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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.

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Related in: MedlinePlus

Anaemia in Lig1 mutants.(A) A Lig1 mutant (mut) with pale orange liver (arrow) compared to the red haemoglobin rich liver of a wildtype (wt) littermate. (B, C) Analysis of peripheral blood in E13.5 embryos by cytospin and Giemsa staining (x40). (B) Lig1 mutant (mut) peripheral blood shows a high proportion of nucleated erythroblasts (arrow) to enucleated erythroblasts (arrowhead) compared with (C) a wild type (wt) littermate.
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pone-0055429-g004: Anaemia in Lig1 mutants.(A) A Lig1 mutant (mut) with pale orange liver (arrow) compared to the red haemoglobin rich liver of a wildtype (wt) littermate. (B, C) Analysis of peripheral blood in E13.5 embryos by cytospin and Giemsa staining (x40). (B) Lig1 mutant (mut) peripheral blood shows a high proportion of nucleated erythroblasts (arrow) to enucleated erythroblasts (arrowhead) compared with (C) a wild type (wt) littermate.

Mentions: Based on these criteria we screened embryos for pale livers and identified E13.5 G3 embryos with visible anaemia in the liver in line 12BCC-22a (Figure 4A). Giemsa staining of peripheral blood verified that the embryos lacked mature enucleated red blood cells (RBC) (Figure 4 B, C). The gene responsible for this blood defect was localised to a 32.6 Mb region on the proximal end of chromosome 7 (rs4226386–rs3724525) (Figure 3 C). This region contained approximately 700 genes. Sequencing of all exons in this region using NGS identified a 1798A>T substitution in DNA ligase 1 (Lig1) (open reading frame of NCBI RefSeq transcript NM_001199310.1) resulting in an Ile600Phe mutation. The structure of Human LIG1 has been solved and we used this to predict that Ile 600 is on the surface that contacts DNA during DNA binding and repair [42]. This mutation alters the charge of this amino acid in the DNA binding and ATP dependant DNA ligase domain of LIG1. This may affect the ability of LIG1 to catalyse the joining of single strand DNA breaks during DNA replication, repair and recombination.


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)

Anaemia in Lig1 mutants.(A) A Lig1 mutant (mut) with pale orange liver (arrow) compared to the red haemoglobin rich liver of a wildtype (wt) littermate. (B, C) Analysis of peripheral blood in E13.5 embryos by cytospin and Giemsa staining (x40). (B) Lig1 mutant (mut) peripheral blood shows a high proportion of nucleated erythroblasts (arrow) to enucleated erythroblasts (arrowhead) compared with (C) a wild type (wt) littermate.
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Related In: Results  -  Collection

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pone-0055429-g004: Anaemia in Lig1 mutants.(A) A Lig1 mutant (mut) with pale orange liver (arrow) compared to the red haemoglobin rich liver of a wildtype (wt) littermate. (B, C) Analysis of peripheral blood in E13.5 embryos by cytospin and Giemsa staining (x40). (B) Lig1 mutant (mut) peripheral blood shows a high proportion of nucleated erythroblasts (arrow) to enucleated erythroblasts (arrowhead) compared with (C) a wild type (wt) littermate.
Mentions: Based on these criteria we screened embryos for pale livers and identified E13.5 G3 embryos with visible anaemia in the liver in line 12BCC-22a (Figure 4A). Giemsa staining of peripheral blood verified that the embryos lacked mature enucleated red blood cells (RBC) (Figure 4 B, C). The gene responsible for this blood defect was localised to a 32.6 Mb region on the proximal end of chromosome 7 (rs4226386–rs3724525) (Figure 3 C). This region contained approximately 700 genes. Sequencing of all exons in this region using NGS identified a 1798A>T substitution in DNA ligase 1 (Lig1) (open reading frame of NCBI RefSeq transcript NM_001199310.1) resulting in an Ile600Phe mutation. The structure of Human LIG1 has been solved and we used this to predict that Ile 600 is on the surface that contacts DNA during DNA binding and repair [42]. This mutation alters the charge of this amino acid in the DNA binding and ATP dependant DNA ligase domain of LIG1. This may affect the ability of LIG1 to catalyse the joining of single strand DNA breaks during DNA replication, repair and recombination.

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