Anja R Ollodart, Chiann-Ling C Yeh, Aaron W Miller, Brian H Shirts, Adam S Gordon, Maitreya J Dunham
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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.</p>","PeriodicalId":12706,"journal":{"name":"Genetics","volume":"218 2","pages":""},"PeriodicalIF":3.3000,"publicationDate":"2021-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8225350/pdf/iyab058.pdf","citationCount":"0","resultStr":"{\"title\":\"Multiplexing mutation rate assessment: determining pathogenicity of Msh2 variants in Saccharomyces cerevisiae.\",\"authors\":\"Anja R Ollodart, Chiann-Ling C Yeh, Aaron W Miller, Brian H Shirts, Adam S Gordon, Maitreya J Dunham\",\"doi\":\"10.1093/genetics/iyab058\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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. 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Multiplexing mutation rate assessment: determining pathogenicity of Msh2 variants in Saccharomyces 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 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.
期刊介绍:
GENETICS is published by the Genetics Society of America, a scholarly society that seeks to deepen our understanding of the living world by advancing our understanding of genetics. Since 1916, GENETICS has published high-quality, original research presenting novel findings bearing on genetics and genomics. The journal publishes empirical studies of organisms ranging from microbes to humans, as well as theoretical work.
While it has an illustrious history, GENETICS has changed along with the communities it serves: it is not your mentor''s journal.
The editors make decisions quickly – in around 30 days – without sacrificing the excellence and scholarship for which the journal has long been known. GENETICS is a peer reviewed, peer-edited journal, with an international reach and increasing visibility and impact. All editorial decisions are made through collaboration of at least two editors who are practicing scientists.
GENETICS is constantly innovating: expanded types of content include Reviews, Commentary (current issues of interest to geneticists), Perspectives (historical), Primers (to introduce primary literature into the classroom), Toolbox Reviews, plus YeastBook, FlyBook, and WormBook (coming spring 2016). For particularly time-sensitive results, we publish Communications. As part of our mission to serve our communities, we''ve published thematic collections, including Genomic Selection, Multiparental Populations, Mouse Collaborative Cross, and the Genetics of Sex.