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Functional evaluation of BRCA1/2 variants of unknown significance with homologous recombination assay and integrative in silico prediction model

Journal of Human Genetics volume 68, pages 849–857 (2023)Cite this article

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Abstract

Numerous variants of unknown significance (VUSs) exist in hereditary breast and ovarian cancers. Although multiple methods have been developed to assess the significance of BRCA1/2 variants, functional discrepancies among these approaches remain. Therefore, a comprehensive functional evaluation system for these variants should be established. We performed conventional homologous recombination (HR) assays for 50 BRCA1 and 108 BRCA2 VUSs and complementarily predicted VUSs using a statistical logistic regression prediction model that integrated six in silico functional prediction tools. BRCA1/2 VUSs were classified according to the results of the integrative in vitro and in silico analyses. Using HR assays, we identified 10 BRCA1 and 4 BRCA2 VUSs as low-functional pathogenic variants. For in silico prediction, the statistical prediction model showed high accuracy for both BRCA1 and BRCA2 compared with each in silico prediction tool individually and predicted nine BRCA1 and seven BRCA2 variants to be pathogenic. Integrative functional evaluation in this study and the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines strongly suggested that seven BRCA1 variants (p.Glu272Gly, p.Lys1095Glu, p.Val1653Leu, p.Thr1681Pro, p.Phe1761Val, p.Thr1773Ile, and p.Gly1803Ser) and four BRCA2 variants (p.Trp31Gly, p.Ser2616Phe, p.Tyr2660Cys, and p.Leu2792Arg) were pathogenic. This study demonstrates that integrative evaluation using conventional HR assays and optimized in silico prediction comprehensively classified the significance of BRCA VUSs for future clinical applications.

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Acknowledgements

JOHBOC Registration Committee [Committee]: Tadashi Nomizu (Hoshi General Hospital), Akihiro Sakurai (Sapporo Medical University), Megumi Ohkawa (St. Luke’s International Hospital), Junko Yotsumoto (International University of Health and Welfare), Kumamaru Hiraku (The University of Tokyo), [Secretariat of JOHBOC]: Shiro Yokoyama, Miyuki Shimoda. We would like to thank all the members of the Department of Oncology, Juntendo University for their valuable discussions.

Funding

This work was supported by AMED under Grant Number JP19kk0305012 (YoM), JSPS KAKENHI Grant Number JP19H03497 (YoM), JP19H01806 (MM) and JP20H04333 (SS).

Author information

Author notes

  1. These authors contributed equally: Qianqian Guo, Shuting Ji.

Authors and Affiliations

  1. Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan

    Qianqian Guo, Shuting Ji, Zeyu Xu, Yoshio Miki & Shigeaki Sunada

  2. Graduate School of Public Health, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605, Japan

    Kazuma Takeuchi, Asuka Suzuki & Masaaki Matsuura

  3. Department of Information Sciences, Tokyo University of Science, 2641 Yamazaki, Noda City, Chiba, 278-8510, Japan

    Wataru Urasaki

  4. Laboratory for Genotyping Development, RIKEN Center for Integrative Medical Sciences, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa, 230-0045, Japan

    Yusuke Iwasaki & Yukihide Momozawa

  5. Department of Genetic Diagnosis, The Cancer Institute, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo, 135-8550, Japan

    Hiroko Saito & Yoshio Miki

  6. Department of Clinical Genetics, Juntendo University, Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan

    Masami Arai

  7. Division of Breast Surgical Oncology, Department of Surgery, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, 142-8555, Japan

    Seigo Nakamura

  8. Department of Cancer Biology; Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryomachi, Aoba-ku, Sendai, 980-8575, Japan

    Natsuko Chiba

  9. Juntendo Advanced Research Institute for Health Science, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan

    Shigeaki Sunada

  10. Department of Oncology, School of Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan

    Shigeaki Sunada

Authors
  1. Qianqian Guo
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  2. Shuting Ji
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  3. Kazuma Takeuchi
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Contributions

Conceptualization: YoM, MM, SS; Data curation: QG, SJ, KT, WU, AS, YI, HS, ZX, YuM, YoM, M.M., SS; Formal analysis: MM; Funding acquisition: YoM, MM, SS; Investigation: QG, SJ, KT, WU; Methodology: MM, SS; Project administration: YoM, MM, SS; Resources: QG, SJ, KT, WU, HS, ZX, MA, SN; Software: KT, WU, YI, MM; Supervision: YuM, NC, YoM, MM, SS; Validation: QG, SJ, KT, WU; Visualization: MM, SS; Writing-original draft: QG, SS; Writing-review & editing: YuM, YoM, MM, SS.

Corresponding authors

Correspondence to Yoshio Miki, Masaaki Matsuura or Shigeaki Sunada.

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The authors declare no competing interests.

Ethics Statement

Approval of the research protocol by an Institutional Reviewer Board: JOHBOC, Tokyo Medical and Dental University, and Teikyo University Ethics Committee approved the study and the use of concealed variant information from clinical databases.—Informed Consent: N/A.—Registry and the Registration No. of the study/trial: N/A.—Animal Studies: N/A.

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Guo, Q., Ji, S., Takeuchi, K. et al. Functional evaluation of BRCA1/2 variants of unknown significance with homologous recombination assay and integrative in silico prediction model. J Hum Genet 68, 849–857 (2023). https://doi.org/10.1038/s10038-023-01194-6

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