Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance

Meryem Ozgencil; Julian Barwell; Marc Tischkowitz; Louise Izatt; Ian Kesterton; Michael Simpson; Paul Sharpe; Paulo de Sepulveda; Edwige Voisset; Ellen Solomon

doi:10.1371/journal.pone.0260852

Peer Review History

Original SubmissionSeptember 1, 2021
6 Oct 2021 Decision Letter - Alvaro Galli, Editor PONE-D-21-28014Assessing BRCA1 activity in DNA damage repair using human induced Pluripotent Stem Cells to assist classification of BRCA1 variants of uncertain significancePLOS ONE Dear Dr. Voisset, 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 Nov 20 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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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? 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Reviewer #1: 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: In this manuscript Ozgencil et al. present an analysis of a subset of BRCA1 variants in a new model of induced pluripotent cells that can form three-dimensional mammospheres. The authors argue that this system constitute an important cell context to evaluate the function of BRCA1 variants. The authors isolate fibroblasts from carriers, induce pluripotency in vitro, and then characterize and analyze the behavior of heterozygous cell lines in their ability to form pluripotent colonies, and in their response to DNA damage. The paper is well written, the results are of high quality and the analysis of variants of uncertain significance is timely. There are a few issues that the authors should consider revising. While I agree that this model is extremely important to add to the analysis of unclassified variants and there are several advantages of this model over existing ones, the claim in the title “to assist classification of BRCA1 variants of uncertain significance” maybe premature. I believe the authors can say that it may be a powerful method to do so, and argue that point in the paper, but at this stage having this in the title suggests that the assay has been validated, which is not the case (need for additional controls to correctly calibrate the assay, for example). The issue of haploinsufficiency of BRCA1 is far from being resolved and whether the presence of a wild-type copy of BRCA1 in these cells affects the results. For example, whether a BRCA1 variant that has no detectable effect in heterozygosis (with a wild type copy), would show a defect when in hemizygozis after the loss of the wt copy. Are there dominant negative effects of any variant? (why does the cell with transfected K381X does not appear to have the wt copy?) The issue of the heterozygote context should be discussed in more detail and not mentioned only in passing. The authors claim that “The three known pathogenic variants studied here, C61G [20], K381X and A1708E 554 [21, 59-61], were correctly classified according to our evaluation.” Needs to be clarified. The authors conduct several assays, and the ‘correct’ classification seems dependent on what do the authors refer to their ‘evaluation’. While the three pathogenic variants mentioned score as loss of function in the relative repair efficiency assay, they do not differ from the wt in the ability to form mammosphere, or expression of markers. The A1708E is not tested in any downstream assays. The authors should define what do they mean by their evaluation. I think the following sentence, which may be referring to explaining the family history, is excessively broad if the authors mean for classifying a variant. Please clarify. “Thus, likewise genetic testing alone is unsatisfactory for the classification of BRCA1 568 VUSs, functional assays are also insufficient regardless of the cellular model used 569 [52, 58].” Finally, I wonder if the authors are selling themselves short on the last assay (ability to form luminal and basal lineage) where they report that there is no qualitative difference. Given the role in BRCA1 in shifting basal/luminal differentiation, I wonder if the authors have looked at it quantitatively, before discounting it as a way to assist in the classification of variants. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Revision 1
8 Nov 2021 Author Response RESPONSES TO THE EDITOR’ COMMENTS Our detailed responses are below, and the highlighted copy of our manuscript shows the changes (in dark red) we have made in response to all comments received. Moreover, in order to fulfil Plos One criteria, we added a supporting information file with uncropped and unadjusted Western-blot images (S1 Raw data), we also added all figures and files that were originally stated as “data not shown”, and renamed our files as requested. We hope that you will find this revised manuscript suitable for publication in Plos One. RESPONSES TO THE REVIEWER’ COMMENTS We have made the changes and amendments suggested by the Reviewer. Our responses to the individual comments are shown below. We hope that the Reviewer will find our responses satisfactory, and that our manuscript will now prove acceptable for publication in Plos One. Review Comments to the Author Reviewer #1: In this manuscript Ozgencil et al. present an analysis of a subset of BRCA1 variants in a new model of induced pluripotent cells that can form three-dimensional mammospheres. The authors argue that this system constitutes an important cell context to evaluate the function of BRCA1 variants. The authors isolate fibroblasts from carriers, induce pluripotency in vitro, and then characterize and analyze the behavior of heterozygous cell lines in their ability to form pluripotent colonies, and in their response to DNA damage. The paper is well written, the results are of high quality and the analysis of variants of uncertain significance is timely. There are a few issues that the authors should consider revising. Point 1: While I agree that this model is extremely important to add to the analysis of unclassified variants and there are several advantages of this model over existing ones, the claim in the title “to assist classification of BRCA1 variants of uncertain significance” maybe premature. I believe the authors can say that it may be a powerful method to do so, and argue that point in the paper, but at this stage having this in the title suggests that the assay has been validated, which is not the case (need for additional controls to correctly calibrate the assay, for example). Answer: We understand the point that the Reviewer is making, and have altered the title of the manuscript to “Assessing BRCA1 activity in DNA damage repair using human induced Pluripotent Stem Cells as an approach to assist classification of BRCA1 variants of uncertain significance”. Point 2: The issue of haploinsufficiency of BRCA1 is far from being resolved and whether the presence of a wild-type copy of BRCA1 in these cells affects the results. For example, whether a BRCA1 variant that has no detectable effect in heterozygosis (with a wild type copy), would show a defect when in hemizygozis after the loss of the wt copy. Are there dominant negative effects of any variant? (why does the cell with transfected K381X does not appear to have the wt copy?) The issue of the heterozygote context should be discussed in more detail and not mentioned only in passing. Answer: We thank the Reviewer for raising this point on LOH, which is indeed quite critical, and that we did not address since we only had heterozygous variant cell lines. We have now added a section on this topic in the discussion (lines 551-560). Concerning the K381X variant- this mutation introduces a premature stop codon resulting in a truncated protein. We were unable to detect the endogenous form of this variant. The WT allele can however be observed in Figure 3a, and the signal is significantly reduced compared to the WT control since only one full-length allele is present. A GFP-tagged construct was then transfected in 293T cells (Figure 3c) to determine whether this variant can be expressed. In order to clarify this point, we have specified it on Figure 3c. Point 3: The authors claim that “The three known pathogenic variants studied here, C61G [20], K381X and A1708E [21, 59-61], were correctly classified according to our evaluation.” Needs to be clarified. The authors conduct several assays, and the ‘correct’ classification seems dependent on what do the authors refer to their ‘evaluation’. While the three pathogenic variants mentioned score as loss of function in the relative repair efficiency assay, they do not differ from the wt in the ability to form mammosphere, or expression of markers. The A1708E is not tested in any downstream assays. The authors should define what do they mean by their evaluation. Answer: We apologize for the lack of clarity. This sentence has now been corrected as follow: “The three known pathogenic variants studied here, C61G [20] and K381X and A1708E [21, 61-63] presented reduced HR effectiveness and increased level of chromosomal aberrations, reflecting strong defects in DNA repair, as expected. Therefore, we validated these two assays to evaluate the four other variants (likely benign and VUS).” Point 4: I think the following sentence, which may be referring to explaining the family history, is excessively broad if the authors mean for classifying a variant. Please clarify. “Thus, likewise genetic testing alone is unsatisfactory for the classification of BRCA1 VUSs, functional assays are also insufficient regardless of the cellular model used [52, 58].” Answer: We apologize for the lack of clarity. This sentence has now been corrected as follow: “Thus, likewise genetic testing alone is unsatisfactory for the classification of BRCA1 VUSs, functional assays appear also insufficient regardless of the cellular model used: establishing a reliable classification of variants will only be possible by integrating data from multiple sources such as family history, gene and protein structure and functional assays [52, 58, 68].” Point 5: Finally, I wonder if the authors are selling themselves short on the last assay (ability to form luminal and basal lineage) where they report that there is no qualitative difference. Given the role in BRCA1 in shifting basal/luminal differentiation, I wonder if the authors have looked at it quantitatively, before discounting it as a way to assist in the classification of variants. Answer: We would like to apologize for the misunderstanding: we performed this assay to assess the ability of all BRCA1 variants (1) to form mammospheres and (2) to go through the differentiation process. However, it is a project per se to investigate the degree of luminal versus basal differentiation in all variants, which would require many additional experiments and deep analyses, that we, unfortunately, are unable to afford. We thank the Reviewer for the suggestion, and we surely think this would be the ideal next step of this project. We have now revised the manuscript to clarify this point (lines 593-594). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0260852.r002
18 Nov 2021 Decision Letter - Alvaro Galli, Editor Assessing BRCA1 activity in DNA damage repair using human induced Pluripotent Stem Cells as an approach to assist classification of BRCA1 variants of uncertain significance PONE-D-21-28014R1 Dear Drs. Voisset, 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, Alvaro Galli 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: I am satisfied with the changes provided in the revised manuscript and all concerns were addressed. ****** 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 https://doi.org/10.1371/journal.pone.0260852.r003
Formally Accepted
22 Nov 2021 Acceptance Letter - Alvaro Galli, Editor PONE-D-21-28014R1 Assessing BRCA1 activity in DNA damage repair using human induced Pluripotent Stem Cells as an approach to assist classification of BRCA1 variants of uncertain significance Dear Dr. Voisset: 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. Alvaro Galli Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0260852.r004

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