LAUSR.org creates dashboard-style pages of related content for over 1.5 million academic articles. Sign Up to like articles & get recommendations!

Multiplexing Mutation Rate Assessment: Determining Pathogenicity of Msh2 Variants in S. cerevisiae.

Despite the fundamental importance of mutation rate as a driving force in evolution and disease risk, common methods to assay mutation rate are time consuming and tedious. Established methods such… Click to show full abstract

Despite the fundamental importance of mutation rate as a driving force in evolution and disease risk, common methods to assay mutation rate are time consuming and tedious. Established methods such as fluctuation tests and mutation accumulation experiments are low-throughput and often require significant optimization to ensure accuracy. We established a new method to determine the mutation rate of many strains simultaneously by tracking mutation events in a chemostat continuous culture device and applying deep sequencing to link mutations to alleles of a DNA-repair gene. We applied this method to assay the mutation rate of hundreds of Saccharomyces cerevisiae strains carrying mutations in the gene encoding Msh2, a DNA repair enzyme in the mismatch repair pathway (MMR). Loss-of-function (LOF) mutations in MSH2 are associated with hereditary non-polyposis colorectal cancer (HNPCC), an inherited disorder that increases risk for many different cancers. However, the vast majority of MSH2 variants found in human populations have insufficient evidence to be classified as either pathogenic or benign. We first benchmarked our method against Luria-Delbrück fluctuation tests using a collection of published MSH2 missense variants. Our pooled screen successfully identified previously characterized non-functional alleles as high mutators. We then created an additional 185 human missense variants in the yeast ortholog, including both characterized and uncharacterized alleles curated from ClinVar and other clinical testing data. In a set of alleles of known pathogenicity, our assay recapitulated ClinVar's classification; we then estimated pathogenicity for 157 variants classified as uncertain or conflicting reports of significance. This method is capable of studying the mutation rate of many microbial species and can be applied to problems ranging from the generation of high-fidelity polymerases to measuring the frequency of antibiotic resistance emergence.

Keywords: msh2 variants; pathogenicity; mutation; mutation rate

Journal Title: Genetics
Year Published: 2021

Link to full text (if available)


Share on Social Media:                               Sign Up to like & get
recommendations!

Related content

More Information              News              Social Media              Video              Recommended



                Click one of the above tabs to view related content.