Molecular modeling of retinoschisin with functional analysis of pathogenic mutations from human X-linked retinoschisis.

Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. RS1 is an adhesive protein which is proposed to preserve the structural and functional integrity of the retina, but there is very little evidence of the mechanism by which protein changes are related to XLRS disease. Here, we report molecular modeling of the RS1 protein and consider perturbations caused by mutations found in human XLRS subjects. In 60 XLRS patients who share 27 missense mutations, we then evaluated possible correlations of the molecular modeling with retinal function as determined by the electroretinogram (ERG) a- and b-waves. The b/a-wave ratio reflects visual-signal transfer in retina. We sorted the ERG b/a-ratios by patient age and by the mutation impact on protein structure. The majority of RS1 mutations caused minimal structure perturbation and targeted the protein surface. These patients' b/a-ratios were similar across younger and older subjects. Maximum structural perturbations from either the removal or insertion of cysteine residues or changes in the hydrophobic core were associated with greater difference in the b/a-ratio with age, with a significantly smaller ratio at younger ages, analogous to the ERG changes with age observed in mice with no RS1-protein expression due to a recombinant RS1-knockout gene. The molecular modeling suggests an association between the predicted structural alteration and/or damage to retinoschisin and the severity of XLRS as measured by the ERG analogous to the RS1-knockout mouse.

Genotyping microarray (gene chip) for the ABCR (ABCA4) gene.

Genetic variation in the ABCR (ABCA4) gene has been associated with five distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), and age-related macular degeneration (AMD). Comparative genetic analyses of ABCR variation and diagnostics have been complicated by substantial allelic heterogeneity and by differences in screening methods. To overcome these limitations, we designed a genotyping microarray (gene chip) for ABCR that includes all approximately 400 disease-associated and other variants currently described, enabling simultaneous detection of all known ABCR variants. The ABCR genotyping microarray (the ABCR400 chip) was constructed by the arrayed primer extension (APEX) technology. Each sequence change in ABCR was included on the chip by synthesis and application of sequence-specific oligonucleotides. We validated the chip by screening 136 confirmed STGD patients and 96 healthy controls, each of whom we had analyzed previously by single strand conformation polymorphism (SSCP) technology and/or heteroduplex analysis. The microarray was >98% effective in determining the existing genetic variation and was comparable to direct sequencing in that it yielded many sequence changes undetected by SSCP. In STGD patient cohorts, the efficiency of the array to detect disease-associated alleles was between 54% and 78%, depending on the ethnic composition and degree of clinical and molecular characterization of a cohort. In addition, chip analysis suggested a high carrier frequency (up to 1:10) of ABCR variants in the general population. The ABCR genotyping microarray is a robust, cost-effective, and comprehensive screening tool for variation in one gene in which mutations are responsible for a substantial fraction of retinal disease. The ABCR chip is a prototype for the next generation of screening and diagnostic tools in ophthalmic genetics, bridging clinical and scientific research.

Assessment of visual function in patients with gyrate atrophy who are considered candidates for gene replacement.

OBJECTIVE: To assess the course of change of visual function outcome variables in 5 patients with gyrate atrophy before a gene replacement therapy clinical trial. METHODS: The outcome variables selected were visual field sensitivity and electroretinogram amplitude. The course of change of these outcome variables was determined by calculation of their half-lives. RESULTS: In the 4 to 6 years during which each patient was followed up for this study, median visual field half-lives were 17.0 years (static perimetry) and 11.4 years (kinetic perimetry). Median electroretinogram half-lives were 16.0 years (maximal response) and 10.7 years (flicker response). CONCLUSIONS: The course of the decline of visual function outcome variables is frequently slow. Thus, a long-term clinical trial would be required to assess the efficacy of the intervention in the preservation of visual function.

Correlation of visual acuity and ocular pigmentation with the 16-bp duplication in the HPS-1 gene of Hermansky-Pudlak syndrome, a form of albinism.

OBJECTIVE: Patients with the Hermansky-Pudlak syndrome (HPS), a form of albinism, were studied. The first purpose of this investigation was to determine if visual acuity was related to the presence or absence of the 16-bp duplication in the HPS-1 gene. The second was to study the correlation between the degree of ocular pigmentation and visual acuity within the two genetic groups described above. DESIGN: Cross-sectional study of a series of consecutive patients. PARTICIPANTS: Forty-nine patients with HPS with or without the 16-bp duplication in HPS-1. METHODS: Best corrected visual acuity (VA) using Early Treatment Diabetic Retinopathy Study (ETDRS) charts, photographic gradings of iris transillumination and of visibility of choroidal vessels in the macula (macular transparency). MAIN OUTCOME MEASURES: Association between VA and the presence or absence of the 16-bp duplication in HPS-1 and correlation between VA and the degree of iris transillumination (iris score) and macular transparency (fundus score), as determined by masked reading of photographs, with respect to the presence or absence of the 16-bp duplication in HPS-1 were the main outcome measures. RESULTS: The VA of the better eye did not differ between the two genetic groups (P = 0.322, two-sided t test). Spearman's rank correlation between VA and iris scores in 39 eyes of 20 patients with the duplication was not statistically significant (P = 0.698) but was statistically significant in 36 eyes of 19 patients without the duplication (P < 0.001). Among all patients, the correlation was statistically significant (r = -0.36 in RE and r = -0.51 in LE). Spearman's rank correlation between VA and fundus scores in 36 eyes of 19 patients with and 34 eyes in 18 patients with and without the duplication was statistically significant (P = 0.035 and P = 0.008, respectively). Among all patients, it was also statistically significant (r = -0.39 in RE and r = -0.45 in LE). CONCLUSIONS: The mean VA of the better eye did not differ in patients with the 16-bp duplication compared with those without the duplication. There were statistically significant associations between VA and the iris score and the fundus score except for the VA and iris scores in patients with the 16-bp duplication. However, because of the variability of VA, these associations were not large enough for useful prediction of VA based on the degree of ocular pigmentation.

Pilot trial of the safety, tolerability, and retinoid levels of N-(4-hydroxyphenyl) retinamide in combination with tamoxifen in patients at high risk for developing invasive breast cancer.

PURPOSE: N-(4-hydroxyphenyl) retinamide (¿4-HPR, Fenretinide; R.W. Johnson Pharmaceutical Research Institute, Springhouse, PA) and tamoxifen (TAM) have synergistic antitumor and chemopreventive activity against mammary cancer in preclinical studies. We performed a pilot study of this combination in women at high risk for developing breast cancer. PATIENTS AND METHODS: Thirty-two women were treated with four cycles of 4-HPR, 200 mg orally (PO) for 25 days of each 28-day cycle, and TAM, 20 mg PO once daily for 23 months beginning after 1 month of 4-HPR alone. Tolerability, dark adaptometry, tissue biopsies, and retinoid plasma concentrations (Cp) were evaluated. RESULTS: Symptomatic reversible nyctalopia developed in two patients (6%) on 4-HPR, but 16 (73%) of 22 patients had reversible changes in dark adaptation, which correlated with relative decrease in Cp retinol (P

Effects of fenretinide (4-HPR) on dark adaptation.

OBJECTIVES: To assess the alterations in dark adaptation induced by low (200 mg/d) doses of fenretinide (4-HPR), to assess whether these effects were cumulative and whether they were reversible, and to attempt to elucidate the mechanism underlying the changes in night vision. DESIGN: Case series. SETTING: Outpatient eye clinic. PATIENTS: Twenty-two women enrolled in a breast cancer chemoprevention trial, and 18 normal control subjects. INTERVENTION: Measurements of absolute luminance thresholds during dark adaptation. MAIN OUTCOME MEASURES: Parameters of an exponential model of the dark-adaptation function before, during, and after administration of fenretinide. RESULTS: The most conspicuous effect of fenretinide on dark adaptation was a significant delay in the timing of the rod-cone break (P<.001). A minimal elevation of the final cone threshold was also observed. These effects were reversible after fenretinide therapy was discontinued and did not seem to be cumulative. An inverse relationship between delay of the rod-cone break and plasma retinol concentration was found. CONCLUSION: The dose of fenretinide used in this study produced clearly measurable, but not severe, changes in night vision, which were rarely symptomatic.

Gallium nitrate optic neuropathy.

PURPOSE: To report a patient with visual loss after systemic administration of gallium nitrate. METHOD: Case report. RESULTS: After receiving intravenous gallium nitrate, a 77-year-old man developed bilateral visual loss and optic neuropathy with central scotomas on visual field testing and diminished P2-wave amplitude on visual evoked potential examination. The condition worsened after oral corticosteroid therapy. Partial recovery of optic nerve function in both eyes was present after 12 months of oral ferrous sulfate administration. CONCLUSIONS: Partially reversible bilateral optic neuropathy may occur after administration of gallium nitrate in the absence of other chemotherapeutic agents. Ophthalmic examinations are indicated in patients who receive gallium nitrate.

Evaluation of visual function following neodymium:YAG laser posterior capsulotomy.

PURPOSE: Improvement in visual acuity is the primary endpoint for successful neodymium:YAG (Nd:YAG) laser posterior capsulotomy for posterior capsule opacification. There is limited information on related parameters of visual function that may also improve after laser treatment. The authors evaluate changes in contrast sensitivity and glare disability, aside from visual acuity, following Nd:YAG laser posterior capsulotomy. METHODS: Measurements of visual acuity, contrast sensitivity (using the Pelli-Robson chart), and glare disability (using the Brightness Acuity Tester [Mentor O & O, Inc., Norwell, MA]) were obtained from 24 consecutive patients before and after Nd:YAG laser posterior capsulotomy. Glare testing was done with both the Pelli-Robson and Early Treatment Diabetic Retinopathy Study (ETDRS) charts. The degree of glare disability was indicated by the difference between visual function with glare (at medium and high settings) and without glare. Prelaser measurements were taken within 2 weeks prior to treatment, and postlaser measurements were obtained within 3 months after treatment. Only one eye per patient was evaluated. RESULTS: Mean differences between prelaser and postlaser measurements were significantly different from zero: (1) Contrast sensitivity, mean difference = 0.24 log units (P < 0.0001); (2) High glare disability using Pelli-Robson chart, mean difference = 0.15 log units (P = 0.004); (3) Visual acuity using ETDRS chart, mean difference = 11 letters (P < 0.0001); 4) High glare disability using ETDRS chart, mean difference = 7 letters (P = 0.005). CONCLUSIONS: Using the above methods for visual function testing, Nd:YAG laser capsulotomy is shown to significantly improve visual acuity, contrast sensitivity, and glare disability measurements as compared with prelaser values. The ophthalmologist may find it helpful to document the last two measurements prior to Nd:YAG laser capsulotomy, especially in patients who have good visual acuity but complain of glare sensitivity.

Association of visual field, cup-disc ratio, and magnetic resonance imaging of optic chiasm.

OBJECTIVE: To assess the association of visual field, vertical cup-disc (VC/D) ratio, and vertical height of optic chiasm. DESIGN: Case series. SETTING: Outpatient eye clinic. PATIENTS: Eighteen patients with low, normal, or elevated intraocular pressure, with or without visual field defects. INTERVENTION: Measurement of visual field, VC/D ratio, and vertical height of optic chiasm. MAIN OUTCOME MEASURES: Association between VC/D ratio and visual field defects compared with association between vertical height of optic chiasm and visual field defects. RESULTS: Visual field defects were graded as 0, 1 to 10, and 11 to 20 (from least to most severe). Group mean VC/D ratios were 0.47 (0), 0.55 (1-10), and 0.69 (11-20) for right eyes and 0.48 (0), 0.57 (1-10), and 0.75 (11-20) for left eyes. The significance level for trend was P = .02 for right eyes and P = .006 for left eyes. Group mean chiasm heights were 3.5 (0), 2.9 (1-10), and 2.2 (11-20) mm for right eyes and 3.5 (0), 2.8 (1-10), and 2.2 (11-20) mm for left eyes. The significance level for trend was P < .001 for right eyes and P = .002 for left eyes. To assess the simultaneous effects of VC/D ratio and chiasm height on the visual field defects groups, we used ordinal logistic regression models. Models with both variables implied that chiasm height was a stronger predictor of visual field defects group than VC/D ratio (for right eyes, P = .04 [VC/D ratio], P = .001 [chiasm height]; for left eyes, P = .11 [VC/D ratio], P = .005 [chiasm height]). CONCLUSIONS: When chiasm and VC/D ratio were analyzed in the same model, chiasm height was a stronger predictor of visual field defects. In advanced visual field defects, the optic chiasm is atrophic.

Clinical biochemical and pathologic correlations in Bietti's crystalline dystrophy.

We examined three affected members of a Chinese-American family with Bietti's crystalline retinopathy. The clinical characteristics of a 24-year-old proband are contrasted to the clinical findings of her grandmother, for whom we have 26 years of follow-up data. Lymphocytes and fibroblasts from a skin biopsy of the grandmother contained crystalline lysosomal material, which supports the diagnosis. Biochemical studies of the crystalline lysosomal material failed to identify the stored compounds but did not show them to be cholesterol or cholesterol ester. Finally, histopathologic studies performed for this condition demonstrated advanced panchorioretinal atrophy, with crystals and complex lipid inclusions seen in choroidal fibroblasts.

Evoked potential analysis of visual pathways in human albinism.

PURPOSE: To explore the abnormal crossing of the retinogeniculate pathways in human albinos with visual-evoked potentials (VEPs). METHODS: The authors studied the symmetry of the topographic distribution of pattern onset/offset VEPs over the posterior part of the scalp elicited by monocular stimulation of each eye in 31 consecutive patients with different types of albinism. RESULTS: Twenty-one patients had asymmetric responses, with the major amplitude recorded over the lateral part of the scalp contralaterally to the stimulated eye in 14 patients and ipsilaterally in 7 patients. In two patients, the responses had a symmetric topographic distribution. In eight patients, the amplitude was so low that studying the symmetry was not possible. CONCLUSIONS: These findings demonstrate a non-uniform pattern of asymmetry as a result of the miswiring of the visual pathways in human albinism. Moreover, the low amplitude of the VEP recorded in a consecutive series of patients shows the difficulty of the study of this phenomenon in a clinical setting.

Glare sensitivity in early cataracts.

In a previous study significant glare sensitivity (using Vistech MCT8000) was found only in patients with posterior subcapsular cataracts (PSC) beyond the very early (LOCS II grade 1) stage. The aim of the present study was to evaluate glare sensitivity in patients with early cataracts. The brightness acuity tester (BAT) was used with the Pelli-Robson chart on 50 patients with early cataracts (LOCS II grade 1 or 2) and on 14 normal volunteers. Only age and PSC were found to be associated with change in contrast sensitivity at high glare. Eyes with grade 1 PSC were not significantly different from eyes with grade 0 PSC after adjusting for age. Eyes with grade 2 PSC had significant glare effect compared with eyes having grade 0 PSC. Thus, glare sensitivity is associated only with early (grade 2) PSC. Other tests still need to be developed to assess visual function changes in patients with early cortical and nuclear cataracts.

Contrast sensitivity in patients with nuclear cataracts.

Spatial contrast sensitivity and lens density were measured in 30 subjects (18 patients with pure nuclear cataracts and 12 age-matched controls). Contrast sensitivity was assessed using two techniques: a conventional monitor method in which gratings were viewed through the cataract (overall spatial contrast sensitivity) and a laser interferometer method in which gratings were formed directly on the retina (interferometric spatial contrast sensitivity), thus reducing the effect of an opaque lens on grating contrast. The degree of lens nuclear opacity was measured by assessing the density of Zeiss Scheimpflug slit-lamp video camera images. A contrast sensitivity loss was found by using both methods; this reduction reached statistical significance only when monitor stimuli were used. There was a significant correlation between lens nuclear density and sensitivity loss at spatial frequencies from 4 to 16 cycles/degree (r = .56 to .79 and P less than .05 to less than .001). A correlation coefficient of .82 (P less than .001) characterized the relationship between visual acuity (log of the minimal angle of resolution) and lens density. Nuclear lens opacity significantly affects contrast sensitivity; pure nuclear cataracts produce spatial visual losses at intermediate and high spatial frequencies.

Attempted enzyme replacement using human amnion membrane implantations in mucopolysaccharidoses.

Amnion membrane implantation has been proposed as an approach to enzyme replacement in mucopolysaccharidoses. Human amnion membranes have been subcutaneously implanted in the abdominal wall in 19 patients with mucopolysaccharidoses (MPS I, II and III). A protocol was developed for the objective evaluation of experimental treatments of these patients. Systematic evaluation of the clinical status before and 6 months after amnion membrane implantation reveals no change in function except improvement in joint mobility. The sum of all joint movements showed improvement from baseline values to 6 months after implantation by ANOVA followed by post-hoc analysis (p less than 0.056). The only specific joint movements to significantly improve after 6 months were shoulder extension (p less than 0.01) and hip internal rotation (p less than 0.05). Serial measurements of the deficient lysosomal enzyme activity in serum and white blood cells did not increase in any patient after amnion membrane implantation. Urinary glycosaminoglycan excretion decreased transiently in 2 of 10 patients after implantation, but a second amnion membrane implantation did not result in any change. Biopsy of the implantation site in 10 patients 6 months after amnion membrane implantation revealed a foreign-body reaction with giant cell formation and fibrosis and no recognizable amnion membrane tissue. We conclude that human amnion membrane implantation is not an effective therapy in mucopolysaccharidoses.

Gyrate atrophy of the choroid and retina. Long-term reduction of ornithine slows retinal degeneration.

Gyrate atrophy of the choroid and retina is an autosomal recessive, chorioretinal dystrophy that begins in childhood and leads to blindness in the fourth to seventh decade of life. The primary defect is deficiency of ornithine-delta-amino-transferase, which results in accumulation of ornithine. We examined six pairs of affected siblings to determine if intrafamilial variability in the phenotype was less than interfamilial, and to determine if long-term (5- to 7-year) reduction of ornithine with an arginine-restricted diet had an effect on the progression of the chorioretinal degeneration. All but one set of siblings underwent periodic ophthalmologic examinations. The clinical diagnosis was confirmed with the demonstration of hyperornithinemia and deficiency of ornithine-delta-aminotransferase. The molecular defects in their ornithine-delta-amino-transferase genes also were determined. The two younger pairs of siblings were given an arginine-restricted diet and followed up for 5 to 7 years. We found strikingly similar phenotypes in affected members of the same pair of siblings. In the young patients receiving the diet, there was substantial reduction of ornithine levels. These children had only modest progression of their ocular disease during this period. Furthermore, a comparison of the outcome of the younger with their older siblings at an equivalent age showed that the younger siblings, who started receiving the diet at an earlier age, had much less ocular disease. We conclude that intrafamilial phenotypic variation in gyrate atrophy is less than interfamilial and, therefore, that genetic heterogeneity plays a role in the phenotypic variability of gyrate atrophy. Furthermore, we conclude that chronic reduction of ornithine with an arginine-restricted diet dramatically slows the progression of the chorioretinal dystrophy.

A randomized placebo-controlled trial of cysteamine eye drops in nephropathic cystinosis.

Eighteen patients with nephropathic cystinosis who were younger than 42 months and 11 patients 4 to 31 years of age were entered into a double-masked, randomized, placebo-controlled trial of topical cysteamine eye drops between November 1985 and September 1989. Eight of the younger patients and 2 of the older patients showed marked clearing of corneal crystals in one eye compared with the fellow eye. When the code was broken, all 10 patients were found to have received cysteamine eye drops in the improved eye. Of the remaining 19 patients 4 were unavailable for follow-up. In 15 patients no marked difference was noted between the two eyes. Eight have presumably been in the protocol for too short a time and several have been poor compliers with the therapy. These results not only demonstrate the potential for primary prevention of corneal crystal deposition but also, for the first time, offer the possibility of reversing the corneal complications of cystinosis in older patients.

Attenuating effects of diazepam on the electroretinogram of normal humans.

Response/log I curves were obtained under conditions of dark-and light-adaptation in seven normal male volunteers. For each subject, a baseline electroretinogram (ERG) was recorded. In the same afternoon, a second ERG was obtained after 0.05 mg/kg diazepam, and a third ERG was recorded following 0.10 mg/kg diazepam. Under conditions of dark-adaptation, the following dose-dependent changes were identified: 1) Diazepam attenuated the rod b-wave peak amplitude and increased its implicit time; 2) The dark-adapted longer wavelength cone a-wave and 'blue cone' b-wave showed an increased implicit time; 3) All oscillatory potentials showed an increased implicit time. Under conditions of light-adaptation, dose-dependent decreases in peak amplitude and delays in implicit time were observed for the a- and b-waves. The effects of diazepam on the human ERG were more profound under conditions of light- than dark-adaptation.