patient should be included, with the emphasis on the clinical scleroderma subtype, autoantibodies present and disease duration. We thank you for the suggestion. The focus of this paper is to highlight the molecular histophysiologal profile of collagen fibrils and to keep the manuscript succinct, we had not included significant details of the clinical phenotype. We have made modifications and included the relevant clinical and laboratory findings, duration and autoantibodies. We have edited the body of the manuscript under the section, clinical cases - Study ID No 4 (labeled as hEDS- Scleroderma throughout the paper) “patient had a 10-year history of inflammatory arthralgias involving PIP and MCP joints, diffuse muscle and joint pains affecting multiple areas of body, secondary diagnosis of fibromyalgia, patchy areas of skin thickening, and Raynaud’s phenomenon. History was negative for uveitis, iritis and psoriasis. Lab testing was positive for Anti-Nuclear Antibody (ANA) and Anti centromere Antibody (ACA) and negative for Anti CCP and rheumatoid factor.” 4. The existing literature addressing the previous use of AFM for connective tissue disorders should be included in introduction. We thank the reviewer for the comment. We have included the following in introduction: AFM has previously used for connective tissue morphometry studies such as Study of the Extracellular Connective Tissue Matrix in Patients with Pelvic Organ Prolapse [34], nanohistological investigation of scleroderma [35], oral submucous fibrosis [36], assessments of extracellular matrix in intervertebral disc and degeneration [37], and many other studies. In this study we have employed AFM to examine ex-vivo skin biopsies obtained from EDS patients to characterize collagen morphometric differences in cEDS and hEDS. 5. minor comments: page 5 line 79 - "genetic variants of EDS" while implying additional genes causative of EDS. Please rephrase Thank you for the comment. The sentence has been edited as follow “hEDS shares several clinical features with EDS subtypes but lacks confirmed genetic markers” page 5 line 85 – typo, “relayed” should be “related” We are sorry for the type. It is now corrected. Attachments Attachment Submitted filename: response letter.docx https://doi.org/10.1371/journal.pone.0307442.r002
5 Jul 2024 Decision Letter - Richard G. Haverkamp, Editor Investigation of dermal collagen nanostructures in Ehlers-Danlos Syndrome (EDS) patients. PONE-D-24-12071R1 Dear Dr. Bozec, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Richard G. Haverkamp, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0307442.r003
Formally Accepted
12 Jul 2024 Acceptance Letter - Richard G. Haverkamp, Editor PONE-D-24-12071R1 PLOS ONE Dear Dr. Bozec, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Richard G. Haverkamp Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0307442.r004

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