Selective loss of a family of gene transcripts in a hereditary murine cataract.

The eye lens of the Fraser mouse contains a dominantly inherited cataract with reduced amounts of seven distinct but homologous gamma crystallins encoded by a family of gamma-crystallin genes. The results of experiments with cultured lenses, cell-free RNA translation, and Northern blot hybridization indicated a specific loss of the family of gamma-crystallin messenger RNA's in the Fraser mouse lens. Southern blot hybridization of genomic DNA's from normal and Fraser mice showed no differences in gamma-crystallin coding sequences.

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 approximately 20kb in size and to consist of 17 exons and 16 introns. The exons are generally small, including exon 16 of 10bp, 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, 15bp 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.

Isolation and characterization of cDNA encoding the gamma-subunit of cGMP phosphodiesterase in human retina.

Cyclic GMP-phosphodiesterase (cGMP-PDE) plays a key role in the normal functioning of retinal rod photoreceptor cells. The enzyme is composed of alpha- and beta-catalytic subunits which are inhibited by two identical gamma-subunits. A cDNA encoding the gamma-subunits (PDE gamma) from human retina has been cloned and sequenced. The 1012-bp cDNA has a coding region of 261 bp which is highly homologous to those of the PDE gamma cDNAs from bovine and mouse retinas. Comparison of the deduced amino acid sequences of the proteins from the three species indicates that PDE gamma has been very well conserved through evolution. The mRNA encoded by the cloned cDNA is 1.0 kb long, is similar in size to the corresponding mRNAs from mouse, dog and bovine retinas and is not detected in ground squirrel retina. The PDEG gene has been assigned to human chromosome 17, probably in the region q21.1.

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.

X-arrestin: a new retinal arrestin mapping to the X chromosome.

We have been using a differential cDNA cloning approach to isolate human retina-specific and retina-enriched genes [1]. A 1,314 bp cDNA was isolated by this approach, representing a highly retina-specific message encoding a 388 amino acid protein showing 58%, 50%, and 49% homology to bovine beta-arrestin, and bovine and human retinal arrestin (S-antigen), respectively. Chromosomal mapping localized this new arrestin gene to the proximal long arm of the X chromosome, hence it was named X-arrestin. In situ hybridization demonstrated its expression in the inner and outer segments and the inner plexiform regions of the retina.

Immunolocalization of X-arrestin in human cone photoreceptors.

X-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 approximately 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 immunolabeling 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.

Analysis of basic fibroblast growth factor in rats with inherited retinal degeneration.

In RCS rats, photoreceptors degenerate between postnatal days 20 and 60, secondary to a genetic defect expressed in the neonatal retinal pigmented epithelium (RPE). Previous work has shown delay of the photoreceptor degeneration in this model by intraocular injection of basic fibroblast growth factor (bFGF). Evidence is presented here, from bFGF immunostaining and Northern analysis of bFGF mRNA, for reduced bFGF expression in uncultured RPE of dystrophic RCS pups. It is also shown that in the mutant eyes angiogenesis in the underlying choroid, which normally occurs between postnatal days 7 and 10, is markedly delayed, with irregular distribution of vessels, consistent with a reduction in this known angiogenesis factor. Mutational analysis of the bFGF transcript and gene by denaturing gradient gel electrophoresis and Southern analysis did not, however, reveal abnormalities in the coding sequence of this gene in RCS rats.

Mechanism of suppression of malignancy in hybrids between Y79 retinoblastoma and NIH3T3 cells.

Hybrids were prepared between Y79 retinoblastoma (RB) and nonmalignant NIH3T3 cells and studied to confirm the presumed recessiveness of RB. Twenty hybrids containing both of the dominant gene markers, pSV2neo and pSV2GPT, initially transfected into the parent cells were isolated. All of the hybrids showed a fibroblastic morphology and anchorage-dependent growth. None of the tested hybrids or the parent NIH3T3 cells showed growth in soft agar; the parent RB cells showed a 15% growth in soft agar. The results indicate suppression of the malignant phenotype in the hybrids, confirming the recessiveness of the malignant phenotype of RB at the cellular functional level. Karyotyping of selected hybrids and the parent cells indicated a cumulative representation of all of the Y79 chromosomes in the hybrids, excluding loss of a specific Y79 chromosome causing the suppression of malignancy. Northern analysis of RNA from the hybrids demonstrated the mRNA of the reported putative mouse RB gene, consistent with the complementation of the Y79 RB gene defect by the normal mouse RB gene in the hybrids. Such a complementation may be a factor in the suppression of the malignant phenotype. Interestingly, the abnormal Y79 RB mRNA was absent in the hybrids, suggesting a possible negative feedback control by the normal mouse RB gene product.

Gene transfer and expression of human ornithine aminotransferase.

A generalized deficiency in the mitochondrial enzyme, ornithine aminotransferase (OAT), is present in the hereditary blinding disease, gyrate atrophy (GA). Because the OAT gene is a multi-gene family, and the native OAT enzyme is an oligomer, it would be important to identify the gene locus actually responsible for the OAT activity and affected in GA. A mammalian expression clone containing a previously characterized human OAT cDNA (pHOAT), corresponding to the OAT gene on chromosome 10, was prepared (pcDHOAT), transfected, and tested for expression in NIH3T3 cells and OAT(-) Chinese hamster ovary (CHO) cells. Incorporation of pcDHOAT and synthesis of human OAT mRNAs and active enzyme were demonstrated in both cell types, confirming the completeness of the coding sequence of pHOAT. The results indicated that the chromosome 10 gene corresponding to the cDNA is the functional gene fully capable of expressing the active oligomeric enzyme by itself and is, therefore, consistent with being the site of the molecular defect in GA.

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

PURPOSE: 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. 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. RESULTS: 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.

Double fluorescent vital assay of phagocytosis by cultured retinal pigment epithelial cells.

PURPOSE: The goal of this study was to develop the first vital assay system for in vitro analysis of phagocytosis of rod outer segments (ROS) by normal retinal pigment epithelial (RPE) cells and for study of the phagocytic defect in RPE of the Royal College of Surgeons (RCS) rat with inherited retinal degeneration. Required features included ability to directly visualize and quantitate the phagocytic process in living RPE cultures, and capability for subsequent quantitative analysis after fixation of the cells at any chosen time after incubation with ROS. METHODS: A double fluorescent method was designed, based on the process of phagosome-lysosome fusion. For vital staining of lysosomes, confluent cultures of rat RPE cells were incubated with sulforhodamine (SR), a red fluorescent lysosomotropic dye. SR-stained cultures were challenged with isolated rat ROS tagged with fluorescein isothiocyanate (FITC), a green fluorescent probe. RESULTS: This method was used to observe all phases of the phagocytic process in the living cells and the kinetics of ROS binding, ingestion, and phagosome-lysosome fusion were determined. Control studies showed no differences in binding, ingestion, or digestion of unstained versus FITC-stained ROS. Additionally, the phagocytic defect in dystrophic RCS rat RPE cells was confirmed using this technique. CONCLUSIONS: This relatively simple new method is useful in that it uses inexpensive, readily available reagents, it enables real-time analysis of phagocytosis experiments, and it does not require termination of the cultures for analysis of phagocytic ability.

Localization of HRG4, a photoreceptor protein homologous to Unc-119, in ribbon synapse.

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.

Chromosomal localization of human ornithine aminotransferase gene sequences to 10q26 and Xp11.2.

Gyrate atrophy is a hereditary chorioretinal degeneration associated with a deficiency of ornithine aminotransferase (OAT). By means of a complementary DNA clone encoding human OAT, the OAT gene sequences were mapped by somatic cell hybrids and in situ hybridization to human chromosome regions 10q26 and Xp11.2. A review of 80 biochemically confirmed cases of gyrate atrophy confirmed the autosomal recessive inheritance of this disease and supported the presence of a functional OAT gene on chromosome 10. Interestingly, the X chromosome OAT gene sequences (Xp11.2) map to the same region as L1.28 (Xp11.0-p11.3), a marker closely linked to X-linked recessive retinitis pigmentosa.

Evaluation of the human gene encoding recoverin in patients with retinitis pigmentosa or an allied disease.

PURPOSE: To determine whether defects in the human recoverin gene cause retinitis pigmentosa (RP) or an allied disease such as Usher syndrome, Leber congenital amaurosis, or the Bardet-Biedl syndrome. METHODS: Single-strand conformation polymorphism analysis and direct genomic sequencing techniques were used to screen 596 unrelated patients, comprising 167 patients with dominant RP, 168 with recessive RP, and 261 with an allied disease. RESULTS: Four sequence variants were discovered. The first was a missense change (Ala200Thr) found in one family with autosomal dominant RP and in one family with autosomal recessive RP; it did not segregate with disease. The second was a silent, single-base variation affecting codon Ser24 with a minor allele frequency of approximately 0.5%. The third was a silent, single-base variation affecting codon Va1122. The fourth was a single-nucleotide substitution in intron 2, 11 bp upstream of exon 3. CONCLUSIONS: The authors found no evidence that mutations in the recoverin gene are a cause of RP or another of the hereditary retinal diseases studied. The human phenotype associated with mutations of the recoverin gene remains unknown.

X-linked retinitis pigmentosa: functional phenotype of an RP2 genotype.

Rod- and cone-mediated function was studied with psychophysics and electroretinography in members of an X-linked retinitis pigmentosa pedigree with the RP2 genotype. An asymptomatic hemizygote with an early stage of the disease had cone dysfunction in the mid-periphery and an abnormal cone electroretinogram (ERG); rod function was normal. Hemizygotes with more advanced disease had cone and rod dysfunction in the mid-peripheral retina and cone dysfunction in the far periphery; cone and rod ERGs were abnormal. At very advanced stages, there was an absolute mid-peripheral scotoma and marked cone and rod dysfunction in the far peripheral and central retina. Cone and rod ERGs were severely abnormal or not detectable. Heterozygotes showed tapetal-like reflexes, patches of pigmentary retinopathy, and a range of functional findings from no detectable abnormalities to moderate levels of retinal dysfunction. There were regions of normal function adjacent to dysfunctional patches that had greater cone than rod sensitivity losses or comparable cone and rod losses. The results suggest that the phenotype of this RP2 genotype of X-linked retinitis pigmentosa, unlike other forms of retinitis pigmentosa, is first expressed as a cone photoreceptor system dysfunction, and as the disease progresses, both rod and cone systems are involved.

Expression defect of ornithine aminotransferase gene in gyrate atrophy.

A generalized deficiency in the mitochondrial enzyme, ornithine aminotransferase (OAT: EC 2.6.1.13), is the hallmark of gyrate atrophy (GA), a hereditary degenerative disease of the choroid and retina of the eye that leads to blindness. A human OAT cDNA, previously constructed and characterized in our laboratory, and anti-human OAT antibody were used as probes to examine the OAT gene, mRNA and protein of GA patients. A blot analysis of the genomic DNAs, RNAs and proteins of 14 GA patients identified a case with a partial heterozygous deletion of the functional OAT gene located on chromosome 10, no detectable OAT mRNA, and a barely detectable level of OAT antibody-reactive protein. The rest of the cases showed grossly normal OAT gene, mRNA, and variably reduced levels of OAT protein. A restriction fragment length polymorphism (RFLP) was identified in the functional OAT gene sequence with EcoRI which may be useful for prenatal diagnosis of GA. RFLPs were also identified in the OAT-related gene sequences located on the X chromosome with Hind III and Pst I which may potentially show linkage to X-linked retinitis pigmentosa locus. The finding of an OAT gene, mRNA, and protein defect in a GA case constitutes the first real demonstration of the molecular genetic defect of OAT in GA.

HRG4 (UNC119) mutation found in cone-rod dystrophy causes retinal degeneration in a transgenic model.

PURPOSE: To investigate the function and pathogenicity of HRG4, a photoreceptor synaptic protein homologous to the Caenorhabditis elegans neuroprotein UNC119. METHODS: HRG4 was screened for mutations in patients with various retinopathies, and a transgenic mouse model was constructed and analyzed based on a mutation found. RESULTS: A heterozygous premature termination codon mutation was found in a 57-year-old woman with late-onset cone-rod dystrophy. In some transgenic mice carrying the identical mutation, age-dependent fundus lesions developed accompanied by electroretinographic changes consistent with defects in photoreceptor synaptic transmission (depressed b-wave, normal c-wave), and retinal degeneration occurred with marked synaptic and possible transsynaptic degeneration. CONCLUSIONS: HRG4, the only synaptic protein known to be highly enriched in photoreceptor ribbon synapses, is now shown to be pathogenic when mutated.

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 1q21-31 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 1OP > 30 mmHg (without treatment) and glaucomatous optic nerve damage or visual field defects. Persons older than 40 years with intraocular pressures < or = 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 1q2I-31. In the family from the Canary Islands, the disease gene was linked to the chromosome 1q2I-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 1q21-31. Our Hispanic families showed genetic heterogeneity with respect to autosomal dominant, juvenile-onset, primary open angle glaucoma.

Genotype-phenotype correlation of a pyridoxine-responsive form of gyrate atrophy.

Two clinical subtypes of gyrate atrophy (GA) have been defined based on in vivo or in vitro evidence of response to vitamin B6 (pyridoxine), which is the cofactor of the enzyme ornithine aminotransferase (OAT) shown to be defective in GA. We identified the E318K mutation in the OAT gene, heterozygously in three patients and homozygously in one patient, all of whom were vitamin B6-responsive by previous in vivo and in vitro studies. Dose-dependent effects of the E318K mutation were observed in the homo- and heterozygotes in the OAT activity, increase of OAT activity in the presence of pyridoxal phosphate, and apparent Km for pyridoxal phosphate. The highest residual level of OAT activity and mildness of clinical disease correlated directly with the dose of the mutant E318K allele present in the patient.

Analysis of phosducin as a candidate gene for retinopathies.

Phosducin, a retina-expressed gene mapped to chromosome 1q25-32.1, was analyzed as a candidate gene for retinopathies. The phosducin gene was cloned and characterized, and PCR primers were designed. Eighty-three patients with various retinopathies and 45 control subjects (24 American, 21 Japanese) were analyzed for mutations in the phosducin gene by PCR, denaturing gradient gel electrophoresis (DGGE), and sequencing. A heterozygous sequence variant changing a glycine to arginine at codon 178 was found in one Usher syndrome type II (USH2) patient, while the other USH2 patients did not show any coding sequence variant. A heterozygous sequence variant changing an asparagine to lysine at codon 174 was found in a patient with a severe retinal degeneration in the category of diseases known as acute zonal occult outer retinopathy (AZOOR). Three non-coding sequence variants were found. Two of these were always present together and found in 20.8% of American and 2.4% of Japanese control subjects, reflecting a difference in population pools. In conclusion, the phosducin gene did not show mutations consistent with it being the causative gene for USH2, but its possible pathogenicity in AZOOR or other retinopathies remains an open question which may be answered by further analysis.