Identification and functional modelling of plausibly causative cis-regulatory variants in a highly-selected cohort with X-linked intellectual disability

Hemant Bengani; Detelina Grozeva; Lambert Moyon; Shipra Bhatia; Susana R. Louros; Jilly Hope; Adam Jackson; James G. Prendergast; Liusaidh J. Owen; Magali Naville; Jacqueline Rainger; Graeme Grimes; Mihail Halachev; Laura C. Murphy; Olivera Spasic-Boskovic; Veronica van Heyningen; Peter Kind; Catherine M. Abbott; Emily Osterweil; F. Lucy Raymond; Hugues Roest Crollius; David R. FitzPatrick

doi:10.1371/journal.pone.0256181

Peer Review History

Original SubmissionApril 14, 2021
14 Apr 2021 Author Response Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. Attachments Attachment Submitted filename: Point by Point Reviewer Response.docx https://doi.org/10.1371/journal.pone.0256181.r001
12 May 2021 Decision Letter - Chaeyoung Lee, Editor PONE-D-21-12396 Functional predictors of causation for cis-regulatory mutations PLOS ONE Dear Dr. FitzPatrick, 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 Jun 26 2021 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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[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 Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A Reviewer #3: 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 Reviewer #3: 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 Reviewer #3: 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 manuscript by Bengani and FitzPatrick used an updated method to determine causality of SNPs found in genomes of patients with genetically inherited intellectual disability (ID). Those are resourceful information to the other peers studying X-linked recessive forms of ID and represent diagnostic values for this particular disorder. The data in this work use a combination of zebra fish and mouse genetics models and functionally validated the clinically identified rare variants on the X chromosomes. Overall, I agree that the study is conclusive and thus recommend for acceptance. I list two minor issues and I hope the authors can address them quickly before publication. 1, An increasing body of evidences have implied brains are highly associated with somatic mutations, which often cause defects either derived from neural degeneration or development. Dr. Christopher Walsh from Harvard University is an expert in this field. The point is brain genomes are not identical to genomes of other tissue types as the method presented in this study used nonbrain tissues for sequencing. Would the authors comments on this point with respect to performance of gnomAD2.1 in the discussion section? 2, The quality/resolution of some figures are low, e.g. fig. 1, 3, 4 and 5. Is there any way to increase the readability of these figures? Reviewer #2: Bengani et al. performed a study for the identification of functional cis-regulatory mutations involved in the development of X-linked intellectual disability. The task is definitely very interesting, but also very challenging and time consuming. Indeed, it took several years and two different in vivo models to find data supporting a causative role for one out of 31 highly conserved CRE initially identified, the FMR1-CRE variant. I think that this manuscript is worth publishing because it has a quite strong rationale behind the selection of the non-coding variants to be tested and it shows that it is possible to make functional sense of mutations in regulatory elements. However, the phenotype for which the authors are trying to find a genetic evidence in CRE is very complex and very hard to dissect. Indeed, it is very difficult to find a straightforward link with a single variant in CRE. The phenotypes observed in the mouse model and the link to the human disease appears quite weak. I think this should be clearly underlined in the manuscript and better explained. Moreover, I think that the title, being very general, overstates the results of the manuscript and it should be changed to make it more related to the findings. The description of the statistical analysis applied by the authors is lacking. Moreover, I would suggest to check the statistical methods used for the analysis, in general, and, in particular, for Fmr1 transcription by qPCR (Fig. S41): some differences between wt and mutants would seem significant (i.e. in Hind brain P7), especially compared to other data reported as significant in the manuscript (i.e. Fig. 4H). Based on these reevaluations, some results and some statement in the paper might need to be revised. Reviewer #3: Manuscript Summary: Identifying causative variants in cis -regulatory elements (CREs) in neurodevelopmental disorders has proven challenging. Bengani, Grozeva, Moyon, and Bhatia et al. used a translational bridge between humans-zebrafish-mice, with the aid of model systems, and circling back to humans to identify and evaluate the pathological implications of chromosome X-resident CRE mutations that cause X-linked intellectual disability (XLID). Overall, this manuscript is perfectly suited for the scope of PLOS ONE. The translational approach that the authors have taken in this study is albeit challenging but particularly well appreciated. Studies such as these would certainly enhance the field's understanding of pathogenic variants implicated in neurodevelopmental disorders. In addition, the methodology employed in this study is thorough and the authors present the data/findings objectively. Major Comments and Questions: 1) 1a) Figures 2F and 2G: The authors use Six3-targeting morpholinos to demonstrate that six3-knockdown rescues the activity of mutant TENM1-CRE, the latter of which is quite convincing. However, there is no validation data provided to demonstrate that the concentration of the six3-morpholino used post-optimizations indeed led to a sufficient knockdown of six3. 1b) Additional note 1 (not required for the acceptance of this manuscript for publication): While I can understand the following might be outside the scope of this manuscript, an IP-seq experiment to pull down six3-bound to either TENM1-WT or TENM1-CRE would provide irrefutable evidence to substantiate the author's hypothesis that TENM1-CRE introduces a novel binding site of the repressor, six3. 1c) Additional note 2: The following could be addressed in the discussion, as per the authors' discretion. While TENM1-CRE led to a consistent loss of tenm1-expression in the mid-brain and hind-brain (table 1), it did not lead to a loss of tenm1-expression in the neural tube. Is six3 and six6 not expressed in the neural tubes and what additional six3/six6-independent mechanisms could be at play here? 2) In the figure legend for Fig. 6C: The authors state that there was "No significant difference was observed in audiogenic seizure incidence in the hemizygous mice with the variant Fmr1CRE compared to wild-type littermates (lines 447 - 449)." However, as per Fig. 6C, the Fmr1-CRE did display a statistically-significant decrease in the predisposition to audiogenic seizures (AGS), starkly contrasting the anticipated results of an increase in AGS derived from Fmr1-null mice. The data and results appears to contradict the figure legend and the authors' interpretation. 3) It is nice that the authors took a bench-to-bedside approach to re-evaluate the individuals within the FMR1-CRE-segregating family. Did these individuals not have any olfactory disorders, which may recapitulate the phenotype observed in Fig. 5A,B? And, following up from the precedence derived from Fig. 6C, did the affected individuals also display a resistance to the development of AGS? 4) The six3 piece of data from the zebrafish study was intriguing (Fig. 2). Was there no correlative evidence of a similar interplay between Six3 and Tenm1 in mice and/or humans? Could murine and human SIX3 exhibit a non-overlapping pattern of expression with TENM1, which might explain the lack of a phenotype in Tenm1-CRE mice? What are the authors thoughts on the Six3/Six6-dependent and -independent mechanisms of tenm1-CRE in zebrafish and how these mechanisms translate to mammalian species (e.g. mice and humans)? Minor Comments and Questions: 1) Formatting: It would be nice to keep the text format used in all figures, either in the main text or in the supplement, consistent in size and style. 2) It would be nice to spell out the full names of the protein abbreviations used, such as FMR1, FMRP, TENM1. All additional major and minor comments from me were either fully or partially overlapping with those raised by the reviewers during the peer-review for the PLOS Genetics submission and have been addressed by the authors. ****** 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 Reviewer #3: 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. 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Revision 1
21 Jul 2021 Author Response Reviewer #1: This manuscript by Bengani and FitzPatrick used an updated method to determine causality of SNPs found in genomes of patients with genetically inherited intellectual disability (ID). Those are resourceful information to the other peers studying X-linked recessive forms of ID and represent diagnostic values for this particular disorder. The data in this work use a combination of zebra fish and mouse genetics models and functionally validated the clinically identified rare variants on the X chromosomes. Overall, I agree that the study is conclusive and thus recommend for acceptance. I list two minor issues and I hope the authors can address them quickly before publication. 1, An increasing body of evidences have implied brains are highly associated with somatic mutations, which often cause defects either derived from neural degeneration or development. Dr. Christopher Walsh from Harvard University is an expert in this field. The point is brain genomes are not identical to genomes of other tissue types as the method presented in this study used nonbrain tissues for sequencing. Would the authors comments on this point with respect to performance of gnomAD2.1 in the discussion section? This is an interesting point. Our experimental design relies on the variants being inherited and co-segregating with the disease in an individual family. gnomAD, likewise, is very focused on cataloging constitutional variation and data is filtered with the specific purpose of excluding somatic mosaic variants. To clarify this we have added the following text to the discussion: “Recently somatic mutations in the brain have been implicated in the causation of neuro developmental disorders {Rodin et al., 2021, #61844}. The fact that we have selected individuals with a positive family history would significantly reduce the prior probability of this disease mechanism in our cohort. For this reason, we have designed our variant filtering strategy to identify constitutional mutations and exclude variants that are likely to be mosaic.” (Line: 460-464) 2, The quality/resolution of some figures are low, e.g. fig. 1, 3, 4 and 5. Is there any way to increase the readability of these figures? We apologise for this and we have increased the resolution of these figures – we also have Adobe Illustrator files available if required. Reviewer #2: Bengani et al. performed a study for the identification of functional cis-regulatory mutations involved in the development of X-linked intellectual disability. The task is definitely very interesting, but also very challenging and time consuming. Indeed, it took several years and two different in vivo models to find data supporting a causative role for one out of 31 highly conserved CRE initially identified, the FMR1-CRE variant. I think that this manuscript is worth publishing because it has a quite strong rationale behind the selection of the non-coding variants to be tested and it shows that it is possible to make functional sense of mutations in regulatory elements. However, the phenotype for which the authors are trying to find a genetic evidence in CRE is very complex and very hard to dissect. Indeed, it is very difficult to find a straightforward link with a single variant in CRE. The phenotypes observed in the mouse model and the link to the human disease appears quite weak. I think this should be clearly underlined in the manuscript and better explained. We have tried not to over-state the link between the mouse model and the human disease. However for the FMR1CRE/Fmr1CRE variant there is clear evidence that this variant results in reduced expression of endogenous Fmr1 in the embryonic brain and abnormal results in neurophysiological tests that are considered to the gold-standard markers of FMRP function in the hippocampus. We agree that the behavioural phenotype is not Fmr1-specific but there are no known phenotypes that are specific to this genotype. We have changed the penultimate sentence in the abstract to address the reviewer’s concerns. It now reads: ”Assigning causative status to any ultra-rare CRE variant remains problematic and requires disease-relevant in vivo functional data from multiple sources.”(Line: 53-55). Moreover, I think that the title, being very general, overstates the results of the manuscript and it should be changed to make it more related to the findings. We have changed the title to be more descriptive: “Identification and functional modelling of plausibly causative cis-regulatory variants in a highly-selected cohort with X-linked intellectual disability.” The description of the statistical analysis applied by the authors is lacking. Moreover, I would suggest to check the statistical methods used for the analysis, in general, and, in particular, for Fmr1 transcription by qPCR (Fig. S41): some differences between wt and mutants would seem significant (i.e. in Hind brain P7), especially compared to other data reported as significant in the manuscript (i.e. Fig. 4H). Based on these reevaluations, some results and some statement in the paper might need to be revised. We have added a section named Statistical analysis (Line 255-260) under Methods in which we have given details of statistical methods used and also added the details of statistics used in the legend section of figures. There was mistake in calculating the standard error in figure S41.We have re done the analysis and updated the figure S41 (now S42 Fig) and result confirm that differences between wt and mutants is not significant. We apologise for this error. Reviewer #3: Manuscript Summary: Identifying causative variants in cis -regulatory elements (CREs) in neurodevelopmental disorders has proven challenging. Bengani, Grozeva, Moyon, and Bhatia et al. used a translational bridge between humans-zebrafish-mice, with the aid of model systems, and circling back to humans to identify and evaluate the pathological implications of chromosome X-resident CRE mutations that cause X-linked intellectual disability (XLID). Overall, this manuscript is perfectly suited for the scope of PLOS ONE. The translational approach that the authors have taken in this study is albeit challenging but particularly well appreciated. Studies such as these would certainly enhance the field's understanding of pathogenic variants implicated in neurodevelopmental disorders. In addition, the methodology employed in this study is thorough and the authors present the data/findings objectively. Major Comments and Questions: 1) 1a) Figures 2F and 2G: The authors use Six3-targeting morpholinos to demonstrate that six3-knockdown rescues the activity of mutant TENM1-CRE, the latter of which is quite convincing. However, there is no validation data provided to demonstrate that the concentration of the six3-morpholino used post-optimizations indeed led to a sufficient knockdown of six3. We have validated the knockdown of Six3 by western blot. We have added a figure in the supplementary data named as S40 Fig and also documented it in the result section.(Line 333-334) 1b) Additional note 1 (not required for the acceptance of this manuscript for publication): While I can understand the following might be outside the scope of this manuscript, an IP-seq experiment to pull down six3-bound to either TENM1-WT or TENM1-CRE would provide irrefutable evidence to substantiate the author's hypothesis that TENM1-CRE introduces a novel binding site of the repressor, six3. We thank the reviewer for this suggestion, and we would endeavour to do those experiments in future follow-up studies. 1c) Additional note 2: The following could be addressed in the discussion, as per the authors' discretion. While TENM1-CRE led to a consistent loss of tenm1-expression in the mid-brain and hind-brain (table 1), it did not lead to a loss of tenm1-expression in the neural tube. Is six3 and six6 not expressed in the neural tubes and what additional six3/six6-independent mechanisms could be at play here? We believe there might be distinct regions of the TENM1-CRE driving expression in the mid-brain/hind-brain and the neural tube. The single-nucleotide change affecting the six3 binding site possibly localises to the region of the CRE driving brain expression. Similar scenarios have been observed in other CREs (Bhatia et al AJHG 2013) 2) In the figure legend for Fig. 6C: The authors state that there was "No significant difference was observed in audiogenic seizure incidence in the hemizygous mice with the variant Fmr1CRE compared to wild-type littermates (lines 447 - 449)." However, as per Fig. 6C, the Fmr1-CRE did display a statistically-significant decrease in the predisposition to audiogenic seizures (AGS), starkly contrasting the anticipated results of an increase in AGS derived from Fmr1-null mice. The data and results appears to contradict the figure legend and the authors' interpretation. The apparent decrease in audiogenic seizures in the Frm1-CRE mice is intriguing but is not statistically significant. Significance was determined using two-tailed Fisher’s exact test (appropriate for analysing nominal data sets). A p-value of < 0.05 was considered statistically significant. The calculated p value is p = 0.063 which is greater than the cut off used. 3) It is nice that the authors took a bench-to-bedside approach to re-evaluate the individuals within the FMR1-CRE-segregating family. Did these individuals not have any olfactory disorders, which may recapitulate the phenotype observed in Fig. 5A,B? And, following up from the precedence derived from Fig. 6C, did the affected individuals also display a resistance to the development of AGS? There was no obvious olfaction anomalies in the affected individuals from this family and no seizure predisposition (Line 393-394). We have added this as a comment to the second last paragraph of the Results section “Fmr1/FMRP-Focused Analyses”. 4) The six3 piece of data from the zebrafish study was intriguing (Fig. 2). Was there no correlative evidence of a similar interplay between Six3 and Tenm1 in mice and/or humans? Could murine and human SIX3 exhibit a non-overlapping pattern of expression with TENM1, which might explain the lack of a phenotype in Tenm1-CRE mice? What are the authors thoughts on the Six3/Six6-dependent and -independent mechanisms of tenm1-CRE in zebrafish and how these mechanisms translate to mammalian species (e.g. mice and humans)? We agree this it would be interesting to explore the relationship between SIX3-binding and TENM1 expression in the mouse mutants but this is obviously a much more challenging experimental task than it is in zebrafish embryos. The zebrafish reporter-transgenic assay used an isolated fragment of human DNA the dramatic effect of Six3 binding on CRE function may be exaggerated compared to the endogenous loci in mice and humans if other enhancers exist with partially overlapping function. Minor Comments and Questions: 1) Formatting: It would be nice to keep the text format used in all figures, either in the main text or in the supplement, consistent in size and style. We have reformatted all the text in the figures and legends in the main text as well as in supplement with consistent size and style. We were not able to change the format of figure S3-S33 (pedigree analysis) as we got the file from the clinical lab long time back and unable to access the original file. We apologise for this. 2) It would be nice to spell out the full names of the protein abbreviations used, such as FMR1, FMRP, TENM1. We have added the details of full gene name/protein abbreviation in the main text FMR1: FMRP translational regulator 1. (Line 96) Tenm1: Teneurin transmembrane protein 1. (Line 184) SIX3: SIX homeobox 3. (Line 323) SIX6: SIX homeobox 6. (Line 324) RFX2: Regulatory factor X2. (Line 338) FMRP: fragile X mental retardation protein. (Line 375) POLA1/PCYT1B: DNA polymerase alpha 1, catalytic subunit. (Line 408) KDM6A: lysine demethylase 6A. (Line 409) ARHGEF6: Rac/Cdc42 guanine nucleotide exchange factor 6. (Line 410) AFF2: AF4/FMR2 family member 2. (Line 410) All additional major and minor comments from me were either fully or partially overlapping with those raised by the reviewers during the peer-review for the PLOS Genetics submission and have been addressed by the authors. Attachments Attachment Submitted filename: Response to Reviewers Comments.docx https://doi.org/10.1371/journal.pone.0256181.r003
2 Aug 2021 Decision Letter - Chaeyoung Lee, Editor Identification and functional modelling of plausibly causative cis-regulatory variants in a highly-selected cohort with X-linked intellectual disability PONE-D-21-12396R1 Dear Dr. FitzPatrick, 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. 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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 Reviewer #3: 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 Reviewer #2: (No Response) Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: (No Response) Reviewer #3: 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 Reviewer #2: (No Response) Reviewer #3: 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 Reviewer #2: (No Response) Reviewer #3: 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: All of my minor concerns have been addressed in this revisedmanuscript. I have no further question. Reviewer #2: (No Response) Reviewer #3: The authors have sufficiently and more than satisfactorily addressed all my comments and questions. Overall, this manuscript is perfectly suited for the scope of PLOS ONE. The translational approach that the authors have taken in this study is albeit challenging but particularly well appreciated. Studies such as these would certainly enhance the field's understanding of pathogenic variants implicated in neurodevelopmental disorders. In addition, the methodology employed in this study is thorough and the authors present the data/findings objectively. ****** 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 Reviewer #3: No https://doi.org/10.1371/journal.pone.0256181.r004
Formally Accepted
5 Aug 2021 Acceptance Letter - Chaeyoung Lee, Editor PONE-D-21-12396R1 Identification and functional modelling of plausibly causative cis-regulatory variants in a highly-selected cohort with X-linked intellectual disability Dear Dr. FitzPatrick: 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 Prof. Chaeyoung Lee Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0256181.r005

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