Photoreceptor rescue by an abbreviated human RPGR gene in a murine model of X-linked retinitis pigmentosa.

The X-linked RP3 gene codes for the ciliary protein RPGR and accounts for over 10% of inherited retinal degenerations. The critical RPGR-ORF15 splice variant contains a highly repetitive purine-rich linker region that renders it unstable and difficult to adapt for gene therapy. To test the hypothesis that the precise length of the linker region is not critical for function, we evaluated whether adeno-associated virus-mediated replacement gene therapy with a human ORF15 variant containing in-frame shortening of the linker region could reconstitute RPGR function in vivo. We delivered human RPGR-ORF15 replacement genes with deletion of most (314 codons, 'short form') or 1/3 (126 codons, 'long form') of the linker region to Rpgr null mice. Human RPGR-ORF15 expression was detected post treatment with both forms of ORF15 transgenes. However, only the long form correctly localized to the connecting cilia and led to significant functional and morphological rescue of rods and cones. Thus the highly repetitive region of RPGR is functionally important but that moderate shortening of its length, which confers the advantage of added stability, preserves its function. These findings provide a theoretical basis for optimizing replacement gene design in clinical trials for X-linked RP3.

Melanoma-associated retinopathy and recurrent exudative retinal detachments in a patient with choroidal melanoma.

PURPOSE: To report a patient who presented with photopsias, night blindness, exudative retinal detachments, and melanoma-associated retinopathy in her right eye 23 years after the left eye was enucleated for a choroidal melanoma. METHODS: Assessment of fundus findings, fluorescein angiograms, and electroretinograms. RESULTS: The patient had recurrent exudative detachments of the macula in her right eye and electroretinogram responses consistent with the diagnosis of melanoma-associated retinopathy. The abdominal computed tomography (CT) scan was negative, but 13 months later, CT scanning revealed many masses in her liver. Fine-needle biopsy confirmed the diagnosis of metastatic melanoma. CONCLUSION: To our knowledge, this is the first report of melanoma-associated retinopathy in a patient with a previous choroidal melanoma.

Evaluation of the myosin VIIA gene and visual function in patients with Usher syndrome type I.

Usher syndrome type I (USH1) is a recessively-inherited disorder consisting of retinitis pigmentosa, profound congenital deafness, and vestibular ataxia. It can be caused by mutations in at least six different loci (USH1A-1F). The gene encoding human myosin VIIA (MYO7A) is the USH1B locus. In this study, 66 unrelated patients with USH1 were evaluated for defects in MYO7A using single-strand conformation polymorphism analysis and direct genomic sequencing. Twenty-nine per cent of cases were found to have likely pathogenic MYO7A mutations. A total of 22 likely pathogenic changes were identified, 18 of which were novel. Cosegregation analysis of mutations in five available families showed that the MYO7A changes segregated with the disease in an autosomal recessive fashion. Average visual function as measured by visual acuity, visual field area, and ERG amplitude was not significantly different between the group of patients with likely pathogenic MYO7A changes and the group in which no likely pathogenic MYO7A changes were detected.

Safety of <7500 RE (<25000 IU) vitamin A daily in adults with retinitis pigmentosa.

BACKGROUND: Vitamin A supplementation is being used successfully to treat some forms of cancer and the degenerative eye disease retinitis pigmentosa. The daily biological need for vitamin A is estimated to be 800 retinol equivalents (RE)/d (2667 IU/d) for adult women and 1000 RE/d (3300 IU/d) for adult men; doses > or = 7500 RE (> or = 25000 IU)/d are considered potentially toxic over the long term. OBJECTIVE: We assessed the safety in adults of long-term vitamin A supplementation with doses above the daily biological need but <7500 RE (<25000 IU)/d. DESIGN: Adults aged 18-54 y with retinitis pigmentosa but in generally good health (n = 146) were supplemented with 4500 RE (15000 IU) vitamin A/d for < or = 12 y (group A) and compared with a similar group (n = 149) that received 23 RE (75 IU)/d (trace group). Mean total consumption of vitamin A in group A was 5583 RE (18609 IU)/d (range: 4911-7296 RE/d, or 16369-24318 IU/d) and that in the trace group was 1053 RE (3511 IU)/d (range: 401-3192 RE/d, or 1338-10638 IU/d). RESULTS: Patients in group A showed an 8% increase in mean serum retinol concentration at 5 y and an 18% increase at 12 y (P < 0.001); no retinol value exceeded the upper normal limit (3.49 micromol/L, or 100 microg/dL). Mean serum retinyl esters were elevated approximately 1.7-fold at 5 y and remained relatively stable thereafter. No clinical symptoms or signs of liver toxicity attributable to vitamin A excess were detected. CONCLUSIONS: Prolonged daily consumption of <7500 RE (<25000 IU) vitamin A/d can be considered safe in this age group.

Evaluation of the human gene encoding recoverin in patients with retinitis pigmentosa or an allied disease.

PURPOSE: To determine whether defects in the human recoverin gene cause retinitis pigmentosa (RP) or an allied disease such as Usher syndrome, Leber congenital amaurosis, or the Bardet-Biedl syndrome. METHODS: Single-strand conformation polymorphism analysis and direct genomic sequencing techniques were used to screen 596 unrelated patients, comprising 167 patients with dominant RP, 168 with recessive RP, and 261 with an allied disease. RESULTS: Four sequence variants were discovered. The first was a missense change (Ala200Thr) found in one family with autosomal dominant RP and in one family with autosomal recessive RP; it did not segregate with disease. The second was a silent, single-base variation affecting codon Ser24 with a minor allele frequency of approximately 0.5%. The third was a silent, single-base variation affecting codon Va1122. The fourth was a single-nucleotide substitution in intron 2, 11 bp upstream of exon 3. CONCLUSIONS: The authors found no evidence that mutations in the recoverin gene are a cause of RP or another of the hereditary retinal diseases studied. The human phenotype associated with mutations of the recoverin gene remains unknown.

Evaluation of patients with retinitis pigmentosa receiving electric stimulation, ozonated blood, and ocular surgery in Cuba.

OBJECTIVE: To evaluate the effect of intervention with electric stimulation, autotransfused ozonated blood, and ocular surgery, performed in Cuba, on the course of the common forms of retinitis pigmentosa. DESIGN: Ocular evaluations over 6 to 8 months before and after intervention in Cuba. SETTING: Evaluations performed at a US clinical research facility. PATIENTS: Ten adult patients aged 25 to 67 years with retinitis pigmentosa. MAIN OUTCOME MEASURES: Visual acuity, visual field area, and electroretinogram (ERG) amplitude. RESULTS: No significant change in visual acuity or visual field area was observed on average between preintervention and postintervention values over a 6- to 8-month interval. Mean 30-Hz cone ERG amplitude declined by 15.5% between preintervention and postintervention values (P = .006). When data on change in visual field area from 1 statistically significant outlier were excluded from the analysis, a significant decline of 12.9% in mean visual field area was observed (P = .025). CONCLUSIONS: These data support the conclusion that the intervention offered in Cuba provides no benefit to patients with retinitis pigmentosa as measured by visual acuity, visual field area, and ERG. The magnitudes of the mean declines observed in ERG amplitude and visual field area over a 6- to 8-month interval, relative to those reported in previous studies, raise the possibility that this intervention may worsen the course of the disease.

Mutation spectrum of the gene encoding the beta subunit of rod phosphodiesterase among patients with autosomal recessive retinitis pigmentosa.

Mutations in the gene encoding the beta subunit of rod cGMP phosphodiesterase are known causes of photoreceptor degeneration in two animal models of retinitis pigmentosa, the rd (retinal degeneration) mouse and the Irish setter dog with rod/cone dysplasia. Here we report a screen of 92 unrelated patients with autosomal recessive retinitis pigmentosa for defects in the human homologue of this gene. We identified seven different mutations that cosegregate with the disease. They were found among four patients with each patient heterozygously carrying two mutations. All of these mutations are predicted to affect the putative catalytic domain, probably leading to a decrease in phosphodiesterase activity and an increase in cGMP levels within rod photoreceptors. Mutations in the gene encoding the beta subunit of rod phosphodiesterase are the most common identified cause of autosomal recessive retinitis pigmentosa, accounting for approximately 4% of cases in North America.

In vivo transfer of a reporter gene to the retina mediated by an adenoviral vector.

PURPOSE: The ability of replication-deficient adenovirus to mediate gene transfer to retinal cells was evaluated. METHODS: A replication-deficient adenoviral vector, AdCMV beta A.ntlacZ, which contains the bacterial beta-galactosidase (lacZ) reporter gene, was injected into the subretinal space of normal, rd, and rds strains of mice at various ages. The efficiency and duration of transgene expression were assessed by histochemical examination and transmission electron microscopy. RESULTS: AdCMV beta A.ntlacZ was effective in mediating gene transfer to the retinal pigment epithelial cells, rod and cone photoreceptor cells, and cells in the inner nuclear layer of the retina for periods of up to 1 month. Gene transfer to retinal pigment epithelial cells occurred at much lower viral titers than was required for gene transfer to photoreceptor cells. The extent to which photoreceptor cells could be transduced varied with the age of the animals and the conditions of the photoreceptor cells: greater numbers of photoreceptor cells were transduced in 5- to 7-day-old pups and in mice at the initial stages of photoreceptor degeneration than in normal adult mice. No evidence of gross pathogenic effects or viremia in recipient mice was observed. CONCLUSIONS: Replication-deficient adenovirus mediates transfer and expression of a foreign gene in retinal pigment epithelial and photoreceptor cells. Gene transfer to photoreceptor cells is enhanced in developing retinas or at the predegenerate stage of photoreceptors in genetically programmed retinal degeneration.

Hyperopia and neovascularization in age-related macular degeneration.

PURPOSE: Refractive errors for phakic eyes of patients referred with age-related macular degeneration were reviewed to determine whether some range of refractive error might be a risk factor for the neovascular form. METHODS: The authors compared refractive errors of 198 patients with unilateral neovascular disease with refractive errors of 129 patients with bilateral dry disease. These groups had comparable distributions with respect to age, sex, and visual acuity of their better eyes. Student's t tests and multiple linear regression analyses were performed to assess group differences in mean refractive error. Contingency table and multiple logistic regression analyses were performed to determine odds ratios for having the neovascular form based on a stratification of refractive error. RESULTS: By comparing better eyes of the two groups, patients with the unilateral neovascular form had an average spherical equivalent that was 1.0 diopter (D) more hyperopic than that of patients with the bilateral dry form (P < 0.001). Patients with a refractive error of +0.75 D or greater were more likely to have the neovascular form compared with patients with other refractive errors (odds ratio, 2.40; 95% confidence interval, 1.53-3.78; P < 0.001). Similar relationships between the two groups of patients were found by comparing worse eyes. CONCLUSION: These findings suggest that hyperopia is a risk factor for choroidal neovascularization among patients referred with age-related macular degeneration.

Recessive mutations in the gene encoding the beta-subunit of rod phosphodiesterase in patients with retinitis pigmentosa.

We have found four mutations in the human gene encoding the beta-subunit of rod cGMP phosphodiesterase (PDE beta) that cosegregate with autosomal recessive retinitis pigmentosa, a degenerative disease of photoreceptors. In one family two affected siblings both carry allelic nonsense mutations at codons 298 and 531. Affected individuals have abnormal rod and cone electroretinograms. PDE beta is the second member of the phototransduction cascade besides rhodopsin that is absent or altered as a cause of retinitis pigmentosa, suggesting that other members of this pathway may be defective in other forms of this disease.

Opsin phosphorylation in retinas from autopsy eyes with retinitis pigmentosa.

Opsin phosphorylation in light was detected in three retinas from autopsy eyes with retinitis pigmentosa (RP) including one with sex-linked RP and two with autosomal recessive RP, that were studied at postmortem intervals of 1-4 hr. In these retinas from RP eyes, opsin phosphorylation in light was reduced compared with that in normal human retinas, a finding that is compatible with reduced amounts of opsin due to extensive loss of photoreceptor cells. ATP and GTP levels, although reduced below normal, also were easily detectable, supporting the idea that the reduction in opsin phosphorylation was due to loss of photoreceptor cells and not to a reduced capacity for energy metabolism. These findings in these RP retinas contrast with those in rd mice and Irish setters with rod-cone dysplasia in which a failure of opsin phosphorylation has been detected prior to onset of photoreceptor cell degeneration.

Paraneoplastic night blindness with malignant melanoma.

A 69-year-old hyperopic man developed acute night blindness and hallucinations of shimmering lights three years after resection of a cutaneous malignant melanoma. There were no metastatic ocular lesions and he had received no medications. His electroretinogram showed abnormalities comparable to those of patients with congenital stationary night blindness with myopia. Metastatic melanoma was recognized several months later. His electroretinographic responses were also identical to those ascribed to vincristine therapy in a previously described patient with malignant melanoma. Our findings showed that acquired night blindness, apparently resulting from interruption of intraretinal rod signal transmission, can be a paraneoplastic effect of a malignant melanoma.

Electroretinograms in English setters with neuronal ceroid lipofuscinosis.

Full-field electroretinograms (ERGs) were recorded from three adult English setters with advanced neuronal ceroid lipofuscinosis and three normal controls. Affected setters showed 30% to 40% reductions in a-wave and b-wave amplitudes, normal cone and rod b-wave implicit times, and slightly elevated a-wave and b-wave thresholds. The ERGs of these affected setters differed from those that have been recorded from humans with neuromal ceroid lipofuscinosis (Batten's disease), humans with hereditary retinitis pigmentosa, Irish setters with rod-cone dysplasia, and miniature French poodles with progressive rod-cone degeneration.

Sex-linked retinitis pigmentosa: ultrastructure of photoreceptors and pigment epithelium.

An ultrastructural study of a postmortem donor eye from a 24-year-old male patient with sex-linked retinitis pigmentosa showed abnormalities in all remaining cones and rods. Central foveal cones were reduced in number by about 50% and had shortened and severely distorted outer segments. Cones from the parafovea through the midperiphery gradually decreased in density and had no organized outer segments. In the far periphery, cones and rods had only slightly shortened outer segments. Photoreceptors equidistant from the fovea in all quadrants showed similar changes. The virtual absence of organized cone outer segments from the parafovea through the midperiphery was conspicuous in that this patient had full visual fields with large test lights 3 weeks prior to death. The pigment epithelium contained abnormally large numbers of melanolysosomes and few free melanin granules from the fovea through the midperiphery and few melanolysosomes and many free melanin granules in the far periphery. Whether or not these observations in the pigment epithelial cells represent a primary defect in this disease or reflect changes secondary to a defect in the photoreceptor cells remains to be defined.

Retinal ultrastructure in advanced retinitis pigmentosa.

An ultrastructural study of the retina of a patient with advanced retinitis pigmentosa revealed an orderly process of cone degeneration with more advanced stages in the perifovea and less advanced stages in the fovea. No rod photoreceptors were seen. Remaining cones had enlarged nuclei with autophagic vacuoles and bundles of parallel undulating 12 nm. filaments in the cytoplasm. In the foveola and fovea, cone outer segments were very truncated or absent, although synaptic pedicles were intact. In the perifovea, many cone cell bodies were rounded with loss of synaptic pedicles. Pigment epithelial cells underlying remaining cones were enlarged with apically displaced nuclei, occasional phagosomes, and large amounts of melanolysosomes. The pigment epithelial cell layer anterior to the perifovea contained flattened pigment epithelial cells without melanolysosomes and macrophage-like cells without pigment. Pigment-laden epithelial cells around atrophic blood vessels in the midperipheral retina showed only round pigment granules unlike the smaller elongated pigment granules observed in retinal pigment epithelial cells in situ. The advanced stage and structural abnormalities in both photoreceptors and pigment epithelial cells preclude assigning a primary site for the defect.

Retinal degeneration in cats fed casein. I. Taurine deficiency.

All cats fed a taurine-free casein diet for at least 23 weeks have shown granularity with a hyper-reflective white zone in the area centralis, nondetectable electroretinograms (ERG's), and structural changes indicating photoreceptor cell degeneration. The present study has demonstrated that cats fed this casein diet have a selective decrease in plasma and retinal taurine concentrations by five weeks; taurine levels were about 4 per cent of normal in plasma, and 60 per cent of normal in retina. After 10 weeks, taurine levels were 2 to 4 per cent of normal in plasma and reached a minimum of 20 to 30 per cent of normal in the retina. These biochemical changes occurred in association with a delay in the cone ERG implicit time at five weeks and reduced cone and rod ERG amplitudes at 10 weeks. During this period, retinal DNA content (as a measure of cell viability) and fundus appearance were normal. By 23 weeks, ERG's were nondetectable, retinal DNA content was reduced, and the fundus showed typical changes in the area centralis. These studies help to establish a biological role for taurine in maintaining photoreceptor cell function and viability in the cat.

Retinal degeneration in cats fed casein. II. Supplementation with methionine, cysteine, or taurine.

All cats fed a taurine-free casein diet for 23 weeks have shown a nondetectable electroretinogram (ERG) in association with a plasma and retinal taurine deficiency. In the present study, the casein diet was supplemented with either taurine or taurine precursors (methionine or cysteine) for 23 weeks to see if retinal function would be preserved. Cats fed the casein diet supplemented with methionine or cysteine showed ERG's reduced in amplitude and delayed in implicit time and had plasma and retinal taurine levels that were well below normal by 23 weeks. Only those cats given taurine in the diet (i.e., those fed chow or casein supplemented with taurine) retained normal ERG function and normal plasma and retinal taurine concentrations. These findings establish a role for taurine in maintaining normal retinal function in the cat.