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1.
PLoS Genet ; 18(6): e1009798, 2022 06.
Article in English | MEDLINE | ID: mdl-35675330

ABSTRACT

Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1rd8/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1rd8 allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. At one month of age, Müller glia and microglia mislocalization at dysplastic lesions in both modifier strains was similar to that in B6.Cg-Crb1rd8/Pjn mice but photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1rd8/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms in humans.


Subject(s)
Nerve Tissue Proteins , Retinal Dysplasia , Rho Guanine Nucleotide Exchange Factors , Animals , Disease Models, Animal , Eye Proteins/genetics , Eye Proteins/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Protein Kinase C/genetics , Protein Kinase C/metabolism , Retina/metabolism , Retinal Degeneration/genetics , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Retinal Dysplasia/genetics , Retinal Dysplasia/metabolism , Retinal Dysplasia/pathology , Rho Guanine Nucleotide Exchange Factors/genetics , Rho Guanine Nucleotide Exchange Factors/metabolism
2.
Int J Mol Sci ; 23(4)2022 Feb 17.
Article in English | MEDLINE | ID: mdl-35216333

ABSTRACT

Fluid and solute transporters of the retinal pigment epithelium (RPE) are core components of the outer blood-retinal barrier. Characterizing these transporters and their role in retinal homeostasis may provide insights into ocular function and disease. Here, we describe RPE defects in tvrm77 mice, which exhibit hypopigmented patches in the central retina. Mapping and nucleotide sequencing of tvrm77 mice revealed a disrupted 5' splice donor sequence in Slc4a5, a sodium bicarbonate cotransporter gene. Slc4a5 expression was reduced 19.7-fold in tvrm77 RPE relative to controls, and alternative splice variants were detected. SLC4A5 was localized to the Golgi apparatus of cultured human RPE cells and in apical and basal membranes. Fundus imaging, optical coherence tomography, microscopy, and electroretinography (ERG) of tvrm77 mice revealed retinal detachment, hypopigmented patches corresponding to neovascular lesions, and retinal folds. Detachment worsened and outer nuclear layer thickness decreased with age. ERG a- and b-wave response amplitudes were initially normal but declined in older mice. The direct current ERG fast oscillation and light peak were reduced in amplitude at all ages, whereas other RPE-associated responses were unaffected. These results link a new Slc4a5 mutation to subretinal fluid accumulation and altered light-evoked RPE electrophysiological responses, suggesting that SLC4A5 functions at the outer blood-retinal barrier.


Subject(s)
Mutation/genetics , RNA Splicing/genetics , Retina/pathology , Retinal Detachment/genetics , Retinal Pigment Epithelium/pathology , Sodium-Bicarbonate Symporters/genetics , Animals , Cells, Cultured , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Inbred C57BL , Retinal Detachment/pathology , Tomography, Optical Coherence/methods
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