Quick Search:

uzh logo
Browse by:
bullet
bullet
bullet
bullet

Zurich Open Repository and Archive 

Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-31457

Trojan, J; Zeuzem, S; Randolph, A; Hemmerle, C; Brieger, A; Raedle, J; Plotz, G; Jiricny, J; Marra, G (2002). Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system. Gastroenterology, 122(1):211-219.

[img] PDF - Registered users only
1MB

Abstract

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 (hMLH1 Delta 9/10) 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 Delta 9/10 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.

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
DDC:570 Life sciences; biology
Language:English
Date:2002
Deposited On:09 Jul 2010 14:20
Last Modified:02 Dec 2013 19:40
Publisher:Elsevier
ISSN:0016-5085
Publisher DOI:10.1053/gast.2002.30296
PubMed ID:11781295
Citations:Web of Science®. Times Cited: 116
Google Scholar™
Scopus®. Citation Count: 119

Users (please log in): suggest update or correction for this item

Repository Staff Only: item control page