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.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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