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

Area of Science:

• Genetics and Molecular Biology
• Ophthalmology
• Biochemistry

Background:

• Mutations in the RPE65 gene are a known cause of inherited retinal dystrophies leading to blindness.
• While typically recessive, a specific RPE65 mutation (c.1430A>G) has been linked to autosomal dominant retinitis pigmentosa (adRP).

Purpose of the Study:

• To investigate the pathogenic mechanism of the human RPE65 c.1430A>G mutation using a mouse model.
• To determine if the mutation causes disease through altered protein function or other molecular mechanisms.

Main Methods:

• Generation of a knock-in (KI) mouse model harboring the RPE65 c.1430A>G mutation using CRISPR/Cas9.
• Phenotypic analysis of heterozygous and homozygous KI mice, including visual function tests and retinal structural examination.
• Investigation of RPE65 mRNA splicing and protein expression in KI mice and via an in vitro Exontrap assay for the human mutation.

Main Results:

• Heterozygous KI mice showed no discernible visual or structural defects.
• Homozygous KI mice exhibited heightened sensitivity to light stress, with retinal degeneration observed under these conditions.
• The c.1430A>G mutation was found to induce aberrant RPE65 mRNA splicing by creating an ectopic splice site, disrupting normal RPE65 protein production.

Conclusions:

• The RPE65 c.1430G mutation's pathogenicity in humans is primarily due to a splicing defect, not solely a missense protein alteration.
• This discovery provides critical insights into the molecular basis of dominant retinitis pigmentosa linked to this mutation.
• Understanding the splicing defect mechanism opens avenues for targeted therapeutic strategies for patients with this specific RPE65 mutation.