Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2genes in yeast

Abstract

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.

Yeast Cells Expressing the Human Mitochondrial DNA Polymerase Reveal Correlations between Polymerase Fidelity and Human Disease Progression

“…The "humanized" yeast system, with the yeast genes replaced by their human orthologs, has been developed to rapidly examine the pathology of human genetic variation, thanks to the powerful genetic tools available for yeast (32)(33)(34)(35). Here, we begin addressing important questions by developing "humanized" yeast expressing human mitochondrial DNA polymerase genes with wild-type and mutant alleles.…”

Non-truncating hMLH1 variants identified in Slovenian gastric cancer patients are not associated with Lynch Syndrome: a functional analysis report

“…Introduction of hMLH1 mutations into humanized yeast strain co-expressing hMLH1 and hPMS2 genes Two MLH1 nucleotide substitutions were introduced into humanized yeast strain according to a previous report [8], using in vivo site-directed mutagenesis approach [11]. The first step of the approach involves integration of a counterselectable reporter (CORE) cassette in the region of interest.…”

“…The yeast Saccharomyces cerevisiae strains MV400 (MATa ade2 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 mlh1::hMLH1 pms1::hPMS2) and MV100 (MATa ade2 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 mlh1D::kanMX) [8] and plasmids pSH91, Yep181 and pCORE were used in the study [9][10][11]. The E. coli strain DH5a [F gyrA96 (Nal r ) recA1 endA1 thi-1 hsdR17 (r k -m k -) gluV44 deoR U80d D(lacZ)M15] [12] was used for plasmid propagation.…”

“…Since limited clinical parameters were obtained, pathogenicity of the identified UVs was impossible to interpret on clinical samples alone. We assessed their functional significance by recently described in vivo yeast-based approach, whose further advantage is that the approach is not limited to mutations located at evolutionary conserved regions and functional domains and therefore enables all variants, found within the coding region of the hMLH1 gene, to be analysed [8].…”

Genetic Instability in Gastric Cancer

“…Finally, it is also not easy to determine sensitivity and specificity of these tests, therefore results should still be utilized with caution and interpreted alongside clinical data of the affected carriers. (Vogelsang et al, 2009). With our approach we have functionally characterized four missense MLH1 variants, which we previously identified in MSI-H positive gastric cancers with limited MLH1 hypermethylation.…”