Disease relevance of RPE65

• Our results suggest that retinal degeneration in these individuals is due to apparently complete paternal isodisomy involving reduction to homoallelism for RPE65 or MERTK loss-of-function alleles [1].
• Mutations in RPE65 cause Leber's congenital amaurosis [2].
• Early-onset severe rod-cone dystrophy in young children with RPE65 mutations [3].
• One father had an ERG compatible with congenital stationary night blindness unrelated to his heterozygous state for the RPE65 mutation [3].
• Moreover, the RPE65 mRNA was detected in three out of four renal tumor cultures analyzed including congenital mesoblastic nephroma and clear cell sarcoma of the kidney [4].

High impact information on RPE65

• Mutations in the human RPE65 gene cause a blinding disease of infancy called Leber congenital amaurosis [5].
• Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium [5].
• Although the function of RPE65 is not yet known, an important role in the RPE/photoreceptor vitamin-A cycle is suggested by the fact that RPE65 associates both with serum retinol-binding protein and with the RPE-specific 11-cis retinol dehydrogenase, an enzyme active in the synthesis of the visual pigment chromophore 11-cis retinal [6].
• In contrast to other genes whose defects have been implicated in degenerative retinopathies, RPE65 is the first disease gene in this group of inherited disorders that is expressed exclusively in the RPE, and may play a role in vitamin-A metabolism of the retina [6].
• We asked whether abnormally thinned RPE65-mutant retina with photoreceptor loss would respond to treatment [7].

Biological context of RPE65

• Retinal dystrophy due to paternal isodisomy for chromosome 1 or chromosome 2, with homoallelism for mutations in RPE65 or MERTK, respectively [1].
• Mutations in RPE65, a gene essential to normal operation of the visual (retinoid) cycle, cause the childhood blindness known as Leber congenital amaurosis (LCA) [7].
• RESULTS: A 35-year-old female carried two RPE65 mutations: a maternal 961A>T (K303X) nonsense mutation and a paternal 1346A>G (Y431C) missense mutation [8].
• METHODS: Haplotype analysis was performed on the DNA of the family members using microsatellite markers against GUCY2D, RPE65, and LCA5 [9].
• Genomic DNA was screened for mutations by means of single-strand conformational polymorphism (SSCP) analysis in exons of the RPE65 and CRX genes [9].

Anatomical context of RPE65

• RPE65 is a protein of unknown function expressed specifically by the retinal pigment epithelium [10].
• Here we show that recombinant RPE65, when expressed in QBI-293A and COS-1 cells, has robust enzymatic activity of the previous unidentified isomerohydrolase, an enzyme converting all-trans retinyl ester to 11-cis retinol in the visual cycle [11].
• Successful, stable restoration of rod and cone photoreceptor function in these dogs has important implications for treatment of human patients affected with Leber congenital amaurosis caused by RPE65 mutations [12].
• In summary, we demonstrate mRNA and protein expression of RPE65 in epidermal keratinocytes [13].
• The RPE65 mRNA was detected in transformed kidney cell lines including the human embryonic kidney cell line HEK293 and the African green monkey kidney cell lines COS-1 and COS-7 by reverse transcription PCR [4].

Associations of RPE65 with chemical compounds

• Mutations in RPE65 cause early-onset blindness, and Rpe65-deficient mice lack 11-cis-retinal but overaccumulate alltrans-retinyl esters in the retinal pigment epithelium (RPE) [14].
• RPE65 is proposed to be a substrate chaperone but may have an enzymatic role because it is closely related to carotenoid oxygenases [14].
• These findings establish a catalytic role, in conjunction with lecithin:retinol acyltransferase, for RPE65 in synthesis of 11-cis-retinol, and its identity as the isomerohydrolase [14].
• Identification of conserved histidines and glutamic acid as key residues for isomerohydrolase activity of RPE65, an enzyme of the visual cycle in the retinal pigment epithelium [15].
• Here, we demonstrated that mutation of any one of the absolutely conserved four histidine and one glutamic acid residues to alanine in RPE65 abolished its isomerohydrolase activity [15].
• This study demonstrates that RPE65 from the cone-dominant chicken RPE possesses significantly higher specific retinol isomerohydrolase activity, when compared with RPE65 from rod-dominant species, consistent with the faster regeneration rates of visual pigments in cone-dominant retinas [16].

Regulatory relationships of RPE65

• RESULTS: We found that the transcriptional activity of the human RPE65 promoter was dramatically enhanced 10-13 fold in RPE cells co-transfected with DNA constructs phR65luc and phR65GV when compared to the human RPE65 promoter alone [17].

Other interactions of RPE65

• We identified an apparently homozygous loss-of-function mutation of RPE65 (1p31) in one retinal dystrophy patient and an apparently homozygous loss-of-function mutation of MERTK (2q14.1) in a second retinal dystrophy patient [1].
• The molecular data for CRB1 and RPE65 support previous hypotheses that LCA can represent the severe end of a spectrum of retinal dystrophies [18].
• The relative contribution of each gene to the total number of mutations was as follows: CRX, 2.8%; GUCY2D, 6.3%; and RPE65, 6.8% [19].
• We suggest that the unusual phenotypic variability in these two siblings with LCA is caused by the modifying effect of a heterozygous GUCY2D mutation observed against the disease background of a homozygous RPE65 mutation [20].
• Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or leber congenital amaurosis [10].

Analytical, diagnostic and therapeutic context of RPE65

• Identifying photoreceptors in blind eyes caused by RPE65 mutations: Prerequisite for human gene therapy success [7].
• 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 [7].
• We performed real-time PCR analysis to evaluate expression of RPE65 mRNA in proliferating and differentiating keratinocytes [13].
• We further show isolation of RPE65 protein by affinity chromatography from lysates of keratinocytes using a retinol-binding protein-matrix gel column [13].
• RESULTS: Sequence analysis places the polymorphic marker, D1S2803, within the RPE65 upstream region and identifies a number of sequences homologous to the gene encoding the cellular retinaldehyde-binding protein [21].

References