A high-throughput functional complementation assay for classification of BRCA1 missense variants

Cancer Discov. 2013 Oct;3(10):1142-55. doi: 10.1158/2159-8290.CD-13-0094. Epub 2013 Jul 18.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antineoplastic Agents / pharmacology
  • BRCA1 Protein / chemistry
  • BRCA1 Protein / genetics*
  • Breast Neoplasms / genetics*
  • Cell Proliferation
  • Cisplatin / pharmacology
  • DNA Repair
  • Drug Screening Assays, Antitumor
  • Embryonic Stem Cells / physiology
  • Female
  • Genes, BRCA1*
  • Genetic Complementation Test*
  • Genetic Variation
  • High-Throughput Screening Assays / methods*
  • Homologous Recombination
  • Humans
  • Mice
  • Mice, Knockout
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation, Missense*
  • Ovarian Neoplasms / genetics*
  • Phthalazines / pharmacology
  • Piperazines / pharmacology
  • Poly(ADP-ribose) Polymerase Inhibitors
  • RING Finger Domains


  • Antineoplastic Agents
  • BRCA1 Protein
  • BRCA1 protein, human
  • Phthalazines
  • Piperazines
  • Poly(ADP-ribose) Polymerase Inhibitors
  • Cisplatin
  • olaparib