Multiplexing mutation rate assessment: determining pathogenicity of Msh2 variants in Saccharomyces cerevisiae
| Autor: | Aaron W. Miller, Adam S. Gordon, Brian H. Shirts, Anja R Ollodart, Chiann Ling C. Yeh, Maitreya J. Dunham |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Developmental and Behavioral Genetics
Mutation rate Saccharomyces cerevisiae Proteins DNA Mutational Analysis Mutation Missense Saccharomyces cerevisiae Biology 03 medical and health sciences 0302 clinical medicine Mismatch Repair Pathway Mutation Rate Genetics Missense mutation Humans Allele DNA Fungal Gene 030304 developmental biology Gene Library 0303 health sciences Drug Resistance Microbial Mutation Accumulation High-Throughput Screening Assays MutS Homolog 2 Protein MSH2 030220 oncology & carcinogenesis Mutation (genetic algorithm) |
| Zdroj: | Genetics |
| ISSN: | 1943-2631 |
| Popis: | 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. Loss-of-function mutations in MSH2 are associated with hereditary nonpolyposis colorectal cancer, 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 nonfunctional 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|