A yeast recombination assay to characterize humanBRCA1missense variants of unknown pathological significance

Abstract

The BRCA1 tumor suppressor gene is found mutated in familial breast cancer. Although many of the mutations are clearly pathological because they give rise to truncated proteins, several missense variants of uncertain pathological consequences have been identified. A novel functional assay to screen for BRCA1 missense variants in a simple genetic system could be very useful for the identification of potentially deleterious mutations. By using two prediction computer programs, Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen), seven nonsynonymous missense BRCA1 variants likely disrupting the gene function were selected as potentially deleterious. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) was used to test these cancer-related missense mutations for their ability to affect cell growth and homologous recombination (HR) at the HIS3 and ADE2 loci. The variants localized in the BRCA1 C-Terminus (BRCT) domain did not show any growth inhibition when overexpressed in agreement with previous results. Overexpression of either wild-type BRCA1 or two neutral missense variants did not increase yeast HR but when cancer-related variants were overexpressed a significant increase in recombination was observed. Results clearly showed that this genetic system can be useful to discriminate between neutral and deleterious BRCA1 missense variants.

A recombination-based method to characterize human BRCA1 missense variants

“…The over-expression of BRCA1-Y179C and BRCA1-A1789T UCVs increased the frequency of random integration as compared to the wild type. It was observed that the over-expression of BRCA1-Y179C induces a hyper-recombination phenotype also in yeast (Table 3) [31]. Moreover, we have previously reported that the in vivo analysis on tumor tissue revealed that the proband carrier of the Y179C showed loss of heterozigosity (LoH) of the wild type allele and the proband carrier of A1789T showed hypermethylation of the wild type allele.…”

“…In our experiments, no BRCA1 missense variant increased HR by 2 fold, therefore we can conclude that this assay does not distinguish between pathogenic mutation and neutral polymorphism. Recently, we have developed a yeast-recombination assay that could be helpful to characterize BRCA1 missense variants [31]. In yeast, the over-expression of pathogenic BRCA1 variants induce HR by 2-4 fold as compare to the wild type or neutral polymorphism [31] (Table 3).…”

“…Recently, we have developed a yeast-recombination assay that could be helpful to characterize BRCA1 missense variants [31]. In yeast, the over-expression of pathogenic BRCA1 variants induce HR by 2-4 fold as compare to the wild type or neutral polymorphism [31] (Table 3).…”

“…Moreover, we have previously reported that the in vivo analysis on tumor tissue revealed that the proband carrier of the Y179C showed loss of heterozigosity (LoH) of the wild type allele and the proband carrier of A1789T showed hypermethylation of the wild type allele. Both LoH and hypermethylation are considered to be indicative of the pathogenicity of the variant [31].…”

Yeast cells reveal the misfolding and the cellular mislocalization of the human BRCA1 protein

“…It has been known for a long time that expression of the full-length human BRCA1 protein in yeast cells compromises cell growth (Humphrey et al, 1997;Caligo et al, 2009;Millot et al, 2011). However, the origin of this growth inhibition is poorly understood.…”