Human BRCA pathogenic variants were originated during recent human history

Evolutionary origin of pathogenic variants in human BRCA1 and BRCA2.


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Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** This brief analysis of BRCA1 and BRCA2 mutation patterns across human pre-history is an interesting analysis of the patterns of emergence of pathogenic mutations of BRCA genes. As noted by the authors, the relatively high frequency of pathogenic or likely pathogenic (PLP) BRCA alleles is not well understood. The authors summarize two possible explanations: "Possible explanations can be 1). BRCA PLPs develop cancer mostly at later repeoduction [sic] or memopause [sic] stage. Before reaching the stage, the mutation has already transmitted to the next generation; 2). The oncogenic effects of BRCA PLPs could be developmental stage-dependent. They could be benefitial [sic] at the reproductive stage but deleterious at later repeoduction [sic] or memopause [sic] stage. As positive selection mostly imposes on reproductive stage, BRCA PLPs may be favorablly [sic] selected." The analysis presented does not resolve this question, yet in the body of the text it is suggested that positive selection for BRCA PLP alleles did indeed occur. It is important to clarify the logic and to stick to one point of view-the correct one being that the issue is unresolved.
As is clear from the excerpt quoted above, the phrasing, spelling and syntax contains numerous errors, making the arguments sometimes difficult to follow. It would be worthwhile to enlist help in rewriting the m/s to make the text clearer and to correct the numerous errors. In other respects, this reviewer found the summary of BRCA gene PLP mutations across human (pre-)history interesting and informative, as was the comparison across species.
Reviewer #2 (Comments to the Authors (Required)): The manuscript describes a highly interesting analysis of pathogenic and likely pathogenic variants in the BRCA1/2 genes across species and in ancient human DNA. The very interesting conclusion is that the mutations are in general young -less than 3500 years. The comprehensive cross specie and ancient analysis is original and interesting. However, there are some major questions to the paper: 1) the authors describe positive selection for PLP variants. This is controversial and should be explained and justified in more detail. Obviously, there is a negative selection against PLPs since dual carriers do not survive or develop Fanconi Anemia. This should be discussed. 2) a more likely explanation for the many different PLPs is that the genes are large. The reason why the mutations are young could be the negative selection. This should also be discussed.
3) why are there many mutations common to other species especially avians, despite not being conserved in evolution? The most likely explanation is mutational hotspots. This may not be expected or loss of function mutations. 4) page 9. Results from phylogenetic analysis are referred to but not shown. It may be obvious, but it would be illustrative to show. 5) page 14: the oncogenes effect of BRCA PLP. BRCA genes are tumor suppressors, not oncogenes. 6) figure 3. Yellow color is hardly visible.
Reviewer #3 (Comments to the Authors (Required)): In this manuscript Li and colleagues examine the origins of BRCA pathogenic/likely pathogenic variants in human history. The paper is essentially divided in two parts. The first of which uses phylogenetic analysis to determine the origins of pathogenic variants; whether they may have originated in common evolutionary ancestors and its presence in current human populations would have been driven by positive selection. The second searches for pathogenic/likely pathogenic variants in ancient human samples of a wide range of dates (dated from 300 to 45,000 years ago).
The manuscript has several issues that need to be addressed.
Overall, the methods, description of results, and underlining data are so brief that it is extremely difficult to understand the rationale behind the approach, what was done with the data and to verify that the conclusions are warranted by the results obtained.
For example, the authors state that they 'searched' for human PLP in the reference (presumably) sequence of other species, but it is unclear what they mean by that. It is not described how the sequences were aligned and the criteria for the inclusion of species, or whether it was disproportionately skewed to mammalian species. There is no information about how the alignments were inspected whether there was manual editing, whether the alignment was also done at the level of amino acid. The authors identify a significant fraction of species that carry variants corresponding to human pathogenic variants. Because these are overwhelmingly premature termination variants and most of them lead to inactive proteins, are we to assume that many species do not have functional BRCA? I couldn't find the alignments (or even representative examples of the alignments) to examine the results. The excel table with a selection of nucleotides that are proposed to represent these variants lacks context and details (accession number, etc).
The few specific examples are problematic: the three pathogenic variants in BRCA1 presented as intronic (pages 7 and 8) are not intronic but rather coding substitutions, and the BRCA2 variant presented as pathogenic shared with 57 species (p.Met1168Ile) is not pathogenic, but rather a VUS. Conclusions are presented in a very confusing way as exemplified by the self-contradictory statements: "We found no evidence for their presence in these species, but the wildtype alleles in BRCA1 185delAG was present across 51 species from Chimpanzee to Armadillo, in BRCA1 5382insC across 83 species from Rhesus to Coelacanth, in BRCA2 6174delT across 87 species from Chimpanzee to Tetraodon." Therefore, the results presented in this section seems unconvincing at best.
The section on ancient DNAs suffers from the same issues and alignments are not provided. It is unclear whether the sequences in which they were identified are part of complete sequence of the locus or from smaller fragments. Here the authors find a very high frequency of individuals with pathogenic variants (1.8%) including findings of variants rare in current populations being found in more than one individual in a subpopulation. One possibility is that this is an extremely small sample to derive conclusions. In this context, statements such as "The PLPs shared between the much early Eurasians and the much later Dominicans implies that these could be inherited from Europeans to Dominicans in recent colonization history." seem unwarranted.
It is unclear how they determined the 'time of origin' as they state that they conducted "haplotyping analysis", but very little information is given. No consideration is given to the possibility that mutations may arise at multiple times in multiple independent populations. Also, no consideration is given to the role of pleiotropy in driving selection of variants whose effects mostly occur in post-reproductive age.

Minor issues
The section on "Data from previous studies" seems dispensable and can be summarized in the introduction.
Please define the criteria ('interpretation'; 'review status'; how to deal with variants that have multiple interpretation, etc) used to extract the data from ClinVar. This is extremely important as it is the basis of the analysis. State the date and version (if applicable) when the data was extracted. 1 Point-to-point response to reviewers' comments Reviewer #1

Question
As noted by the authors, the relatively high frequency of pathogenic or likely pathogenic (PLP) BRCA alleles is not well understood. The authors summarize two possible explanations: "Possible explanations can be 1). BRCA PLPs develop cancer mostly at later repeoduction or memopause stage. Before reaching the stage, the mutation has already transmitted to the next generation; 2). The oncogenic effects of BRCA PLPs could be developmental stage-dependent. They could be benefitial at the reproductive stage but deleterious at later repeoduction or memopause stage. As positive selection mostly imposes on reproductive stage, BRCA PLPs may be favorablly selected." The analysis presented does not resolve this question, yet in the body of the text it is suggested that positive selection for BRCA PLP alleles did indeed occur. It is important to clarify the logic and to stick to one point of view-the correct one being that the issue is unresolved. Answer Thanks for the comments. Indeed, our study doesn't provide answer for the two possible explanations.
In the revision, the sentences were included in Discussion to clarify the issue: Possible explanations can be 1). The cancer caused by BRCA PLPs occurs mostly at later repeoduction stage. Before reaching the stage, the mutation has already been transmitted to the next generation; 2). The loss of tumor suppressing function of BRCA due to BRCA PLPs could be developmental stage-dependent that the PLPs could be benefitial at the reproductive stage but deleterious at later repeoduction stage. Positive selection imposed on reproductive stage can select the BRCA PLPs not deleterious at the stage. However, our current study does not determine whether one of the two explanations or both could contribute to the higher frequency of BRCA PLP in human population.

Question
As is clear from the excerpt quoted above, the phrasing, spelling and syntax contains numerous errors, making the arguments sometimes difficult to follow. It would be worthwhile to enlist help in rewriting the m/s to make the text clearer and to correct the numerous errors. Answer Thanks for the comments. In the revision, we have made extensive correction and a native English speaker colleague has polished the final manuscript. We hope the resubmitted version has improved the quality of English writing.

Reviewer #2
Question 1) the authors describe positive selection for PLP variants. This is controversial and should be explained and justified in more detail. Obviously, there is a negative selection against PLPs since dual carriers do not survive or develop Fanconi Anemia. This should be discussed. Answer Thanks for the comments. Rich data from BRCA evolution study in the past 2 decades has well demonstrated the presence of two types of evolution selection in BRCA: the positive selection in human, chimp and bonobo, and the negative/neutral selections in all other species. As described in our manuscript "Studies revealed that BRCA is evolutionarily highly conserved across wide-range species, including primates, by negative selection in reflecting its essential roles in maintaining genome stability; it has also observed that BRCA is rapidly evolving by positive selection in humans and its close living relatives of the Chimpanzee and Bonobo (Huttley et al. 2000;Fleming et al. 2003;Abkevich et al. 2004;Pavlicek et al. 2004;Burk-Herrick et al. 2006;O'Connell 2010;Lou et al. 2014). Besides our current study in human BRCA, we also did a parallel evolution study in mouse Brca. Our study showed that negative/neutral selection is present in mouse Baca (Wang and Wang 2021). The positive selection likely reflects the new function of BRCA gained in these three species of human, chimp and bonobo (Huttley et al. 2000)". It is true that "dual carriers do not survive or develop Fanconi Anemia". This is more likely due to the embryonic lethal effects of homozygotic BRCA mutation as BRCA is essential for proper development (Xu, X. et al. Nat Genet 22, 37-43, 1999).
In the revision, the paragraph has been revised as the following paragraph to clarify better the issue: Studies revealed that BRCA across wide-range species including most of the primates is evolutionarily highly conserved by negative selection in reflecting its essential roles in maintaining genome stability. In contrast, however, BRCA in humans and its closest living relatives of the Chimpanzees and Bonobos is rapidly evolving by positive selection ( The following paragraph is included under Discussion: Homozygotic BRCA PLP carriers seldom survive or develop Fanconi Anemia (Seo et al. 2018). This is due to the embryonic lethal effect of homozygotic BRCA mutation as BRCA is essential for development rather than negative selection against BRCA PLPs.
Question 2) a more likely explanation for the many different PLPs is that the genes are large. The reason why the mutations are young could be the negative selection. This should also be discussed. Answer Thanks for the comments. Based on current knowledge, human BRCA is under positive selection but not negative selection (Huttley et al. 2000;Fleming et al. 2003;Abkevich et al. 2004;Pavlicek et al. 2004;Burk-Herrick et al. 2006;O'Connell 2010;Lou et al. 2014). This is well demonstrated by the Figure 2 in the pioneer BRCA phylogenic study, showing the positive selection in human and chimpanzee BRCA1, and negative selection in other species (Huttley GA et al. Nat Genet 25: 410-413, 2000). The observation has been confirmed by many following reports including ourselves.
Human BRCA1 has over 1,863 residues, and human BRCA2 has over 3,418 residues. While the large size of human BRCA can be a factor, it is very unlikely that it can be a major factor contributing to the large quantity of PLPs. As shown by our own study, mouse Brca is highly stable without much variation although the size of mouse Brca (Brca1 1,812 residues, Brca2 3,329 residues) are very close to the human ones.
In the revision, the following paragraph has been included in the Discussion to explain this issue: Other factors may also contribute to the rich human BRCA PLPs. Human BRCA1 has 1,863 residues and BRCA2 has 3,418 residues. Although gene with large size can produce more genetic variants, it is unlikely that the size in BRCA can be a major factor for the large quantity of BRCA PLPs. Our study showed that mouse Brca does not contain much variation although the size of mouse Brca (Brca1 1,812 residues, Brca2 3,329 residues) is very close to the human BRCA (Wang et al, 2021).
Question 3) why are there many mutations common to other species especially avians, despite not being conserved in evolution? The most likely explanation is mutational hotspots. This may not be expected or loss of function mutations.

Answer
We do not have explanation for the "far distance BRCA PLP conservation" we named between human and Aves and Sarcopterygii. A study (Gao L and Zhang J. Why are some human disease-associated mutations fixed in mice? TRENDS in Genetics. 19.12: 678-681, 2003) compared multiple human deleterious mutations, including several mutations in BRCA, shared with mouse. They evaluated multiple theories including "Founder effect", "Fixations of slightly deleterious mutations", "Relaxed selection on late-onset phenotypes" and "Compensatory changes", they were in favor of the "compensation theory", which states that "compensatory mutations at other sites of the same or a different protein render the deleterious mutations neutral", to explain the conservation of human deleterious mutation across distant species. We consider that the compensation theory can also be used to explain the sharing of human BRCA PLPs in aves, although data is not available to validate the explanation.
In the revision, the entire paragraph has been modified to clarify the issue: Many human BRCA PLPs were shared with species in Aves and Sarcopterygii clads. This could happen by chance rather than by cross-species evolution conservation, as it is unlikely that the conservation would allow the presence of the gaps across such wide distances. Alternatively, it could also be related with species-specific pathogenicity of the same BRCA variants that the BRCA PLPs in humans may not be pathogenic in non-human species (Gao and Zhang, 2003). There is lack of evidence to link the cancers in non-human species with their shared human BRCA PLPs. For example, Tasmanian devil has high risk to develop facial cancer but there is no evidence to link the disease with the multiple human BRCA1 PLPs its shared (Hamilton 1966). A study analyzed multiple human deleterious mutations, including several mutations in BRCA, shared with mouse (Gao and Zhang. 2003). Among multiple theories including "Founder effect", "Fixations of slightly deleterious mutations", "Relaxed selection on late-onset phenotypes" and "Compensatory changes", they considered that the "compensation changes", which stated that "compensatory mutations at other sites of the same or a different protein render the deleterious mutations neutral", can be the best to explain the conservation of human deleterious mutation with the distant species of mouse. We consider that the compensation theory can also be used to explain the sharing of human BRCA PLPs with the species in aves and Sarcopterygii clads, although direct evidence is not available to validate the explanation.
Question 4) page 9. Results from phylogenetic analysis are referred to but not shown. It may be obvious, but it would be illustrative to show. Answer Sorry for the confusion. The sentence "The results from the phylogenetic analysis didn't support evolution conservation as the main source of human BRCA PLPs." is to make conclusion for the phylogenetic study.
In the revision, the sentence has been revised as: The results from our phylogenetic analysis above didn't support evolution conservation as the source of human BRCA PLPs.
Question 5) page 14: the oncogenes effect of BRCA PLP. BRCA genes are tumor suppressors, not oncogenes. Answer Sorry for the confusion. The originial sentence "The oncogenic effects of BRCA PLPs could be developmental stage-dependent." refers to the oncogenic effected by the mutated BRCA but not the wildtype BRCA.
In the revision, the sentence has been changed as: The loss of tumor suppressing function of BRCA due to BRCA PLPs could be developmental stage-dependent.
Question 6) figure 3. Yellow color is hardly visible.

Answer
To make better visible for the figure, the color range used in the figure has been changed from yellow-to-blue to red-to-blue. In this way, the original yellow spot is changed to red spot.

Reviewer #3 Question
Overall, the methods, description of results, and underlining data are so brief that it is extremely difficult to understand the rationale behind the approach, what was done with the data and to verify that the conclusions are warranted by the results obtained. For example, the authors state that they 'searched' for human PLP in the reference (presumably) sequence of other species, but it is unclear what they mean by that. It is not described how the sequences were aligned and the criteria for the inclusion of species, or whether it was disproportionately skewed to mammalian species. There is no information about how the alignments were inspected whether there was manual editing, whether the alignment was also done at the level of amino acid. Answer Thanks for the comments. Indeed, the methodological description in the original manuscript was rather brief, as we thought that many methodologies we used followed the well-established approaches by others, such as phylogenic analysis and archeological analyses. Obviously, too briefs can cause difficulties for readers to follow up the details as indicated by the comments made by the reviewer.
In the revision, we expanded the description including more technique details and adding citations/links to help readers to better understand the rationale and conditions used in the analysis.

Question
The authors identify a significant fraction of species that carry variants corresponding to human pathogenic variants. Because these are overwhelmingly premature termination variants and most of them lead to inactive proteins, are we to assume that many species do not have functional BRCA? I couldn't find the alignments (or even representative examples of the alignments) to examine the results. The excel table with a selection of nucleotides that are proposed to represent these variants lacks context and details (accession number, etc). Answer Thanks for the comments. In the revision, we have added the actual human BRCA PLP variants and ClinVar allele ID at the top of Supplemental Table 3 (BRCA1) and Supplemental Table 4 (BRCA2) to allow direct comparison between species; we have also generated Supplementary  Figure 1 showing examples of the alignments.

Question
The few specific examples are problematic: the three pathogenic variants in BRCA1 presented as intronic (pages 7 and 8) are not intronic but rather coding substitutions, the BRCA2 variant presented as pathogenic shared with 57 species (p.Met1168Ile) is not pathogenic, but rather a VUS. Answer Very sorry for the mistake: "Intronic" is a typo. In the revision, the sentence has been revised as: "For example, BRCA1 c.3268C>T (p.Gln1090Ter) was shared with 19 species from Rock pigeon in Aves to Lizard in Sarcopterygii, c.2498T>A (p.Leu833Ter) with 18 species from Saker falcon in Aves to Spiny softshell turtle in Sarcopterygii, and c.2138C>A (p.Ser713Ter) with 18 species from Rock pigeon in Aves to Lizard in Sarcopterygii".
In the revision, we used the pathogenic BRCA2 c.8933C>A (p.Ser2978Ter) to replace BRCA2 c.3504G>T (p.Met1168Ile) to avoid this trouble, and the sentence is revised as: c.8933C>A (p.Ser2978Ter) with 36 species from Lesser Egyptian jerboa in Euarchontoglires to Opossum in Mammal, and the most diatal species shared with this variant was Spotted gar in Fish.

Question
Conclusions are presented in a very confusing way as exemplified by the self-contradictory statements: "We found no evidence for their presence in these species, but the wildtype alleles in BRCA1 185delAG was present across 51 species from Chimpanzee to Armadillo, in BRCA1 5382insC across 83 species from Rhesus to Coelacanth, in BRCA2 6174delT across 87 species from Chimpanzee to Tetraodon." Therefore, the results presented in this section seems unconvincing at best. Answer Very sorry for the confusion here. What we want to say is that the wildtype bases in the three typical BRCA PLP are highly conserved without variation.
In the revision, the paragraph has been revised as the followings: As control, we searched BRCA1 c. 68_69del, BRCA1 c.5266dup and BRCA2 c.5946del, the three BRCA founder PLPs in Ashkenazi Jews population, in the 100 vertebrates (Levy-Lahad et al. 1997). We found no evidence for their presence in these species, but the wildtype AG at the position of 68-69 in BRCA1 was present across 51 species from Chimpanzee to Armadillo, the wildtype C at the position of 5266 in BRCA1 was present across 83 species from Rhesus to Coelacanth, the wildtype T at the position of 5946 of BRCA2 was present across 87 species from Chimpanzee to Tetraodon.

Question
The section on ancient DNAs suffers from the same issues and alignments are not provided. It is unclear whether the sequences in which they were identified are part of complete sequence of the locus or from smaller fragments. Here the authors find a very high frequency of individuals with pathogenic variants (1.8%) including findings of variants rare in current populations being found in more than one individual in a subpopulation. One possibility is that this is an extremely small sample to derive conclusions. In this context, statements such as "The PLPs shared between the much early Eurasians and the much later Dominicans implies that these could be inherited from Europeans to Dominicans in recent colonization history." seem unwarranted.

Answer
Following the comments, we generated Supplementary Figure 1A to include multiple alignment examples between modern and ancient BRCA PLP. Indeed, the number of ancient cases being sequenced is much smaller than the number of modern cases being sequenced. This is exemplified by the availability of only 3 Neanderthal and 2 Denisovan genomes over 40,000 year-old.
"It is unclear whether the sequences in which they were identified are part of complete sequence of the locus or from smaller fragments.": As shown in Supplementary Table 2, of the 2,972 BRCA1 PLP, less than 10 were indels with >50base change, and most were single base variation or smaller indel with a few bases; similar situation was present in 3,652 BRCA2 PLP. Therefore, the sequences in which they were identified are mostly single base changes.
"The very high frequency of individuals with pathogenic variants (1.8%) including findings of variants rare in current populations being found in more than one individual in a subpopulation": Indeed, the match is much likely by chance rather than statistically significant events.
In the revision, the sentence "The PLPs shared between the much early Eurasians and the much later Dominicans implies that these could be inherited from Europeans to Dominicans in recent colonization history." has been revised to let the observation less conclusive: The PLP shared between the much early Eurasians and the much later Dominicans suggests that these might be inherited from Europeans to Dominicans in recent colonization history.

Question
It is unclear how they determined the 'time of origin' as they state that they conducted "haplotyping analysis", but very little information is given. Answer Thanks for the comments that we should have made better explanation on the issue. The haplotyping assay is widely used in BRCA founder mutation identification with their age determined, for example, the age of the three BRCA founder mutations (BRCA1 c. 68_69del and c.5266dup and BRCA2 c.5946del in Ashkenazi Jews population. Haplotyping assay ensures that the variant is inherited by referring the targeted variant with other neighboring variants together as a block. In our study, we directly used the haplotyping assay-determined BRCA founder mutations from literature but we didn't perform haplotyping testing ourselves. The principle of using haplotyping to estimate age of genetic variants was developed decades ago. Here, we cite the description in a paper (PMID: 17902052) outlining how it works: "this method estimates the number of generations between the present time and the most recent common ancestor of all the mutation-bearing individuals as a function of the observed haplotypes, the recombination fraction between the mutation and the marker loci (and markers themselves), the rate of mutation at the microsatellite and SNP marker loci, and the allele frequencies of the marker loci. …. The 95% confidence interval for estimated allele age was calculated …. from the maximum likelihood estimate.". Our study observed that the known human BRCA founder mutations were arisen 3,225 to 140 years ago.
In the revision, the following sentences are included in "Dated BRCA founder PLPs and ethnicspecific distribution of BRCA PLPs" under Results: Many BRCA PLPs have been determined as BRCA founder variants in different ethnic population, their arisen times were determined by haplotyping analysis. For example, the three BRCA founder mutations (BRCA1 c. 68_69del and c.5266dup and BRCA2 c.5946del in Ashkenazi Jews population were determined being arisen 1,720, 1,800, and 580 years ago respectively (Table 3).

Question
No consideration is given to the possibility that mutations may arise at multiple times in multiple independent populations. Answer Thanks for the valuable comments. It is certainly possible that some BRCA PLP could be arisen by chance at the same genetic locations in different populations.
In the revision, the following sentences are added to indicate the possibility under Discussion: Another factor related with ethnic-specific PLPs may be that the same BRCA PLP could arose by chance in different populations at different times. For example, the BRCA1 68-69del founder mutation in Ashikenazi Jews is also present in other populations at very low frequencies, and BRCA1 3607C>T shared between the Slovania case dated 4971±450 ago and Dominica case dated 921±500 ago (Table 2). However, this unlikely played a major role in contributing to human BRCA PLPs.

Question
Also, no consideration is given to the role of pleiotropy in driving selection of variants whose effects mostly occur in post-reproductive age. Answer This is certainly possible. Geoge William's pleiotrophy theory indeed provide another anchor to look at the origin of evolution selection for human BRCA PLPs, particularly for these selected in post-reproductive age.
In the revision, we added the following sentences to reflect this factor under Discussion: In addition, pleiotrophy effects may also exist that variation in none-BRCA genes may contribute to the selection of BRCA PLPs, particularilly for these selected in the post-reproductive age (Williams. 1957). This possibility is interesting as BRCA PLP mainly cause high cancer risk after reproduction age.

Question
The section on "Data from previous studies" seems dispensable and can be summarized in the introduction. Answer Fully agree. In the revision, the entire section is relocated to the introduction part.

Question
Please define the criteria ('interpretation'; 'review status'; how to deal with variants that have multiple interpretation, etc) used to extract the data from ClinVar. This is extremely important as it is the basis of the analysis. State the date and version (if applicable) when the data was extracted. Answer This is certainly an important issue for the study. The BRCA PLP data used in our study (Supplementary Table 2) was originated from ClinVar. We only used the BRCA PLP with single classification as P or LP. These with conflict interpretation, multiple interpretation, and uncertain classification were all excluded from our study to ensure the reliability of the analysis.
In the revision, the following sentence is added to clarify the issue: The variants with conflict classifications were excluded from our study to ensure the reliability of the analysis.

Question
Please define what is being considered 'Pathogenic' and 'Likely Pathogenic' (ClinVar? ENIGMA consortium data?). Answer ClinVar classification of 'Pathogenic' and 'Likely Pathogenic' is based on a consortium activity, involving the evidence from ENIGMA, expert panel, ACMG guidelines, Clinical testing labs, etc. ENIGMA is one of the major sources but not the only one. Thank you for submitting your revised manuscript entitled "Human BRCA pathogenic variants were originated during recent human history" to Life Science Alliance. The manuscript has been seen by two of the original reviewers whose comments are appended below. While the reviewers continue to be overall positive about the work in terms of its suitability for Life Science Alliance, some important issues remain. As you will note from the reviewers' comments below, Reviewer 3 still has some remaining concerns that need to be addressed before resubmission. In the light of this, we encourage you to make a more careful update of the references and argument better why you think there is a high prevalence of PLPs. Also toning down the conclusions would be beneficial to avoid being misleading.
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Reviewer #3 (Comments to the Authors (Required)): While the paper was improved in adding additional information about the methods. However, many of the reviewers questions were not satisfactorily answered.
In particular, the question about whether these species that carry inactivating mutations have no functional BRCA was not answered.
Many citations are not directly related to the statements.
The treatment of variants that are considered pathogenic is not reassuring. The premise that the frequency of BRCA PLPs are high is, in my view, incorrect, and no evidence is shown to support the numbers. In fact, only in high risk cohorts the prevalence approaches the numbers cited (and implicitly stated to correspond to unselected cohorts).
The approach to compare large population datasets with sequence of homologs as presented seems to lack rigor and the conclusions are not compelling. The authors continue to make broad-stroke conclusions with references that are clearly inappropriate. The passage referring to the Tasmanian Devil transmissible face tumor (which has been well characterized and is a unrelated tumor) with a reference from thirty years before the cloning of BRCA1, is just one example.

Reply to editor's comments
Reviewer 3 still has some remaining concerns that need to be addressed before resubmission. In the light of this, we encourage you to 1. make a more careful update of the references Answer We have double-checked all references, replaced the less relevant ones with more relevant ones, and added new ones to the topics as marked in the revision.
2. argument better why you think there is a high prevalence of PLPs.

Answer
We have increased the scope in Discussion to further address the issue.
3. Also toning down the conclusions would be beneficial to avoid being misleading. Answer Thanks for indicting the issue. We have modified the wording for the conclusion to avoid overstate the data.

Reply to reviewer #3 comments
Question whether these species that carry inactivating mutations have no functional BRCA was not answered.

Answer
There are rich evidence showing the consequences caused by the inactivating mutations in non-human BRCA, as well reflected by the studies in Brca1 knockout mouse studies performed in the past 3 decades by different laboratories around the world. The conventional Brca1 knockout in mice caused post-implantation development (1), embryonic cellular proliferation (2), embryonic lethal (3), neuroepithelial abnormalities (4); and the conditional Brca1 knockout in mice led to gamma-irradiation hypersensitivity and genetic instability (5), abnormal T cell development (6, 7), and tumorigenesis (8,9).
In the revision, the following sentences and references are included to address the issue: Extensive animal model studies have provided rich evidence in showing the pathogenic consequences by BRCA PLP in non-human species, especially from mouse studies. Brca1 knockout-mice showed abnormal post-implantation development and embryonic proliferation (Liu et al. 1996, Hakem et al. 1996, embryonic lethality (Ludwig et al. 1997), neuroepithelial abnormalities (Gowen et al. 1996), irradiation hypersensitivity and genetic instability (Shen et al. 1998), abnormal T cell development (Mak et al. 2000;Xu et al. 2001), and tumorigenesis (Xu et al. 1999;Brodie et al. 2001). Without the presence of other mutated genes such as the mutated TP53, however, the mutated Brca1 alone is not sufficient to directly cause cancer (Xu et al.2001).

Question
Many citations are not directly related to the statements. Answer In this 2 nd revision, we have checked each reference to ensure their relevance to the related topics. For these less relevant, we either remove or replace them with more relevant references, and new references are also included to related topics as marked in the revision.

Question
The treatment of variants that are considered pathogenic is not reassuring.

Answer
We addressed this issue in our previous revision: "The BRCA PLP data used in our study (Supplementary Table 2) was originated from ClinVar. We only used the BRCA PLP with single classification as P or LP. These with conflict interpretation, multiple interpretation, and uncertain classification were all excluded from our study to ensure the reliability of the analysis". Among all BRCA databases, the BRCA variation and classification data from ClinVar is currently the golden standard in guiding clinical interpretation of BRCA test results around the world. In terms of quality and quantity, we believe the use of the BRCA PLPs from ClinVar is the best assurance to ensure that the BRCA variants used in our study are pathogenic and likely pathogenic, and the results really reflect the origins of human BRCA PLP, rather than BRCA Introduction …..Data from our study provide evidence to show that human BRCA PLP was highly unlikely originated from cross-species evolution conservation but most likely arisen by positive selection in the humans, during recent human history after migrating out-of-Africa and expansion of human population.

Discussion
….. Based on these observations, we propose that human BRCA PLP was most likely arisen in recent human history, possibly within a few thousand years, after the latest human out-of-Africa migration and settlement at different global destinations. We consider that the positive selection on human BRCA could play a major role, and the population expansion could further increase the prevalence of human BRCA PLP in modern human population (Figure 4).

Question
The passage referring to the Tasmanian Devil transmissible face tumor (which has been well characterized and is a unrelated tumor) with a reference from thirty years before the cloning of BRCA1, is just one example. Answer Fully agree. It has been determined that the disease is related with the fusion of chromosome 1 and X, but not related with BRCA mutations (Taylor et al, 2017). We have added three more recent references for the issue: In the revision, the section has been revised as the following: For example, Tasmanian devil shared 20 human BRCA PLP variants. Tasmanian has high risk of developing facial cancer, which is related with the fusion of chromosome 1 and X (Hawkins et al, 2006;Murchison et al, 2010;Bender et al. 2014;Taylor et al. 2017), but no evidence to show that the cancer is related with the BRCA PLP variants shared with human.