A High-Throughput Functional Complementation Assay for Classification of BRCA1 Missense Variants
Published version of a preprint
Abstract
Mutations in BRCA1 and BRCA2 account for the majority of hereditary breast and ovarian cancers, and therefore sequence analysis of both genes is routinely conducted in patients with early-onset breast cancer. Besides mutations that clearly abolish protein function or are known to increase cancer risk, a large number of sequence variants of uncertain significance (VUS) have been identified. Although several functional assays for BRCA1 VUSs have been described, thus far it has not been possible to conduct a high-throughput analysis in the context of the full-length protein. We have developed a relatively fast and easy cDNA-based functional assay to classify BRCA1 VUSs based on their ability to functionally complement BRCA1-deficient mouse embryonic stem cells. Using this assay, we have analyzed 74 unclassified BRCA1 missense mutants for which all predicted pathogenic variants are confined to the BRCA1 RING and BRCT domains. Significance: BRCA1 VUSs are frequently found in patients with hereditary breast or ovarian cancer and present a serious problem for clinical geneticists. This article describes the generation, validation, and application of a reliable high-throughput assay for the functional classification of BRCA1 sequence variants of uncertain significance. Cancer Discov; 3(10); 1142–55. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 1083
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BRCT domains are frequently affected by clinically important mutations, and most of these mutations disrupt the BRCT domain's phosphopeptide-binding surface.
“BRCT domains are frequently targeted by many clinically important mutations and most of these mutations disrupt the binding surface of the BRCT domains to phosphorylated peptides (Bouwman et al., 2013; Glover, 2006).”
From The BRCA1-Interacting Protein Abraxas Is Required for Genomic Stability and Tumor Suppression (2014) · cites this paper as Bouwman et al., 2013
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References 50 · Cited by 140
- Authors
- Bouwman, P., van der Gulden, H., van der Heijden, I., Drost, R., Klijn, C. N., Prasetyanti, P., Pieterse, M., Wientjens, E., Seibler, J., Hogervorst, F. B. L., & Jonkers, J.
- Affiliations
- The Netherlands Cancer Institute (NL)
- Authors' Affiliations: 1Division of Molecular Pathology and Cancer Genomics Centre and 2Department of Pathology, The Netherlands Cancer Institute Amsterdam, The Netherlands; and 3TaconicArtemis GmbH, Cologne, Germany
- ORCID
- Venue
- Cancer Discovery
- Published
- 2013-10-01
- Volume
- 3
- Issue
- 10
- Pages
- 1142-1155
- ISSN
- 2159-8274 (ISSN-L)
- 2159-8290
- OpenAlex
- W2162502512
- Cited by
- 140
- References
- 50
- Citations by year
- 2026: 8 · 2025: 5 · 2024: 10 · 2023: 8 · 2022: 3 · 2021: 24 · 2020: 13 · 2019: 10 · 2018: 10 · 2017: 7 · 2016: 13 · 2015: 18 · 2014: 8 · 2013: 3
- FWCI
- 9.29
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Cancer Discovery (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › BRCA gene mutations in cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
- Health Sciences › Medicine › Pathology and Forensic Medicine › Genetic factors in colorectal cancer
- Keywords
- BRCA1, missense mutations, variants of uncertain significance, high-throughput functional assay, RING domain, BRCT domain, hereditary breast and ovarian cancer, mouse embryonic stem cells, variant pathogenicity classification
- MeSH terms
- Poly(ADP-ribose) Polymerase Inhibitors, Amino Acid Sequence, Animals, Antineoplastic Agents, Breast Neoplasms, Cisplatin, DNA Repair, Drug Screening Assays, Antitumor, Female, Genetic Complementation Test, Humans, Molecular Sequence Data, Ovarian Neoplasms, Phthalazines, Piperazines, Genetic Variation, Mutagenesis, Site-Directed, Mice, Knockout, BRCA1 Protein, Genes, BRCA1, Mutation, Missense, Cell Proliferation, Mice, Embryonic Stem Cells, RING Finger Domains, High-Throughput Screening Assays, Homologous Recombination
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