Real-time resolution of point mutations that cause phenovariance in mice

Tao Wang, Xiaowei Zhan, Chun Hui Bua, Stephen Lyona, David Pratta, Sara Hildebrand, Jin Huk Choi, Zhao Zhang, Ming Zeng, Kuan Wen Wang, Emre E Turer, Zhe Chen, Duan-Wu Zhang Ph.D., Tao Yue, Ying Wang, He-Xin Shi, Jianhui Wang, Lei Sun, Jeff SoRelle, William McAlpineNoelle Hutchins, Xiaoming Zhan, Maggy Fin, Rochelle Gobert, Jiexia Quan, McKensie Kreutzer, Stephanie Arnett, Kimberly Hawkins, Ashley Leach, Christopher Tate, Chad Daniel, Carlos Reyna, Lauren Prince, Sheila Davis, Joel Purrington, Rick Bearden, Jennifer Weatherly, Danielle White, Jamie L Russell, Qihua Sun, Miao Tang, Xiaohong Li, Lindsay Scott, Eva Marie Y Moresco, Gerald M. McInerney, Gunilla B. Karlsson Hedestam, Yang Xie, Bruce A Beutler

Research output: Contribution to journalArticle

36 Scopus citations

Abstract

With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.N-ethyl-N-nitrosourea, genetic mapping, forward genetics, mutagenesis, massively parallel sequencing.

Original languageEnglish (US)
Pages (from-to)E440-E449
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number5
DOIs
StatePublished - Feb 3 2015

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    Wang, T., Zhan, X., Bua, C. H., Lyona, S., Pratta, D., Hildebrand, S., Choi, J. H., Zhang, Z., Zeng, M., Wang, K. W., Turer, E. E., Chen, Z., Zhang Ph.D., D-W., Yue, T., Wang, Y., Shi, H-X., Wang, J., Sun, L., SoRelle, J., ... Beutler, B. A. (2015). Real-time resolution of point mutations that cause phenovariance in mice. Proceedings of the National Academy of Sciences of the United States of America, 112(5), E440-E449. https://doi.org/10.1073/pnas.1423216112