Retinal degeneration in the mouse mutant, rd, was previously shown to be a disorder of cyclic nucleotide metabolism involving a deficiency in the activity of the rod photoreceptor cGMP phosphodiesterase (PDE). We have characterized the normal and rd PDE beta-subunit gene, and their respective transcripts, by PCR and direct sequence analysis. We show that the gene consists of at least 22 exons ranging in size from 48 base pairs to several hundred base pairs, covering greater than 25 kilobases. Within a 67-base-pair exon of the rd PDE beta-subunit gene, we identified a nonsense ochre mutation (a C----A transversion in codon 347) that truncates the normal gene product, eliminating more than one-half of the peptide chain, including the putative catalytic domain. The consequences of the truncation are consistent with the observed phenotypes in rd mice heterozygous and homozygous for the disorder. The nonsense mutation was also found in another related and in six unrelated strains displaying the rd phenotype, indicating that the rd allele arose from a single genetic event. The results strongly argue for the nonsense mutation being responsible for retinal degeneration in the rd mouse.

Irish setter dogs affected with a rod/cone dysplasia (locus designation, rcd1) display markedly elevated levels of retinal cGMP during postnatal development. The photoreceptor degeneration commences approximately 25 days after birth and culminates at about 1 year when the population of rods and cones is depleted. A histone-sensitive retinal cGMP phosphodiesterase (PDE; EC 3.1.4.35) activity, a marker for photoreceptor PDEs, was shown previously to be present in retinal homogenates of immature, affected Irish setters. Here we report that, as judged by HPLC separation, this activity originates exclusively from cone photoreceptors, whereas rod PDE activity is absent. An immunoreactive product the size of the PDE alpha subunit, but none the size of the beta subunit, can be detected on immunoblots of retinal extracts of affected dogs, suggesting a null mutation in the PDE beta-subunit gene. Using PCR amplification of Irish setter retinal cDNA, we determined the complete coding sequence of the PDE beta subunit in heterozygous and affected animals. The affected PDE beta-subunit mRNA contained a nonsense amber mutation at codon 807 (a G-->A transition converting TGG to TAG), which was confirmed to be present in putative exon 21 of the affected beta-subunit gene. The premature stop codon truncates the beta subunit by 49 residues, thus removing the C-terminal domain that is required for posttranslational processing and membrane association. These results suggest that the rcd1 gene encodes the rod photoreceptor PDE beta subunit and that a nonsense mutation in this gene is responsible for the production of a nonfunctional rod PDE and the photoreceptor degeneration in the rcd1/rcd1 Irish setter dogs.

Mutations in RPE65 , a gene essential to normal operation of the visual (retinoid) cycle, cause the childhood blindness known as Leber congenital amaurosis (LCA). Retinal gene therapy restores vision to blind canine and murine models of LCA. Gene therapy in blind humans with LCA from RPE65 mutations may also have potential for success but only if the retinal photoreceptor layer is intact, as in the early-disease stage-treated animals. Here, we use high-resolution in vivo microscopy to quantify photoreceptor layer thickness in the human disease to define the relationship of retinal structure to vision and determine the potential for gene therapy success. The normally cone photoreceptor-rich central retina and rod-rich regions were studied. Despite severely reduced cone vision, many RPE65 -mutant retinas had near-normal central microstructure. Absent rod vision was associated with a detectable but thinned photoreceptor layer. We asked whether abnormally thinned RPE65 -mutant retina with photoreceptor loss would respond to treatment. Gene therapy in Rpe65 ^-/- mice at advanced-disease stages, a more faithful mimic of the humans we studied, showed success but only in animals with better-preserved photoreceptor structure. The results indicate that identifying and then targeting retinal locations with retained photoreceptors will be a prerequisite for successful gene therapy in humans with RPE65 mutations and in other retinal degenerative disorders now moving from proof-of-concept studies toward clinical trials.