Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system
Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system
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DOI：
10.1053/gast.2002.30296
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发表时间：
2002-01-01
期刊：
影响因子：
29.4
通讯作者：
Marra, G
中科院分区：
文献类型：
--
作者：
Trojan, J;Zeuzem, S;Marra, G
背景目标; DNA错配修复（MMR）基因hMLH 1和hMSH 2的种系突变与遗传性非息肉病性结直肠癌（HNPCC）的易感性相关。由于很大比例的hMLH 1突变是错义的，因此评估其致病作用可能是困难的。迄今为止，错义突变的功能分析主要在酿酒酵母中进行。本研究的目的是检查在人类表达系统中的hMLH 1蛋白变体的生化特性。研究方法：克隆了HNPC相关hMLH 1突变T117 M、V185 G、R217 C、G244 D、R265 C、V326 A和K618 T，多态性I219 V和R265 H，以及缺失外显子9和10的hMLH 1剪接变体（hMLH 1 triangle 9/10）。在将这些构建体转染入人293 T细胞（其由于启动子超甲基化而不表达hMLH 1）后，通过Western印迹和MMR测定分析hMLH 1蛋白变体。结果：所有hMLH 1构建体均成功转染。正如预期的那样，突变K618 T和T117 M影响hMLH 1的高度保守结构域，这些结构域分别是与hPMS 2相互作用或三磷酸腺苷（ATP）结合所必需的，它们影响蛋白质稳定性或其补充MMR缺陷型293 T细胞提取物的能力。V185 G、G244 D和triangle 9/10变体也不能在293 T细胞中补充MMR，而携带I219 V、R265 H、R265 C、R217 C和V326 A突变的hMLH 1蛋白是MMR活性的。结论：这些数据表明，hMLH 1错义突变和剪接变体的致病作用可以通过分析其蛋白产物在同源表达系统中的生化特性来评估。
Background Aims; Germline mutations in the DNA mismatch repair (MMR) genes hMLH1 and hMSH2 are associated with susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC). Because a significant proportion of hMLH1 mutations are missense, the assessment of their pathogenic role may be difficult. To date, functional analysis of missense mutations has been performed primarily in Saccharomyces cerevisiae. The aim of this study was to examine the biochemical properties of hMLH1 protein variants in a human expression system. Methods: The HNPCC-related hMLH1 mutations T117M, V185G, R217C, G244D, R265C, V326A, and K618T, the polymorphisms I219V and R265H, and a hMLH1 splicing variant lacking exon 9 and 10 (hMLH1triangle9/10) were cloned. On transfection of these constructs into human 293T cells, which do not express hMLH1 because of promoter hypermethylation, the hMLH1 protein variants were analyzed by Western blotting and in a MMR assay. Results: Transfection was successful for all hMLH1 constructs. As anticipated, the mutations K618T and T117M, which affect the highly conserved domains of hMLH1 that are necessary for interaction with hPMS2 or for adenosine triphosphate (ATP) binding, respectively, affected protein stability or its ability to complement MMR-deficient 293T-cell extracts. The V185G, G244D, and triangle9/10 variants were also unable to complement MMR in 293T cells, whereas hMLH1 proteins carrying the I219V, R265H, R265C, R217C, and V326A mutations were MMR competent. Conclusions: These data show that the pathogenic role of hMLH1 missense mutations and splicing variants can be assessed by analyzing the biochemical properties of their protein products In a homologous expression system.