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Hum Mutat. 2019 Apr;40(4):426-443. doi: 10.1002/humu.23706. Epub 2019 Jan 25.

Aberrant RNA splicing is the major pathogenic effect in a knock-in mouse model of the dominantly inherited c.1430A>G human RPE65 mutation.

Author information

1
Laboratory of Retinal Cell & Molecular Biology, National Eye Institute, NIH, Bethesda, Maryland.
2
Neurobiology-Neurodegeneration & Repair Laboratory, National Eye Institute, NIH, Bethesda, Maryland.
3
Visual Function Core, National Eye Institute, NIH, Bethesda, Maryland.
4
Genetic Engineering Core, National Eye Institute, NIH, Bethesda, Maryland.
5
National Center for Biotechnology Information, National Library of Medicine, NIH, Bethesda, Maryland.

Abstract

Human RPE65 mutations cause a spectrum of retinal dystrophies that result in blindness. While RPE65 mutations have been almost invariably recessively inherited, a c.1430A>G (p.(D477G)) mutation has been reported to cause autosomal dominant retinitis pigmentosa (adRP). To study the pathogenesis of this human mutation, we have replicated the mutation in a knock-in (KI) mouse model using CRISPR/Cas9-mediated genome editing. Significantly, in contrast to human patients, heterozygous KI mice do not exhibit any phenotypes in visual function tests. When raised in regular vivarium conditions, homozygous KI mice display relatively undisturbed visual functions with minimal retinal structural changes. However, KI/KI mouse retinae are more sensitive to light exposure and exhibit signs of degenerative features when subjected to light stress. We find that instead of merely producing a missense mutant protein, the A>G nucleotide substitution greatly affects appropriate splicing of Rpe65 mRNA by generating an ectopic splice site in comparable context to the canonical one, thereby disrupting RPE65 protein expression. Similar splicing defects were also confirmed for the human RPE65 c.1430G mutant in an in vitro Exontrap assay. Our data demonstrate that a splicing defect is associated with c.1430G pathogenesis, and therefore provide insights in the therapeutic strategy for human patients.

KEYWORDS:

RNA splicing; RPE65; autosomal dominant; autosomal recessive retinitis pigmentosa; retina; retinal pigment epithelium

PMID:
30628748
PMCID:
PMC6425930
[Available on 2020-04-01]
DOI:
10.1002/humu.23706

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