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


CONCLUSIONS
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.
Department for Biosynthesis and Biotransformation, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia. matjaz.vogelsang@ki.si
Matjaz
M
Vogelsang
Aleksandra
A
Comino
Neja
N
Zupanec
Petra
P
Hudler
Radovan
R
Komel
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D053843:DNA Mismatch Repair; D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D045643:DNA Repair Enzymes