Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

Abstract

Background: Loss 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.

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

Results: The 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.

Conclusion: Results 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.

Figure 1

Constructing humanized yeast strains with delitto perfetto system. A. Replacing yeast MMR (yMMR) genes (i.e. yMLH1 and yPMS1) with their human orthologs (hMMR). First, the CORE cassette was introduced into yeast chromosome (line) after the stop codon of a yeast MMR gene, enabling the yeast gene to be still active during the second step of the process. Next, together with CORE cassette, the yeast gene was replaced with its human ortholog. B. Introducing missense alterations into hMLH1 gene in strain co-expressing hMLH1 and hPMS2 genes. First, the CORE cassette was introduced into hMLH1, replacing nucleotide of interest (black vertical line). Next, the CORE cassette was replaced by DNA fragment harbouring a single-nucleotide alteration (yellow vertical line). Genetic technique, delitto perfetto, is based on recombination event (grey area) between two identical strand of DNA.

Figure 2

The MMR efficiency in yeast strains harbouring endogenous and/or human MMR genes. Each datum is the mean, with a 95% confidence interval indicated, of three independent experiments. Mean mutation rates are: Wild-type 2.19 × 10^-6 ; MV100 1.74 × 10⁴; MV200 6.05 × 10^-5; MV300 8.86 × 10^-5; MV400 2.25 × 10^-6. Genotypes of strains are boxed. All prepared strains originated from W303 background [17].