Limits...
Genome-wide ENU mutagenesis in combination with high density SNP analysis and exome sequencing provides rapid identification of novel mouse models of developmental disease.

Caruana G, Farlie PG, Hart AH, Bagheri-Fam S, Wallace MJ, Dobbie MS, Gordon CT, Miller KA, Whittle B, Abud HE, Arkell RM, Cole TJ, Harley VR, Smyth IM, Bertram JF - PLoS ONE (2013)

Bottom 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne, Australia. georgina.caruana@monash.edu

ABSTRACT

Background: Mice 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 findings: ENU 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/significance: In 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.

Show MeSH

Related in: MedlinePlus

Linkage Analysis.(A) Based on the phenotype, a targeted approach was used to establish linkage to Fras1 in the bfb strain. Polymorphic markers flanking the four previously characterised blebs genes (open boxes) were used to identify a candidate for sequencing. Strong linkage was observed with the Fras1 gene (grey box). (B) A genome-wide scan established linkage to chromosome 17 in the cauli strain. Chromosome 17-specific markers were then used to refine linkage to a 7 Mb region between rs3667809 and rs3684506 (grey boxed region) containing 218 genes. No additional informative markers were available to further refine the linkage region. (C) A genome-wide scan established linkage to chromosome 7 in the 12BCC-22a line. Subsequent analysis using chromosome 7-specific markers refined linkage to a 32 Mb region containing approximately 700 genes. Numbers to the left in each diagram denote individual mutant embryos except in (C) where both mutant and unaffected embryos are shown. Black boxes denote homozygous C57BL/6 alleles, grey boxes denote C57BL6/C3H heterozygosity, white boxes denote untested markers.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585849&req=5

pone-0055429-g003: Linkage Analysis.(A) Based on the phenotype, a targeted approach was used to establish linkage to Fras1 in the bfb strain. Polymorphic markers flanking the four previously characterised blebs genes (open boxes) were used to identify a candidate for sequencing. Strong linkage was observed with the Fras1 gene (grey box). (B) A genome-wide scan established linkage to chromosome 17 in the cauli strain. Chromosome 17-specific markers were then used to refine linkage to a 7 Mb region between rs3667809 and rs3684506 (grey boxed region) containing 218 genes. No additional informative markers were available to further refine the linkage region. (C) A genome-wide scan established linkage to chromosome 7 in the 12BCC-22a line. Subsequent analysis using chromosome 7-specific markers refined linkage to a 32 Mb region containing approximately 700 genes. Numbers to the left in each diagram denote individual mutant embryos except in (C) where both mutant and unaffected embryos are shown. Black boxes denote homozygous C57BL/6 alleles, grey boxes denote C57BL6/C3H heterozygosity, white boxes denote untested markers.

Mentions: Line 11BC-5 was originally identified as a strain that exhibited isolated cleft secondary palate at E18.5. Additional screening revealed a range of phenotypes including blood filled blisters on the head and feet at E13.5 and preaxial polydactyly, open eye lids (Figure 2A–D) and renal agenesis (data not shown) at E18.5. Based on the phenotype observed in E13.5 embryos, this strain was named blood filled blisters (bfb). This collection of phenotypic features is characteristic of the blebs mutants that arise following mutation of Fras1, Frem 1, 2 and Grip1[35]. Targeted SNP analysis using markers polymorphic between C57BL/6 and C3H were used to investigate the possibility of linkage of the bfb phenotype to one of these four blebs genes using a cohort of 9 phenotypically mutant embryos. Markers flanking Frem1, Frem2 and Grip1 exhibited mixed genotypes (Figure 3A). In contrast, markers flanking Fras1 were homozygous for the C57BL/6 allele in 9/9 samples, indicating clear linkage of the bfb phenotype to Fras1. Sequencing of all 75 exons and flanking sequences from genomic DNA of a single mutant identified a 10762T>C substitution (open reading frame of NCBI RefSeq transcript NM_175473.3) causing a Ser3588Pro mutation in FRAS1. PolyPhen-2 analysis predicts that this change is “probably damaging” and SIFT predicts that it is “not tolerated”.


Genome-wide ENU mutagenesis in combination with high density SNP analysis and exome sequencing provides rapid identification of novel mouse models of developmental disease.

Caruana G, Farlie PG, Hart AH, Bagheri-Fam S, Wallace MJ, Dobbie MS, Gordon CT, Miller KA, Whittle B, Abud HE, Arkell RM, Cole TJ, Harley VR, Smyth IM, Bertram JF - PLoS ONE (2013)

Linkage Analysis.(A) Based on the phenotype, a targeted approach was used to establish linkage to Fras1 in the bfb strain. Polymorphic markers flanking the four previously characterised blebs genes (open boxes) were used to identify a candidate for sequencing. Strong linkage was observed with the Fras1 gene (grey box). (B) A genome-wide scan established linkage to chromosome 17 in the cauli strain. Chromosome 17-specific markers were then used to refine linkage to a 7 Mb region between rs3667809 and rs3684506 (grey boxed region) containing 218 genes. No additional informative markers were available to further refine the linkage region. (C) A genome-wide scan established linkage to chromosome 7 in the 12BCC-22a line. Subsequent analysis using chromosome 7-specific markers refined linkage to a 32 Mb region containing approximately 700 genes. Numbers to the left in each diagram denote individual mutant embryos except in (C) where both mutant and unaffected embryos are shown. Black boxes denote homozygous C57BL/6 alleles, grey boxes denote C57BL6/C3H heterozygosity, white boxes denote untested markers.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585849&req=5

pone-0055429-g003: Linkage Analysis.(A) Based on the phenotype, a targeted approach was used to establish linkage to Fras1 in the bfb strain. Polymorphic markers flanking the four previously characterised blebs genes (open boxes) were used to identify a candidate for sequencing. Strong linkage was observed with the Fras1 gene (grey box). (B) A genome-wide scan established linkage to chromosome 17 in the cauli strain. Chromosome 17-specific markers were then used to refine linkage to a 7 Mb region between rs3667809 and rs3684506 (grey boxed region) containing 218 genes. No additional informative markers were available to further refine the linkage region. (C) A genome-wide scan established linkage to chromosome 7 in the 12BCC-22a line. Subsequent analysis using chromosome 7-specific markers refined linkage to a 32 Mb region containing approximately 700 genes. Numbers to the left in each diagram denote individual mutant embryos except in (C) where both mutant and unaffected embryos are shown. Black boxes denote homozygous C57BL/6 alleles, grey boxes denote C57BL6/C3H heterozygosity, white boxes denote untested markers.
Mentions: Line 11BC-5 was originally identified as a strain that exhibited isolated cleft secondary palate at E18.5. Additional screening revealed a range of phenotypes including blood filled blisters on the head and feet at E13.5 and preaxial polydactyly, open eye lids (Figure 2A–D) and renal agenesis (data not shown) at E18.5. Based on the phenotype observed in E13.5 embryos, this strain was named blood filled blisters (bfb). This collection of phenotypic features is characteristic of the blebs mutants that arise following mutation of Fras1, Frem 1, 2 and Grip1[35]. Targeted SNP analysis using markers polymorphic between C57BL/6 and C3H were used to investigate the possibility of linkage of the bfb phenotype to one of these four blebs genes using a cohort of 9 phenotypically mutant embryos. Markers flanking Frem1, Frem2 and Grip1 exhibited mixed genotypes (Figure 3A). In contrast, markers flanking Fras1 were homozygous for the C57BL/6 allele in 9/9 samples, indicating clear linkage of the bfb phenotype to Fras1. Sequencing of all 75 exons and flanking sequences from genomic DNA of a single mutant identified a 10762T>C substitution (open reading frame of NCBI RefSeq transcript NM_175473.3) causing a Ser3588Pro mutation in FRAS1. PolyPhen-2 analysis predicts that this change is “probably damaging” and SIFT predicts that it is “not tolerated”.

Bottom 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne, Australia. georgina.caruana@monash.edu

ABSTRACT

Background: Mice 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 findings: ENU 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/significance: In 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.

Show MeSH
Related in: MedlinePlus