Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis

Abstract

Germ line inactivating mutations in BRCA1 confer susceptibility for breast and ovarian cancer. However, the relevance of the many missense changes in the gene for which the effect on protein function is unknown remains unclear. Determination of which variants are causally associated with cancer is important for assessment of individual risk. We used a functional assay that measures the transactivation activity of BRCA1 in combination with analysis of protein modeling based on the structure of BRCA1 BRCT domains. In addition, the information generated was interpreted in light of genetic data. We determined the predicted cancer association of 22 BRCA1 variants and verified that the common polymorphism S1613G has no effect on BRCA1 function, even when combined with other rare variants. We estimated the specificity and sensitivity of the assay, and by meta-analysis of 47 variants, we show that variants with <45% of wild-type activity can be classified as deleterious whereas variants with >50% can be classified as neutral. In conclusion, we did functional and structure-based analyses on a large series of BRCA1 missense variants and defined a tentative threshold activity for the classification missense variants. By interpreting the validated functional data in light of additional clinical and structural evidence, we conclude that it is possible to classify all missense variants in the BRCA1 COOH-terminal region. These results bring functional assays for BRCA1 closer to clinical applicability.

Figure 1

Location of variants in BRCA1. Location of variants (closed black triangles) and of negative (open triangles, underlined) and positive (closed gray triangles, gray font) controls. Gray boxes, BRCT domains; DBD, GAL4 DNA binding domain.

Figure 2

Functional analysis of missense variants in BRCA1. A, quantitative transcriptional assay in yeast cells. Cells were cotransformed with a LexA-responsive β-galactosidase reporter gene (diagram shown above the graph) and a LexA DBD fusion to residues 1,396 to 1,863 of wt BRCA1, or the same fragment carrying various UCVs. We used the wt and the S1613G neutral polymorphism as positive controls (+). Deleterious mutations M1775R and Y1853X were used as negative controls (−). Three independent yeast clones were tested in triplicates. The activity of the construct with wt BRCA1 was expressed as 100%, with the other results placed on this scale. B, quantitative transcriptional assay in mammalian cells. Cells were cotransfected with a GAL4-responsive firefly luciferase reporter gene (diagram shown above the graph), a Renilla luciferase driven by a constitutive promoter (internal control, not shown), and a GAL4 DNA binding domain (DBD) fusion to residues 1,396 to 1,863 of wt BRCA1 (WT), or the same fragment carrying various UCVs. Controls are the same as described above but fused to GAL4 DBD. Measurements were done in triplicates and normalized against the internal transfection controls. The activity of the construct with wt BRCA1 was expressed as 100%, with the other results placed on this scale. To control for possible variations in protein expression levels, samples were analyzed by Western blot with rabbit anti-LexA DBD polyclonal antibody in yeast extracts (C) or mouse anti-GAL4 DBD monoclonal antibody in mammalian cell extracts (D).

Figure 3

Functional analysis of double mutants in BRCA1. A, quantitative assay in human 293T HEK cells done as described in Fig. 2. No difference was found in the activity between pairs of rare variant and rare variant combined with S1613G polymorphism. B, protein levels determined by Western blot with mouse anti-GAL4 DBD monoclonal antibody.

Figure 4

Missense variants and risk. A, hypothetical correlation between protein activity and cancer risk assuming a continuous relationship. B, hypothetical correlation between protein activity and cancer risk assuming a discrete relationship. C, functional analysis of 47 variants tested in a quantitative and standardized fashion plotted as percent of wt activity. Squares, average activity of the variant; bars, range of interexperiment (replicates in the same experiment) and intraexperiment (assays done at different times) variability. Red and blue squares, variants that have been classified by genetic and/or integrated methods as deleterious or neutral, respectively (Table 2). Red and blue arrows, deleterious variant with highest activity and neutral variant with lowest activity, respectively.