DETECTION OF HETEROZYGOUS TRUNCATING MUTATIONS IN THE BRCA1 AND APC GENES BY USING A RAPID SCREENING ASSAY IN YEAST

The detection of inactivating mutations in tumor suppressor genes is c ritical to their characterization, as well as to the development of di agnostic testing, Most approaches for mutational screening of germ-lin e specimens are complicated by the fact that mutations are heterozygou s and that missense mutations are difficult to interpret in the absenc e of information about protein function. We describe a novel method us ing Saccharomyces cerevisiae for detecting protein-truncating mutation s in any gene of interest, The PCR-amplified coding sequence is insert ed by homologous recombination into a yeast URA3 fusion protein, and t ransformants are assayed for growth in the absence of uracil, The high efficiency of homologous recombination in yeast ensures that both all eles are represented among transformants and achieves separation of al leles, which facilitates subsequent nucleotide sequencing of the mutat ed transcript. The specificity of translational initiation of the URA3 gene leads to minimal enzymatic activity in transformants harboring a n inserted stop codon, and hence to reliable distinction between speci mens with wild-type alleles and those with a heterozygous truncating m utation. This yeast-based stop codon assay accurately detects heterozy gous truncating mutations in the BRCA1 gene in patients with early ons et of breast cancer and in the APC gene in patients with familial aden omatous polyposis. This approach offers a rapid and reliable method fo r genetic diagnosis in individuals at high risk for germ-line mutation s in cancer susceptibility genes.