Whole genome sequencing identifies novel structural variant in a large Indian family affected with X-linked agammaglobulinemia

Abhinav Jain; Geeta Madathil Govindaraj; Athulya Edavazhippurath; Nabeel Faisal; Rahul C. Bhoyar; Vishu Gupta; Ramya Uppuluri; Shiny Padinjare Manakkad; Atul Kashyap; Anoop Kumar; Mohit Kumar Divakar; Mohamed Imran; Sneha Sawant; Aparna Dalvi; Krishnan Chakyar; Manisha Madkaikar; Revathi Raj; Sridhar Sivasubbu; Vinod Scaria

doi:10.1371/journal.pone.0254407

Peer Review History

Original SubmissionDecember 9, 2020
16 Mar 2021 Decision Letter - Obul Reddy Bandapalli, Editor PONE-D-20-38741 Whole genome sequencing identifies novel structural variant in a large Indian family affected with X-linked agammaglobulinemia PLOS ONE Dear Dr. Scaria, 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. Based on internal evaluation and external review this manuscript was found to be interesting. However the reviewers raised points that needs to be addressed carefully, especially the claim that WES did not give any casual variants was not taken well by one of the reviewers and suggested to look for CNVs before concluding. Please try to address all those points by performing the analysis or write rebuttal putting forward your arguments. 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[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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 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: The authors report the detection of a large deletion in the BTK gene in a large family with X-linked agammaglobulinemia. More than 500 mutations in this gene have previously been identified to cause the disease, so the paper adds one more mutation to the list. The paper is clearly written and the data analysis is well described. The authors have attempted to show that WGS is needed for identifying the pathogenic variant, however, the data presented does not support this claim (see below). My main criticism is that the authors performed whole-exome sequencing on four affected individuals but say that did not find "any causal variant using whole-exome sequencing". However, this is not well justified since the authors only looked at small variants identified from exome data. Given the strong association of mutations in BTK with the phenotype, the authors could easily have looked for copy number variants specifically in the BTK gene (the deletion would easily be seen as zero read depth on the X chromosome in males). There are many tools for detection of CNVs from exome data that could also have been used. Simply visualizing read depth using the IGV viewer can reveal deletions that overlap coding regions. Therefore, the claim that WGS was needed to detect the deletion in the BTK gene is not supported. WGS is definitely beneficial for identifying the precise breakpoints but exome sequencing is still 3-4 times cheaper than WGS. I would recommend that the authors report the analysis of read depth from exome data. The rationale for performing chromosome painting analysis of the region around the deletion is not clear to me. The large deletion identified in this family cannot be expected to be seen in controls since it would cause a severe disease in males. Therefore, it cannot be expected to have a specific population origin. The individuals studied in this paper are from India and it is not surprising that they have South Asian ancestry. This should be true for their entire genome, rather than just the region around the deletion. Reviewer #2: A. Is the manuscript technically sound, and the data support the conclusions? The manuscript by Jain et al. describes about the identification of large deletion (5,296 bp) encompassing exons 3-5 of the BTK gene of the patients with X - linked agammaglobulinemia (XLA) using the whole-genome sequencing (WGS). The authors have performed whole-exome sequencing (WES) and WGS of the four and two patients with XLA, respectively. Also, the authors have performed several assays including multiplex-ligation dependent probe amplification (MLPA) assay. The manuscript can be accepted after the following minor concerns have been addressed. In abstract section, the authors must rephrase the following sentence as they concluded (in the results section) that they could not find any variant which could correlate with the clinical features: “Whole genome sequencing led to identification of the accurate genetic mutation which could help in early diagnosis leading to improved outcomes, prevention of permanent organ damage and improved quality of life, as well as enabling prenatal diagnosis”. In the methodology section the authors can use multiple algorithms for the identification of alterations including copy number alterations using WES and WGS data of the patients with XLA. B. Has the statistical analysis been performed appropriately and rigorously? The statistical analysis has not been performed by the authors. C. Have the authors made all data underlying the findings in their manuscript fully available? The authors have provided analyzed data in the figures/tables and supplementary information in the manuscript wherever necessary. The raw data can be accessed through the convener of the Institutional Human Ethics Committee of the institute. D. Is the manuscript presented in an intelligible fashion and written in standard English? Overall manuscript is okay written, however the authors can rephrase the sentences in methodology section for example, “small indels, small insertion, and small deletion”. ****** 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. 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Revision 1
13 May 2021 Author Response 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 report the detection of a large deletion in the BTK gene in a large family with X-linked agammaglobulinemia. More than 500 mutations in this gene have previously been identified to cause the disease, so the paper adds one more mutation to the list. The paper is clearly written and the data analysis is well described. The authors have attempted to show that WGS is needed for identifying the pathogenic variant, however, the data presented does not support this claim (see below). My main criticism is that the authors performed whole-exome sequencing on four affected individuals but say that they did not find "any causal variant using whole-exome sequencing". However, this is not well justified since the authors only looked at small variants identified from exome data. Given the strong association of mutations in BTK with the phenotype, the authors could easily have looked for copy number variants specifically in the BTK gene (the deletion would easily be seen as zero read depth on the X chromosome in males). There are many tools for detection of CNVs from exome data that could also have been used. Simply visualizing read depth using the IGV viewer can reveal deletions that overlap coding regions. Therefore, the claim that WGS was needed to detect the deletion in the BTK gene is not supported. WGS is definitely beneficial for identifying the precise breakpoints but exome sequencing is still 3-4 times cheaper than WGS. I would recommend that the authors report the analysis of read depth from exome data. Response: We agree with the reviewer the point regarding visualizing the whole exome sequencing (WES) reads on the IGV could potentially identify the deletion. However in the case of WES, one cannot be sure whether such a loss of coverage could be due to chromosomal deletion or the efficiency of probes. Additionally the tools used for calling CNV using WES dataset are not accurate and robust and lead to false positive calls even in normal cases. In this context, WGS is beneficial to identify the precise breakpoint in the deletion as well as have sensitive and specific tools for structural variant calling - given it is independent of the probes, and additionally the paired-end reads can accurately identify not just the deletion but also the breakpoints. In addition, WGS provides us with an appropriate basis to perform further analysis like haplotypes and ancestry assessments which are not possible using WES dataset. The rationale for performing chromosome painting analysis of the region around the deletion is not clear to me. The large deletion identified in this family cannot be expected to be seen in controls since it would cause a severe disease in males. Therefore, it cannot be expected to have a specific population origin. The individuals studied in this paper are from India and it is not surprising that they have South Asian ancestry. This should be true for their entire genome, rather than just the region around the deletion. Reviewer: We apologize to the reviewer for not making it clear in the manuscript. The purpose of performing the haplotype ancestry for the patients in our study affected with PID. It is previously described in literature that a number of variants in PID have a founder effect and could be mapped to specific ancestry (PMID: 20414676, 27896283, 32822427) . The basic purpose of performing the haplotype ancestry is to identify whether the variant in the South Indian family might have a specific ancestry and also verify whether this could be associated with a founder effect in the Indian population. We have included the statement in the revised manuscript to make it clear for the reader also. Reviewer #2: A. Is the manuscript technically sound, and the data support the conclusions? The manuscript by Jain et al. describes about the identification of large deletion (5,296 bp) encompassing exons 3-5 of the BTK gene of the patients with X - linked agammaglobulinemia (XLA) using the whole-genome sequencing (WGS). The authors have performed whole-exome sequencing (WES) and WGS of the four and two patients with XLA, respectively. Also, the authors have performed several assays including multiplex-ligation dependent probe amplification (MLPA) assay. The manuscript can be accepted after the following minor concerns have been addressed. In abstract section, the authors must rephrase the following sentence as they concluded (in the results section) that they could not find any variant which could correlate with the clinical features: “Whole genome sequencing led to identification of the accurate genetic mutation which could help in early diagnosis leading to improved outcomes, prevention of permanent organ damage and improved quality of life, as well as enabling prenatal diagnosis”. Response: We thank the reviewer for the comment, we have included the statement in the Abstract section that “We could not identify any variant from the WES dataset that correlates with the clinical feature of the patient”. Also we have rephrased the concluding statement of the Abstract section. In the methodology section the authors can use multiple algorithms for the identification of alterations including copy number alterations using WES and WGS data of the patients with XLA. Response: We completely agree with the reviewer on using multiple tools for identification of CNV using WES and WGS. However the tools for CNV calling in the WES dataset are not robust and results in the false negative CNV calling. While using the WGS dataset, there are multiple tools with high sensitivity and specificity for CNV calling. In our study, we have used a tool LUMPY (version 0.2.13) for structural variant calling that identify the large deletion in our patient B. Has the statistical analysis been performed appropriately and rigorously? The statistical analysis has not been performed by the authors. Response: Since the study involved individual cases , no statistical analysis was performed C. Have the authors made all data underlying the findings in their manuscript fully available? The authors have provided analyzed data in the figures/tables and supplementary information in the manuscript wherever necessary. The raw data can be accessed through the convener of the Institutional Human Ethics Committee of the institute. Response: Since the WGS/WES constitute data that can be potentially identifiable and contains patient sensitive information, the data is available only on request. Access to the data could be requested by mailing to Dr. Jyoti Yadav (ni.ser.bigi@vaday.j) who is the convener of the Institutional Human Ethics Committee of CSIR-IGIB. D. Is the manuscript presented in an intelligible fashion and written in standard English? Overall manuscript is okay written, however the authors can rephrase the sentences in the methodology section for example, “small indels, small insertion, and small deletion”. Response: We thank the reviewer we have rephrased by removing the small indels in the statement. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0254407.r002
28 Jun 2021 Decision Letter - Obul Reddy Bandapalli, Editor Whole genome sequencing identifies novel structural variant in a large Indian family affected with X-linked agammaglobulinemia. PONE-D-20-38741R1 Dear Dr. Scaria, 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, Obul Reddy Bandapalli, MSc, PhD 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 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 #1: (No Response) Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) Reviewer #2: N/A ****** 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: (No Response) 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 #1: (No Response) 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 #1: (No Response) Reviewer #2: (No Response) ****** 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: No Reviewer #2: No https://doi.org/10.1371/journal.pone.0254407.r003
Formally Accepted
1 Jul 2021 Acceptance Letter - Obul Reddy Bandapalli, Editor PONE-D-20-38741R1 Whole genome sequencing identifies novel structural variant in a large Indian family affected with X-linked agammaglobulinemia Dear Dr. Scaria: 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. Obul Reddy Bandapalli Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0254407.r004

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