The functional impact of variants of uncertain significance in BRCA2

Abstract

Purpose: Genetic testing has uncovered large numbers of variants in the BRCA2 gene for which the clinical significance is unclear. Cancer risk prediction of these variants of uncertain significance (VUS) can be improved by reliable assessment of the extent of impairment of the tumor suppressor function(s) of BRCA2.

Methods: Here, we evaluated the performance of the mouse embryonic stem cell (mESC)-based functional assay on an extensive set of BRCA2 missense variants.

Results: Whereas all 20 nonpathogenic (class 1/2) variants were able to complement the cell lethal phenotype induced by loss of endogenous mouse Brca2, only 1 out of 15 pathogenic (class 4/5) variants (p.Gly2609Asp) was able to do so. However, in this variant the major tumor suppressive activity of BRCA2, i.e., homology directed repair (HDR), was severely abrogated. Among 43 evaluated VUS (class 3), 7 were unable to complement the lethal phenotype of mouse Brca2 loss while 7 other variants displayed a more severe reduction of HDR activity than observed for class 1/ 2 variants.

Conclusion: The mESC-based BRCA2 functional assay can reliably determine the functional impact of VUS, distinguish between pathogenic and nonpathogenic variants, and may contribute to improved cancer risk estimation for BRCA2 VUS carriers.

Keywords: BRCA2; Functional assays; Homology directed repair.; Variants of uncertain significance.

Fig. 1

Position of BRCA2 variants. Schematic representation of the position of class 1/2 variants (green dots), class 4/5 variants (red dots), and BRCA2 variants of uncertain significance (VUS) (gray dots) in the BRCA2 gene. Ta transcriptional activation domain, P phosphorylation site, H helical domain, DBD DNA binding domain, OB oligonucleotide binding fold, T Tower domain, TR2 C-terminal RAD51 binding site, NLS nuclear localization signal, aa amino acids^,,

Fig. 2

Representative images of complementation phenotypes and controls. Brca2^-/loxPPim1^DR-GFP/WT cells expressing WT BRCA2 or BRCA2 variants were transfected with a Cre-GFP expression plasmid to induce loss of the conditional Brca2 allele and restore the Hprt gene. Upon Cre-recombinase expression cells become Brca2 deficient, which is lethal unless complemented by the expression of a (partially) functional BRCA2 variant. Untransfected cells that still contain the conditional Brca2 allele lack Hprt expression and will subsequently not survive HAT selection as shown in the –Cre-recombinase control. Thirteen days post Cre-GFP transfection culture dishes were stained with methylene blue

Fig. 3

Homology directed repair (HDR) activity of BRCA2 variants relative to wild type (WT) BRCA2 activity. GFP signal was measured in I-Sce1 expressing cells 2 days post transfection by flow cytometry for (a) classified BRCA2 missense variants and (b) BRCA2 variants of uncertain significance (VUS). Relative HDR activity is expressed as the ratio between the percentage of GFP-positive cells observed in BRCA2 variant expressing cells and the percentage of GFP-positive cells in WT BRCA2-expressing cells (green line). Brca2 (green bar) represents the conditional Brca2^-/loxPPim1^DR-GFP/WT cell line expressing endogenous Brca2. The upper gray box represents the HDR range of class 1/2 BRCA2 variants. The lower gray box represents the HDR range associated with >95% probability of pathogenicity as reported by Guidugli et al. (see also Fig. S6b). The black bars correspond to the mean HDR activity and error bars indicate the SD of at least six independent GFP measurements per variant as represented by the dots (purple = class 1/2, red = class 4/5)