title: Real-time resolution of point mutations that cause phenovariance in mice.
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.
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390; Quantitative Biomedical Research Center, Department of Clinical Science, University of Texas Southwestern Medical Center, Dallas, TX 75390; and.
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390; Quantitative Biomedical Research Center, Department of Clinical Science, University of Texas Southwestern Medical Center, Dallas, TX 75390; and.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zhan
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Chun-Hui
authors.initials: CH
authors.lastname: Bu
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Stephen
authors.initials: S
authors.lastname: Lyon
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: David
authors.initials: D
authors.lastname: Pratt
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Sara
authors.initials: S
authors.lastname: Hildebrand
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Jin Huk
authors.initials: JH
authors.lastname: Choi
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Zhao
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Ming
authors.initials: M
authors.lastname: Zeng
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Kuan-wen
authors.initials: KW
authors.lastname: Wang
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Emre
authors.initials: E
authors.lastname: Turer
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Zhe
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Duanwu
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Tao
authors.initials: T
authors.lastname: Yue
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Hexin
authors.initials: H
authors.lastname: Shi
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Jianhui
authors.initials: J
authors.lastname: Wang
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Lei
authors.initials: L
authors.lastname: Sun
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Jeff
authors.initials: J
authors.lastname: SoRelle
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: William
authors.initials: W
authors.lastname: McAlpine
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Noelle
authors.initials: N
authors.lastname: Hutchins
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Zhan
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Maggy
authors.initials: M
authors.lastname: Fina
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Rochelle
authors.initials: R
authors.lastname: Gobert
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Jiexia
authors.initials: J
authors.lastname: Quan
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: McKensie
authors.initials: M
authors.lastname: Kreutzer
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Stephanie
authors.initials: S
authors.lastname: Arnett
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Kimberly
authors.initials: K
authors.lastname: Hawkins
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Ashley
authors.initials: A
authors.lastname: Leach
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Christopher
authors.initials: C
authors.lastname: Tate
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Chad
authors.initials: C
authors.lastname: Daniel
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Carlos
authors.initials: C
authors.lastname: Reyna
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Lauren
authors.initials: L
authors.lastname: Prince
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Sheila
authors.initials: S
authors.lastname: Davis
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Joel
authors.initials: J
authors.lastname: Purrington
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Rick
authors.initials: R
authors.lastname: Bearden
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Jennifer
authors.initials: J
authors.lastname: Weatherly
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Danielle
authors.initials: D
authors.lastname: White
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Jamie
authors.initials: J
authors.lastname: Russell
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Qihua
authors.initials: Q
authors.lastname: Sun
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Miao
authors.initials: M
authors.lastname: Tang
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Li
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Lindsay
authors.initials: L
authors.lastname: Scott
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors.forename: Eva Marie Y
authors.initials: EM
authors.lastname: Moresco
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171, 77 Stockholm, Sweden.
authors.forename: Gerald M
authors.initials: GM
authors.lastname: McInerney
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171, 77 Stockholm, Sweden.
authors.forename: Gunilla B
authors.initials: GB
authors.lastname: Karlsson Hedestam
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390; Quantitative Biomedical Research Center, Department of Clinical Science, University of Texas Southwestern Medical Center, Dallas, TX 75390; and.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX 75390; Bruce.Beutler@UTSouthwestern.edu.
authors.forename: Bruce
authors.initials: B
authors.lastname: Beutler
mesh_terms: D000483:Alleles; D000818:Animals; D005260:Female; D005804:Genes, Lethal; D008040:Genetic Linkage; D008297:Male; D051379:Mice; D010375:Pedigree; D010641:Phenotype; D017354:Point Mutation; D040641:Quantitative Trait Loci
keywords: N-ethyl-N-nitrosourea; forward genetics; genetic mapping; massively parallel sequencing; mutagenesis