Massively parallel sequencing of the mouse exome to accurately identify rare, induced mutations: an immediate source for thousands of new mouse models

Abstract

Summary Accurate identification of sparse heterozygous single-nucleotide variants (SNVs) is a critical challenge for identifying the causative mutations in mouse genetic screens, human genetic diseases and cancer. When seeking to identify causal DNA variants that occur at such low rates, they are overwhelmed by false-positive calls that arise from a range of technical and biological sources. We describe a strategy using whole-exome capture, massively parallel DNA sequencing and computational analysis, which identifies with a low false-positive rate the majority of heterozygous and homozygous SNVs arising de novo with a frequency of one nucleotide substitution per megabase in progeny of N -ethyl- N -nitrosourea (ENU)-mutated C57BL/6j mice. We found that by applying a strategy of filtering raw SNV calls against known and platform-specific variants we could call true SNVs with a false-positive rate of 19.4 per cent and an estimated false-negative rate of 21.3 per cent. These error rates are small enough to enable calling a causative mutation from both homozygous and heterozygous candidate mutation lists with little or no further experimental validation. The efficacy of this approach is demonstrated by identifying the causative mutation in the Ptprc gene in a lymphocyte-deficient strain and in 11 other strains with immune disorders or obesity, without the need for meiotic mapping. Exome sequencing of first-generation mutant mice revealed hundreds of unphenotyped protein-changing mutations, 52 per cent of which are predicted to be deleterious, which now become available for breeding and experimental analysis. We show that exome sequencing data alone are sufficient to identify induced mutations. This approach transforms genetic screens in mice, establishes a general strategy for analysing rare DNA variants and opens up a large new source for experimental models of human disease.

Systems-guided forward genetic screen reveals a critical role of the replication stress response protein ETAA1 in T cell clonal expansion

“…Etaa1 ΔEx2/ΔEx2 and Etaa1 C166X/C166X mice were chosen from a list of random single-nucleotide variants generated by ENU mutagenesis (databases.apf.edu.au/mutations) and bred to homozygosity at generation 3 (17). C57BL/6 NCrl (C57BL/6), B6.SJL-Ptprc a Pepc b /BoyJ (CD45.1), and B6.129S7-Rag1 tm1Mom /J (Rag1 −/− ) mice were purchased from the Australian Phenomics Facility.…”

“…A systems-guided forward genetics screen was conducted on de novo heterozygous point mutations revealed by exome sequencing of hundreds of G1 offspring of ENU-treated C57BL/6 male mice (16,17). G1 mouse IGL1100 had an Etaa1 point mutation in the intron between exons 2 and 3, predicted to be damaging because it substituted a C nucleotide for the essential +2 T of the splice donor consensus sequence (Fig.…”

“…Because Etaa1 mRNA was most abundant in T and B lymphocytes among all mouse tissues except embryonic stem cells (BioGPS data for probe 1453064_at; biogps.org) (15) and encoded part of the "dark proteome," it was a good candidate to test for immune system phenotypes in a systems-guided forward genetics screen. The screen starts by exome sequence analysis to identify large numbers of heterozygous de novo point mutations in the mouse genome induced at random in first-generation (G1) offspring of inbred C57BL/6 mice treated with ethylnitrosourea (ENU) (16,17). Computational filtering then identifies mutations that are predicted to be damaging in genes of unknown function, focusing on genes with indirect systems-level evidence of a role in the immune system.…”

Mouse Genome Database: From sequence to phenotypes and disease models

“…These include the International Gene Trap Consortium, producing ≈190,000 gene-trapped ES cell lines, (Nord et al ., 2006 1 ), a number of N-ethyl-N-nitrosourea (ENU)-mutagenesis projects producing over 3,300 defined mutations (for example, see Andrews et al ., 2012; Boles et al , 2009; Clark et al ., 2004), and the International Knockout Mouse Consortium (IKMC) generating targeted mutations in all mouse protein-coding genes in ES cell lines, with over 14,000 genes successfully targeted to date (Bradley et al ., 2012). Genetic engineering methodologies including CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated sequences) engineering (Wang et al ., 2013) and elegant, technologies that allow spatiotemporal control of gene ablation and regulation are providing new insight into gene function through the use of conditional mutagenesis (Murray et al ., 2012).…”

Can the impact of human genetic variations be predicted?

“…In a comprehensive study, Miosge et al (6) studied homozygous mice after mutagenesis by N-ethyl-N-nitrosourea (ENU). They focused on 30 missense mutations in 23 immunity genes, each found in a single mouse substrain (8). These genes were selected because other mice, homozygous for null mutations in these genes, have a clear immunological phenotype.…”