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Mutations in zebrafish lrp2 result in adult-onset ocular pathogenesis that models myopia and other risk factors for glaucoma.

Veth KN, Willer JR, Collery RF, Gray MP, Willer GB, Wagner DS, Mullins MC, Udvadia AJ, Smith RS, John SW, Gregg RG, Link BA - PLoS Genet. (2011)

Bottom Line: Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes.This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis.Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

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Both bugeyemw1 and bugeyep5bnc mutants have non-sense mutations in lrp2.A Genetic and corresponding physical map of the critical interval for bugeyemw1 and bugeyep5bnc locus on chromosome 9. Associated number of recombination events per 270 meioses are shown for each polymorphic marker. SSR, Simple sequence repeat. B Sequence comparisons of lrp2 revealed distinct non-sense mutations in bugeyemw1 and bugeyep5bnc. In mw1, the cysteine at amino acid position 23 is changed to a stop codon by a T>A mutation; in p5bnc, the glutamine at 413 is changed to a stop by a C>T mutation. In both, heterozygous genotypes show both alleles. C Model of Lrp2 protein structural domains, with the locations of the identified mutations indicated by arrows. The bulk of the protein is extracellular with ligand binding domains, while the intracellular domain contains an NPXY endocytosis sequence motif. D-E Immunostaining for Lrp2 in 56-hpf pigmentation-blocked embryos. Lrp2 immunoreactivity was robust in the retina pigmented epithelium (RPE) of wild-types (D), but absent in bugeye embryos (E). Insets in D and E are magnified in D' and E'. Scale bars  = 25 µm; circles show placement of the lenses. F Images of ethidium bromide stained agarose gels show restriction fragment length polymorphism (RFLP) genotypes: homozygous mutant (−/−), heterozygote (+/−) and wild-type (+/+) genotype for each mutation.
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pgen-1001310-g002: Both bugeyemw1 and bugeyep5bnc mutants have non-sense mutations in lrp2.A Genetic and corresponding physical map of the critical interval for bugeyemw1 and bugeyep5bnc locus on chromosome 9. Associated number of recombination events per 270 meioses are shown for each polymorphic marker. SSR, Simple sequence repeat. B Sequence comparisons of lrp2 revealed distinct non-sense mutations in bugeyemw1 and bugeyep5bnc. In mw1, the cysteine at amino acid position 23 is changed to a stop codon by a T>A mutation; in p5bnc, the glutamine at 413 is changed to a stop by a C>T mutation. In both, heterozygous genotypes show both alleles. C Model of Lrp2 protein structural domains, with the locations of the identified mutations indicated by arrows. The bulk of the protein is extracellular with ligand binding domains, while the intracellular domain contains an NPXY endocytosis sequence motif. D-E Immunostaining for Lrp2 in 56-hpf pigmentation-blocked embryos. Lrp2 immunoreactivity was robust in the retina pigmented epithelium (RPE) of wild-types (D), but absent in bugeye embryos (E). Insets in D and E are magnified in D' and E'. Scale bars  = 25 µm; circles show placement of the lenses. F Images of ethidium bromide stained agarose gels show restriction fragment length polymorphism (RFLP) genotypes: homozygous mutant (−/−), heterozygote (+/−) and wild-type (+/+) genotype for each mutation.

Mentions: To map the mutant locus, progeny from single pair backcross matings were used for whole-genome linkage analysis. Co-segregation for markers on chromosome 9 and the mutant phenotype was found (Figure 2A). Informatively, no other linkage in the genome was noted, consistent with the single recessive causative mutation hypothesis. Public databases revealed that the lrp2 gene was within the critical recombinant interval. Given the similarity of the bugeye phenotype to those caused by LRP2 mutations in humans, we sequenced this candidate gene. Analysis of lrp2 cDNA from bugeyemw1 mutants revealed a T to A conversion that changes a cysteine to a stop codon at predicted amino acid position 23 (C23X) (Figure 2B, 2C). Through an independent genetic screen we identified a second large eye mutant that like the bugeyemw1 allele, presented in adulthood and showed reduced penetrance. Intercrosses between this mutant (allele p5bnc) and bugeyemw1 were non-complementing and suggested that lrp2 may also be affected in the p5bnc mutant. Indeed, sequencing of p5bnc cDNA revealed a separate non-sense mutation, also very early in the coding region of lrp2 (bugeyep5bnc, Q413X) (Figure 2B, 2C). To test whether somatic reversion or alternate splicing around the non-sense mutations might underlie the reduced penetrance or variability often observed between the left and right eyes, we sequenced ocular cDNA in affected and unaffected eyes. However, we did not find evidence of mosaicism or alternate splicing surrounding the mutations, suggesting the penetrance and phenotype variability is influenced by other genes, epigenetics, and/or unpredictable changes in physiology which affects the phenotypes.


Mutations in zebrafish lrp2 result in adult-onset ocular pathogenesis that models myopia and other risk factors for glaucoma.

Veth KN, Willer JR, Collery RF, Gray MP, Willer GB, Wagner DS, Mullins MC, Udvadia AJ, Smith RS, John SW, Gregg RG, Link BA - PLoS Genet. (2011)

Both bugeyemw1 and bugeyep5bnc mutants have non-sense mutations in lrp2.A Genetic and corresponding physical map of the critical interval for bugeyemw1 and bugeyep5bnc locus on chromosome 9. Associated number of recombination events per 270 meioses are shown for each polymorphic marker. SSR, Simple sequence repeat. B Sequence comparisons of lrp2 revealed distinct non-sense mutations in bugeyemw1 and bugeyep5bnc. In mw1, the cysteine at amino acid position 23 is changed to a stop codon by a T>A mutation; in p5bnc, the glutamine at 413 is changed to a stop by a C>T mutation. In both, heterozygous genotypes show both alleles. C Model of Lrp2 protein structural domains, with the locations of the identified mutations indicated by arrows. The bulk of the protein is extracellular with ligand binding domains, while the intracellular domain contains an NPXY endocytosis sequence motif. D-E Immunostaining for Lrp2 in 56-hpf pigmentation-blocked embryos. Lrp2 immunoreactivity was robust in the retina pigmented epithelium (RPE) of wild-types (D), but absent in bugeye embryos (E). Insets in D and E are magnified in D' and E'. Scale bars  = 25 µm; circles show placement of the lenses. F Images of ethidium bromide stained agarose gels show restriction fragment length polymorphism (RFLP) genotypes: homozygous mutant (−/−), heterozygote (+/−) and wild-type (+/+) genotype for each mutation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040661&req=5

pgen-1001310-g002: Both bugeyemw1 and bugeyep5bnc mutants have non-sense mutations in lrp2.A Genetic and corresponding physical map of the critical interval for bugeyemw1 and bugeyep5bnc locus on chromosome 9. Associated number of recombination events per 270 meioses are shown for each polymorphic marker. SSR, Simple sequence repeat. B Sequence comparisons of lrp2 revealed distinct non-sense mutations in bugeyemw1 and bugeyep5bnc. In mw1, the cysteine at amino acid position 23 is changed to a stop codon by a T>A mutation; in p5bnc, the glutamine at 413 is changed to a stop by a C>T mutation. In both, heterozygous genotypes show both alleles. C Model of Lrp2 protein structural domains, with the locations of the identified mutations indicated by arrows. The bulk of the protein is extracellular with ligand binding domains, while the intracellular domain contains an NPXY endocytosis sequence motif. D-E Immunostaining for Lrp2 in 56-hpf pigmentation-blocked embryos. Lrp2 immunoreactivity was robust in the retina pigmented epithelium (RPE) of wild-types (D), but absent in bugeye embryos (E). Insets in D and E are magnified in D' and E'. Scale bars  = 25 µm; circles show placement of the lenses. F Images of ethidium bromide stained agarose gels show restriction fragment length polymorphism (RFLP) genotypes: homozygous mutant (−/−), heterozygote (+/−) and wild-type (+/+) genotype for each mutation.
Mentions: To map the mutant locus, progeny from single pair backcross matings were used for whole-genome linkage analysis. Co-segregation for markers on chromosome 9 and the mutant phenotype was found (Figure 2A). Informatively, no other linkage in the genome was noted, consistent with the single recessive causative mutation hypothesis. Public databases revealed that the lrp2 gene was within the critical recombinant interval. Given the similarity of the bugeye phenotype to those caused by LRP2 mutations in humans, we sequenced this candidate gene. Analysis of lrp2 cDNA from bugeyemw1 mutants revealed a T to A conversion that changes a cysteine to a stop codon at predicted amino acid position 23 (C23X) (Figure 2B, 2C). Through an independent genetic screen we identified a second large eye mutant that like the bugeyemw1 allele, presented in adulthood and showed reduced penetrance. Intercrosses between this mutant (allele p5bnc) and bugeyemw1 were non-complementing and suggested that lrp2 may also be affected in the p5bnc mutant. Indeed, sequencing of p5bnc cDNA revealed a separate non-sense mutation, also very early in the coding region of lrp2 (bugeyep5bnc, Q413X) (Figure 2B, 2C). To test whether somatic reversion or alternate splicing around the non-sense mutations might underlie the reduced penetrance or variability often observed between the left and right eyes, we sequenced ocular cDNA in affected and unaffected eyes. However, we did not find evidence of mosaicism or alternate splicing surrounding the mutations, suggesting the penetrance and phenotype variability is influenced by other genes, epigenetics, and/or unpredictable changes in physiology which affects the phenotypes.

Bottom Line: Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes.This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis.Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals--but not all--develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease.

Show MeSH
Related in: MedlinePlus