Detection of heterozygous truncating mutations in the BRCA1 and APC genes by using a rapid screening assay in yeast

Abstract

The detection of inactivating mutations in tumor suppressor genes is critical to their characterization, as well as to the development of diagnostic testing. Most approaches for mutational screening of germ-line specimens are complicated by the fact that mutations are heterozygous and that missense mutations are difficult to interpret in the absence of information about protein function. We describe a novel method using Saccharomyces cerevisiae for detecting protein-truncating mutations in any gene of interest. The PCR-amplified coding sequence is inserted by homologous recombination into a yeast URA3 fusion protein, and transformants are assayed for growth in the absence of uracil. The high efficiency of homologous recombination in yeast ensures that both alleles are represented among transformants and achieves separation of alleles, which facilitates subsequent nucleotide sequencing of the mutated transcript. The specificity of translational initiation of the URA3 gene leads to minimal enzymatic activity in transformants harboring an inserted stop codon, and hence to reliable distinction between specimens with wild-type alleles and those with a heterozygous truncating mutation. This yeast-based stop codon assay accurately detects heterozygous truncating mutations in the BRCA1 gene in patients with early onset of breast cancer and in the APC gene in patients with familial adenomatous polyposis. This approach offers a rapid and reliable method for genetic diagnosis in individuals at high risk for germ-line mutations in cancer susceptibility genes.

Germline mutations in PTEN are an infrequent cause of genetic predisposition to breast cancer

“…Yeast transformants were scored for their ability to grow in the absence of uracil, indicating that the inserted PTEN coding region contains an open reading frame, or for failure to grow, resulting from the presence of a stop codon within the PTEN sequence that disrupts translation of the downstream URA3 transcript. In this assay, which has been used to detect truncating mutations in APC and BRCA1 Ishioka et al, 1997), specimens with homozygous wildtype transcript yield 485% ura+ colonies, while those with a heterozygous truncating mutation yield 40 ± 60% ura+ colonies, and those with a homozygous mutation produce 510% ura+ colonies. The absence of truncating PTEN mutations was con®rmed by yeastassay in the 28 cases that had been analysed by direct nucleotide sequencing.…”

“…Lymphoblast specimens from 28 women diagnosed 5 age 30 were Figure 1 Schematic representation of yeast-based protein truncation assay. The yeast-based stop codon assay , involving construction of a chimeric fusion between the yeast URA3 gene and a PCR-ampli®ed coding sequence, has been used to detect heterozygous germline truncating mutations in the APC and BRCA1 genes Ishioka et al, 1997). To facilitate analysis of PTEN, and any other gene of interest, we have modi®ed this technique by using a synthetic 45-nucleotide tail at the 5' end of the oligonucleotide primers, which is sucient to allow homologous recombination in yeast.…”

Identification and evaluation of 55 genetic variations in the BRCA1 and the BRCA2 genes of patients from 50 Japanese breast cancer families

“…Among these SNPs, we found nine protein-truncating mutations (four in BRCA1and five in BRCA2) in ten patients containing six novel mutations that were not found in the BIC database (http://research.nhgri.nih.gov/bic/) ( Table 1). The existence of mutations were also confirmed by the stop codon assay in yeast (Ishioka et al 1997;Sakayori et al 2003). The percentage of protein-truncating mutations in the examined families was 20% (ten of 50), comparable with several reports describing the frequency of protein-truncating mutations of the two genes in Japanese breast cancer families (Inoue et al 1995;Takano et al 1997;Inoue et al 1997;Ikeda et al 2001).…”

Identification and Genetic Mapping of Genes for Hereditary Breast Cancer and Ovarian Cancer in Families Unlinked to BRCA1

“…In one example, a mother had a frameshift mutation and her daughter had a different frameshift mutation inherited from her father (Stoppa- . Ishioka et al (1997) have been able to detect heterozygoustruncating mutations in both the BRCAI and APC genes. In their system, a PCR amplified coding sequence is inserted via homologous recombination into the URA3 gene in yeast and growth is assayed.…”