A spontaneously occurring mouse Rp1 missense mutation causing outer segment shortening and progressive retinal degeneration

Abstract

Purpose: To characterize mice with a novel missense mutation in the mouse retinitis pigmentosa 1 (RP1) gene.

Methods: Photoreceptor degeneration was detected in a small colony of C57BL/6J mice maintained at UPenn for 5 years. To identify the causative mutation, mutant mice were crossed to WT DBA mice and F1 heterozygotes were intercrossed. To chromosomally localize the mutation, whole genomic DNA was extracted from mouse tails, and DNA pools from affected and unaffected mice genotyped genome-wide with SSLP markers. Mutant and control DNA was directly sequenced from cDNA using primers encompassing Rp1, a candidate gene mapping to the region. Outer segments were assessed by histology and optical coherence tomography (OCT). Retinal function was evaluated by electroretinography (ERG). Western analysis was performed to evaluate protein expression.

Results: Two adjacent point mutations: T402C and G403T, resulting in a missense L66P mutation located in exon 2 of the Rp1 gene were identified. Mice homozygous but not heterozygous for the mutations had several retinal abnormalities. OCT imaging demonstrated diffuse high reflectivity in the inner and outer segment regions, with loss of the thin, highly reflective IS/OS junction band visible in WT mice. Histology revealed shortening and disorganization of the inner and outer segments, as well as age-dependent progressive thinning of the outer nuclear layer. ERG rod a and b wave amplitudes were significantly decreased with increasing age, but cone b-wave amplitudes were not diminished. Western analysis with an anti-C-terminal Rp1 antibody confirmed that a normal sized Rp1 protein was produced in quantities similar to WT.

Conclusions: The L66P mutation in the doublecortin domain of the Rp1 gene is sufficient to impair Rp1 protein function, and leads to morphologic abnormalities in photoreceptor outer segments. It is predicted that this mutation may impair protein-protein interactions. This mutant line provides a useful model for studies of Rp1 function and potential therapeutic interventions.

Keywords: 534 gene mapping • 563 inner retina dysfunction: hereditary • 604 mutations