Immunolocalization of X-arrestin in human cone photoreceptors

AbstractX-arrestin is a recently identified retina-specific gene of unknown function. Affinity-purified anti-peptide antibody to human X-arrestin was prepared, and used in Western blot analysis of human retinal proteins and for immunohistochemistry on human retinal sections. By Western blot analysis, the antibody specifically bound to an ≈47 kDa protein, and by indirect immunofluorescence specifically labeled cone photoreceptors with greatest intensity in their outer segments. In single and double label experiments, the localization of X-arrestin immunoreactivity was compared with immunolabelling patterns obtained with antibodies to red/green cone opsin, rhodopsin, and S-antigen. The results showed that X-arrestin is expressed in red-, green- and blue-sensitive cones in the human retina

Spectrum of Mutations in the RPGR Gene That Are Identified in 20% of Families with X-Linked Retinitis Pigmentosa

SummaryThe RPGR (retinitis pigmentosa GTPase regulator) gene for RP3, the most frequent genetic subtype of X-linked retinitis pigmentosa (XLRP), has been shown to be mutated in 10%–15% of European XLRP patients. We have examined the RPGR gene for mutations in a cohort of 80 affected males from apparently unrelated XLRP families, by direct sequencing of the PCR-amplified products from the genomic DNA. Fifteen different putative disease-causing mutations were identified in 17 of the 80 families; these include four nonsense mutations, one missense mutation, six microdeletions, and four intronic-sequence substitutions resulting in splice defects. Most of the mutations were detected in the conserved N-terminal region of the RPGR protein, containing tandem repeats homologous to those present in the RCC-1 protein (a guanine nucleotide-exchange factor for Ran-GTPase). Our results indicate that mutations either in as yet uncharacterized sequences of the RPGR gene or in another gene located in its vicinity may be a more frequent cause of XLRP. The reported studies will be beneficial in establishing genotype-phenotype correlations and should lead to further investigations seeking to understand the mechanism of disease pathogenesis

Spectrum of Mutations in the RPGR Gene That Are Identified in 20% of Families with X-Linked Retinitis Pigmentosa

SummaryThe RPGR (retinitis pigmentosa GTPase regulator) gene for RP3, the most frequent genetic subtype of X-linked retinitis pigmentosa (XLRP), has been shown to be mutated in 10%–15% of European XLRP patients. We have examined the RPGR gene for mutations in a cohort of 80 affected males from apparently unrelated XLRP families, by direct sequencing of the PCR-amplified products from the genomic DNA. Fifteen different putative disease-causing mutations were identified in 17 of the 80 families; these include four nonsense mutations, one missense mutation, six microdeletions, and four intronic-sequence substitutions resulting in splice defects. Most of the mutations were detected in the conserved N-terminal region of the RPGR protein, containing tandem repeats homologous to those present in the RCC-1 protein (a guanine nucleotide-exchange factor for Ran-GTPase). Our results indicate that mutations either in as yet uncharacterized sequences of the RPGR gene or in another gene located in its vicinity may be a more frequent cause of XLRP. The reported studies will be beneficial in establishing genotype-phenotype correlations and should lead to further investigations seeking to understand the mechanism of disease pathogenesis

Expression of human ornithine aminotransferase (OAT) in OAT-deficient Chinese hamster ovary cells and fibroblasts of gyrate atrophy patient

Gyrate atrophy is a hereditary chorioretinal degenerative disease caused by a deficiency of the mitochondrial enzyme, ornithine aminotransferase (OAT). Recent investigations have demonstrated the molecular genetic defects of OAT in gyrate atrophy patients. We constructed a eukaryotic expression vector (pcDHOAT) which contains the SV40 promoter and human OAT cDNA. We used OAT(-) Chinese hamster ovary (CHO) cells, which have negligible OAT activity, and fibroblasts from a gyrate atrophy patient (GA35 cell), which have negligible OAT mRNA and enzyme. Incorporation of pcDHOAT and synthesis of human OAT mRNAs and active enzyme were demonstrated in both cell types. The level of expression of human OAT was low in the GA35 cells in comparison to the CHO cells. Despite the limited success, the ability to express active OAT in these OAT-deficient cells using an expression vector offers possibilities of replacement gene therapy for gyrate atrophy

Inherited retinal degeneration: basic FGF induces phagocytic competence in cultured RPE cells from RCS rats

In RCS rats, the retinal pigment epithelium (RPE) is defective in phagocytosis of photoreceptor membranes. We have previously shown reduced expression of basic fibroblast growth factor (bFGF) in the RPE of 7-10-day-old RCS rats. This study using primary RPE cultures from rats of this age demonstrates that the phagocytic defect in the mutant RPE can be overcome by treatment with bFGF, by a mechanism involving gene transcription and that normal RPE phagocytosis, also requiring transcription, is blocked by a bFGF neutralizing antibody. The combined data point to a role for bFGF in the normal mechanism of RPE phagocytosis and the RCS defect

Rapid and efficient molecular analysis of gyrate atrophy using denaturing gradient gel electrophoresis

A generalized biochemical deficiency of the mitochondrial matrix enzyme ornithine aminotransferase (OAT) is the inborn error in gyrate atrophy (GA), an autosomal recessive blinding disease of the retina and choroid of the eye. Because mutations in the OAT gene show a high degree of molecular heterogeneity in GA, the authors set out to determine the mutations by rapid and efficient methods. The mutations in the OAT gene were determined by a combination of polymerase chain reaction (PCR) amplification of gene sequences, analysis by denaturing gradient gel electrophoresis (DGGE), and direct DNA sequencing. Eleven different mutations in 21 (95.5%) out of 22 mutant OAT alleles from 11 patients were identified: six missense mutations, three nonsense mutations, one 2 bp-deletion, and one splice acceptor mutation. A silent polymorphism of Asn (AAC)378 to Asn (AAT) was also observed. CONCLUSIONS. The combination of PCR amplification of the gene sequences, DGGE analysis, and direct sequencing is a rapid and efficient method for detection of mutations in GA cases. The diversity of the mutations attests to the enormous genetic heterogeneity in this disease