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A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen.

ABSTRACT: A genome-wide ethyl-N-nitrosourea (ENU) mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here, we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the Cpox gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX). A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red blood cell phenotype in heterozygous mice. Urinary and faecal porphyrins in female RBC16 heterozygotes were significantly elevated compared with that of wild-type littermates, particularly coproporphyrinogen III, whereas males were biochemically normal. Attempts to induce acute porphyric crises were made using fasting and phenobarbital treatment on females. While fasting had no biochemical effect on RBC16 mice, phenobarbital caused significant elevation of faecal coproporphyrinogen III in heterozygous mice. This is the first known investigation of a mutagenesis mouse model with genetic and biochemical parallels to hereditary coproporphyria.

SUBMITTER: Conway AJ

PROVIDER: S-EPMC5560062 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen.

Conway Ashlee J AJ Brown Fiona C FC Fullinfaw Robert O RO Kile Benjamin T BT Jane Stephen M SM Jane Stephen M SM Curtis David J DJ

Disease models & mechanisms 20170609 8

A genome-wide ethyl-N-nitrosourea (ENU) mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here, we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the <i>Cpox</i> gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX). A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red blo ...[more]

PMID: 28600349

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Project description:Despite considerable progress understanding genes that affect the HDL particle, its function, and cholesterol content, genes identified to date explain only a small percentage of the genetic variation. We used N-ethyl-N-nitrosourea mutagenesis in mice to discover novel genes that affect HDL cholesterol levels. Two mutant lines (Hlb218 and Hlb320) with low HDL cholesterol levels were established. Causal mutations in these lines were mapped using linkage analysis: For line Hlb218 within a 12 Mbp region on Chr 10; and for line Hlb320 within a 17 Mbp region on Chr 7. High-throughput sequencing of Hlb218 liver RNA identified a mutation in Pla2g12b. The transition of G to A leads to a cysteine to tyrosine change and most likely causes a loss of a disulfide bridge. Microarray analysis of Hlb320 liver RNA showed a 7-fold downregulation of Hpn; sequencing identified a mutation in the 3M-bM-^@M-2 splice site of exon 8. Northern blot confirmed lower mRNA expression level in Hlb320 and did not show a difference in splicing, suggesting that the mutation only affects the splicing rate. In addition to affecting HDL cholesterol, the mutated genes also lead to reduction in serum non-HDL cholesterol and triglyceride levels. Despite low HDL cholesterol levels, the mice from both mutant lines show similar atherosclerotic lesion sizes compared to control mice. These new mutant mouse models are valuable tools to further study the role of these genes, their affect on HDL cholesterol levels, and metabolism. Mutant mice were generated as part of The Jackson LaboratoryM-bM-^@M-^Ys Heart, Lung, Blood, and Sleep Disorder Mutagenesis Program by treating male C57BL/6J (B6) mice with N-ethyl-N-nitrosourea (ENU). Third generation (G3) mice were phenotyped to ensure capture of both dominant and recessive mutations. Two unique G3 animals with low HDL cholesterol levels were then used to establish new inbred lines (Hlb218 and Hlb320) by mating them with B6 mice and intercrossing the offspring with low HDL cholesterol for 7 generations. Livers from 3 Hlb218, 3 Hlb320 males, and 6 B6 male controls were obtained for gene expression analysis. The samples were randomized over Illumina Mouse-6 Expression 1.1 BeadChips .

Dataset Information

A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen.

Publications

A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen.

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