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.

A recombination-based method to characterize human BRCA1 missense variants

“…However, both the I1766S and the mutation M1775R affected the transcriptional activation ability of BRCA1 both in yeast and mammalian cells [21]. The A1789T variant also, in addition to its effect in the NHEJ assay, showed to abrogate the BRCA1 transcriptional activity (Guidugli unpublished results), suggesting to be potentially pathogenic.…”

“…The mother of the proband, affected by breast and ovarian cancer diagnosed at 46 and 50 years of age, respectively, was found to be a carrier of the variant (figure 2a). The I1766S was classified as a deleterious amino acidic change by Carvalho et al [21]. It was found in one family: the proband had ovarian carcinoma diagnosed at 42 years of age.…”

Multimodel assessment of BRCA1 mutations in Taiwanese (ethnic Chinese) women with early-onset, bilateral or familial breast cancer

“…Some specific deleterious or benign genetic variants have been documented, such as S1613G (benign polymorphism) and M1775R (deleterious mutation). 16 For VUS, we used multimodel assessment to evaluate the pathogenicity (Figure 1). …”

“…Transcription-based assay could characterize the deleterious mutations of BRCA1 COOH-terminus. 16 To establish this functional assay, we constructed a wildtype cDNA of BRCA1 into pcDNA3.1+ vector, then introduced the BRCA1 Cterminus (cDNA 4186-5792, amino acid 1396-1863) to fuse with GAL4 DBD (DNA binding domain) into pGBT9 vector by the restriction site of BamHI and EcoRI. Then, the fusion segment of GAL4 DBD-BRCA1 C-terminus was subcloned into pcDNA 3.1+ vector using the restriction site of HindIII and BamHI.…”

“…15 This large gene displays several thousand different alleles, and most of them are missense changes according to the Breast Cancer Information Core (BIC) database. Categorizing missense changes as deleterious mutations associated with breast cancer development vs benign polymorphisms is difficult 16 and poses significant obstacles in genetic studies. 17 Currently, these mutations are generally classified as deleterious, benign polymorphisms or variants of uncertain significance (VUS).…”