PONE-D-21-10014

Nationwide screening for Fabry disease in unselected stroke patients

PLOS ONE

Dear Dr. Tomek,

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.

Your paper was evaluated by an expert in clinical human genetics and myself. The topic is interesting, and the study design and data support the conclusion. However, several points need to be clarified. Please read the comment carefully and address the issues accordingly. 

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We look forward to receiving your revised manuscript.

Kind regards,

Tomohiko Ai, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

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

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2. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

3. Thank you for stating the following in the Financial Disclosure section:

[AT, JPS, PR, GD, AL have received speaker’s fees from Takeda. The required laboratory tests for FD were financed through a grant from Takeda (formerly Shire) to the Czech Neurological Society. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There are no other relevant conflicts to report. The authors have no financial relationships pertinent to this article to disclose.]. 

We note that you received funding from a commercial source: Takeda (formerly Shire)

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4. One of the noted authors is a group or consortium [National Stroke Research Network, part of Czech Clinical Research Infrastructure Network (CZECRIN) and Czech Neurological Society, Cerebrovascular Section]. In addition to naming the author group, please list the individual authors and affiliations within this group in the acknowledgments section of your manuscript. Please also indicate clearly a lead author for this group along with a contact email address.

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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

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

Reviewer #1: I Don't Know

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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

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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

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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 manuscript entitled: “Nationwide screening for Fabry disease in unselected stroke patients” presents an interesting analysis about the prevalence of Fabry disease in an unselected population of 986 consecutive individuals affected by stroke in the Czech Republic. The main findings are that, although rare, molecular signature leading to the diagnosis of FD was achieved in 0.2%, in patients younger than 50 y/o and that the pseudodefiency allele c.937G>T (p.Asp313Tyr) (aka D313Y) could represent a risk allele for increased stroke susceptibility.

The manuscript is clear, the experiments were carried appropriately, and the limitations carefully delineated. As the authors stated, although theirs is not the first study addressing FD prevalence in stroke patients, it appears to be the first in the Czech Republic.

There are few minor points the authors should address

• The authors should specify that the discriminant is not the age per se, but the age of onset of the stroke event/s. So, it would read better if they would specify that FD screening is appropriate for all subjects that had a stroke event before 50 years of age

• Given the limitation of the algorithm employing enzymatic activity and globotriaosylsphingosine (lyso-Gb3) quantification before genetic analysis in males, should they consider suggesting doing genetic testing for GLA in all subjects that had a stroke event before 50 years of age irrespective of biochemical analysis?

• In the Methods section, the authors stated that they used: “The GLA gene was analyzed by PCR and sequencing (NGS-Illumina) of the entire coding region and the highly conserved exon-intron splice junctions”. Could they provide more details about the platform and technical specifics used (i.e.: MiSeq, HiSeq, depth of coverage, bioinformatic analysis, etc.) and if copy number variants (CNV) have been analyzed. CNV are rare but can be identified in up to 5% of cases (see: https://www.ncbi.nlm.nih.gov/books/NBK1292/#fabry.Summary). If CNV was not done, please add to the limitations

• While the GLA variant c.1235_1236del (p.Thr412Serfs*) is an established pathogenic change (https://www.ncbi.nlm.nih.gov/clinvar/variation/198402/), for the c.973G>A (p.Gly325Ser) (aka G325S) in ClinVar there is conflicting classification (https://www.ncbi.nlm.nih.gov/clinvar/variation/92573/), If considered Pathogenic or Likely Pathogenic, please provide your classification and rationale using the ACMG 2015 guidelines (PMID: 25741868) and any professional updated guidelines

• Although still erroneously use in official publication and mistakenly tolerated by scientific journals, the official term is variant, not mutation (see: PMID: 25741868 under “Terminology”)

• The authors should specify that the c.937G>T (p.Asp313Tyr) (aka D313Y) is a common pseduodeficiency allele that does not cause disease, but individuals with this variant can exhibit low alpha-galactosidase activity during enzyme analysis. As far as the other variants, the authors correctly identified them as potentially benign or variants of uncertain significance (VUS). In the case of the two novel GLA variants, p.Arg30Lys and p.Arg38Gly, although the age at testing (please provide the age at stroke event onset, if known) is favoring a more tolerated role, the authors should discuss the fact that, most females (not only with regard to those two variants, but in general), harboring a GLA variant, may or may not present the whole collection of symptoms, due to absolute gene dosage (heterozygous versus hemizygous in males) and the phenomenon of X-inactivation

• Was all the participant ancestry background uniform, or were there a significant ancestry background admixture? Some variants may be common in population isolates due to founder effect

• Please use the most current human genome variation nomenclature (https://varnomen.hgvs.org/)

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Reviewer #1: Yes: Matteo Vatta

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Nationwide screening for Fabry disease in unselected stroke patients

PONE-D-21-10014R1

Dear Dr. Tomek,

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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Kind regards,

Tomohiko Ai, M.D., Ph.D.

Academic Editor

PLOS ONE

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

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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

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

Reviewer #1: N/A

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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

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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

**********

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 adequately answered to all reviewer's comments and the manuscript's overall quality was improved.

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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.

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Reviewer #1: No