Dear Dr Li,

Thank you very much for submitting your Research Article entitled 'ENPP1 variants in patients with GACI and PXE expand the clinical and genetic heterogeneity of heritable disorders of ectopic calcification' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address in a revised manuscript

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. 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 us at figures@plos.org.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

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.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Melissa Wasserstein, MD

Associate Editor

PLOS Genetics

Gregory Barsh

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors reports the clinical, laboratory, and molecular evaluations of ten GACI and two PXE patients from five and two unrelated families registered in GACI Global and PXE International databases, respectively. The authors conclude that the phenotypic spectrum of ENPP1-deficiency is much broader than was previously anticipated. It is known that GACI and PXE are complex disease, and the genes involved have many modifiers, e.g. doi: 10.3389/fcell.2021.612581. The authors further state that the correlation of plasma PPi and severity of GACI and PXE may not hold. The content of the paper could be greatly increased if the authors discuss alternative disease mechanisms beside the long-held association of extracellular PPi and calcification inhibition. Circulating PPi may be a poor proxy of the local PPi concentrations, which may actually determine the cellular calcification milieu. Also, cleavage of each ATP releases AMP along with PPi. What ist known about the role of AMP signalling in these diseases? Adenine, and specific ribonucleosides that disrupt pyrimidine synthesis may regulate the severity of GACI and PXE by affecting cell survival. (Li et al https://doi.org/10.1172/JCI149711). Please discuss.

li 151 "The Family #7 had one adopted 27-year-old male, patient #12, of Caucasian and African American descent (Fig. 1g). He also seeks support from PXE International." The fact that an unrelated adopted child acquired similar affection to me suggests the contribution of environmental or nutritional factors. Is this possible or was the child adopted BECAUSE it was affected? Please explain.

li 272 "elevated serum FGF23 levels in several GACI patients in the current study support this hypothesis" FGF-23 is a phosphatonin. Elevated levels may suggest phosphate and calciprotein particle toxicity with consequences for cell ageing and cell death (https://doi.org/10.1016/j.kint.2019.10.019 and papers cited therein). Please discuss.

Reviewer #2: Authors of the work entitled “ENPPI variants in patients with GACI and PXE expand the clinical and genetic heterogeneity of heritable disorders of ectopic calcification” present a thorough and interesting article describing how mutations in ENPP1, classically described in cases of GACI, are also present in patients with PXE. While GACI is seen as a severe form of soft tissue calcification disorder, PXE is considered “less severe” given its later onset in life and relatively reduced impact on patient morbidity and mortality. This work demonstrates that mutations in ENPP1 can also lead to phenotypes indicative of “less severe” PXE, illustrating clear genetic heterogeneity and resulting phenotypes across patients.

A clear highlight of this work is how the authors utilize two patient networks to obtain data and support detailed genetic and molecular analysis. This work would not have been possible without the support of these networks and illustrates an important role they play in the scientific community.

Minor edits to the work are as follows:

1. For readers less familiar with this pathway, please provide a graphical representation for the pathways of interest in the introduction highlighting ABCC6, ENPP1, Pyrophosphate, ATP, etc.

2. In the introduction both GACI1 and GACI are utilized- please keep consistent

3. On line 170 it indicates that patients 7, 8, and 9 are siblings. From the diagram in figure 1, is patient 9 a sibling or cousin? Please correct.

4. An additional paragraph in the results section connection PPi levels to phenotype is warranted. This is a main idea of the abstract and could be better discussed in the results, along side the PPi level measures. Are Phenodex type of values available for GACI patients?

5. Please change blue arrows in Figure 2 to another color- maybe yellow, to increase visibility

6. Please provide scale bars throughout Figure 2

7. At the end of figure 2 legend, it denotes d, dead; a, alive – where is this denoted in the figure? All patients included alive, correct?

8. Please provide scale bars for 3d.

9. Please indicate how many biological or technical replicated were performed in Figure 3.

10. Please expand in the methods the concentration of antibody used for both western and fluorescent cell-based analysis. 1:100? 1:1000? Additional details to help other reproduce such work is needed.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: Willi Jahnen-Dechent

Reviewer #2: No

Dear Dr Li,

We are pleased to inform you that your manuscript entitled "ENPP1 variants in patients with GACI and PXE expand the clinical and genetic heterogeneity of heritable disorders of ectopic calcification" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about making your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Melissa Wasserstein, MD

Associate Editor

PLOS Genetics

Gregory Barsh

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

----------------------------------------------------

Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly: 

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-21-01563R1

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

----------------------------------------------------

Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org.