A Missense Mutation in Canine CNGA3 Eliminates Retinal Cone Function: A Novel Model for Achromatopsia

Abstract

Purpose: Achromatopsia is an autosomal recessive retinal disorder causing day-blindness, poor visual acuity and monochromacy as a result of impaired signal transduction in cone photoreceptor cells. Transmission of light-evoked visual signals requires cyclic nucleotide-gated (CNG) channels on cone outer segment membranes. We identified a one-year-old German shepherd dog with the clinical manifestations of day-blindness. The aim of this study was to identify and characterize the genetic basis, visual phenotype and molecular pathogenesis of the disease.

Methods: ERG recordings under light and dark-adapted conditions were evoked for the day-blind dog and compared to normal ERGs recorded under identical conditions. Cone phototransduction cascade genes CNGB3, CNGA3, GNAT2, PDE6C, and PDE6H were selected for mutation screening. For expression studies, cDNA was ligated into a YFP expression vector and the CNGA3-R424W plasmid was generated by site-directed mutagenesis. HEK cells were transfected with either WT or CNGA3-R424W-YFP construct. Excised patch clamping was performed to monitor cAMP and cGMP activation at -60mV to +60mV. Cellular localization of YFP-tagged CNGA3 protein was visualized using fluorescence microscopy. The amino acid topology of the canine CNGA3 transmembrane segments (S1-S6) was analyzed in silico.

Results: Behavioral study of the affected German shepherd indicated complete day-blindness and ERG recordings confirmed loss of cone function. Phenotype-directed candidate gene analysis revealed a R424W (C1270T) missense mutation in exon 7 of canine CNGA3. HEK cells expressing CNGA3-R424W-YFP showed no cyclic nucleotide-activated current with patch-clamp recordings in comparison to WT. Furthermore, mutant homomeric channels showed abnormal cellular localization indicating that the mutation alters subunit trafficking; the nature of this mislocalization is still under investigation. According to amino acid alignment with Kv1.2/2.1, the R424 residue is located in the C-terminal end of S6. Homology modeling in silico is underway to predict noncovalent interactions of this residue.

Conclusions: We report the first canine model for CNGA3-achromatopsia and characterize its molecular mechanism. Based on our findings, we propose the R424W as a valuable model for studying CNG channel malfunction and potentially developing therapies for cone cell signal transduction diseases caused by CNGA3 mutations.

Keywords: 569 ion channels • 604 mutations • 714 signal transduction