Gastroenterology

Gastroenterology

Volume 122, Issue 1, January 2002, Pages 211-219
Gastroenterology

Basic Research
Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system*,**

https://doi.org/10.1053/gast.2002.30296Get rights and content

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▵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 ▵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.

GASTROENTEROLOGY 2002;122:211-219

Section snippets

hMLH1 variants

The majority of hMLH1 missense mutations associated with HNPCC lead to structural changes within the amino- or the carboxy-terminal regions containing the ATPase and the hPMS2-interaction domains, respectively (Figure 1).

. Schematic representation of germline hMLH1 missense mutations found in patients with HNPCC based on the ICG-HNPCC mutation database (www.nfht.nl). hMLH1 variants studied in this report are shown in bold. Alleles described as pathogenic mutations are shown in the upper part of

Characterization of MMR efficiency in 293T cells

To establish a suitable human expression system for hMLH1 and its variants, we screened different cell lines for hMLH1 expression. The human embryonic kidney fibroblast cell line 293T was found to lack hMLH1 as well as hPMS2, whereas hMSH2 and hMSH6 were normally expressed (Figure 2A).

. Characterization of DNA MMR efficiency of human 293T-cell extracts. TK6, MMR-proficient control; HCT116, MMR-deficient control lacking hMLH1 (hMLH1−/−) and hPMS2. (A) Western blot analysis of hMSH6, hMSH2, hPMS2,

Discussion

Germline mutations of hMLH1 and hMSH2 account for the majority of MMR gene mutations identified in HNPCC families.23 Many of these mutations result in truncated proteins and are predicted to predispose their carriers to colon cancer. However, more than 30% of the reported HNPCC-linked hMLH1 mutations are missense, and, in this case, single amino-acid changes may not impair protein function or may be only partially inactivating. Functional testing of the mutant hMLH1 alleles is therefore

Acknowledgements

The authors thank Dr. Markus Raeschle for discussion.

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    *

    Address requests for reprints to: Joerg Trojan, M.D., Medizinische Klinik II, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. e-mail: [email protected]; fax: (49) 69-6301-6448.

    **

    Supported in part by grants from the University of Frankfurt (F15/01) and the Swiss National Science Foundation. J.T. is supported by a Harald-Goebel scholarship of the Deutsche Gesellschaft für Verdauungs- und Stoffwechselerkrankungen.

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