[Clinical and genetic findings in a patient with fundus albipunctatus]. / Klinische und molekulargenetische Befunde bei einer Patientin mit Fundus albipunctatus.

METHODS: The 38-year-old index patient was examined by visual acuity testing, perimetry, dark adaptometry, funduscopy, electroretinogram (ERG), and multifocal ERG. She was screened for mutations in exons 2-5 and exon/intron boundaries of the 11- cis retinol dehydrogenase gene by direct sequencing. RESULTS: Visual acuity was 1.0, but perimetry revealed paracentral scotomas associated with reading problems. The optic discs were normal. After 45 min of darkness there was nearly no increase of light sensitivity. After 30 min of dark adaptation, the scotopic ERG showed reduced amplitudes, but after 60 min a nearly normal level was reached. The 30-Hz flicker response of the cone ERG showed borderline implicit times, but no reduction of amplitudes. However, multifocal ERG clearly disclosed a paracentral amplitude reduction as the reason for the visual field defects. The fundus was typical for fundus albipunctatus. The patient is a compound heterozygote carrying a Ile33Asn and a Arg157Trp mutation. CONCLUSIONS: The paracentral visual field defects were due to cone dysfunction. So far the patient exhibits no cone dystrophy.

Null mutation in the human 11-cis retinol dehydrogenase gene associated with fundus albipunctatus.

PURPOSE: Recent studies show that mutations in the gene encoding 11-cis retinol dehydrogenase are associated with fundus albipunctatus. The authors wanted to investigate whether additional, more severe, mutations in the 11-cis retinol dehydrogenase gene might be responsible for more severe forms of hereditary retinal diseases. DESIGN: Case-control molecular genetics study. PARTICIPANTS AND CONTROLS: Two index patients, 7 relatives, and 50 control individuals. METHODS: The authors screened two index patients diagnosed with fundus albipunctatus for mutations in exons 2 to 5 and exon/intron boundaries of the 11-cis retinol dehydrogenase gene by direct sequencing. Control individuals were screened for the presence of the mutations using allele-specific oligonucleotide hybridization. MAIN OUTCOME MEASURES: Mutations in exons 2 to 5 and exon/intron boundaries of the 11-cis retinol dehydrogenase gene. RESULTS: In a compound heterozygote, two novel mutations were found: a 4 bp insertion in exon 2 and a missense mutation Cys267Trp in exon 5. In a second pedigree, a homozygous frameshift mutation in codon 43 (Arg42ct[1-bpdel]) was detected. In both families, the mutations segregate with the disease. The mutations were not found in 50 control individuals. CONCLUSIONS: On the basis of our observations, it is unlikely that mutations in the 11-cis retinol dehydrogenase gene are associated with other, possibly more severe, retinal pathologic conditions/dystrophies or syndromic diseases in which the retina is also affected.

Retinoic acid delays transcription of human retinal pigment neuroepithelium marker genes in ARPE-19 cells.

The effect of retinoic acid on the differentiation of a human retinal pigment epithelium-derived cell line ARPE-19 was studied. Differentiation of ARPE-19 cells is delayed by retinoic acid. The minimum all-trans-retinoic acid concentration needed for delay of ARPE-19 differentiation is 1 microM. A delay of differentiation was also observed using 1 microM 9-cis or 13-cis-retinoic acid. Differentiation at the molecular level was studied by analyzing transcription of two RPE-marker genes, RPE65 and peropsin. In the presence of 1 microM retinoic acid the onset of transcription of both genes was delayed by 2-3 weeks. We conclude that all-trans-, 9-cis-, and 13-cis-retinoic acid delay differentiation of ARPE-19 cells into cells that phenotypically resemble cells from the human retinal pigment epithelium.

Disruption of the 11-cis-retinol dehydrogenase gene leads to accumulation of cis-retinols and cis-retinyl esters.

To elucidate the possible role of 11-cis-retinol dehydrogenase in the visual cycle and/or 9-cis-retinoic acid biosynthesis, we generated mice carrying a targeted disruption of the 11-cis-retinol dehydrogenase gene. Homozygous 11-cis-retinol dehydrogenase mutants developed normally, including their retinas. There was no appreciable loss of photoreceptors. Recently, mutations in the 11-cis-retinol dehydrogenase gene in humans have been associated with fundus albipunctatus. In 11-cis-retinol dehydrogenase knockout mice, the appearance of the fundus was normal and punctata typical of this human hereditary ocular disease were not present. A second typical symptom associated with this disease is delayed dark adaptation. Homozygous 11-cis-retinol dehydrogenase mutants showed normal rod and cone responses. 11-cis-Retinol dehydrogenase knockout mice were capable of dark adaptation. At bleaching levels under which patients suffering from fundus albipunctatus could be detected unequivocally, 11-cis-retinol dehydrogenase knockout animals displayed normal dark adaptation kinetics. However, at high bleaching levels, delayed dark adaptation in 11-cis-retinol dehydrogenase knockout mice was noticed. Reduced 11-cis-retinol oxidation capacity resulted in 11-cis-retinol/13-cis-retinol and 11-cis-retinyl/13-cis-retinyl ester accumulation. Compared with wild-type mice, a large increase in the 11-cis-retinyl ester concentration was noticed in 11-cis-retinol dehydrogenase knockout mice. In the murine retinal pigment epithelium, there has to be an additional mechanism for the biosynthesis of 11-cis-retinal which partially compensates for the loss of the 11-cis-retinol dehydrogenase activity. 11-cis-Retinyl ester formation is an important part of this adaptation process. Functional consequences of the loss of 11-cis-retinol dehydrogenase activity illustrate important differences in the compensation mechanisms between mice and humans. We furthermore demonstrate that upon 11-cis-retinol accumulation, the 13-cis-retinol concentration also increases. This retinoid is inapplicable to the visual processes, and we therefore speculate that it could be an important catabolic metabolite and its biosynthesis could be part of a process involved in regulating 11-cis-retinol concentrations within the retinal pigment epithelium of 11-cis-retinol dehydrogenase knockout mice.

Retinoic acid receptors and retinoid X receptors in the mature retina: subtype determination and cellular distribution.

PURPOSE: In the mature retina retinoic acid (RA) biosynthesis was reported to be regulated by light. RA is of vital importance for proper function of the retina. RA activity is mediated by interaction with nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). The purpose of this study was to determine if and which RARs and RXRs are present in the mature retina, and to determine their location within the retina. METHODS: The reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify cDNA fragments encoding RARalpha, RARbeta, RARgamma, RXRalpha, RXRbeta, and RXRgamma from human retinal RNA. RT-PCR products were cloned, sequenced, and used in Northern blot experiments. Antibodies directed against each receptor subtype were used for immunocytochemical analysis. RESULTS: RT-PCR and Northern blot analysis indicated that all RAR and RXR subtypes are present in the mature retina. Western blot analysis, using a cytoplasmic protein fraction isolated from the bovine and human neural retina, showed the presence of RXRalpha. Immunocytochemical analysis of the human, bovine, and rat retina showed that RARalpha is highly expressed in the outer segments of cone photoreceptor cells. RXRalpha expression was observed in the rod inner segment layer. RXRgamma was detected in the nuclei and outer segments of cone photoreceptor cells. CONCLUSIONS: The retinal expression pattern of RARs and RXRs is heterogeneous. The observation that RXRalpha is present in rods whereas RARalpha is present in cones, suggests a mechanism by which RA that is produced upon bleaching, could generate different responses in the two photoreceptor cell subtypes.

Cloning and structural analysis of the murine GCN5L1 gene.

We recently cloned the murine 11-cis retinol dehydrogenase gene. A second gene, the murine GCN5L1 gene, was found to be situated upstream of the murine 11-cis retinol dehydrogenase gene. We have isolated and sequenced the complete coding sequence of the murine GCN5L1 gene. The distance between the 3'-end of the murine GCN5L1 gene and the 5'-end of the 11-cis retinol dehydrogenase gene is only 776 nt. The murine GCNSL1 gene consists of four exons encompassing approximately 3.5 kb of genomic DNA. Intron/exon splice sites conform to the GT/AG rule. The open reading frame consists of 375 nucleotides encoding a 14 kDa protein. The murine GCN5L1, like the human GCN5L1 protein, displays weak homology (27%) to yeast GCN5. The distance between the murine, human and bovine GCN5L1 and 11-cis retinol dehydrogenase genes appeared to be conserved.

Cloning and expression of a cDNA encoding bovine retinal pigment epithelial 11-cis retinol dehydrogenase.

PURPOSE: Identification of a 32-kd protein in the bovine retinal pigment epithelium. METHODS: A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage lambda ZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated. RESULTS: The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus-infected insect cells synthesized the 21-C3/AV antigen. This protein showed 11-cis retinol dehydrogenase activity. CONCLUSIONS: Homology to the human D-beta-hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD. Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde.

Characterization of monoclonal antibodies recognizing retinal pigment epithelial antigens.

PURPOSE: To purify and characterize retinal pigment epithelial proteins exhibiting uveitogenic characteristics after immunization of Lewis rats, a broad panel of monoclonal antibodies directed against retinal pigment epithelial (RPE) protein antigens, was isolated. METHODS: Bovine RPE detergent extracts were used to isolate monoclonal antibodies against RPE antigens. Species and tissue specificity within the eye was tested through immunocytochemical analysis. Western blot analysis was used to determine the molecular weight of the RPE antigens. RESULTS: Several RPE-reactive antibodies were obtained. At least four monoclonal antibodies were isolated that reacted with different RPE antigen types. Against most of the antigens more than one hybridoma cell line was isolated. Two hybridoma lines were isolated producing antibodies, which on immunocytochemical analysis showed strong reactivity with the RPE and eye muscle tissue. The latter monoclonal antibody recognizes a 32 kD protein in RPE cells. Five monoclonal antibodies recognize a protein with an apparent molecular weight of 65 kD. One cell line was isolated that produced antibodies showing an irregular reaction pattern with both iris and ciliary body. CONCLUSIONS: RPE cells, striated eye muscle and smooth muscle cells share a 32 kD antigenic protein. This antigen is present in almost all ocular epithelial cells. Based on reaction patterns on Western blot and immunocytochemical analysis, there are at least three different 65 kD RPE antigens, two of which are RPE-specific and one of which is also present in the kidney epithelium.