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Background: Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly characterized by retinal pigment epithelium (RPE) degeneration with accumulation of abnormal intracellular deposits (lipofuscin) and photoreceptor death. RPE is vital for the retina and integrity of photoreceptors through its phagocytic function which is closely linked to formation of lipofuscin through daily phagocytosis of discarded photoreceptor outer segments (POS). Although phagocytosis has been implicated in AMD, it has not been directly shown to be altered in AMD. RPE phagocytic defect was previously shown to be rescued by subretinal injection of human umbilical tissue derived cells (hUTC) in a rodent model of retinal degeneration (RCS rat) through receptor tyrosine kinase (RTK) ligands and bridge molecules. Here, we examined RPE phagocytic function directly in the RPE from AMD patients and the ability and mechanisms of hUTC to affect phagocytosis in the human RPE., Methods: Human RPE was isolated from the post-mortem eyes of normal and AMD-affected subjects and cultured. RPE phagocytic function was measured in vitro using isolated POS. The effects of hUTC conditioned media, recombinant RTK ligands brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and glial cell-derived neurotrophic factor (GDNF), as well as bridge molecules milk-fat-globule-EGF-factor 8 (MFG-E8), thrombospondin (TSP)-1, and TSP-2 on phagocytosis were also examined in phagocytosis assays using isolated POS. RNA was isolated from normal and AMD RPE treated with hUTC conditioned media and subjected to transcriptome profiling by RNA-Seq and computational analyses., Results: RPE phagocytosis, while showing a moderate decline with age, was significantly reduced in AMD RPE, more than expected for age. hUTC conditioned media stimulated phagocytosis in the normal human RPE and significantly rescued the phagocytic dysfunction in the AMD RPE. RTK ligands and bridge molecules duplicated the rescue effect. Moreover, multiple molecular pathways involving phagocytosis, apoptosis, oxidative stress, inflammation, immune activation, and cholesterol transport were affected by hUTC in the RPE., Conclusions: We demonstrated for the first time RPE phagocytic dysfunction in AMD, highlighting its likely importance in AMD, and the ability of hUTC to correct this dysfunction, providing insights into the therapeutic potential of hUTC for AMD.

Retinal pigment epithelium (RPE) cells perform many functions crucial for retinal preservation and vision. RPE cell dysfunction results in various retinal degenerative diseases, such as retinitis pigmentosa and age-related macular degeneration (AMD). Currently, there are no effective treatments for retinal degeneration except for a small percentage of individuals with exudative AMD. Cell therapies targeting RPE cells are being developed in the clinic for the treatment of retinal degeneration. Subretinal injection of human umbilical tissue-derived cells (hUTC) in the Royal College of Surgeons (RCS) rat model of retinal degeneration was shown to preserve photoreceptors and visual function. However, the precise mechanism remains unclear. Here, we demonstrate that hUTC rescue phagocytic dysfunction in RCS RPE cells in vitro. hUTC secrete receptor tyrosine kinase (RTK) ligands brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and glial cell-derived neurotrophic factor (GDNF), as well as opsonizing bridge molecules milk-fat-globule-epidermal growth factor 8 (MFG-E8), growth arrest-specific 6 (Gas6), thrombospondin (TSP)-1, and TSP-2. The effect of hUTC on phagocytosis rescue in vitro is mimicked by recombinant human proteins of these factors and is abolished by siRNA-targeted gene silencing in hUTC. The bridge molecules secreted from hUTC bind to the photoreceptor outer segments and facilitate their ingestion by the RPE. This study elucidates novel cellular mechanisms for the repair of RPE function in retinal degeneration through RTK ligands and bridge molecules, and demonstrates the potential of using hUTC for the treatment of retinal degenerative diseases., (© 2015 AlphaMed Press.)

UNC119 is widely expressed among vertebrates and other phyla. We found that UNC119 recognized the acylated N terminus of the rod photoreceptor transducin α (Tα) subunit and Caenorhabditis elegans G proteins ODR-3 and GPA-13. The crystal structure of human UNC119 at 1.95-Å resolution revealed an immunoglobulin-like β-sandwich fold. Pulldowns and isothermal titration calorimetry revealed a tight interaction between UNC119 and acylated Gα peptides. The structure of co-crystals of UNC119 with an acylated Tα N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119's hydrophobic cavity. UNC119 bound Tα-GTP, inhibiting its GTPase activity, thereby providing a stable UNC119-Tα-GTP complex capable of diffusing from the inner segment back to the outer segment after light-induced translocation. UNC119 deletion in both mouse and C. elegans led to G protein mislocalization. Thus, UNC119 is a Gα subunit cofactor essential for G protein trafficking in sensory cilia.

HRG4 (UNC119) is a photoreceptor protein predominantly localized to the photoreceptor synapses and to the inner segments to a lesser degree. A heterozygous truncation mutation in HRG4 was found in a patient with late onset cone-rod dystrophy, and a transgenic (TG) mouse expressing the identical mutant protein developed late onset retinal degeneration, confirming the pathogenic potential of HRG4. Recently, the dominant negative pathogenic mechanism in the TG model was shown to involve increased affinity of the truncated mutant HRG4 for its target, ARL2, which leads to a delayed decrease in its downstream target, mitochondrial ANT1, mitochondrial stress, synaptic degeneration, trans-synaptic degeneration, and whole photoreceptor degeneration by apoptosis. In this study, the mouse HRG4 (MRG4) gene was cloned and targeted to construct a knock-out (KO) mouse model of HRG4 in order to study the effects of completely inactivating this protein. The KO model was examined by genomic Southern blotting, Western blotting, immunofluorescence, funduscopy, LM and EM histopathology, ERG, and TUNEL analyses. The KO model developed a slowly progressive retinal degeneration, characterized by mottling in the fundus, mild thinning of the photoreceptor layer, and increase in apoptosis as early as 6 months, dramatic acceleration at approximately 17 months, and virtual obliteration of the photoreceptors by 20 months. When compared to retinal degeneration in the TG model, significant differences existed in the KO consisting of more severe and early photoreceptor death without evidence of early synaptic and trans-synaptic degeneration as seen in the TG, confirmed by LM and EM histopathology, ERG, and Western blotting of synaptic proteins. The results indicated a dysfunction in the KO outside the synapses in the distal end of photoreceptors where MRG4 is also localized. Differences in the phenotypes of retinal degeneration in the KO and TG models reflect a dysfunction in the two opposite ends of photoreceptors, i.e., the distal inner/outer segments and proximal synapses, respectively, indicating a second function of MRG4 in the distal photoreceptor and dual functionality of MRG4. Thus, inactivation of MRG4 by gene targeting resulted in a retinal degeneration phenotype quite different from that previously seen in the TG, attesting to the multiplicity of MRG4 function, in addition to the importance of this protein for normal retinal function. These models will be useful in elucidating the functions of HRG4/MRG4 and the mechanism of slow retinal degeneration.

Purpose: To characterize the time course of apoptosis and degeneration in a transgenic mouse model of retinal degeneration based on truncated mutant HRG4; to investigate the nature of binding of the mutant HRG4 to its target, ADP-ribosylation factor-like (ARL)2; to study its effects on the downstream molecules Binder-of-ARL2 (BART) and adenine nucleotide transporter (ANT)-1 and on the induction of apoptosis., Methods: Saturation binding, microscopic morphometric, Western blot, immunofluorescence, and TUNEL analyses were used., Results: Increased apoptosis did not occur until 20 months in the transgenic retina, consistent with the delayed-onset degeneration in this model. The truncated HRG4 protein exhibited approximately threefold greater affinity for ARL2 than the wild-type HRG4, likely resulting in nonfunctional sequestration of ARL2. A significant decrease in ARL2 was present by 20 months, accompanied by a 50% decrease in ANT-1 in the photoreceptor synaptic mitochondria, with evidence of mitochondrial dysfunction. Preapoptotic degeneration in the photoreceptor synapse was demonstrated with cytochrome c release and caspase 3 activation within the synapse-without evidence of TUNEL-positive apoptosis in the photoreceptor cell body-indicating an initial event in the synapse leading to apoptosis. Caspase 3 was activated in the accompanying secondary neuron, consistent with transsynaptic degeneration., Conclusions: The results support a novel mechanism of retinal degeneration in which preapoptotic degeneration starts in the photoreceptor synapse because of a deficiency in ANT-1 and spreads to the secondary neuron transsynaptically, followed by apoptosis and degeneration in the cell body of the photoreceptor.

X-arrestin (arrestin-3) is an arrestin present specifically in the outer segments of red-, green-, and blue-cone photoreceptors. The X-arrestin gene is on Xcen-q22, and consists of 17 exons with a promoter containing a TATA box and elements important for photoreceptor expression, including three CRX and one PCE-1-like element. In order to delineate the promoter structure necessary for the pan-cone-specific expression of X-arrestin, the expression of the gene in retinoblastoma cell lines was investigated, and a structure-function analysis of the promoter was conducted in the appropriate cellular substrate. Expression of X-arrestin was detected at a low level in the Y79 retinoblastoma cell line but not in the WERI retinoblastoma cell line. Truncation and expression analysis of the X-arrestin promoter in Y79 showed maximal activity in the proximal 378-bp region containing the CRX and PCE-1-like elements upstream of the TATA and CAAT boxes and a negative regulator in the distal 1-2-kbp region. Mutagenesis of the three CRX and PCE-1-like elements and expression analysis demonstrated complete elimination of the promoter activity. Mutagenesis of the TATA box and PCE-1-like element individually resulted in similar decrease in promoter activity, but the decrease in the promoter activity was greater when the CRX elements were mutagenized with a 5' to 3' spatial gradient in the negative effect, suggesting a cooperative effect of the three CRX elements. The regulation of expression from this promoter may involve the binding of a multi-protein enhanceosome complex at the CRX triplet and the PCE-1-like element, resulting in the recruitment and activation of the RNA polymerase II complex at the downstream TATA box.

Human retinal gene 4 (HRG4) (UNC119) is a photoreceptor synaptic protein of unknown function, shown when mutated to cause retinal degeneration in a patient and in a confirmatory transgenic model. ADP-ribosylation factor-like protein 2 (ARL2) was identified as an interactor of HRG4 by the yeast two-hybrid strategy. The presence of ARL2 in the retina and co-localization with HRG4 was confirmed by Western blot and double immunofluorescence analysis, respectively. The interaction of ARL2 with HRG4 was further confirmed by co-immunoprecipitation and direct binding analysis. Phosphodiesterase delta (PDEdelta) is an ARL2-binding protein homologous to HRG4. Amino acid residues of PDEdelta involved in binding ARL2 and forming a hydrophobic pocket were shown to be highly conserved in HRG4, suggesting similarity in binding mechanism and function.

Purpose: To determine whether measurements of the a-wave latency of the electroretinogram (ERG) can be made as reliably as that of the implicit time (IT) in rats. In addition, to determine the relationship between the potential level selected for the latency and the baseline potential level., Methods: ERGs, elicited by different stimulus intensities, were recorded from Long-Evans rats. The a-wave latency was determined by measuring the time between the stimulus onset and the beginning of the negative-going a-wave, and the IT was measured as the time between the stimulus onset and the peak of the a-wave. To test the reliability of the measurements of the latency, the a-wave latency and the IT were measured by three independent observers for the same 15 ERGs., Results: The mean a-wave latency was approximately 14 milliseconds, and the mean a-wave implicit time was approximately 36 milliseconds. The mean of the a-wave latency and the IT, as measured by the three observers, were within 1 millisecond of each other. The coefficient of variation was as good for the latency as for the IT of the a-wave. The potential level selected for the latency was lower than the mean baseline potential level by 1 to 2 standard deviations., Conclusions: Selection of the a-wave latencies can be made as reliably as that for the IT. Because the a-wave latency is not affected by the activity of the second order neurons, the latency is a better measure than the IT of the time course of the a-wave.

Purpose: HRG4 (UNC119) is a photoreceptor synaptic protein, a truncation mutant of which has been shown to cause late-onset cone-rod dystrophy in a patient and retinal degeneration with marked synaptic degeneration in a transgenic model. To investigate the mechanism of the retinal degeneration, the effect of the mutant protein expression on the other synaptic proteins was examined., Methods: The status of 12 known synaptic proteins in the retinas of 5-month- and 13-month-old HRG4 transgenic and control mice was examined by Western blot analysis. Three selected proteins were analyzed by immunofluorescence in the 13-month-old retinas. The 12 proteins were tested for binding to HRG4 by a direct-binding assay and Western blot analysis., Results: A decrease in three synaptic vesicle proteins and an increase in five cytoplasmic and plasma membrane proteins was detected by Western blot analysis in the older but not the younger transgenic retinas. These changes were demonstrated in both the outer and inner plexiform layers of the retina by immunofluorescence, along with a significant reduction in the thickness of the inner plexiform layer. A 23-kDa specie was found to bind to HRG4, but none of the 12 synaptic proteins matched it, according to immunoblot analysis., Conclusions: The expression of a mutant HRG4 protein in the photoreceptor synapses of the transgenic model had an intrasynaptic and transsynaptic effect, resulting in a decrease in three synaptic vesicle proteins, an increase in five cytoplasmic and plasma membrane proteins, and a significant reduction in the thickness of the inner plexiform layer. These changes were age dependent, similar to the pathologic phenotype of the transgenic model and the patient, and supported a close relationship of HRG4 with other participants in synaptic vesicle function. This interaction was not mediated by a direct coupling of HRG4 with any of the tested synaptic proteins but possibly through interaction with a 23-kDa protein.

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.

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.

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 discovered Caenorhabditis elegans gene, 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., (Copyright 1999 Academic Press.)

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.

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.

The 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.

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.

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.

We examined whether primary cultures of rat retinal pigment epithelial (RPE) cells and RPE cells of an immortalized rat cell line, BPEI-1, would be responsive to the neurokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), which are known to be potent trophic factors for neuronal cells. Primary RPE cell cultures were characterized by indirect immunofluorescence and exhibited positive immunoreactivity for RET-PE2, a monoclonal antibody that recognizes RPE cells, and for the intermediate filaments cytokeratin and vimentin. The survival of cultured RPE cells in serum-free defined medium in the presence of CNTF or LIF was investigated during a 0- to 5-day period. Both CNTF and LIF, at concentrations of 1-50 ng/mL (4-200 pM), markedly enhanced RPE cell survival. Bromodeoxyuridine labelling of RPE cells revealed an increased mitotic activity in cell cultures treated with either CNTF or LIF in comparison to untreated serum-free cultures. Increases in cell survival and proliferation after neurokine treatment were also observed with the BPEI-1 cell line. However, in comparison to the primary RPE cultures, LIF was more effective than CNTF in promoting survival of the cell line over a 5-day treatment period. These studies demonstrate that the neurokines CNTF and LIF are potent trophic factors for mammalian RPE cells in vitro and may serve as candidate therapeutic agents in degenerative conditions that affect the retina and RPE.

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.

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., Conclusion: The eight Japanese OAT mutations represent a group of heterogenous mutations unique to a specific population pool.

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.

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.

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.