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.N-ethyl-N-nitrosourea | genetic mapping | forward genetics | mutagenesis | massively parallel sequencing P henotypic variation in mice can be induced with N-ethyl-Nnitrosourea (ENU), which creates single base pair substitutions in germ line DNA. However, the positional cloning of ENU-induced mutations causative for phenotypes of interest has historically been a time-consuming process, beginning with generation of an outcrossed recombinant mapping population of phenotypically mutant and WT mice, genotyping individual mice at genetic markers across the genome to create a linkage map, and finally targeted sequencing to identify the causative mutation within the critical region. The advent of massively parallel sequencing techniques has given rise to more rapid "mapping-bysequencing" methods in which genome-wide marker genotyping and DNA sequencing are combined into a single step applied to either individual or pooled groups of organisms (1). For ENUmutagenized mice, early experiments used massively parallel sequencing for mutation identification within a critical region defined by traditional or bulk segregation mapping using recombinant mapping populations produced by outcrossing the mutant to another inbred laboratory strain and backcrossing or intercrossing a second time (2-4). Later reports demonstrated mapping with the identified sequence variants themselves as markers, which eliminated...

Insulin resistance and diabetes caused by genetic or diet-induced KBTBD2 deficiency in mice

“…trait under a recessive model of inheritance ( Fig. 2A) (14). No tny phenotypes were observed in heterozygous mice ( Fig.…”

“…A phenotype observed among third-generation (G3) C57BL/6J mice homozygous for mutations induced by N-ethyl-N-nitrosourea (ENU), termed teeny (tny) (14), was characterized by reduced body size and weight within the first few days and throughout postnatal life ( Fig. 1 A and B and Fig.…”

Creatine maintains intestinal homeostasis and protects against colitis

“…Whole-exome sequencing and mapping were performed as described (48). Briefly, exome-enriched DNA from all G1 mice was sequenced using the Illumina HiSeq 2500 platform.…”

“…Briefly, exome-enriched DNA from all G1 mice was sequenced using the Illumina HiSeq 2500 platform. All G3 mice were genotyped across coding mutations according to their pedigree using Ion Torrent AmpliSeq custom panels as previously described (48). To correlate mice with the genotyping results, we used the percentage of original weight loss as a continuous variable.…”

“…Blood cells were isolated and incubated as previously described (48). Data were acquired on a LSRFortessa cell analyzer (BD Biosciences) and analyzed with FlowJo software (TreeStar).…”