Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2genes in yeastReportar como inadecuado

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BMC Cancer

, 9:382

First Online: 28 October 2009Received: 19 December 2008Accepted: 28 October 2009DOI: 10.1186-1471-2407-9-382

Cite this article as: Vogelsang, M., Comino, A., Zupanec, N. et al. BMC Cancer 2009 9: 382. doi:10.1186-1471-2407-9-382


BackgroundLoss of DNA mismatch repair MMR in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer HNPCC. Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells.

MethodsYeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested.

ResultsThe yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants A92P, S93G, I219V, K618R and K618T were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic.

ConclusionResults of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2407-9-382 contains supplementary material, which is available to authorized users.

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Autor: Matjaz Vogelsang - Aleksandra Comino - Neja Zupanec - Petra Hudler - Radovan Komel


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