Autosomal Dominant Retinal Degeneration in ENU Mutagnized Mice Caused By a Cys185Arg Mutation in Rhodopsin

Abstract

Abstract: : Purpose: By screening mice with ENU–induced ocular mutations, we identified a line, BEMR3 (R3), with a dominant retinal degeneration (RD). We have mapped the chromosomal location of this mutation and determined the specific mutation. We are studying the mechanism of retinal degeneration that results from this mutation. Methods: A genome–wide linkage analysis was used to map the chromosomal location of the mutation by using F2 mice generated between strains C57BL/6J (B6) and C3H. F2 mice were phenotyped by indirect ophthalmoscopy. Candidate genes were identified from the mouse genome database based on the chromosomal location of the mutation. The mutation was verified by DNA sequencing. Retinal phenotypes of R3 mice were examined by histopathology, immunocytochemistry, biochemical assays, and electroretinograms (ERG). Results: The BEMR3 mutation causes a dominant retinal degeneration characterized by hypopigmented spots and retinal vessel attenuation visible in the fundus within 3 postnatal weeks. Histopathology demonstrates a progressive thinning of the outer nuclear layer (ONL), concomitant with a reduction in the ERG a– and b– waves. The ONL is reduced to a single layer by two months. The mutation was fine mapped to Chr 6 in close proximity to the rhodopsin gene. DNA sequence analysis of the RT–PCR products of rhodopsin transcripts of heterozygous mice verified a point mutation of T to C resulting in a Cys–185 to Arg substitution. Conclusions: Rapid retinal degeneration observed in BEMR3 mice results from expression of the C185R mutant rhodopsin. This mutation is located in the second extracellular loop of rhodopsin (EII) between the transmembrane helices IV and V. The EII loop is hypothesized to be critical for modulating interactions with retinal in the ground and excited states of rhodopsin. We hypothesize that the C185R mutation introduces a positive charge that perturbs the chromophore binding pocket and also acts as a dominant negative inhibitor for the endogenous wild type Rhodopsin in heterozygous mice. We are currently comparing the molecular pathology of photoreceptor degeneration in rhodopsin C185R point mutation mice to rhodopsin null mice.

Keywords: retinal degenerations: cell biology • opsins • protein structure/function