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

Characterization of the Gene for HRG4 (UNC119), a Novel Photoreceptor Synaptic Protein Homologous to Unc-119

HRG4 (HGMW-approved symbol UNC119) is a novel human photoreceptor-enriched gene coding for a 240-amino-acid protein. Initially, HRG4 was shown to be 57% homologous to a newly discoveredCaenorhabditis elegansgene, mutated in a coordination mutant and involved in chemosensation. Recently, HRG4 has been localized to the photoreceptor synapses in the outer plexiform layer of the retina. The HRG4 gene was cloned and characterized to facilitate its analysis as a potential pathogenic gene. The gene consisted of five coding exons, spread over approximately 8 kb of genomic DNA. The transcriptional start site was 14 bp upstream of the cDNA, 68 bp upstream of the putative translational initiation codon. Five GC boxes were identified in a 100-bp upstream region, along with a photoreceptor conserved element 1-like sequence at −603. Another photoreceptor gene-associated sequence, Ret-1, was present in intron 1, 71 bp downstream of the exon 1/intron 1 border. A CpG island encompassing exon 1 and sequences just before and after it was present. The gene was fine mapped to 17q11.2, facilitating its future consideration as a candidate for retinal diseases mapped to the same region

Analysis of the human ornithine aminotransferase gene family

Ornithine aminotransferase is a mitochondrial matrix enzyme that is deficient in patients with gyrate atrophy, an autosomal recessive disease of the eye. Southern blots of human DNA probed with a previously characterized OAT cDNA showed a complex pattern of gene fragments, suggesting a gene family. Hybridization of these blots with 5′ and 3′ OAT cDNA probes indicated that there are at least three to four copies of the OAT (approximately 22 kbp) and OAT-related gene sequence(s). We have isolated and partially characterized human OAT gene clones from total genomic and X-chromosome DNA libraries. Sequence analysis confirmed the following previously reported findings on the functional OAT gene: 11 exons, ten introns, an atypical TATA box (TTTAA), two CCAAT boxes, several GC-rich binding sites, 5′ sequence homologous to SV40 enhancer core sequence (GTGGA/GA/GA/GG) and promoter region of three urea cycle enzymes (GTATCCTGCCCTC). In addition, we extended the OAT gene sequence in both the 5′ and 3′ directions and found its promoter region also contained a sequence homologous to the progesterone receptor (TGTTCA/TCC/T), several of the glucocorticoid responsive element (AGAACA), a cyclic AMP-responsive element (TGACGTCG), and recognition motifs for transcription factors AP-2, NF1 and Sp1. Partial sequence analyses of X-chromosome clones demonstrated an intron-less pseudogene with 77% identity to the functional OAT gene. These results demonstrate that the OAT gene is a gene family that contains both functional and related OAT gene sequence(s)

Isolation of human retinal genes: Recoverin cDNA and gene

A human retina cDNA library enriched for retina-specific clones was prepared by subtraction with a non-retina population of cDNA in combination with polymerase chain reaction (PCR) amplifications. A highly retina-specific cDNA clone (1190 bp) was obtained through this library encoding a 200 amino acid protein with three calcium binding sites and 87% homology to the bovine photoreceptor protein, recoverin, which has been shown to mediate the recovery of the dark current after photoactivation, and 58% homology to the calcium-binding chick cone protein, visinin. Analysis of the gene indicated a 9–10 kb single-copy gene with at least three exons and two introns. The three exons contained the entire coding sequence, and all of the calcium-binding EF-hand regions were in putative exon 1. The recoverin gene was mapped to human chromosome 17 by hybridization to a panel of human-rodent hybrid DNAs

Genetic heterogeneity in Hispanic families with autosomal dominant juvenile glaucoma

A gene for autosomal dominant, juvenile-onset, primary open angle glaucoma (glcia) has been previously mapped to Iq21-31 in several Caucasian pedigrees. We studied two Hispanic families with this disease to determine if their disease genes also map to this region. Individuals were considered as being affected if they had iop > 30 mmHg (without treatment) and glaucomatous optic nerve damage or visual field defects. Persons older than 40 years with intraocular pressures 21 mmHg and no evidence of optic nerve damage or visual field loss were scored as unaffected. Individuals not falling into these two categories were considered unknown. Genomic dna was extracted from blood samples and subjected to PCR-based microsatellite marker analysis. Computer-based linkage analysis was used to determine if the disease gene mapped to chromosome Iq21-31. In the family from the Canary Islands, the disease gene was linked to the chromosome Iq21-31 region previously identified by other researchers. Markers D1S212 and D1S218 produced maximum lod scores of 3·38 and 2.99, respectively. In the family from the Balearic Islands, the disease gene was excluded from this region by genetic linkage analysis. Haplotype analysis also excluded the disease gene from chromosome Iq21-31. Our Hispanic families showed genetic heterogeneity with respect to autosomal dominant, juvenile-onset, primary open angle glaucoma

Isolation and characterization of the human X-arrestin gene

Arrestins are signal transduction modulators that quench the activated state of receptors. X-arrestin (ARRX) is specifically expressed in the red-, green-, and blue-sensitive cone photoreceptors, and is most likely a modulator of cone phototransduction. The human gene for X-arrestin at Xcen–Xq22 has been shown to be ∼20 kb in size and to consist of 17 exons and 16 introns. The exons are generally small, including exon 16 of 10 bp, and are clustered into three groups, separated by the two largest introns. This gene structure is generally similar to that of S-antigen, the rod photoreceptor arrestin. There is remarkable similarity, however, among the individual exons between the two genes in that 10 of the exons are identical in size. The 5′ upstream region of the X-arrestin gene contains TATA and CAAT boxes, typical of genes expressed in a tissue-specific manner, in contrast to the S-antigen gene, which lacks these promoter sequences. The promoter elements, common to both the X-arrestin and S-antigen genes, include the Ret-1/PCE-1 (PCE-1-like in X-arrestin), CRX, and the thyroid hormone/retinoic acid-responsive sequences, the former two being present in a number of photoreceptor-expressed genes. Three CRX-binding elements, 15 bp apart, are present in a cluster. The common promoter elements between the cone-expressed genes, X-arrestin and color opsins, include the TATA box, PCE-1, and CRX-binding sequences, the combination of which might be important for directing cone-specific expression

Heterogeneity and uniqueness of ornithine aminotransferase mutations found in Japanese gyrate atrophy patients

Purpose. To identify mutations in ornithine aminotransferase (OAT) in seven Japanese families with gyrate atrophy (GA), an autosomal recessive chorioretinal degeneration of the eye caused by a generalized biochemical deficiency in OAT; mutations in the OAT gene have shown a high degree of molecular heterogeneity. Methods. DNA was prepared from patients' fibroblasts and analyzed by polymerase-chain-reaction amplification of the OAT gene sequence, denaturing gradient gel electrophoresis, and direct sequencing for identification of the mutations. Results. Eight different mutations were identified in seven unrelated Japanese GA patients with hyperornithinemia, confirming the high genetic heterogeneity of this disease. Five of these mutations were new, including one causing a pyridoxine-responsive disease, and all eight mutations have been found only in Japanese GA patients. Consistent with some similarity between the Japanese and Finnish populations in genetic isolation and homogeneity, there was a preponderance of homozygous mutations (five out of seven patients) as was previously reported for 16 Finnish GA pedigrees. Conclusions. The eight Japanese OAT mutations represent a group of heterogenous mutations unique to a specific population pool

Inana G. Localization of HRG4, a photoreceptor protein homologous to

PURPOSE. TO characterize further HRG4, a novel photoreceptor protein recently identified by subtractive cDNA cloning, by sequence analysis and immunolocalization. METHODS. The rat homolog of HRG4, RRG4 was expressed and used to prepare an antibody. The antibody was used in Western blot analysis, and immunofluorescent localization at the light and electron microscopic levels of HRG4-RRG4 protein. The HRG4-RRG4 sequence was also analyzed for homologies. RESULTS. HRG4-RRG4 showed 57% homology with unc-119, a Caenorhabditis elegans neuroprotein causing defects in locomotion, feeding, and chemosensation when mutated. By Western blot analysis, the HRG4-RRG4 protein was demonstrable only in retina and was soluble in nature. Immunofluorescence microscopic study of human and rat retinas, using the HRG4-RRG4 antibody, and other rod and cone photoreceptor-specific antibodies showed that the HRG4-RRG4 protein is localized in the outer plexiform layer of the retina in the synaptic termini of rod and cone photoreceptors. Electron microscopic immunolocalization showed the protein in the cytoplasm and on the presynaptic membranes of the photoreceptor synapses. CONCLUSIONS. The homology to unc-119 and localization to the photoreceptor synapse are suggestive of a function for HRG4-RRG4 in photoreceptor neurotransmission. HRG4 is the first photoreceptor-enriched synaptic protein to be reported, suggesting that its function may be unique to the specialized ribbon synapses formed between photoreceptors and the horizontal and bipolar cells of the retina. (Invest Ophthalmol Vis Set. 1998;39:690-698) T he recently identified HRG4 is a novel human photoreceptor cDNA encoding a 240 amino acid protein. 1 The HRG4 cDNA was one of the clones isolated by a subtractive cDNA cloning strategy to obtain novel human retina-specific cDNAs. Recently, it has been determined that HRG4 is related to a gene discovered in Caenorhabditis elegans. The C. elegans gene, unc-119 was isolated in a mutant nematode with defects in locomotion, feeding behavior, and chemosensation. 3 The defect in locomotion was shown to be based in the nervous system, most likely involving sensory and motor neurons. Expression of unc-119 was seen in many neurons, consistent with the abnormal phenotype of the worm. Because unc-119 appears to be involved in nematode neuronal function, including chemosensation, the homology of HRG4 with unc-119 is suggestive that the novel photoreceptor protein might also be involved in neuronal signal induction or transmission in the photoreceptors. In this report, the production of an antibody to HRG4-RRG4 and its localization to the synaptic termini of rod and cone photoreceptors are described. Of the many known synaptic proteins expressed in photoreceptors, so far only HRG4 has been found to be mainly in photoreceptors, suggestive of a specialized function in visual neurotransmission. From the 'Bascom Palmer Eye Institute and the departmen