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 advanceddisease 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.visual cycle ͉ Leber congenital amaurosis ͉ rod ͉ cone ͉ retinal imaging

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 (3-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 >25 kilobases. Within a 67-base-pair exon of the rd PDE (3-subunit gene, we identified a nonsense ochre mutation (a C -3 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 rdphenotype, 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.

Mutations in the retinal pigment epithelium gene encoding RPE65 are a cause of the incurable early-onset recessive human retinal degenerations known as Leber congenital amaurosis. Rpe65-deficient mice, a model of Leber congenital amaurosis, have no rod photopigment and severely impaired rod physiology. We analyzed retinoid flow in this model and then intervened by using oral 9-cis-retinal, attempting to bypass the biochemical block caused by the genetic abnormality. Within 48 h, there was formation of rod photopigment and dramatic improvement in rod physiology, thus demonstrating that mechanismbased pharmacological intervention has the potential to restore vision in otherwise incurable genetic retinal degenerations.

Leber's congenital amaurosis (LCA, MIM 204,000), the earliest and most severe form of inherited retinopathy, accounts for at least 5% of all inherited retinal dystrophies. This autosomal recessive condition is usually recognized at birth or during the first months of life in an infant with total blindness or greatly impaired vision, normal fundus and extinguished electroretinogram (ERG). Nystagmus (pendular type) and characteristic eye poking are frequently observed in the first months of life (digito-ocular sign of Franceschetti). Hypermetropia and keratoconus frequently develop in the course of the disease. The observation by Waardenburg of normal children born to affected parents supports the genetic heterogeneity of LCA. Until now, however, little was known about the pathophysiology of the disease, but LCA is usually regarded as the consequence of either impaired development of photoreceptors or extremely early degeneration of cells that have developed normally. We have recently mapped a gene for LCA to chromosome 17p13.1 (LCA1) by homozygosity mapping in consanguineous families of North African origin and provided evidence of genetic heterogeneity in our sample, as LCA1 accounted for 8/15 LCA families in our series. Here, we report two missense mutations (F589S) and two frameshift mutations (nt 460 del C, nt 693 del C) of the retinal guanylate cyclase (RETGC, GDB symbol GUC2D) gene in four unrelated LCA1 probands of North African ancestry and ascribe LCA1 to an impaired production of cGMP in the retina, with permanent closure of cGMP-gated cation channels.

Summary Defects in primary cilia lead to devastating disease due to their roles in sensation and developmental signaling, but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain three-dimensional maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wildtype and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.

Irish setter dogs affected with a rod/cone dysplasia (locus designation, rcdl) display markedly elevated levels of retinal cGMP during postnatal development. The photoreceptor degeneration commences =25 days after birth and culminates at about 1 year when the population of rods and cones is depleted. A histone-sensitive retinal cGMP phospho- subunit by 49 residues, thus removing the C-terminal domain that is required for posttranslational processing and membrane association. These results suggest that the redl gene encodes the rod photoreceptor PDE 13 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 rcdl/rcdl Irish setter dogs.The Irish setter rod/cone dysplasia leading to a rapidly progressing loss of photoreceptors (1) is inherited as an autosomal recessive trait (genetic locus, rcdl). The early onset of the photoreceptor degeneration has been well defined by morphological and biochemical studies (2-4). Clin-ically, the disorder is grouped within a famnily of related canine retinal degenerations which are termed progressive retinal atrophies (5). In affected dogs, retina and photoreceptor development appears normal until 13 days of age (4), but subsequent development of rod photoreceptor cells is arrested. Rod photoreceptor degeneration is evident by 1 month of age; nearly all of the rod photoreceptors have degenerated by 5 months, and cone photoreceptor degeneration is completed by about 1 year (for review, see refs. 6 and 7).The earliest known biochemical manifestation of the rcdl phenotype is a rapid accumulation of cGMP to levels that are about 10-fold above those of age-matched controls (2-4, 8). These features are reminiscent of the phenotype seen in the rd mouse in which a nonsense mutation in exon 7 ofthe cGMP phosphodiesterase (PDE) (-subunit gene prevents the formation of a functional enzyme (9) leading to elevated cGMP levels and a rapid rod photoreceptor degeneration. Earlier studies of retinas from normal and affected Irish setters showed that the a and 'y mRNAs of affected dog PDE were of normal size and abundance (10), whereas the /-subunit mRNA level appeared to be reduced (11,12). This finding, similar to that in the rd mouse, suggests a defect in the dog PDE l3-subunit gene. Since a previous study showed the presence of a histone sensitive cGMP PDE activity in affected, immature Irish setter retinas (13), we reinvestigated its origin by an HPLC method which is capable of separating rod and cone PDEs (14). In this paper we identify the residual PDE activity in affected Irish setter retinas as comigrating with cone PDE, whereas rod PDE activity is completely absent. We further provide evidence that a functional /3 subunit is not produced in affected retinas and that a nonsense mutation near the C-terminal end of the /-subunit gene leads to truncation and destabilization of the gene product,

Retinitis pigmentosa (RP) constitutes a group of genetically heterogeneous progressive photoreceptor degenerations leading to blindness and affecting 50,000-100,000 people in the U.S. alone. Over 20 different RP loci have been mapped, of which six have been identified. Three of these encode members of the rod photoreceptor visual transduction cascade: rhodopsin, the rod cGMP-gated cation channel alpha subunit, and the beta subunit of cGMP-phosphodiesterase (PDEB). As null mutations in PDEB cause some cases of RP and since both alpha and beta subunits are required for full phosphodiesterase activity, we examined the gene encoding the alpha subunit of cGMP phosphodiesterase (PDEA) in 340 unrelated patients with RP. We found three point mutations in PDEA in affected members of two pedigrees with recessive RP. Each mutation alters an essential functional domain of the encoded protein and likely disrupts its catalytic function. PDEA is the seventh RP gene identified, highlighting the extensive genetic heterogeneity of the disorder and encouraging further investigation into the role of other members of the phototransduction cascade in RP.

Ion flow into the rod photoreceptor outer segment (ROS) is regulated by a member of the cyclic-nucleotide-gated cation-channel family; this channel consists of two subunit types, α and β. In the rod cells, the Cngb1 locus encodes the channel β-subunit and two related glutamic-acid-rich proteins (GARPs). Despite intensive research, it is still unclear why the β-subunit and GARPs are coexpressed and what function these proteins serve. We hypothesized a role for the proteins in the maintenance of ROS structural integrity. To test this hypothesis, we created a Cngb1 5′-knockout photoreceptor null (Cngb1-X1). Morphologically, ROSs were shorter and, in most rods that were examined, some disks were misaligned, misshapen and abnormally elongated at periods when stratification was still apparent and degeneration was limited. Additionally, a marked reduction in the level of channel α-subunit, guanylate cyclase I (GC1) and ATP-binding cassette transporter (ABCA4) was observed without affecting levels of other ROS proteins, consistent with a requirement for the β-subunit in channel assembly or targeting of select proteins to ROS. Remarkably, phototransduction still occurred when only trace levels of homomeric α-subunit channels were present, although rod sensitivity and response amplitude were both substantially reduced. Our results demonstrate that the β-subunit and GARPs are necessary not only to maintain ROS structural integrity but also for normal disk morphogenesis, and that the β-subunit is required for normal light sensitivity of the rods.