Kiyoshi Akeo

A novel Cys-214-Ser mutation in the peripherin/RDS gene in a Japanese family with autosomal dominant retinitis pigmentosa

We have screened for possible disease-causing mutations in the peripherin/retinal degeneration slow (RDS) gene in 13 Japanese families with autosomal dominant retinitis pigmentosa (ADRP). Using polymerase chain reaction-single strand conformation polymorphism analysis, a novel mutation at codon 214 was found in which the highly conserved cysteine was replaced with a serine in one family. The mutation at codon 214 was found in all three affected siblings of this family, but none of the 40 normal control individuals had this mutation. These results strongly suggest, that the mutation is pathogenic for RP in this family. The clinical phenotype for this family is a late-onset form of ADRP.

Autosomal dominant retinitis pigmentosa: A mutation in codon 1 81 (Glu → Lys) of the rhodopsin gene in a Japanese family

The PCR/restriction endonuclease digestion (RE) assay and PCR/SSCP analysis of the rhodopsin gene in 13 Japanese families with autosomal dominant retinitis pigmentosa (ad RP) revealed a G-A substitution of the first nucleotide of codon 181, replacing Glu (GAG) with Lys (AAG), in one family. The proband showed an early onset of symptoms in childhood with a diffuse loss of rod and cone function and a relatively good preservation of cone function, corresponding to the type with relatively rapid progression to blindness (type I category of ad RP).

Dopa and oxygen inhibit proliferation of retinal pigment epithelial cells, fibroblasts and endothelial cells in vitro

Some quinones catalyze superoxide formation in a futile cycle involving electron transfer to molecular oxygen. If the quinolic precursors of melanin participated in the futile cycle, the high ambient oxygen surrounding postnatal RPE would make continued melanogenesis risk for the retina. To probe the possibility that arrest of melanogenesis in postnatal RPE is a protective mechanism, we assayed the growth rates of RPE cells, aortic endothelial cells and skin fibroblasts exposed to 0, 10, 50, 100 and 250 microM dopa. To assess the contribution of the futile cycle, we studied the effects of oxygen concentration and the antioxidants, superoxide dismutase and catalase. We found that all three cell types were significantly and dose-dependently inhibited by dopa and that the effects of dopa were oxygen dependent. The powerful inhibition of RPE cells by dopa was counteracted by inclusion of superoxide dismutase and catalase, but not by an inhibitor of dopa oxidase (phenylthiourea), indicating that the mechanism of growth suppression did not involve melanogenesis but, rather, dopa-dependent formation of superoxide in the media. Endothelial cells were more sensitive to the dopa-mediated oxidative damage than were RPE cells or fibroblasts. Fibroblasts were most affected by oxygen alone, and least affected by dopa. These data suggest that suppression of melanogenesis in the postnatal RPE may be an important mechanism for preventing oxidative damage to the retina and the choriocapillaris We propose that the generation of oxygen radicals by the quinone futile cycle is a viable model of the damage of cells in culture by dopa.

Superoxide dismutase activity and growth of retinal pigment epithelial cells are suppressed by 20% oxygen in vitro

Despite knowledge of the toxicity of oxygen to the retina, its effects on the retinal pigment epithelium have not been considered. We examined the effect of 20%, 10% and 5% oxygen on growth and superoxide dismutase (SOD) activity of porcine retinal pigment epithelial cells (RPE). Growth of RPE cells was very significantly lower in 20% oxygen than in either 10% or 5%; optimal growth occurred at 10% oxygen, the concentration most like their environment in vivo. Inclusion of SOD and catalase in the media very significantly stimulated growth in 20% oxygen. The SOD activity of RPE cells was significantly related to ambient oxygen. In first passage (P1) cells, SOD activity was 44% lower on day 7 than on day 1 of culture in 20% oxygen (p less than or equal to 0.05). Transfer of cells growing in 20% oxygen to 5% oxygen arrested the decrease in SOD and resulted in significantly higher SOD levels. In fourth passage (P4) cells grown in 20% oxygen, SOD was 25% and 44% lower than cells in 10% and 5% oxygen, respectively. After one week, SOD levels in the P4 cells were significantly higher than in P1. A statistical model of SOD activity in RPE cells indicated significant negative correlations with both oxygen concentration and the cell number. Growth of RPE cells was significantly influenced by oxygen level, days of culture and passage number, but not SOD activity. We conclude that traditional culture conditions support generation of free radicals in tissue culture media that suppress both growth and superoxide dismutase activity.

Comparative effects of linoleic acid and linoleic acid hydroperoxide on growth and morphology of bovine retinal pigment epithelial cells in vitro

PURPOSE Outer segments of the photoreceptor rods that are phagocytized by the retinal pigment epithelial (RPE) cells contain a high proportion of polyunsaturated fatty acids (PUFA). PUFA are susceptible to lipid peroxidation. We hypothesized that the resulting peroxides could injure RPE cells leading to retinal degeneration. Accordingly, we compared the effects of linoleic acid (LA) and its hydroperoxide (LHP) on the growth and morphology of RPE cells using laser scanning microscopy and transmission microscopy. METHODS We counted the number of RPE cells after incubation for 24 and 48 hrs with concentrations of LA or LHP of 0.035, 0.175, and 0.35 mM. To observe the actin filaments, cultured RPE cells were stained with rhodamine phalloidin. The cells were prefixed with 2% glutaraldehyde and postfixed in 1% osmium tetroxide. Specimens were embedded in Epon 812 after dehydration, and the ultrathin sections were doubly stained with 2% uranyl acetate and 2% lead acetate for examination by transmission electron microscopy. RESULTS Exposure to LA or LHP produced dose-dependent damage to RPE cells with a significantly greater effects of LHP than LA. After incubation for 24 hrs with 0.35 mM LA, the number of vacuoles in RPE cells exceeded that observed in control RPE cells by 365 nm laser microscopy. Exposure to 0.35 mM LHP for 24 hrs produced a pycnotic nucleus, with diffuse and granular autofluorescences observed in and around it. Exposure of RPE cells to 0.35 mM LA for 24 hrs showed that the LA incorporated into the lysosomes was digested and released extracellularly from lysosomes via exocytotic vesicles. However, such exposure to LHP damaged the RPE cells, including the membranes in the pinocytotic vesicles. The packed membranes resembled myelin. CONCLUSIONS While the LA incorporated into the lysosomes was released extracellularly, LHP persisted in the RPE cells, being observed as autofluorescent lipofuscin-like materials. LHP was cytotoxic, and caused damage to the membranes of pinocytotic vesicles and lysosomes.

VISUAL FUNCTION AND GENE ANALYSIS IN A FAMILY WITH OGUCHI'S DISEASE

A family with 1 case of retinitis pigmentosa (III-1) and 2 cases of Oguchi’s disease (III-2, 3) was examined in terms of electrophysiology as well as molecular biology. The proband (III-3), a 42-year-old female, and 2 older brothers (III-1, 2, aged 52 and 45 years) and 2 unaffected members in the same family participated in this study. Corrected visual acuities of the individuals with Oguchi’s disease (III-2, 3) were 1.2. On funduscopy, blood vessels stood out in relief against a metallic-appearing background and a Mizuo-Nakamura phenomenon was evident. Full-field electroretinograms (ERGs) recorded from the proband were indicative of rod dystrophy, but results of other electrophysiological examinations (multifocal ERG, pattern ERG and visual-evoked cortical potential recordings) were within normal limits. Patient III-1 had corrected visual acuities of RE 20 cm/m.m. and LE 30 cm/n.d., severe chorioretinal atrophy in both fundi, and full-field ERG revealed rod-cone dystrophy. Mutation of the arrestin gene (1147de1A) was detected in all 3 cases, but no mutation was observed for the rhodopsin gene. A homozygous deletion 1147 (1147de1A) in codon 309 of the arrestin gene was commonly observed in all 3 patients. Visual function in each patient coincides with that of retinitis pigmentosa or Oguchi’s disease, respectively.

Multicenter genetic study of retinitis pigmentosa in Japan: I. Genetic heterogeneity in typical retinitis pigmentosa☆

A nationwide, multicenter study of typical retinitis pigmentosa (RP) was carried out in collaboration with 18 hospitals throughout Japan to obtain current information for genetic counseling. We analyzed the genetic heterogeneity of RP based on the parental consanguinity of 434 probands registered during a 6-month period in 1990. A gradual decline in the frequency of consanguineous marriage was recognized among the normal parents of RP patients. The relative frequencies of inheritance patterns were estimated as: autosomal recessive, 25.2%; autosomal dominant, 16.9%; X-linked, 1.6%; and simplex, 56.3%. A comparison of these results with previous reports in Japan revealed a decline in the relative frequency of autosomal recessive cases and an increase in simplex cases. This suggests a decrease in the incidence of autosomal recessive retinitis pigmentosa in Japan, as well as the necessity for exhaustive investigations aimed at identifying inheritance patterns for RP patients seeking genetic counseling.

Comparison of Effects of Oxygen and Antioxidative Enzymes on Cell Growth between Retinal Pigment Epithelial Cells and Vascular Endothelial Cells in vitro

We assayed the proliferation of porcine retinal pigment epithelial (RPE) cells, bovine melanotic and amelanotic RPE cells, and bovine aortic endothelial cells exposed to 20, 10 and 5% oxygen and compared their responses to oxygen and antioxidative enzymes (superoxide dismutase and catalase). Irrespective of the cell type, the cell growth was optimal in 10% oxygen that is most closely approximating to the oxygen concentration prevailing in the cellular environment of the choroid and the retina in vivo. However, the effects of oxygen concentrations were cell specific because bovine endothelial cells were influenced by lowering of oxygen concentrations more significantly than bovine and porcine RPE cells. Moreover, addition of antioxidative enzymes caused significant improvement in growth of porcine RPE cells, but had no significant effects on bovine RPE cells. On the contrary, the bovine vascular endothelial cells represented the only one cell type significantly inhibited by antioxidative enzymes, i.e., a decrease in reactive intermediates of oxygen was seen in the media. Our results show that responses of vascular endothelial cells to reactive species of oxygen were distinctly different from those of RPE cells and more easily influenced by the environment related to hypoxia than RPE cells.

Missense mutation of rhodopsin gene codon 15 found in Japanese autosomal dominant retinitis pigmentosa

SummaryHeterozygous missense mutation in codon 15 of the rhodopsin gene was detected in a patient with autosomal dominant retinitis pigmentosa (ADRP), where a transition of adenine to guanine at the second nucleotide in codon 15 (AAT→AGT), corresponding to a substitution of serine residue for asparagine residue (Asn-15-Ser) was detected. None of the remaining unrelated 42 ADRP, 24 autosomal recessive RP (ARRP) and 34 normal individuals had this alteration. Her funduscopic findings were sectorial in type similar to that of the patients with the same mutation found in an Australian pedigree (Sullivan et al., 1993). This study shows phenotypic similarities in patients with the same mutation of a different ancestry.

Correlation between Contrast Sensitivity and Visual Acuity in Retinitis pigmentosa Patients

Purpose: High-contrast figures such as Landolt rings are insufficient to evaluate the function of the foveal cones of retinitis pigmentosa (RP) patients. We investigated the correlation between visual function as determined with Landolt rings and with the Vistech Contrast Sensitivity Function Test (VCTS) at various spatial frequencies, in addition to the Cambridge Low Contrast Grating (CLCG). Methods: The study included 30 retinitis pigmentosa patients (53 eyes). All patients were assessed with Landolt rings, the Vistech method, and the CLCG. We estimated the relative contribution of contrast sensitivity to visual acuity by VCTS at each spatial frequency and by CLCG by simple linear regression analysis. Results: The results of the regression analysis of VCTS at 1.5, 3.0, and 6.0 cycles/degree showed a significant correlation between Landolt rings and VCTS and between CLCG and VCTS that was strongest at 6.0 cycles/degree. There was no significant correlation between Landolt rings and VCTS or between CLCG and VCTS at 12.0 and 18.0 cycles/degree. Patients with a visual acuity of 20/25 and CLCG greater than 100 were divided into two groups according to their contrast sensitivity at 18.0 cycles/degree on VCTS. Conclusions: The VCTS at the highest frequency was useful for evaluating the foveal visual function in RP patients having good visual acuity with the Landolt rings. Thus, contrast sensitivity should be useful in detecting minute impairment or improvement of visual function in RP.