Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system

Gastroenterology. 2002 Jan;122(1):211-9. doi: 10.1053/gast.2002.30296.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Base Pair Mismatch / genetics*
  • Carrier Proteins
  • Cells, Cultured
  • Colorectal Neoplasms, Hereditary Nonpolyposis / genetics*
  • DNA Mutational Analysis
  • DNA Repair / genetics*
  • DNA-Binding Proteins*
  • Fibroblasts / cytology
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Kidney / cytology
  • MutL Protein Homolog 1
  • MutS Homolog 2 Protein
  • Mutation, Missense / genetics*
  • Neoplasm Proteins / genetics*
  • Nuclear Proteins
  • Polymorphism, Genetic / genetics
  • Proto-Oncogene Proteins / genetics
  • Recombinant Proteins / genetics
  • Transfection


  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • DNA-Binding Proteins
  • MLH1 protein, human
  • Neoplasm Proteins
  • Nuclear Proteins
  • Proto-Oncogene Proteins
  • Recombinant Proteins
  • MSH2 protein, human
  • MutL Protein Homolog 1
  • MutS Homolog 2 Protein