A Clinical Qualification Protocol Highlights Overlapping Genomic Influences and Neuro-Autonomic Mechanisms in Ehlers–Danlos and Long COVID-19 Syndromes

Round 1

Reviewer 1 Report

The manuscript is interesting but it is too long (it looks like a review) and should be shortened and better reorganized. 

Lines 60-66: The complexity of COVID-19 disease deserves to be highlighted since it has been reported that SARS-CoV2 infection can lead to respiratory but also non-respiratory disease as recently review (PMID: 35943095, 35114008, 36072173).

Figure 2 is unreadable

Figure 5 can be improved

Manuscript is too long and should be shortened

Author Response

I have greatly condensed the manuscript to 8074 words in response to this and other reviewers' comments about its being too long. I have focused on the clinical qualification protocol of DNA variants and its enabling of long COVID19-EDS comparison that were the most important parts of the initial article. This focus narrows the research design and results presentation that the reviewer thought must be improved and eliminates former Fig. 2 that was a concern. I retain the mentioned Fig. 5 as Fig. 3 and was not sure how to improve it as suggested--I will certainly adhere to any specific suggestions for this revision

Reviewer 2 Report

Your paper starts out with a linguistic flourish with its implied reference to Marshall McLuhan's "medium is the message". It is generally very well written. However, I have four problems with it, the gravity of each I will leave to you and the editors.

First, you don't state this clearly, but this is an observational study. You have selected patients for the study not with reference to a set of theoretically relevant inclusion criteria, but because they were patients in your clinical practice. If patients seek you out as a physician because you have a point of view about EDS, then it is more than likely you will find here the associations you are seeking. You need to be much clearer about why we, the readers, who do not share your practice speciality, should pay attention. I agreed to review the paper because our team is thinking a lot about long COVID and I wanted to see what your insights were. I have to wade through too much to get to the point in the paper as currently structured.

Second, your statements in Section 2.3 about patient awareness and agreement to participate and your IRB approval leave me with a number of concerns. First the approval comes from 2014 from other work with the same patient cohort as I read it. How about now? What was the nature of the patient agreement to participation in this particular study? Not stated. I believe you should return to the IRB and seek an updated approval.

Third, in a similar area of concern, there is no standard Table 1 that lays out who the participants were. Especially when you are undertaking an observational study of a mass of people, I would like to know who they are--age ranges, distribution by gender, race, socio-economic status and other variables of importance that could affect their exposure to or severity of COVID symptoms.

Fourth, you have a number of papers buried in this single paper and would do better to separate them into a number of submissions. For example, your Figure diagram really deserves a paper of its own as original work.

The real focused subject of this paper should be what the association is between EDS people and the COVID symptoms they can suffer and as a Discussion section, what this can mean for potential long-COVID treatments.

You will want to remember that readers (and reviewers) have limited time and ability to focus as a practical matter and papers should address 1 point and address it well. A paper this large will mainly go unread and unappreciated for its level of detail. 

Finally, a minor point. I suggest taking on a co-author who is a biostatistician. Your use of Excel as the main statistical tool weakens the presentation of the numbers. Using either a specialized statistical program like Prism or SPSS or a statistical language like R would give you much richer numerical results.

It is written by native speakers and is fine.

Author Response

I have greatly condensed the manuscript to 8074 words in response to this and other reviewers' comments about its being too long. I have focused on the clinical qualification protocol of DNA variants as suggested by this reviewer and the long COVID19-EDS comparison that its mechanistic focus makes possible. I hope that this focus will address several concerns of this very helpful and insightful reviewer:

1) The introduction, results presentation, research design, and results presentation have been shortened and re-organized consistent with the restricted focus.

2) Two topics among the many in the prior article are addressed as the reviewer suggested.

3) I have greatly condensed the clinical description of EDS patients and hope that the explicit statements about this being an observational study with referral bias will satisfy those concerns. 4)

4) Detailed description of the EDS patient population will be more relevant to the planned paper describing EDS-related genes in detail and I hope that the revised Table 1 (selecting data from former Tables 1 and 2) is a sufficient description of the EDS population (it was also detailed in the preliminary reports or references 2 and 11).

5) Now the supplementary data Table S2 only includes the genes variant in EDS patients without specifying the actual variants (those will be provided in a subsequent paper, gathered with consents in my clinic and on the GeneDx requisitions. The concern about the consent process for listing patient DNA results should be muted for this paper; I cannot resubmit the IRB application as suggested in any case since that protocol was closed and I am not longer at its constituent institution. I would point out that the assembly of patient records into a de-identified database is generally considered exempt by IRB committees as mine was, that only professionals accessing supplementary Excel files in my subsequent published articles along with receipt of my 1261EDSGW1-23 database will be able to match the sparse demographic data (sex, age-range) with actual variants, and that ~65% of these variants have already been submitted to databases by GeneDx.

6. I hope that the limited clinical and genetic data will also mitigate statistical concerns since a few simple calculations of means and standard deviations with t-test comparisons are now employed. I could see sophisticated statistical analyses concerning the combinations of clinical symptoms and genetic changes that would pertain to the subsequent paper about EDS but feel that is far beyond what is needed for the subsequent descriptive paper or certainly for this one.  I would welcome sharing of this huge welter of data (~150 descriptors for each of 1260 plus 64 EDS plus No EDS patients, then the filtering of an average 12000 exome variants to yield those listed) with a bioinformatics expert in the future, their analysis worthy of independent publication.

Reviewer 3 Report

Some autonomic symptoms are common between EDS and long COVID-19. The authors tried to reveal gene networks involved in these common symptoms between EDS and long COVID-19. The authors described overlapping gene networks between EDS causative genes and genes modulating the severity of COVID-19. Some genes including F2, LIFR, NLRP3, STAT1, T1CAM1, TNFRSF13B, were extracted for the gene networks.

Statistical analyses were not efficiently performed in this study. Please conduct statistical analyses on the data shown in the present study.

The manuscript was too long. Please reduce the length of Results and Discussion sections.

Author Response

I have greatly condensed the manuscript to 8074 words in response to this and other reviewers' comments about its being too long. I have focused on the clinical qualification protocol of DNA variants and its enabling of long COVID19-EDS comparison that were the most important parts of the initial article. The welter of data on EDS patient symptoms and their companion variants will be mostly addressed in a subsequent paper, this one only listing the variant genes in EDS patients and comparing them to those moderating COVID19 severity. This focus narrows the statistics to simple calculations of means and standard deviations and a few t test comparisons that I hope will satisfy the reviewer.

Round 2

Reviewer 1 Report

the manuscript has been significantly improved and can be accepted in the present form 

Author Response

I appreciate the positive response to revision of reviewer 1

Reviewer 2 Report

Thank you for your improvements to the manuscript. This version is much better and more understandable. Your Figure 1 will have readers puzzling through it as it did me, but I now think I understand it. 

Author Response

I appreciate the positive comments of reviewer 2--I will look at the presentation of results when submitting the final revision, the only area that reviewer 2 cites as needing improvement