Development of a novel method for genetic diagnosis of hereditary colon cancer syndromes using yeast.

1.错配修复基因和APC基因杂合突变检测方法的发展。(1)错配修复基因杂合突变检测方法的发展。我们开发了一种能够检测杂合功能缺失突变的新方法。该方法从患者淋巴细胞中提取RNA，利用RT-PCR技术从RNA中扩增hMLH1 cDNA。将扩增得到的hMLH1 cDNA导入体内，同源组合到间隙载体中，在酵母中表达。当野生型hMLH1表达时，酵母转化子表现出突变表型，可能是由于宿主错配修复系统的干扰（显性突变效应）。我们已经证明，在HNPCC家族中检测到的大多数hMLH1种系突变都失去了这种功能，这表明该系统有可能检测到未知的杂合hMLH1功能丧失突变。(2)建立了检测APC杂合突变的方法。我们还开发了一种新的方法，称为停止密码子（SC）测定，该方法能够检测APC基因中的ptotein截断突变，APC基因是家族性腺瘤性息肉病（FAP）中突变的基因。采用SC法，通过PCR扩增出APC片段，并将其导入URA3基因标记的体内缺口载体。表达的融合蛋白由氨基端apc衍生的多肽和羧基端ura3衍生的多肽组成。该融合蛋白能够补充酵母ura3表型，因此转化能够在缺乏Uracil的培养皿上生长。任何破坏APC序列读框的突变，如无义突变和移码突变，都可以被检测为无法补充酵母ura3表型，酵母转化子不能在相同的板上生长。使用(1)和(2)中描述的新方法，我们已经检测到FAP作为HNPCC家族的杂合hMLH1突变和杂合蛋白截断突变。少

1.Development of methods for detection of heterozygous mutations in the mismatch repair genes and the APC gene.(1) Development of methods for detection of heterozygous mutations in the mismatch repair genes. We developed a novel method which has the ability to detect heterozygous loss-of-function mutations. Using this method, RNA was extracted from patient lymphocytes and the hMLH1 cDNA was amplified from the RNA by RT-PCR technique. The amplified hMLH1 cDNA was introduced, homologously combined into a gap vector in vivo and was expressed in yeast Saccharomyces cerevisiae. When the wild-type hMLH1 was expressed, yeast transformants showed mutator phenotype, possibly, due to the iterferance of host mismatch repair system (dominant mutator effect). We have shown that most of the hMLH1 germline mutations detected in HNPCC families lose this function, suggesting that this system has potentially detect unknown heterozygous hMLH1 loss-of-function mutations.(2) Development of methods for dete … More ction of heterozygous APC mutations.We also developed a novel method, called stop codon (SC) assay which has the ability to detect ptotein truncating mutations in APC gene, a gene mutated in familial adenomatous polyposis (FAP), from patient lymphocytes. Using the SC assay, an APC fragment was amplified by PCR and the fragment was introduced into a URA3 gene-tagged gap vector in vivo. The plasmid expressed the fusion protein consisted of the APC-derived polypeptide at the amino terminus and the URA3-derived polypeptide at the carboxy terminus. The fosion protein was able to compliment yeast ura3-phenotype therefore the transformant was able to grow on the plate lacking Uracil. Any mutations that disrupt the reading frame of the APC sequence, such as nonsense and frameshift mutations, could detect as inability to compliment yeast ura3-phenotype and the yeast transformants cannot grow on the same plates.2.Using the new methods described in (1) and (2), we have detected heterozygous loss-of-function hMLH1 mutation and heterozygous protein truncating mutations from FAP as HNPCC families. Less