A guide for functional analysis of BRCA1 variants of uncertain significance

Abstract

Germline mutations in the tumor suppressor gene BRCA1 confer an estimated lifetime risk of 56-80% for breast cancer and 15-60% for ovarian cancer. Since the mid 1990s when BRCA1 was identified, genetic testing has revealed over 1,500 unique germline variants. However, for a significant number of these variants, the effect on protein function is unknown making it difficult to infer the consequences on risks of breast and ovarian cancers. Thus, many individuals undergoing genetic testing for BRCA1 mutations receive test results reporting a variant of uncertain clinical significance (VUS), leading to issues in risk assessment, counseling, and preventive care. Here, we describe functional assays for BRCA1 to directly or indirectly assess the impact of a variant on protein conformation or function and how these results can be used to complement genetic data to classify a VUS as to its clinical significance. Importantly, these methods may provide a framework for genome-wide pathogenicity assignment.

Figure 1. The landscape of BRCA1 as assessed by functional assays

A. Diagram of BRCA1 protein. RING domain (aa 8–96), NES, nuclear export signal (aa 22–30 and aa 81–99), NLS, nuclear localization signal (aa 503–508 and 606–615); AAD, auxiliary activation region (aa 1396–1559); TAD, transcriptional activation domain (aa 1560–1863), BRCT, BRCA1 C-terminal domains (aa 1646–1736 and 1760–1855). B. Sequences used by the different functional assays are indicated in grey. Limits of the aa sequence interrogated by the assays are indicated on the right. CA, centrosome amplification; ESC, embryonic stem cell; FL, full length; HDR, homology-directed recombination; RRR, restoration of radiation resistance; SCP, small colony phenotype; TA, transcription activation; YLP, yeast localization phenotype. Black box, full length sequence is used but assessment restricted to this region only.

Figure 2. Structural domains in BRCA1

A. Ring domain of BRCA1. Ribbon representation of the BRCA1-BARD1 heterodimer complex (pdb: 1jm7). BARD1 RING finger is colored in light grey. Black and dark grey indicates BRCA1 residues involved in the interaction to BARD1 or to UbcH5c, respectively. B. BRCT domains. Ribbon representation of the BRCA1 BRCT domains (pdb: 1jnx) and the interaction with phosphopeptides from BACH1 (pdb: 1t15), CtIP (pdb: 1y98), and ACC1 (pdb: 3coj). Residues common to the binding to all three phosphorylated peptides are shown in black (Ser1655, Gly1656, Arg1699, Leu1701, Lys1702, Phe1704, Asn1774, Met1775, and Leu1839). Variants Arg1699Trp, Arg1699Gln, Met1775Arg and Met1775Lys have been classified as IARC Class 5 (Lindor et al. 2012). C. Ribbon representation of the BRCA1 BRCT domains. BRCT residues corresponding to variants that have already been classified listed in Table 2 are shown in black.