Whole-exome sequencing in a Japanese multiplex family identifies new susceptibility genes for intracranial aneurysms

Tatsuya Maegawa; Hiroyuki Akagawa; Hideaki Onda; Hidetoshi Kasuya

doi:10.1371/journal.pone.0265359

Peer Review History

Original SubmissionNovember 3, 2021
28 Dec 2021 Decision Letter - Avaniyapuram Kannan Murugan, Editor PONE-D-21-35037Whole-exome sequencing in a Japanese multiplex family identifies new susceptibility genes for intracranial aneurysmsPLOS ONE Dear Dr. Akagawa, 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 submit your revised manuscript by Feb 11 2022 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. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Additional Editor Comments: The study is well appreciated. Further, reviewers do support the manuscript yet, raise many concerns and those points to be addressed prior processing the manuscript further. Particularly, the comments from Rev. 1. 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: I Don't Know 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 study brings forth causal variants in two genes NPNT and CBY2 for intracranial aneurysms. The authors have examined families and singletons and conducted expression studies for these genes. The focus has been on CBY2, within the locus identified in a different population and highly expressed in testis. I have the following comments about this manuscript: Major comments: 1. Did the authors consider separate population frequency cut offs for predicted AD AR inheritance for each? 2. What population frequency database were referred to? I would include gnomAD, KAVIAR and any other population specific public database if available. 3. In table 1, include population frequency for the alternate allele from gnomAD/ALFA like in Suppl table S1 etc 4. The authors discuss the possibility of a complex pattern of inheritance including reduced penetrance for the multiplex families. I suggest that the authors include an inheritance pattern (autosomal dominant, recessive etc) explicitly in the Discussion to make their case stronger. 5. Can the authors also include a hypothesis about the functional effect of the variants? In supplementary information, the data for splice site variant is shown for NPNT. It would be good to know if the CBY2 and NPNT variants are acting as loss or function or gain of function mutation in IA. 6. The authors mention 'gene product' on many occasions throughout the manuscript. By that, do they mean mRNA or protein? If the latter, then it needs to be modified in the manuscript. Minor comments: 1. Correct syntax errors: Ex: Remove g for DNA sample of sister in line 267; Remove gDNA in line 386 etc. 2. I would suggest moving Figure S1 to the main manuscript. Reviewer #2: I genuinely appreciate the effortful and highly systematic methodologies conducted in revealing novel susceptibility genes for intracranial aneurysms in the Japanese family. I do encourage the authors to conduct this well-elaborated methodology at a higher scale with increasing sample sizes and incorporating other non-Japanese candidates as possible in the near future. ****** 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: Yes:** MM 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. https://doi.org/10.1371/journal.pone.0265359.r001
Revision 1
23 Feb 2022 Author Response RESPONSE TO REVIEWER #1: We wish to express our appreciation to the Reviewer for his/her insightful comments, which have helped us to considerably improve our manuscript. Reviewer #1: This study brings forth causal variants in two genes NPNT and CBY2 for intracranial aneurysms. The authors have examined families and singletons and conducted expression studies for these genes. The focus has been on CBY2, within the locus identified in a different population and highly expressed in testis. I have the following comments about this manuscript: Comment 1: Did the authors consider separate population frequency cut offs for predicted AD AR inheritance for each? Response: Thank you for this comment. We have added the following text (revised text lines 254-257): “There were no homozygous or compound heterozygous candidates shared by the affected sisters (III-1, III-4, and III-5) with allelic frequencies less than 3% in the gnomAD and the Human Genome Variation Database (HGVD, https://www.hgvd.genome.med.kyoto-u.ac.jp/)” Comment 2: What population frequency database were referred to? I would include gnomAD, KAVIAR and any other population specific public database if available. Response: As we responded in comment 1 and presented in the Table S1, the Human Genetic Variation Database (HGVD, https://www.hgvd.genome.med.kyoto-u.ac.jp/) in the Japanese general population was referred to in addition to the gnomAD. Comment 3: In table 1, include population frequency for the alternate allele from gnomAD/ALFA like in Suppl table S1 etc. Response: Based on the reviewer’s comment, we have added the HGVD and gnomAD data in Table 1. Comment 4: The authors discuss the possibility of a complex pattern of inheritance including reduced penetrance for the multiplex families. I suggest that the authors include an inheritance pattern (autosomal dominant, recessive etc) explicitly in the Discussion to make their case stronger. Response: Based on the reviewer’s comment, we have changed the following text from (revised text lines 408-411): “The accumulated genetic data from many other large pedigrees with IA will facilitate our understanding of the polygenic architecture of IA susceptibility.” to “Accumulated genetic data from many other large pedigrees with IA will facilitate our understanding of the polygenic architecture of IA susceptibility, which vastly differs from Mendelian inheritance such as autosomal dominant and recessive patterns.” Comment 5: Can the authors also include a hypothesis about the functional effect of the variants? In supplementary information, the data for splice site variant is shown for NPNT. It would be good to know if the CBY2 and NPNT variants are acting as loss or function or gain of function mutation in IA. Response: Based on the reviewer’s comment, we have added the following text (revised text lines 361-363) and reference to describe the presumed effect of the CBY2 variant : “Protein aggregation is involved in the etiology of various human diseases due to the loss of protein function, cytotoxicity, or acquisition novel aggregation-specific functions [44]. ” “44. De Baets G, Van Doorn L, Rousseau F, Schymkowitz J. Increased aggregation is more frequently associated with human disease-associated mutations in neutral polymorphisms. PLoS Comput Biol. 2015;11(9):e1004374.” The NPNT variants identified in our IA cases were loss-of-function, as mentioned in the discussion (revised text lines 353-355). Comment 6: The authors mention 'gene product' on many occasions throughout the manuscript. By that, do they mean mRNA or protein? If the latter, then it needs to be modified in the manuscript. Response: We strictly adhered to the guidelines for human gene nomenclature established by the HUGO Gene Nomenclature Committee (HGNC) throughout the manuscript, which is also recommended in the submission guidelines of PLOS ONE (https://journals.plos.org/plosone/s/submission-guidelines). The HGNC endorses the use of italics to denote genes, alleles and RNAs to distinguish them from proteins, and recommends that “protein and gene symbols should use the same abbreviation”. They further suggested that proteins should be referenced using non-italicized gene symbols to distinguish them from genes. Please see the following paper from HGNC. Bruford EA, Braschi B, Denny P, Jones TEM, Seal RL, Tweedie S. Guidelines for human gene nomenclature. Nat Genet. 2020 Aug;52(8):754-758. doi: 10.1038/s41588-020-0669-3. PMID: 32747822; PMCID: PMC7494048. Comment 7: Correct syntax errors: Ex: Remove g for DNA sample of sister in line 267; Remove gDNA in line 386 etc. Response: We sincerely apologize for these errors. They have been corrected in the revised manuscript. Comment 8: I would suggest moving Figure S1 to the main manuscript. Response: Based on the reviewer’s comment, we moved Figure S1 to the main manuscript as Figure 2. We wish to thank the Reviewer again for the valuable comments. RESPONSE TO REVIEWER #2: We wish to express our appreciation to the reviewer for his/her insightful comments, which have helped us to considerably improve our manuscript. Reviewer #2: I genuinely appreciate the effortful and highly systematic methodologies conducted in revealing novel susceptibility genes for intracranial aneurysms in the Japanese family. I do encourage the authors to conduct this well-elaborated methodology at a higher scale with increasing sample sizes and incorporating other non-Japanese candidates as possible in the near future. Response: We thank the reviewer for this important suggestion regarding our future research plan. Attachments Attachment Submitted filename: Response_to_Reviewers.docx https://doi.org/10.1371/journal.pone.0265359.r002
1 Mar 2022 Decision Letter - Avaniyapuram Kannan Murugan, Editor Whole-exome sequencing in a Japanese multiplex family identifies new susceptibility genes for intracranial aneurysms PONE-D-21-35037R1 Dear Dr. Hiroyuki, 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, Avaniyapuram Kannan Murugan, M.Phil., Ph.D. Academic Editor PLOS ONE 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 #2: 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: 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 #2: 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 #2: 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 #2: As discussed before, I would encourage the authors to generalize the results to non-Japanese population via multicenter-based studies. ****** 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 #2: Yes:** MM https://doi.org/10.1371/journal.pone.0265359.r003
Formally Accepted
8 Mar 2022 Acceptance Letter - Avaniyapuram Kannan Murugan, Editor PONE-D-21-35037R1 Whole-exome sequencing in a Japanese multiplex family identifies new susceptibility genes for intracranial aneurysms Dear Dr. Akagawa: 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. Avaniyapuram Kannan Murugan Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265359.r004

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