PONE-D-24-36034Prevalence and spectrum of germline BRCA1 and BRCA2 mutations in multiethnic cohort of breast cancer patients in Brunei DarussalamPLOS ONE

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We sincerely acknowledge all the doctors, nurses and staff from TBCC for their cooperation and also to CRM and First Base Laboratories Sdn Bhd for their contribution to the study. We would like to thank our undergraduate students; Jacinda Lim Xin Yan and Hon Kar Yee; who have taken a small project under this study. Finally, we would like to express our gratitude to all patients who have participated in our research study.  MRWHAH received a grant from Universiti Brunei Darussalam  to fund the initial part of this study [UBD/PNC2/2/RG/1(186))] 

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1:  This manuscript investigated the germline BRCA1/2 mutations among Brunei breast cancer patients and aimed to characterize the genetic contribution to breast cancer based on the mutational profiles. The authors used both NGS and Sanger sequencing to characterize the BRCA genes using peripheral blood samples. The variants were carefully examined and annotated and the association between genetic variation and demographical/clinical features of the patients was then examined. The manuscript was well written, and the supporting evidence and information was very well documented. Although the conclusion was based on small sample size, I believe this manuscript would be of interests to the scientific community.

Major comments:

- For the variants reported, could the author also report whether these were identified with Sanger or NGS, and comment on the sensitivity of mutation detection between the two platforms (within this study and/or in general)?

- Over 30% of novel variants seem a lot. Can the authors comment on the QC applied to the variant calling? Maybe adding the quality metrics of from the callers would help interpret the results.

- Copy number variation of BRCA has been reported. Did authors plan to look into that as well?

- Line 243: Was the conclusion that these variants have been reported based on the population frequency database only? Were they also found in other studies based on similar population in SEA?

- Line 309: citation needed

Minor comments:

- Line 235: space between “29BRCA1” and “66BRCA2”

Reviewer #2:  Dear Dr. Matusin and collaborators,

The manuscript titled “Prevalence and Spectrum of Germline BRCA1 and BRCA2 Mutations in a Multiethnic Cohort of Breast Cancer Patients in Brunei Darussalam” offers valuable insights into an understudied population. However, it needs substantial revisions before it can be considered for publication. I hope the following comments will be helpful.

1. To help ensure that the ideas and concepts in the manuscript are easily understood, it would be beneficial to conduct a thorough review of the English grammar. This will enhance clarity and make the content more approachable for readers.

2. Introduction:

a) The last paragraph on page 4 requires clearer conceptual explanation. It’s important to understand that finding recurrent variants of clinical significance in African or European populations, as well as in other Asian populations, does not necessarily mean that these variants aren't originally from Africa or Europe. Population structures can shift due to various factors, including social influences and significant events like war, immigration, and emigration. As a result, the frequencies of genetic variants—whether benign or pathogenic—can also change. Thus, haplotype analysis is crucial in determining the true origin of these recurrent variants.

b) In the first paragraph on page 5, the objective was to evaluate potential associations between demographic characteristics and the profiles of BRCA1 and BRCA2. However, it would be helpful to present this data more clearly to improve understanding and interpretation, as the data was not provided.

3. Materials and Methods

a) In the section “study population”, it was noted that terminally ill patients were excluded from the study. The reason for excluding these patients was not specified.

b) In the section titled “Multiplex Ligation-Dependent Probe Amplification (MLPA)” on page 9, it states that only the samples from batch 1 were subjected to MLPA analysis. However, it does not explain why samples from the other batches were not included in this screening, especially since Next-Generation Sequencing (NGS) has significant limitations in detecting large genomic rearrangements.

4. Results

a) The "Personal history of cancer" section in Table 1 currently provides details for only 10% of the studied cases. It would be helpful to gather and include information about the remaining 90% to provide a more comprehensive overview.

b) In Table 1, the row labeled "Number of 2nd relatives with breast cancer" indicates a sum greater than 100%. It may be beneficial to review this data for accuracy.

c) The first paragraph on page 12 needs a thorough review, as clarifying its content could enhance comprehension for readers.

d) To enhance clarity in the legends for Figure S2, it would be helpful to provide descriptions for the acronyms used. This addition could improve understanding for readers.

e) In the S1 Table, I recommend reviewing the ACMG criteria codes, as there appears to be an error in the application of the supporting codes. These variants have strong evidence of pathogenicity and have been reported previously.

f) In Table S2, it would be beneficial to revisit the ACMG codes used and the classification of the variants for clarity. Additionally, I would appreciate insights on the decision why not applying the BP1 code.

g) I recommend revising the title of the S3 table to enhance clarity and engagingness.

h) In Table S5, I recommend replacing the year of diagnosis or death with the patient's age. This adjustment would provide more clinically relevant information.

i) The section “Prevalence of pathogenic mutations and VUS” on page 13 requires significant revision.

5. Discussion

a) In the second paragraph on page 18, it is noted that two carriers of the BRCA2: c.5164_5165delAG are related. Providing more information about the total number of related and unrelated patients in the cohort would enhance the clarity of the discussion. This additional context could help in better understanding the study's findings.

b) The first paragraph on page 19 mentions that a Malay patient carried two founder mutations in the BRCA2 gene: one from French-Canadian origin and the other from Colombian origin. While family history provides compelling evidence, a valid conclusion regarding the potential origin of the BRCA2:c.3170_3174delAGAAA and BRCA2:c.1763_1766delATAAA mutations can only be reached through haplotype analysis.

c) I believe it would be beneficial to rework the last paragraph on page 19 and the first paragraph on page 20 for improved clarity and impact.

d) Page 21 requires rewriting and a review of the concept. The manuscript does not describe the functional analysis of the BRCA2 mutation: c.8524C>T. Relying solely on family history is insufficient.

6. Conclusion

a) I suggest revisiting the conclusions to enhance their clarity and accuracy. While it is noteworthy that only one BRCA2 founder mutation was identified in individuals of Chinese descent, this does not necessarily indicate that the mutation found in the Brunei patient is the same as this particular founder mutation. Furthermore, it’s valuable to acknowledge that other BRCA2 founder mutations from European and African populations were also observed in the Brunei population studied. To further improve the conclusions, the authors could explore conducting a haplotype analysis or offer a more straightforward interpretation of the current findings.

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Reviewer #1: No

Reviewer #2: No

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Prevalence and spectrum of germline BRCA1 and BRCA2 mutations in multiethnic cohort of breast cancer patients in Brunei Darussalam

PONE-D-24-36034R1

Dear Dr. Haji Abdul Hamid,

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