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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >DETECTION OF HETEROZYGOUS TRUNCATING MUTATIONS IN THE BRCA1 AND APC GENES BY USING A RAPID SCREENING ASSAY IN YEAST
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DETECTION OF HETEROZYGOUS TRUNCATING MUTATIONS IN THE BRCA1 AND APC GENES BY USING A RAPID SCREENING ASSAY IN YEAST

机译：快速酵母分析检测BRCA1和APC基因中的异质性截短突变

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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. [References: 17]
机译：肿瘤抑制基因失活突变的检测对于其表征以及诊断测试的发展至关重要。种系标本突变筛选的大多数方法由于突变是杂合的且错义突变很困难而变得复杂在缺乏有关蛋白质功能的信息时进行解释。我们描述了一种使用酿酒酵母检测感兴趣的任何基因中的蛋白截短突变的新方法，通过同源重组将PCR扩增的编码序列插入酵母URA3融合蛋白中，并在没有尿嘧啶的情况下测定转化子的生长，酵母中同源重组的高效率确保了两个等位基因都在转化体中代表并实现了等位基因的分离，这有利于随后对突变的转录本进行核苷酸测序。 URA3基因翻译起始的特异性导致带有插入的终止密码子的转化体中的酶活性最小，因此可以可靠地区分具有野生型等位基因的标本和具有杂合性截断突变的标本。这种基于酵母的终止密码子测定可准确检测乳腺癌早期发作患者的BRCA1基因和家族性腺瘤性息肉病患者的APC基因的杂合截短突变。这种方法为癌症易感基因种系突变高风险个体的遗传诊断提供了一种快速可靠的方法。 [参考：17]

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