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  1. Evaluation Summary:

    This study provides a thorough functional analysis of three mutations in the BRCA2 gene that do not seem to necessarily cause breast cancer. The authors use functional assays in cancer cells and with recombinant proteins to determine that two BRCA2 variants, S1221P and T1980I, are indeed pathogenic, while the T13461 variant is fully functional and benign. The strength of the study is the rigorous assessment of these mutations in a variety of established assays for BRCA2. The work is likely to have a broad impact in the breast cancer field.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    The manuscript "BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair" by Jimenez-Sainz et al focuses on the characterization of three BRCA2 mutants that were previously classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. Mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers and are responsible for nearly half of all hereditary breast cancers and ovarian cancers. Identification of truly pathogenic BRCA2 missense mutations is a challenging but very important task for early cancer diagnostics. In this study, the authors developed a methodology for the identification of pathogenic BRCA2 mutations. They performed comprehensive analyses of three BRCA2 mutations including S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, and T1346I, located within the spacer region between BRC2 and BRC3 repeats. Using an impressive array of cellular and biochemical approaches they demonstrated that the first two BRCA2 mutants have a detrimental effect on RAD52-dependent DNA repair, and therefore likely to be pathogenic. In contrast, T1980I seems to have no effect on DNA repair in various tested assays and is likely to be a passenger mutation.

    Overall, I found the presented study of high quality. The developed methodology can be applied for analyses of other potentially pathogenic mutations in BRCA1, BRCA2, or other genes involved in DNA double-strand break repair. The work may have a broad impact on the biomedical field. The presentation quality is good as well.

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  3. Reviewer #2 (Public Review):

    In the manuscript by Jimenez-Sainz J. et al., the authors investigate 3/1,388 BRCA2 missense mutations found in the BRC domain of BRCA2 that are currently classified as variants of unknown significance. The authors focused on three mutations found in the BRC repeats of BRCA2 which are within BRC2/7 (mutations S1221P and T1980I) and a mutation found in a patient treated for colorectal cancer from Yale whose mutation is within a spacer region between BRC2/3 (mutation T1346I). First, the authors investigated the conservation of these three mutations and show BRCA2 mutations S1221 and T1980 are highly conserved by comparing sequences to the known FxTASGK motif important for RAD51 binding from various species. Then the authors speculate on the location and importance of the residues based on a BRC4 protein structure (Pellegrini et al., 2002 that was used as a starting point for unknown modeling algorithms. The authors speculate from their modeling that S1221P and T1980I either result in steric clashes or protein misfolding. They then go on to compliment human colorectal adenocarcinoma DLD-1 BRCA2-/- cells stably with each mutation and show that while each mutant is able to localize to the nucleus the S1221P and T1980I mutation are unable to complement sensitivity to MMC, Cisplatin, Olaparib, or Talazoparib. Interestingly, T1346I was able to rescue response to all agents tested. Consistent with their inability to compliment S1221P and T1980I also exhibited decreased RAD51 foci following IR treatment and increased micronuclei formation. HDR assays show S1221P and T1980I have diminished clover-positive cells and show diminished fork protection after treatment with HU. The authors then show that the BRC2-S1221P and BRC7-T19801 constructs mutants are unable to interact with RAD51- by co-ip, EMSA's, and biotinylated ssDNA binding assays supporting their diminished functions in the in vivo assays (HDR and fork protection). Finally, the authors show that BRCA2-T1980I has decreased strand exchange activity adding to the functional and mechanistic conclusions drawn from these VUS. The authors have performed a rigorous and thorough in vivo and in vitro analysis of three missense mutations in BRCA2 important for personalized cancer treatment therapies. This paper is important to the field of genome stability, high-fidelity repair mechanisms as well as classification of VUS.

    Strengths:

    • These studies beautifully pair in vivo functional assays with in vitro biochemical mechanistic studies of three missense mutations listed as variants of unknown significance in BRCA2 for classification and targeted cancer treatment.

    • The authors identify two mutations (S1221P and T1980I) within BRC repeats that are defective in HDR and fork protection, while T1346I is functional and a benign mutation.

    Weaknesses:

    • The S1221P mutation was unable to be purified.

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  4. Reviewer #3 (Public Review):

    In this report, the authors examined 3 mutations in the BRC-repeat region of BRCA2 in a series of functional assays. They found that two of the mutants showed severe defects in BRCA2 function, whereas the third mutant had no clear phenotype. The two mutants with functional defects are tested most thoroughly. The assays used here a numerous and have been validated and performed with appropriate controls and statistics. There are no concerns about the experiments themselves or the conclusions. So, the strength of the study is the number of assays performed in a rigorous manner.

    However, the weakness of the study is that it is unclear why these results are impactful. Several reports over the years, including some recent studies mentioned at the end of the Discussion, have involved parallel functional analysis of hundreds of alleles of BRCA2, with a clear end goal of improving medical decision-making for carriers of these BRCA2 alleles. Certainly, these studies have usually focused on other domains of BRCA2, like the DNA binding domain, but nonetheless, since these studies have typically involved testing hundreds of BRCA2 alleles, it is unclear how this manuscript studying 3 alleles fits into a broader population science effort to categorize BRCA2 variants of unknown significance (VUS). Perhaps the authors would argue that their study involves a comprehensive analysis of the 3 alleles, whereas other studies typically involve one or two functional assays. However, if that is the case, then is the argument that multiple assays are needed for accurate characterization of VUS? If so, what is the evidence for that assertion? Are there particular assays that are more likely to be predictive of pathogenicity based on their analysis?

    The mechanistic insight of the study is also unclear. These alleles are in conserved residues of the BRCA2 BRC repeats, which have been established as being important for BRCA2 function. Indeed, in the Discussion, it appears that the findings here are largely confirmatory for other mechanistic studies of the BRC repeats of BRCA2. What new information has been determined about the role of the BRC2 and BRC7 repeats from this study?

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  5. Author Response:

    We would like to thank the reviewers for a very thorough and careful analysis of our manuscript. All the comments and suggestions were taken to heart, and we feel that our revised manuscript is vastly improved because the reviewers clearly put in a significant effort to help us interpret and clarify our conclusions. We appreciate that the reviewers took the time to help us convey our results within the context of the field. Understanding and working with BRCA2 variants of uncertain significance is a challenging and complex, and we strive to report accurate and solid data that will facilitate to predict cancer risk and targeted therapies for patients.

    We appreciate the comment by reviewer 1 stating: “Identification of truly pathogenic BRCA2 missense mutations is a challenging but very important task for early diagnostics.” Our goal is to define cancer risk to prevent tumor formation and to promote personalized medicine with targeted therapies for homologous recombination deficient tumors. In future studies, we will expand these analyses to potentially pathogenic mutations of BRCA1 and PALB2.

    In reviewer 1 public text we have noticed a few mistakes in the text:

    1. In the text it says RAD52, but it should say RAD51. This is the sentence: “_Using an impressive array of cellular and biochemical approaches they demonstrated that the first two BRCA2 mutants have a detrimental effect on RAD52-dependent DNA repair, and therefore likely to be pathogenic_”
      
    2. In the text it says T1980I instead of T1346I. This is the sentence: “_In contrast, T1980I seems to have no effect on DNA repair in various tested assays and is likely to be a passenger mutation_.”
      

    We thank reviewer 2 for the thoughtful comments and questions and we agree that this paper is important to the field of homologous recombination, replication, genome stability maintenance, DNA double strand break repair and classification of VUS. We wish that BRCA2 S1221P full length mutant could have been purified to provide us with deeper mechanistic insights about how this mutant affects BRCA2 biochemical functions.

    We appreciate the constructive question about the impact of the results raised by reviewer 3, and we acknowledge the immense efforts from different laboratories to classify VUS over the years with different approaches (segregation studies, protein prediction algorithms, viability analysis in ES, HDR reporters, in vitro analysis, etc.). However, we see the need of rigorous and thorough in vitro and in cells analysis to understand BRCA2 fundamental biology and better classification of VUS with a more comprehensive analysis of altered BRCA2 functions.

    To answer the comments and questions raised by reviewer 3, we have incorporated more elaborated introduction, results, and discussion in the manuscript to cover this. In brief, our comprehensive analysis of three independent variants located in the BRC repeats of BRCA2 highlight the importance of using multiple analysis to understand the altered BRCA2 functions because there is not a unique assay to measure BRCA2 tumor suppression activity. Our goals were two: 1) to identify potentially pathogenic variants with important clinical implications for cancer risk in patients and 2) to leverage deleterious variants to uncover the specific functions carried out by individual BRC repeats.

    As an example of our answers to the questions of reviewer 3, we incorporate the last paragraph of our discussion here:

    Clinical integration of functional assays into the genetic counseling setting is an important goal but should be met with caution until we fully understand how specific variants impact the tumor suppressor functions of BRCA2. The use of robust and accurate functional assays will be essential to correctly evaluate BRCA2 VUS. Our study demonstrated that novel pathogenic variants exist not only in the DBD domain of BRCA2 but also in the BRC region leading to defects in RAD51 binding, activity, and subsequent HDR deficiencies. Mechanistic studies leveraging patient variants will continue to reveal the many functions of BRCA2.”

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