title: Rapid identification of a disease allele in mouse through whole genome sequencing and bulk segregation analysis.
abstract: In a pedigree of C57BL/6J mice homozygous for germline mutations induced by the mutagen N-ethyl-N-nitrosourea (ENU), numerous animals died under specific pathogen-free (SPF) conditions between 6 and 7 months of age. Death was caused by nephritic syndrome, which progressed to renal failure associated with focal segmental glomerulosclerosis. To identify the mutation responsible for renal disease, we sequenced genomic DNA from an affected animal using the Applied Biosystems SOLiD sequencing platform. Approximately 74% of the nucleotides comprising coding sequences and splice junctions in the mouse genome were covered at least three times. Within this portion of the genome, 64 discrepancies were flagged as potential homozygous mutations and 82 were flagged as potential heterozygous mutations. A total of 10 of these calls, all homozygous, were validated by capillary sequencing. One of the validated mutations disrupted splicing of the Col4a4 transcript. Genetic mapping by bulk segregation analysis excluded all mutations but this one as the cause of renal disease in Aoba mice. Col4a4 has not been targeted in the mouse, and this strain, named Aoba, represents the first functionally null allele in this species. Our study demonstrates the speed and utility of whole genome sequencing coupled with low resolution meiotic mapping as a means of identifying causative mutations induced by ENU.
authors.affiliation: Department of Genetics, Institute for Childhood and Neglected Disease, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Carrie N
authors.initials: CN
authors.lastname: Arnold
authors.forename: Yu
authors.initials: Y
authors.lastname: Xia
authors.forename: Pei
authors.initials: P
authors.lastname: Lin
authors.forename: Charles
authors.initials: C
authors.lastname: Ross
authors.forename: Martin
authors.initials: M
authors.lastname: Schwander
authors.forename: Nora G
authors.initials: NG
authors.lastname: Smart
authors.forename: Ulrich
authors.initials: U
authors.lastname: Müller
authors.forename: Bruce
authors.initials: B
authors.lastname: Beutler
mesh_terms: D000483:Alleles; D000818:Animals; D002874:Chromosome Mapping; D020090:Chromosome Segregation; D024141:Collagen Type IV / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005038:Ethylnitrosourea / Q000633:toxicity*; D016678:Genome; D055106:Genome-Wide Association Study / Q000379:methods*; D018095:Germ-Line Mutation / Q000235:genetics*; D005923:Glomerulosclerosis, Focal Segmental / Q000235:genetics; D006579:Heterozygote; D006720:Homozygote; D051379:Mice; D008810:Mice, Inbred C57BL; D011507:Proteinuria / Q000139:chemically induced / Q000235:genetics; D022821:RNA Splice Sites; D051437:Renal Insufficiency / Q000139:chemically induced / Q000235:genetics*
chemical_list: D024141:Collagen Type IV; D022821:RNA Splice Sites; D005038:Ethylnitrosourea