Strong functional data for pathogenicity or neutrality classify BRCA2 DNA-binding-domain variants of uncertain significance

Am J Hum Genet. 2021 Mar 4;108(3):458-468. doi: 10.1016/j.ajhg.2021.02.005. Epub 2021 Feb 19.


Determination of the clinical relevance of rare germline variants of uncertain significance (VUSs) in the BRCA2 cancer predisposition gene remains a challenge as a result of limited availability of data for use in classification models. However, laboratory-based functional data derived from validated functional assays of known sensitivity and specificity may influence the interpretation of VUSs. We evaluated 252 missense VUSs from the BRCA2 DNA-binding domain by using a homology-directed DNA repair (HDR) assay and identified 90 as non-functional and 162 as functional. The functional assay results were integrated with other available data sources into an ACMG/AMP rules-based classification framework used by a hereditary cancer testing laboratory. Of the 186 missense variants observed by the testing laboratory, 154 were classified as VUSs without functional data. However, after applying protein functional data, 86% (132/154) of the VUSs were reclassified as either likely pathogenic/pathogenic (39/132) or likely benign/benign (93/132), which impacted testing results for 1,900 individuals. These results indicate that validated functional assay data can have a substantial impact on VUS classification and associated clinical management for many individuals with inherited alterations in BRCA2.

Keywords: ACMG/AMP; BRCA2; breast cancer; functional assay; predisposition gene; variant of uncertain significance.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • BRCA2 Protein / genetics*
  • Breast Neoplasms / genetics*
  • Breast Neoplasms / pathology
  • Female
  • Genetic Predisposition to Disease*
  • Genetic Variation / genetics
  • Humans
  • Mutation, Missense / genetics
  • Recombinational DNA Repair / genetics*
  • Structure-Activity Relationship


  • BRCA2 Protein
  • BRCA2 protein, human