Ellison Aaron R, Lofing Joan, Bitter Grant A
BitTech, Inc., Westlake Village, CA 91361, USA.
Nucleic Acids Res. 2004 Oct 8;32(18):5321-38. doi: 10.1093/nar/gkh855. Print 2004.
Germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1 are responsible for the majority of hereditary non-polyposis colorectal cancer (HNPCC), an autosomal-dominant early-onset cancer syndrome. Genetic testing of both MSH2 and MLH1 from individuals suspected of HNPCC has revealed a considerable number of missense codons, which are difficult to classify as either pathogenic mutations or silent polymorphisms. To identify novel MLH1 missense codons that impair MMR activity, a prospective genetic screen in the yeast Saccharomyces cerevisiae was developed. The screen utilized hybrid human-yeast MLH1 genes that encode proteins having regions of the yeast ATPase domain replaced by homologous regions from the human protein. These hybrid MLH1 proteins are functional in MMR in vivo in yeast. Mutagenized MLH1 fragments of the human coding region were synthesized by error-prone PCR and cloned directly in yeast by in vivo gap repair. The resulting yeast colonies, which constitute a library of hybrid MLH1 gene variants, were initially screened by semi-quantitative in vivo MMR assays. The hybrid MLH1 genes were recovered from yeast clones that exhibited a MMR defect and sequenced to identify alterations in the mutagenized region. This investigation identified 117 missense codons that conferred a 2-fold or greater decreased efficiency of MMR in subsequent quantitative MMR assays. Notably, 10 of the identified missense codons were equivalent to codon changes previously observed in the human population and implicated in HNPCC. To investigate the effect of all possible codon alterations at single residues, a comprehensive mutational analysis of human MLH1 codons 43 (lysine-43) and 44 (serine-44) was performed. Several amino acid replacements at each residue were silent, but the majority of substitutions at lysine-43 (14/19) and serine-44 (18/19) reduced the efficiency of MMR. The assembled data identifies amino acid substitutions that disrupt MLH1 structure and/or function, and should assist the interpretation of MLH1 genetic tests.
DNA错配修复（MMR）基因MSH2和MLH1中的种系突变是导致大多数遗传性非息肉病性结直肠癌（HNPCC）的原因，HNPCC是一种常染色体显性早发性癌症综合征。对疑似患有HNPCC的个体进行MSH2和MLH1基因检测时发现了大量错义密码子，这些密码子难以归类为致病突变或沉默多态性。为了鉴定损害MMR活性的新型MLH1错义密码子，在酿酒酵母中开展了一项前瞻性基因筛查。该筛查利用了杂交人-酵母MLH1基因，这些基因编码的蛋白质具有被人源蛋白质同源区域取代的酵母ATP酶结构域区域。这些杂交MLH1蛋白在酵母体内的MMR中具有功能。通过易错PCR合成人编码区的诱变MLH1片段，并通过体内缺口修复直接克隆到酵母中。构成杂交MLH1基因变体文库的所得酵母菌落最初通过半定量体内MMR测定进行筛选。从表现出MMR缺陷的酵母克隆中回收杂交MLH1基因并进行测序，以鉴定诱变区域中的改变。这项研究鉴定出117个错义密码子，在随后的定量MMR测定中，这些密码子使MMR效率降低了2倍或更多。值得注意的是，鉴定出的10个错义密码子等同于先前在人群中观察到的密码子变化，并与HNPCC有关。为了研究单个残基处所有可能的密码子改变的影响，对人MLH1密码子43（赖氨酸-43）和44（丝氨酸-44）进行了全面的突变分析。每个残基处的几个氨基酸替换是沉默的，但赖氨酸-43（14/19）和丝氨酸-44（18/19）处的大多数替换降低了MMR效率。汇总的数据鉴定出破坏MLH1结构和/或功能的氨基酸替换，并且应该有助于解释MLH1基因检测结果。
