Complement factor H polymorphisms in Japanese population with age-related macular degeneration.

PURPOSE: To study the frequency of five haplotypes previously reported in the complement factor H (CFH) gene for Japanese patients with age-related macular degeneration (AMD). METHODS: Genomic DNA was isolated from peripheral blood samples taken from 96 Japanese AMD patients and 89 age-matched controls. All patients were diagnosed as having exudative (wet-type) AMD. The amplified polymerase chain reaction (PCR) products of CFH exons 2, 9, and 13, and intron 6 were analyzed by temperature gradient capillary electrophoresis (TGCE) and by direct sequencing. The haplotypes were identified, and their frequencies were calculated and compared with reported results. RESULTS: Five haplotypes were identified in the Japanese population including four already reported in the American population. The frequencies of these haplotypes were significantly different between Japanese and American in both control and case groups. The haplotype containing Y402H, which was previously reported to be associated with AMD, was only 4% in the control and case population, with a p value of 0.802. However, two other haplotypes were found as risk factors, which gave an increased likelihood of AMD of 1.9 and 2.5 fold (95% CI 1.12-3.69 and 1.42-6.38). One protective haplotype that decreased the likelihood of AMD by 1.6 fold (95% CI 0.26-0.67) was identified. CONCLUSIONS: The frequencies for five haplotypes previously identified were analyzed in a Japanese population with AMD. Four previously found haplotypes were identified and one additional haplotype was found. The frequencies of each haplotype were significantly different from that in found Americans affected with AMD. Two of the haplotypes were identified as risk factors and one was considered protective.

Molecular composition of drusen and possible involvement of anti-retinal autoimmunity in two different forms of macular degeneration in cynomolgus monkey (Macaca fascicularis).

We have previously reported a cynomolgus monkey (Macaca fascicularis) pedigree with early onset macular degeneration that develops drusen at 2 yr after birth. In this study, the molecular composition of drusen in monkeys affected with late onset and early onset macular degeneration was both characterized. Involvement of anti-retinalautoimmunity in the deposition of drusen and the pathogenesis of the disease was also evaluated. Funduscopic and histological examinations were performed on 278 adult monkeys (mean age=16.94 yr) for late onset macular degeneration. The molecular composition of drusen was analyzed by immunohistochemistry and/or direct proteome analysis using liquid chromatography tandem mass spectroscopy (LC-MS/MS). Anti-retinal autoantibodies in sera were screened in 20 affected and 10 age-matched control monkeys by Western blot techniques. Immunogenic molecules were identified by 2D electrophoresis and LC-MS/MS. Relative antibody titer against each antigen was determined by ELISA in sera from 42 affected (late onset) and 41 normal monkeys. Yellowish-white spots in the macular region were observed in 90 (32%) of the late onset monkeys that were examined. Histological examination demonstrated that drusen or degenerative retinal pigment epithelium (RPE) cells were associated with the pigmentary abnormalities. Drusen in both late and early onset monkeys showed immunoreactivities for apolipoprotein E, amyloid P component, complement component C5, the terminal C5b-9 complement complex, vitronectin, and membrane cofactor protein. LC-MS/MS analyses identified 60 proteins as constituents of drusen, including a number of common components in drusen of human age-related macular degeneration (AMD), such as annexins, crystallins, immunoglobulins, and complement components. Half of the affected monkeys had single or multiple autoantibodies against 38, 40, 50, and 60 kDa retinal proteins. The reacting antigens of 38 and 40 kDa were identified as annexin II and mu-crystallin, respectively. Relative antibody titer against annexin II in affected monkeys was significantly higher than control animals (P<0.01). Significant difference was not observed in antibody titer against mu-crystallin; however, several affected monkeys showed considerably elevated titer (360-610%) compared with the mean for unaffected animals. Monkey drusen both in late and early onset forms of macular degeneration had common components with drusen in human AMD patients, indicating that chronic inflammation mediated by complement activation might also be involved in the formation of drusen in these affected monkeys. The high prevalence of anti-retinalautoantibodies in sera from affected monkeys demonstrated an autoimmune aspect of the pathogenesis of the disease. Although further analyses are required to determine whether and how autoantibodies against annexin II or mu-crystallin relate to the pathogenesis of the disease, it could be hypothesized that immune responses directed against these antigens might trigger chronic activation of the complement cascade at the site of drusen formation.

Early-onset macular degeneration with drusen in a cynomolgus monkey (Macaca fascicularis) pedigree: exclusion of 13 candidate genes and loci.

PURPOSE: To describe hereditary macular degeneration observed in the cynomolgus monkey (Macaca fascicularis), which shares phenotypic features with age-related macular degeneration in humans, and to test the involvement of candidate gene loci by mutation screening and linkage analysis. METHODS: Ophthalmic examinations with fundus photography, fluorescein angiography (FA), indocyanine green angiography (IA), electroretinography (ERG), and histologic studies were performed on both affected and unaffected monkeys in the pedigree. The monkey orthologues of the human ABCA4, VMD2, EFEMP1, TIMP3, and ELOVL4 genes were cloned and screened for mutations by single-strand conformation polymorphism (SSCP) analysis or denaturing high-performance liquid chromatography (DHPLC) and direct sequencing in six affected and five unaffected monkeys from the pedigree and in six unrelated, unaffected monkeys. Subsequently, 13 human macular degeneration loci including these five genes were analyzed to test for linkage with the disease. Nineteen affected and seven unaffected monkeys in the pedigree were analyzed by using human microsatellite markers linked to the 13 loci. RESULTS: Yellowish white spots were observed in the macula and fovea centralis, and in some cases the spots scattered to the peripheral retina along the blood vessels. FA showed hyperfluorescence corresponding to the dots except in the foveola. No anomalies were found by IA and ERG. Histologic studies demonstrated that the spots were drusen. Mutation analysis of the ABCA4, VMD2, EFEMP1, TIMP3, and ELOVL4 genes identified a few sequence variants, but none of them segregated with the disease. Linkage analysis with markers linked to these five genes and an additional eight human macular degeneration loci failed to establish linkage. Haplotype analysis excluded the involvement of the 13 candidate loci for harboring the gene associated with macular degeneration in the monkeys. CONCLUSIONS: Significant homology was identified between monkey and human orthologues of the five macular degeneration genes. Thirteen loci associated with macular degeneration in humans or harboring macular degeneration genes were excluded as causal of early-onset macular degeneration in the monkeys. It is likely that none of these loci, but rather a novel gene, is involved in causing the observed phenotype in this monkey pedigree.

Characterization of AOC2 gene encoding a copper-binding amine oxidase expressed specifically in retina.

We have previously cloned a human, retina-specific, amine oxidase gene (RAO, gene symbol: AOC2), a member of the copper-binding amine oxidase super family. AOC2 shares sequence identity with the human kidney amine oxidase gene (KAO, gene symbol: AOC1) and the vascular adhesion protein-1 gene (VAP-1, gene symbol: AOC3). For further analysis of AOC2, the sequences surrounding the human AOC2 and the complete mouse and partial rat homologue of AOC2 were cloned for characterization. Real-time quantitative PCR, in situ hybridization, and immunohistochemistry were performed to determine the specific expression of AOC2 in the mouse retina and especially in the retinal ganglion cells. Our results demonstrated that the copper-binding motif and the enzyme active site of AOC1 and AOC3 were both conserved in mouse AOC2. The human and mouse AOC2 was flanked by two genes, the Psme3 gene for PA-28 gamma subunit and, surprisingly, the AOC3 gene. Rat AOC2 contained a stop codon that terminated the peptide length to 127 amino acids. The presence of human and rat AOC pseudogene in this region, in addition to the tandemly positioned two AOC genes, indicates the possibility of successful AOC3 replication to retina-specific AOC2 for human and mouse but unsuccessful for rat.

Molecular cloning of ELOVL4 gene from cynomolgus monkey (Macaca fascicularis).

ELOVL4, elongation factor of very long chain fatty acids-4, is known to be responsible for autosomal dominant macular degeneration and Stargardt-like macular degeneration. In this study, we cloned the monkey homologue of ELOVL4 and determined the cellular and tissue distribution of the gene product. Sequence analysis of the monkey ELOVL4 gene revealed a high degree of homology between human and monkey. The cloned full-length cDNA of monkey ELOVL4 encoded 314 amino acids, the same length as human and two amino acids longer than mouse. The monkey ELOVL4 conserved the characteristics typical of the super family of ELO enzymes involved in the metabolism of membrane-bound fatty acid elongation. Real-time quantitative PCR demonstrated that the monkey ELOVL4 gene was highly expressed in restricted tissue-specific fashion, not only in the retina but also in the skin (90% of retina) and thymus (111% of retina). Immunohistochemical analysis detected signals predominantly in the photoreceptor layer of the monkey retina.

Comparative proteomic analyses of macular and peripheral retina of cynomolgus monkeys (Macaca fascicularis).

The central region of the primate retina is called the macula. The fovea is located at the center of the macula, where the photoreceptors are concentrated to create a neural network adapted for high visual acuity. Damage to the fovea, e.g., by macular dystrophies and age-related macular degeneration, can reduce central visual acuity. The molecular mechanisms leading to these diseases are most likely dependent on the proteins in the macula which differ from those in the peripheral retina in expression level. To investigate whether the distribution of proteins in the macula is different from the peripheral retina, proteomic analyses of tissues from these two regions of cynomolgus monkeys were compared. Two-dimensional gel electrophoresis and mass spectrometry identified 26 proteins that were present only in the macular gel spots. The expression levels of five proteins, cone photoreceptor specific arrestin-C, gamma-synuclein, epidermal fatty acid binding protein, tropomyosin 1alpha chain, and heterogeneous nuclear ribonucleoproteins A2/B1, were significantly higher in the macula than in the peripheral retina. Immunostaining of macula sections by antibodies to each identified protein revealed unique localization in the retina, retinal pigment epithelial cells and the choroidal layer. Some of these proteins were located in cells with higher densities in the macula. We suggest that it will be important to study these proteins to determine their contribution to the pathogenesis and progression of macula diseases.

Immunohistochemical analysis of aldehyde-modified proteins in drusen in cynomolgus monkeys (Macaca fascicularis).

Protein modifications resulting from reactive aldehydes are thought to be involved in the pathogenesis of various degenerative diseases. Aged cynomolgus monkey (Macaca fascicularis) spontaneously develop drusen in the macula, consistent with the phenotype observed in early-stage age-related macular degeneration (AMD), indicating that this animal is an optimum model for AMD. In retinal sections from three monkeys with macular degeneration, regardless of their size, drusen were consistently positive with immunohistochemical labeling against protein modifications by 4-hydroxynonenal and 4-hydroxyhexenal, end products of non-enzymatic oxidation of n-6 and n-3 polyunsaturated fatty acids, respectively. Positive labeling for both modifications was observed in the ganglion cell layer, the inner nuclear layer, the outer nuclear layer, and the retinal pigment epithelium. However, no consistent differences in location or intensity of the labeling were observed between monkeys with normal macula and macular degeneration. The results suggest a possible association between drusen formation and protein modifications by aldehydes in the pathogenesis of AMD.