Functional assays for classification of BRCA2 variants of uncertain significance

Abstract

The assessment of the influence of many rare BRCA2 missense mutations on cancer risk has proved difficult. A multifactorial likelihood model that predicts the odds of cancer causality for missense variants is effective, but is limited by the availability of family data. As an alternative, we developed functional assays that measure the influence of missense mutations on the ability of BRCA2 to repair DNA damage by homologous recombination and to control centriole amplification. We evaluated 22 missense mutations from the BRCA2 DNA binding domain (DBD) that were identified in multiple breast cancer families using these assays and compared the results with those from the likelihood model. Thirteen variants inactivated BRCA2 function in at least one assay; two others truncated BRCA2 by aberrant splicing; and seven had no effect on BRCA2 function. Of 10 variants with odds in favor of causality in the likelihood model of 50:1 or more and a posterior probability of pathogenicity of 0.99, eight inactivated BRCA2 function and the other two caused splicing defects. Four variants and four controls displaying odds in favor of neutrality of 50:1 and posterior probabilities of pathogenicity of at least 1 x 10(-3) had no effect on function in either assay. The strong correlation between the functional assays and likelihood model data suggests that these functional assays are an excellent method for identifying inactivating missense mutations in the BRCA2 DBD and that the assays may be a useful addition to models that predict the likelihood of cancer in carriers of missense mutations.

Figure 1

Expression of BRCA2 missense mutant constructs. A, BRCA2 gene showing the location of the various missense variants in the DBD. B, BRCA2 wild-type and mutant proteins are equivalently expressed following transient transfection of constructs into HEK293T cells. Protein levels were determined by immunoprecipitation (IP)-immunoblotting of FLAG-tagged BRCA2 full-length mutants. C, the mutant BRCA2 proteins retain the ability to form a complex with Rad51. Rad51 protein immunoprecipitated with FLAG-tagged BRCA2 wild-type and mutant proteins was visualized by Western blotting with anti-Rad51 antibody.

Figure 2

Epidemiologic and genetic evaluation of BRCA2 VUS. A, pedigree data for the R3052W BRCA2 variant. Type of cancer diagnosis and age of onset is shown. Quarter shaded circles, a diagnosis of breast cancer. +, presence of the R3052W variant. B, RT-PCR studies of mRNA isolated from the MCF-7 cell line and lymphoblastoid cell lines derived from patients harboring BRCA2 missense mutations using primers in the flanking exons. The PCR product from Flag-tagged wild-type BRCA2 was included in the last lane. C and D, purified alternatively sized RT-PCR products from B, with sequence analysis of RT-PCR products from R2659T cDNA (C) and E2663V cDNA (D) showing exon skipping.

Figure 3

The influence of BRCA2 variants on homology-directed DNA damage repair. A, scatter plot showing fluorescence-activated cell sorting analysis to detect GFP-positive V-C8 cells following repair of I-Sce1–induced DNA breaks. GFP-expressing cells that are indicative of homology-directed repair are located above the diagonal line in each scatter plot. B, quantization of homology-directed repair in V-C8 cells in response to transiently transfected wild-type and mutant forms of BRCA2. The proportion of GFP-expressing cells for each construct relative to vector control is shown. All values were normalized for transfection efficiency. Dotted lines, upper and lower boundaries of SE for inactivating and neutral variants. Columns, mean from three experiments; bars, SE.

Figure 4

The influence of BRCA2 variants on centriole amplification. A, examples of centriole/centrosome phenotypes in different BRCA2 missense mutants. Wild-type BRCA2 and neutral variant K2729N have the normal complement of either two or four centrioles. Deleterious mutants L2647P and T2722R give rise to supernumerary centrioles. Red, centrin-2; blue, 4′,6-diamidino-2-phenylindole (DAPI). B, graphical representation of centriole amplification induced by BRCA2 missense variants. The percentage of cells with more than four centrioles per cell was plotted for each mutant. Dotted lines, upper and lower boundaries of SE for inactivating and neutral variants. Columns, values from two experiments; bars, SE. C, rescue of centrosome amplification by BRCA2 missense mutants in the BRCA2-null V-C8 cell line. The percentage of cells with more than four centrioles per cell was plotted for a selection of mutants. Columns, mean from two experiments; bars, SE.