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.

Figure 1.—

Elevated blood urea nitrogen levels in Aoba mice. Levels of blood urea nitrogen in wild-type and Aoba mice at the indicated ages. Each point represents the mean ± SEM for 3–10 mice per group, except for the data for 1-month-old mice, which was determined for only one mouse of each genotype.

Figure 2.—

Focal segmental glomerulosclerosis in Aoba mice. Serial kidney sections, 3 μm thick, were stained with PAS (A–C) or Trichrome (D–F) and imaged at ×100 (A and D) or ×200 (B and C, E and F). (A) Pitting of the cortical surface, crowding of glomeruli, tubular atrophy, and dilated tubules in 5-month-old Aoba mouse. (B) Completely sclerosed glomeruli (arrows) in 5-month-old Aoba mouse. (C) Normal staining in 5-month-old wild-type mouse. (D) Extensive collagen deposition and scarring in 5-month-old Aoba mouse. (E) Scarring extending into medulla in 5-month-old Aoba mouse. (F) Normal staining in 5-month-old wild-type mouse.

Figure 3.—

Aoba mice have a mutation in Col4a4. (A) A single nucleotide transition (G→A, marked with black arrowheads above each trace file) destroys the splice donor site in intron 8 of the Col4a4 gene. The alternative splice donor site used in the Aoba Col4a4 gene is marked with a red arrowhead. (B) Reverse transcriptase PCR across the junction between exons 8 and 9 reveals a deletion of eight nucleotides in the Aoba Col4a4 transcript. The normal and altered junctions between exons 8 and 9 are indicated above each trace file. (C) Schematic highlighting the eight nucleotides deleted from the Aoba Col4a4 transcript in yellow. The deletion results in a frameshift error and premature stop codon in Col4a4, indicated by the translated sequences for wild type and Aoba Col4a4. (D) Schematic showing the site of the premature stop codon in the Col4a4 collagenous domain in Aoba mice.

Figure 4.—

Proteinuria was mapped to chromosome 1 using bulk segregation analysis. (A) Homozygous Aoba males were outcrossed to C57BL/10J females, the F₁ progeny were intercrossed, and proteinuria was measured in 4- to 5-month-old F₂ mice. BSA was performed with genomic DNA from 17 mutant and 28 control F₂ mice. The combined LOD score vs. 124 autosomal SNP markers that distinguish C57BL/6J and C57BL/10J mice is shown. The names of the chromosomal markers used for mapping were omitted for clarity, but can be accessed in (Xia et al. 2010). (B) Individual mutant and control F₂ mice were genotyped for the C57BL/6J and C57BL/10J alleles of the peak markers on chromosome 1. The C57BL/6J and C57BL/10J alleles are represented in black or white, respectively, and heterozygosity is represented by striped boxes.

Figure 5.—

Sensorineural hearing loss in Aoba mice. (A) Representative ABR recordings in response to click stimuli in a 5-month-old wild-type mouse and two 5-month-old Aoba mice, one of which (mutant 1) had a normal ABR threshold and one of which (mutant 2) had an elevated ABR threshold. (B) Scatter plot showing the ABR thresholds for individual 5-month-old wild-type and Aoba mice on a pure C57BL/6J background. Each point represents data from one mouse, and the bar indicates the mean of all values.