Discovery of candidate disease genes in ENU-induced mouse mutants by large-scale sequencing, including a splice-site mutation in nucleoredoxin

Abstract

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.

Figure 1. Synteny map of mouse Chromosome 11.

Mouse Chromosome 11 is aligned with human chromosomes 22, 7, 2, 5, 1, and 17. Syntenic regions are represented in the same color and lines indicate breaks of synteny. The mouse Chromosome 11 ENU mutagenesis screen was targeted to the Trp53-Wnt3 (69 Mb–103.5 Mb) interval within the region most conserved with human Chromosome 17, indicated by the black lines.

Figure 2. Conservation analysis of sequence surrounding ENU–induced lesions.

A graph of match scores shows that exons and some non-coding elements are highly conserved. The match score is based on a 100 base pair comparison across seven vertebrates: mouse, human, rat, Rhesus monkey, horse, dog, and chicken. Red = lesion occurred in an exon, blue = lesion occurred in a 5′ or 3′ UTR, green = lesion occurred in an intron, and yellow = lesion occurred downstream of a gene.

Figure 3. Mutation of nucleoredoxin in l11Jus13 (Nxn^J13/J13).

(A) Haplotype map of 239 unaffected and 83 affected mice used for meiotic mapping. The location of each marker and Nxn is displayed (Ensembl v52). The mutation lies in a 6Mb region located between rs3702197 and rs13481117. (B) The Nxn locus is depicted with introns 6 and 7 boxed. (C) The boxed region is expanded to illustrate the consequence of splicing in the wild type and mutant. A transversion (T to A) abolishes a consensus splice donor site leading to aberrant RNA splicing. The six base pair cryptic splice site used in the mutant is underlined. (D) Aberrant splicing in Nxn^J13/J13 predicts an in frame insertion of 10 amino acids, GMELEGKWKA, (white), which occurs within the acidic region (black) of Nxn. (E) RT–PCR using primers flanking exon 6–7 from pools of three E14.5 heads of wild-type, heterozygous, and homozygous mutants demonstrates aberrant splicing in the mutant allele. (F) Western blots of Nxn and Actin from wild-type, heterozygous and homozygous mutants at E12.5 show reduced protein in the homozygous mutant. A reduction in protein was also observed at E15.5 and E18.5, and a polyclonal antibody against the N-terminus gave similar results (data not shown).

Figure 4. Nxn^J13/J13 mutants have cleft palates and small mandibles.

Homozygous Nxn^J13/J13 embryos were compared to Nxn^J13+/+Inv control littermates. (A) Gross morphology at E18.5. Nxn^J13/J13 mutants (right) have a shortened snout compared to control littermates (left). (B) The palate (inside the dotted lines) was examined at E18.5, and a cleft palate (yellow arrow) occurred in Nxn^J13/J13 embryos (right) but not in control littermates (left). (C) Skeletal preparations were carried out at E18.5. The mandible is shorter in length in Nxn^J13/J13 embryos (bottom) than in control littermates (top). (D) The mean and standard error are plotted to show the difference in mandible length. Mutant mandibles are significantly (p<0.001) shorter than controls (n = 10 mandibles per genotype).