Genome-Wide ENU Mutagenesis in Combination with High Density SNP Analysis and Exome Sequencing Provides Rapid Identification of Novel Mouse Models of Developmental Disease

Abstract

BackgroundMice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU).Methodology/Principal FindingsENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1. Conclusions/SignificanceIn this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

A Mouse Splice-Site Mutant and Individuals with Atypical Chromosome 22q11.2 Deletions Demonstrate the Crucial Role for Crkl in Craniofacial and Pharyngeal Development

“…The identification of snoopy embryos from a large-scale ENU mutagenesis programme has been reported previously [Caruana et al, 2013]. All mouse procedures were approved by the Royal Children's Hospital Animal Ethics Committee, RCH AEEC No.…”

“…Generation of snoopy progeny, DNA extraction from affected snoopy embryos and exome enrichment was performed as described previously [Andrews et al, 2012;Caruana et al, 2013].…”

“…We have identified an ethylnitrosourea (ENU) mutant mouse strain, which we named 'snoopy' [Caruana et al, 2013], harbouring a point mutation in the Crkl gene. The snoopy strain exhibits severe craniofacial malformation, including maxillary and mandibular hypoplasia, microstomia, aglossia, pharyngeal occlusion, and holoprosencephaly with markedly variable expressivity and reduced penetrance.…”

A Novel Ataxic Mutant Mouse Line Having Sensory Neuropathy Shows Heavy Iron Deposition in Kidney

“…Chemical mutagenesis combined with exome sequencing has been performed to generate novel mouse models [3,4,5]. The most widely used chemical mutagen is N-ethyl-N-nitrosourea (ENU).…”

“…Andrews et al [4] applied exome sequencing to identify a causative mutation of ENU-mutated mice and showed that exome sequencing data alone were sufficient to identify induced mutations. Because 99% of ENU mutations occur in splice sites and exons, the exome sequencing should identify almost all ENU-induced mutations [5]. …”