Variable patterns of mutation density among NaV1.1, NaV1.2 and NaV1.6 point to channel-specific functional differences associated with childhood epilepsy

Alejandra C. Encinas; Joseph C. Watkins; Iris Arenas Longoria; J. P. Johnson Jr; Michael F. Hammer

doi:10.1371/journal.pone.0238121

Peer Review History

Original SubmissionMarch 17, 2020
19 Jun 2020 Decision Letter - Randall Lee Rasmusson, Editor PONE-D-20-07729 Variable patterns of mutation density among NaV1.1, NaV1.2 and NaV1.6 point to channel-specific functional differences associated with childhood epilepsy PLOS ONE Dear Dr. Hammer, 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. The manuscript needs an expanded discussion along the lines of reviewer 1's notes, with particular emphasis on how this potentially translates into clinical practice and particularly the emerging field of precision medicine. Please submit your revised manuscript by Aug 03 2020 11:59PM. 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If the need for consent was waived by the ethics committee, please include this 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 paper compared patterns of variation in patients’ and public databases to test the hypothesis that regions of known functional significance within the main voltage-gated sodium channels show an increased burden of deleterious variants. The authors demonstrated that patient variant density was higher in regions known to play a role in channel function, e.g. the pore region, which is quite intuitive. On the other hand, they did also found that channel-specific patterns of patient burden may reflect different roles played by the NaV 1.6 inactivation gate in action potential propagation, and by NaV 1.1 S5-S6 linkers in loss of function and haploinsufficiency. Main comments This is an original study that could potentially impact the interpretation of the variants in the genes involved in different epilepsy types, in particular, to infer the pathogenicity of variants of unknown significance. The manuscript is well written and it is technically a quite sound piece of scientific research with data that partially supports the conclusions. Experiments have been conducted rigorously, with appropriate sample sizes. The statistical analysis has been performed rigorously. This reviewer has the following comments: 1) The authors assessed the mutational burden in different regions of the NaV channels considering (1) the excess variants in the public databases and (2) the cumulative distribution of variants throughout the different genes. However, this approach is quite arbitrary. Moreover, there was a clear discrepancy in the choice of the different patients’ database used for this study (i.e., Japanese patients with Dravet syndrome (DS); the literature data for the NaV1.2 missense variants, the patient’s registry for SCN8A mutations. This choice must be discussed as it did potentially affect the results, at least in part. For instance, Nav 1.1 mutations are associated with a spectrum of severity, ranging from autosomal dominant GEFS+ to de novo mutations causing DS. Thus, as honestly discussed by the authors, the adoption of the Japanese register did exclude a priori all the milder form of epilepsy associated with mutations in SCN1A. 2) Overall, pathogenic variants in these genes have been associated with a wide spectrum of epilepsy phenotypes, ranging from benign epilepsies to epileptic encephalopathies with variable severity. Furthermore, a few patients with intellectual disability (ID) or movement disorders without epilepsy have been reported (see, for instance, Johannesen KM, The spectrum of intermediate SCN8A-related epilepsy. Epilepsia. 2019;60(5):830-844). 3) The case of NaV 1.1 variants is even more complicated if we consider that some variants can promote inclusion of a "poison" exon that leads to reduced amounts of full-length SCN1A protein, a mechanism is likely to be broadly relevant to human disease (Carvill GL, Aberrant Inclusion of a Poison Exon Causes Dravet Syndrome and Related SCN1A-Associated Genetic Epilepsies. Am J Hum Genet. 2018;103(6):1022-1029). Reviewer #2: There are no overall issues with the data or conclusions. The authors clearly describe the data bases used in the statistical analysis and point out the potential shortcomings. It is an interesting approach to identifying differences between the various sodium channels with respect to the relationships between the function/role of the sodium channel in neuronal firing and the region/effects of a given mutation. This approach would allow researchers to study more clinically-relevant mutations in ion channels and give greater insight into the relationship between ion channel structure/function and consequences on neuronal activity. ****** 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: No 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. Attachments Attachment Submitted filename: Review of PONE-D-20-07729.pdf https://doi.org/10.1371/journal.pone.0238121.r001
Revision 1
2 Aug 2020 Author Response Reviewer #1: 1a) The authors assessed the mutational burden in different regions of the NaV channels considering (1) the excess variants in the public databases and (2) the cumulative distribution of variants throughout the different genes. However, this approach is quite arbitrary. Response: We not sure what the reviewer means by “arbitrary”; however, the cumulative function distribution contains all the information necessary for the statistical tests (i.e., sufficient for all the statistical tests performed in these analyses). 1b) Moreover, there was a clear discrepancy in the choice of the different patients’ database used for this study (i.e., Japanese patients with Dravet syndrome (DS); the literature data for the NaV1.2 missense variants, the patient’s registry for SCN8A mutations. This choice must be discussed as it did potentially affect the results, at least in part. For instance, Nav 1.1 mutations are associated with a spectrum of severity, ranging from autosomal dominant GEFS+ to de novo mutations causing DS. Thus, as honestly discussed by the authors, the adoption of the Japanese register did exclude a priori all the milder form of epilepsy associated with mutations in SCN1A. Response: We completely agree and thank the reviewer for noting the discrepancy. We have now performed an analysis of ALL SCN1A variants—including an additional 94 patients with milder symptoms in addition to the the Dravet Syndrome patients. While including the milder patients did not alter our main results in a significant way (i.e., variants in the milder group tended to be found in similar segments, domains, linkers and loops as variants with Dravet Syndrome), we did discover that variants associated with these patients tended to accumulate more in the N- and C- terminus. This raises new questions about the functionality of these regions of the gene, which we now discuss in the last section of the manuscript. Future analyses that include greater numbers of variants will have additional statistical power to identify sites in the channel with patient burden. These studies will need to include associations with patient phenotype (i.e., along the spectrum of disease outcome). 2) Overall, pathogenic variants in these genes have been associated with a wide spectrum of epilepsy phenotypes, ranging from benign epilepsies to epileptic encephalopathies with variable severity. Furthermore, a few patients with intellectual disability (ID) or movement disorders without epilepsy have been reported (see, for instance, Johannesen KM, The spectrum of intermediate SCN8A-related epilepsy. Epilepsia. 2019;60(5):830-844). Response: We now refer to this broader spectrum and cite the Johannessen et al. reference. And restate the last sentence of the first paragraph of the introduction to clarify this point. 3) The case of NaV 1.1 variants is even more complicated if we consider that some variants can promote inclusion of a "poison" exon that leads to reduced amounts of full-length SCN1A protein, a mechanism is likely to be broadly relevant to human disease (Carvill GL, Aberrant Inclusion of a Poison Exon Causes Dravet Syndrome and Related SCN1A-Associated Genetic Epilepsies. Am J Hum Genet. 2018;103(6):1022-1029). Response: We agree that this is an interesting finding. However, we feel it is beyond the scope of this study our focus on variants across the entire gene in patient and public databases while variants in the abovementioned exon occur outside the annotated coding regions. Reviewer #2: There are no overall issues with the data or conclusions. The authors clearly describe the data bases used in the statistical analysis and point out the potential shortcomings. It is an interesting approach to identifying differences between the various sodium channels with respect to the relationships between the function/role of the sodium channel in neuronal firing and the region/effects of a given mutation. This approach would allow researchers to study more clinically-relevant mutations in ion channels and give greater insight into the relationship between ion channel structure/function and consequences on neuronal activity. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0238121.r002
11 Aug 2020 Decision Letter - Randall Lee Rasmusson, Editor Variable patterns of mutation density among NaV1.1, NaV1.2 and NaV1.6 point to channel-specific functional differences associated with childhood epilepsy PONE-D-20-07729R1 Dear Dr. Hammer, 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, Randall Lee Rasmusson 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 paper has been improved by implementing the discussion. It is a nice addition to the literature and can support genotype-phenotype correlations. ****** 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: Yes:** Pasquale Striano https://doi.org/10.1371/journal.pone.0238121.r003
Formally Accepted
12 Aug 2020 Acceptance Letter - Randall Lee Rasmusson, Editor PONE-D-20-07729R1 Variable patterns of mutation density among Na_V1.1, Na_V1.2 and Na_V1.6 point to channel-specific functional differences associated with childhood epilepsy Dear Dr. Hammer: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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 Dr. Randall Lee Rasmusson Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0238121.r004

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