Abstract
Purpose: :
Canine multifocal retinopathy 1 (cmr1), caused by a C73T/R25X change in the bestrophin gene (VMD2), has been proposed as a relevant animal model for human Best disease. To assess the molecular consequences of this substitution, RPE from normal, heterozygous and homozygous affected animals was evaluated at the transcriptional and translational level. Moreover, to compare the pathogenetic outcomes of cmr1 to other VMD2 mutations, an in vitro model mimicking cmr is currently being developed.
Methods: :
Dogs carrying the cmr1 bestrophin mutation were introduced into a breeding colony and utilized to produce either carrier or affected offspring. Eyes were collected immediately after euthanasia at the disease-relevant ages, dissected, and tissues were frozen in liquid nitrogen. Total RNA was isolated from RPE/choroid, reverse transcribed, and evaluated by semi-quantitative RT PCR and qRT PCR. Protein expression levels were determined by Western blotting and immunohistochemistry. Mutant clones were produced from full-length VMD2 cDNA by site-directed mutagenesis. ARPE-19 cells used for in vitro studies are being transfected with VMD2 vectors expressing wild-type or mutant cDNAs.
Results: :
The results obtained from the in vivo studies suggest that the protein expression level is reduced in R25X carriers, and no protein can be detected in affected animals, either by western analysis or immunohistochemistry. However, no differences were observed at the RNA level either with disease, nor based on topographic distribution, indicating that nonsense mediated mRNA decay does not occur with this mutation. All animals were tested for VMD2L1 mRNA expression as a control, and no changes were found. Parallel in vitro studies are now in progress.
Conclusions: :
The C73T mutation does not have any effect on the mRNA level in the canine RPE, but alters protein expression in heterozygous and homozygous affected animals. There is no evidence that VMD2L1 expression is altered due to the mutation. To validate these data, and correlate them to observed phenotypes, a cmr in vitro model is being developed to investigate the consequences of other VMD2 mutations.
Keywords: mutations • retinal pigment epithelium • gene/expression