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Peter James Francis, Michael L KleinMacular Degeneration Center, Casey Eye Institute, Oregon Health and Science University, Portland, OR, USAAbstract: Age-related macular degeneration (AMD), akin to other common age-related diseases, has a complex pathogenesis and arises from the interplay of genes, environmental factors, and personal characteristics. The past decade has seen very significant strides towards identification of those precise genetic variants associated with disease. That genes encoding proteins of the (alternative) complement pathway (CFH, C2, CFB, C3, CFI) are major players in etiology came as a surprise to many but has already lead to the development of therapies entering human clinical trials. Other genes replicated in many populations ARMS2, APOE, variants near TIMP3, and genes involved in lipid metabolism have also been implicated in disease pathogenesis. The genes discovered to date can be estimated to account for approximately 50% of the genetic variance of AMD and have been discovered by candidate gene approaches, pathway analysis, and latterly genome-wide association studies. Next generation sequencing modalities and meta-analysis techniques are being employed with the aim of identifying the remaining rarer but, perhaps, individually more significant sequence variations, linked to disease status. Complementary studies have also begun to utilize this genetic information to develop clinically useful algorithms to predict AMD risk and evaluate pharmacogenetics. In this article, contemporary commentary is provided on rapidly progressing efforts to elucidate the genetic pathogenesis of AMD as the field stands at the end of the first decade of the 21st century.Keywords: genes, complex disease, susceptibility, AMD

[This corrects the article DOI: 10.1371/journal.pone.0025598.].

Importance: There are no approved drug treatments for autosomal dominant retinitis pigmentosa, a relentlessly progressive cause of adult and childhood blindness., Objectives: To evaluate the potential efficacy and assess the safety of orally administered valproic acid (VPA) in the treatment of autosomal dominant retinitis pigmentosa., Design, Setting, and Participants: Multicenter, phase 2, prospective, interventional, placebo-controlled, double-masked randomized clinical trial. The study took place in 6 US academic retinal degeneration centers. Individuals with genetically characterized autosomal dominant retinitis pigmentosa were randomly assigned to receive treatment or placebo for 12 months. Analyses were intention-to-treat., Interventions: Oral VPA 500 mg to 1000 mg daily for 12 months or placebo., Main Outcomes and Measures: The primary outcome measure was determined prior to study initiation as the change in visual field area (assessed by the III4e isopter, semiautomated kinetic perimetry) between baseline and month 12., Results: The mean (SD) age of the 90 participants was 50.4 (11.6) years. Forty-four (48.9%) were women, 87 (96.7%) were white, and 79 (87.8%) were non-Hispanic. Seventy-nine participants (87.8%) completed the study (42 [95.5%] received placebo and 37 [80.4%] received VPA). Forty-two (46.7%) had a rhodopsin mutation. Most adverse events were mild, although 7 serious adverse events unrelated to VPA were reported. The difference between the VPA and placebo arms for mean change in the primary outcome was -150.43 degree2 (95% CI, -290.5 to -10.03; P = .035)., Conclusions and Relevance: This negative value indicates that the VPA arm had worse outcomes than the placebo group. This study brings to light the key methodological considerations that should be applied to the rigorous evaluation of treatments for these conditions. This study does not provide support for the use of VPA in the treatment of autosomal dominant retinitis pigmentosa., Trial Registration: ClinicalTrials.gov Identifier: NCT01233609.

Purpose: To analyze static visual field sensitivity with topographic models of the hill of vision (HOV), and to characterize several visual function indices derived from the HOV volume., Methods: A software application, Visual Field Modeling and Analysis (VFMA), was developed for static perimetry data visualization and analysis. Three-dimensional HOV models were generated for 16 healthy subjects and 82 retinitis pigmentosa patients. Volumetric visual function indices, which are measures of quantity and comparable regardless of perimeter test pattern, were investigated. Cross-validation, reliability, and cross-sectional analyses were performed to assess this methodology and compare the volumetric indices to conventional mean sensitivity and mean deviation. Floor effects were evaluated by computer simulation., Results: Cross-validation yielded an overall R 2 of 0.68 and index of agreement of 0.89, which were consistent among subject groups, indicating good accuracy. Volumetric and conventional indices were comparable in terms of test-retest variability and discriminability among subject groups. Simulated floor effects did not negatively impact the repeatability of any index, but large floor changes altered the discriminability for regional volumetric indices., Conclusions: VFMA is an effective tool for clinical and research analyses of static perimetry data. Topographic models of the HOV aid the visualization of field defects, and topographically derived indices quantify the magnitude and extent of visual field sensitivity., Translational Relevance: VFMA assists with the interpretation of visual field data from any perimetric device and any test location pattern. Topographic models and volumetric indices are suitable for diagnosis, monitoring of field loss, patient counseling, and endpoints in therapeutic trials.

Optogenetics is a research field that uses gene therapy to deliver a gene encoding a light-activated protein to cells providing light-regulated control of targeted cell pathways. The technology is a popular tool in many fields of neuroscience, used to transiently switch cells on and off, for example, to map neural circuits. In inherited retinal degenerative diseases, where loss of vision results from the loss of photoreceptors, optogenetics can be applied to either augment the function of surviving photoreceptors or confer light sensitivity to naturally nonlight sensitive retinal cells, such as a bipolar cells. This can be achieved either by the light sensitive protein integrating with native internal signaling pathways, or by using a dual function membrane protein that integrates light signaling with an ion channel or pump activity. Exposing treated cells to light of the correct wavelength activates the protein, resulting in cellular depolarization or hyperpolarization that triggers neurological signaling to the visual cortex. While there is a lot of interest in optogenetics as a pan-disease clinical treatment for end-stage application in the inherited degenerative diseases of the retina, research to date has been limited to nonhuman clinical studies. To address the clinical translational needs of this technology, the Foundation Fighting Blindness and Massachusetts Eye and Ear Infirmary cohosted an International Optogenetic Therapies for Vision Workshop, which was held at Massachusetts Eye and Ear Infirmary, Boston, Massachusetts on June 1, 2012.

Age-related macular degeneration (AMD) is a common cause of blindness in older individuals. To accelerate the understanding of AMD biology and help design new therapies, we executed a collaborative genome-wide association study, including >17,100 advanced AMD cases and >60,000 controls of European and Asian ancestry. We identified 19 loci associated at P < 5 × 10(-8). These loci show enrichment for genes involved in the regulation of complement activity, lipid metabolism, extracellular matrix remodeling and angiogenesis. Our results include seven loci with associations reaching P < 5 × 10(-8) for the first time, near the genes COL8A1-FILIP1L, IER3-DDR1, SLC16A8, TGFBR1, RAD51B, ADAMTS9 and B3GALTL. A genetic risk score combining SNP genotypes from all loci showed similar ability to distinguish cases and controls in all samples examined. Our findings provide new directions for biological, genetic and therapeutic studies of AMD.

Retinal dystrophies are predominantly caused by mutations affecting the visual phototransduction system and cilia, with few genes identified that function to maintain photoreceptor survival. We reasoned that growth factors involved with early embryonic retinal development would represent excellent candidates for such diseases. Here we show that mutations in the transforming growth factor-β (TGF-β) ligand Growth Differentiation Factor 6, which specifies the dorso-ventral retinal axis, contribute to Leber congenital amaurosis. Furthermore, deficiency of gdf6 results in photoreceptor degeneration, so demonstrating a connection between Gdf6 signaling and photoreceptor survival. In addition, in both murine and zebrafish mutant models, we observe retinal apoptosis, a characteristic feature of human retinal dystrophies. Treatment of gdf6-deficient zebrafish embryos with a novel aminopropyl carbazole, P7C3, rescued the retinal apoptosis without evidence of toxicity. These findings implicate for the first time perturbed TGF-β signaling in the genesis of retinal dystrophies, support the study of related morphogenetic genes for comparable roles in retinal disease and may offer additional therapeutic opportunities for genetically heterogeneous disorders presently only treatable with gene therapy.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Although recent studies have demonstrated strong genetic associations between AMD and SNPs in a number of genes, other modes of regulation are also likely to play a role in the etiology of this disease. We identified a significantly decreased level of methylation on the IL17RC promoter in AMD patients. Furthermore, we showed that hypomethylation of the IL17RC promoter in AMD patients led to an elevated expression of its protein and messenger RNA in peripheral blood as well as in the affected retina and choroid, suggesting that the DNA methylation pattern and expression of IL17RC may potentially serve as a biomarker for the diagnosis of AMD and likely plays a role in disease pathogenesis., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: We characterize the in vivo changes over time in the retinal structure of wild-type mice alongside two lines of mice deficient in the β-subunit of phosphodiesterase (rd1 and rd10 mice) using spectral domain optical coherence tomography (SD-OCT)., Methods: SD-OCT images were obtained using the Bioptigen spectral domain ophthalmic imaging system (SDOIS). Wild-type C57BL/6J, rd1 and rd10 mice ranging in age from P14 to P206 were sedated with 1% isoflurane. Horizontal and vertical linear scans through the optic nerve, and annular scans around the optic nerve were obtained., Results: SD-OCT imaging of wild-type mice demonstrated visibility of the inner segment/outer segment (IS/OS) junction, external limiting membrane (ELM), outer nuclear layer (ONL), and outer plexiform layer (OPL). At P14, most rd10 mice exhibited normal SD-OCT profiles, but some displayed changes in the IS/OS junction. At the same time point, rd1 mice had severe outer retinal degeneration. In rd10 mice, imaging revealed loss of the IS/OS junction by P18, hyperreflective changes in the ONL at P20, hyperreflective vitreous opacities, and shallow separation of the neural retina from the RPE. Retinal separations were not observed in rd1 mice. Segmentation analysis in wild-type mice demonstrated relatively little variability between animals, while in rd10 and rd1 mice there was a steady decline in outer retinal thickness. Histologic studies demonstrated correlation of retinal features with those seen on SD-OCT scans. Segmentation analysis provides a quantitative and reproducible method for measuring in vivo retinal changes in mice., Conclusions: SD-OCT provides a non-invasive method of following long-term retinal changes in mice in vivo. Although rd10 and rd1 mice have mutations in the same gene, they demonstrate significantly different features on SD-OCT.

Purpose: To evaluate whether bromfenac eyedrops and ranibizumab intravitreal injections would provide added efficacy over ranibizumab alone., Methods: This was a single-site, multiinvestigator, prospective, open-label, interventional, Phase II study of patients with new or recurrent exudative/neovascular age-related macular degeneration. Thirty eyes were enrolled consecutively and were randomized in a ratio of 2:1 to combination therapy with intravitreal ranibizumab and topical bromfenac, and ranibizumab alone. All patients received ranibizumab monthly therapy for 4 months then as needed monthly in accordance with standard of care. Patients receiving bromfenac self-administered 1 drop twice a day for 12 months. Patients were followed for 12 months., Results: There were no safety concerns with the combination therapy. No statistically significant differences were identified in Early Treatment Diabetic Retinopathy Study best-corrected visual acuity or the number of injections required. However, the mean 12-month change in central macular thickness in the combination group was -81.56 μm while in the ranibizumab group alone the change was -42.50 μm (P = 0.03). The proportion of eyes experiencing a decrease in CMT of 50 μm or more was also significantly higher in those receiving combination therapy (P = 0.046)., Conclusion: This pilot study is the first to prospectively identify a biologic signal that may indicate combination therapy with an easily administered well-tolerated eyedrop and ranibizumab is efficacious for the treatment of neovascular age-related macular degeneration. Further studies are warranted to validate this finding.

Age-related macular degeneration (AMD) is the most common cause of incurable visual impairment in high-income countries. Previous studies report inconsistent associations between AMD and apolipoprotein E (APOE), a lipid transport protein involved in low-density cholesterol modulation. Potential interaction between APOE and sex, and smoking status has been reported. We present a pooled analysis (n = 21,160) demonstrating associations between late AMD and APOε4 (odds ratio [OR] = 0.72 per haplotype; confidence interval [CI]: 0.65-0.74; P = 4.41×10(-11) ) and APOε2 (OR = 1.83 for homozygote carriers; CI: 1.04-3.23; P = 0.04), following adjustment for age group and sex within each study and smoking status. No evidence of interaction between APOE and sex or smoking was found. Ever smokers had significant increased risk relative to never smokers for both neovascular (OR = 1.54; CI: 1.38-1.72; P = 2.8×10(-15) ) and atrophic (OR = 1.38; CI: 1.18-1.61; P = 3.37×10(-5) ) AMD but not early AMD (OR = 0.94; CI: 0.86-1.03; P = 0.16), implicating smoking as a major contributing factor to disease progression from early signs to the visually disabling late forms. Extended haplotype analysis incorporating rs405509 did not identify additional risks beyond ε2 and ε4 haplotypes. Our expanded analysis substantially improves our understanding of the association between the APOE locus and AMD. It further provides evidence supporting the role of cholesterol modulation, and low-density cholesterol specifically, in AMD disease etiology., (© 2011 Wiley-Liss, Inc.)

Objective: To design a risk assessment model for development of advanced age-related macular degeneration (AMD) incorporating phenotypic, demographic, environmental, and genetic risk factors., Methods: We evaluated longitudinal data from 2846 participants in the Age-Related Eye Disease Study. At baseline, these individuals had all levels of AMD, ranging from none to unilateral advanced AMD (neovascular or geographic atrophy). Follow-up averaged 9.3 years. We performed a Cox proportional hazards analysis with demographic, environmental, phenotypic, and genetic covariates and constructed a risk assessment model for development of advanced AMD. Performance of the model was evaluated using the C statistic and the Brier score and externally validated in participants in the Complications of Age-Related Macular Degeneration Prevention Trial., Results: The final model included the following independent variables: age, smoking history, family history of AMD (first-degree member), phenotype based on a modified Age-Related Eye Disease Study simple scale score, and genetic variants CFH Y402H and ARMS2 A69S. The model did well on performance measures, with very good discrimination (C statistic = 0.872) and excellent calibration and overall performance (Brier score at 5 years = 0.08). Successful external validation was performed, and a risk assessment tool was designed for use with or without the genetic component., Conclusions: We constructed a risk assessment model for development of advanced AMD. The model performed well on measures of discrimination, calibration, and overall performance and was successfully externally validated. This risk assessment tool is available for online use.

Purpose: Age-related macular degeneration (AMD) has a complex etiology arising from genetic and environmental influences. This past decade have seen several genes associated with the disease. Variants in five genes have been confirmed to play a major role. The objective of this study was to evaluate whether genes influence treatment response to ranibizumab for neovascular AMD. The hypothesis was that an individual's genetic variation will determine treatment response., Methods: The study was a two-site prospective open-label observational study of patients newly diagnosed with exudative (neovascular) AMD receiving intravitreal ranibizumab therapy. Treatment-naïve patients were enrolled at presentation and received monthly "as needed" therapy. Clinical data was collected monthly and DNA extracted. Genotyping was performed using the Illumina (San Diego, California) 660-Quad single-nucleotide polymorphism (SNP) chip. Regression analyses were performed to identify SNPs associated with treatment-response end points., Results: Sixty-five patients were enrolled. No serious adverse events were recorded. The primary outcome measure was change in ETDRS visual acuity at 12 months. A SNP in the CFH gene was found to be associated with less improvement in visual acuity while receiving ranibizumab therapy. The C3 gene, among others, was associated with reduced thickening and improved retinal architecture. VEGFA, FLT1, and CFH were associated with requiring fewer ranibizumab injections over the 12-month study., Conclusions: This study is one of the first prospective pharmacogenetic study of intravitreal ranibizumab. Although preliminary, the results identify a number of putative genetic variants, which will be further examined by replication and functional studies to elucidate the complete pharmacogenetic architecture of therapy for AMD.

Variation in the apolipoprotein E gene (APOE) has been reported to be associated with longevity in humans. The authors assessed the allelic distribution of APOE isoforms ε2, ε3, and ε4 among 10,623 participants from 15 case-control and cohort studies of age-related macular degeneration (AMD) in populations of European ancestry (study dates ranged from 1990 to 2009). The authors included only the 10,623 control subjects from these studies who were classified as having no evidence of AMD, since variation within the APOE gene has previously been associated with AMD. In an analysis stratified by study center, gender, and smoking status, there was a decreasing frequency of the APOE ε4 isoform with increasing age (χ(2) for trend = 14.9 (1 df); P = 0.0001), with a concomitant increase in the ε3 isoform (χ(2) for trend = 11.3 (1 df); P = 0.001). The association with age was strongest in ε4 homozygotes; the frequency of ε4 homozygosity decreased from 2.7% for participants aged 60 years or less to 0.8% for those over age 85 years, while the proportion of participants with the ε3/ε4 genotype decreased from 26.8% to 17.5% across the same age range. Gender had no significant effect on the isoform frequencies. This study provides strong support for an association of the APOE gene with human longevity.

Complement factor H shows very strong association with Age-related Macular Degeneration (AMD), and recent data suggest that multiple causal variants are associated with disease. To refine the location of the disease associated variants, we characterized in detail the structural variation at CFH and its paralogs, including two copy number polymorphisms (CNP), CNP147 and CNP148, and several rare deletions and duplications. Examination of 34 AMD-enriched extended families (N = 293) and AMD cases (White N = 4210 Indian = 134; Malay = 140) and controls (White N = 3229; Indian = 117; Malay = 2390) demonstrated that deletion CNP148 was protective against AMD, independent of SNPs at CFH. Regression analysis of seven common haplotypes showed three haplotypes, H1, H6 and H7, as conferring risk for AMD development. Being the most common haplotype H1 confers the greatest risk by increasing the odds of AMD by 2.75-fold (95% CI = [2.51, 3.01]; p = 8.31×10(-109)); Caucasian (H6) and Indian-specific (H7) recombinant haplotypes increase the odds of AMD by 1.85-fold (p = 3.52×10(-9)) and by 15.57-fold (P = 0.007), respectively. We identified a 32-kb region downstream of Y402H (rs1061170), shared by all three risk haplotypes, suggesting that this region may be critical for AMD development. Further analysis showed that two SNPs within the 32 kb block, rs1329428 and rs203687, optimally explain disease association. rs1329428 resides in 20 kb unique sequence block, but rs203687 resides in a 12 kb block that is 89% similar to a noncoding region contained in ΔCNP148. We conclude that causal variation in this region potentially encompasses both regulatory effects at single markers and copy number.

Purpose: Cataracts are the most common cause of blindness worldwide. Inherited cataract is a clinically and genetically heterogeneous disease. Here we report a novel mutation in the paired-like homeodomain 3 (PITX3) gene segregating in a four generation English family with an isolated autosomal dominant posterior polar cataract., Methods: A genome-wide linkage was performed by means of single nucleotide polymorphism (SNP) and microsatellite markers. Linkage analyses were performed with the GeneHunter and MLINK programs. Direct sequencing of PCR products was performed to detect mutation in the gene, using the BigDye version 3.1 and analyzed using Sequence analysis version 5.2., Results: Genome-wide linkage analysis with SNP markers, identified a disease-haplotype interval on chromosome 10q. Two point positive logarithm of odds (LOD) scores was obtained with markers D10S205 (Z=3.10 at θ=0.00), flanked by markers D10S1709 and D10S543, which harbors the homeobox gene PITX3. Sequence analysis of PITX3 revealed a 1-bp deletion that cosegregated with all the affected members of this family which resulted in a frameshift in codon 181 and likely to produce an aberrant protein consisting of 127 additional residues., Conclusions: The 542delC is a novel mutation in PITX3 causing an isolated posterior polar cataract., (© 2011 Molecular Vision)

Objective: To report the detailed clinical findings of patients with retinal toxicity that developed secondary to the use of hydroxychloroquine sulfate (n = 13), chloroquine phosphate (n = 2), or a combination of the agents (n = 1)., Methods: Ophthalmologic examination, fundus photography, visual field testing, and detailed electrophysiologic assessment were undertaken in all 16 affected patients. Selected patients also had spectral domain optical coherence tomography (n = 6) and fundus autofluorescence imaging (n = 4)., Results: Sixteen women (mean age, 67 years; range, 44-85) were monitored for 7 years. The mean duration of hydroxychloroquine therapy was 13 years (range, 2-20). In patients in whom the daily dosage of hydroxychloroquine could be estimated (12 of 13), when using actual body weight, 8 were taking 6.5 mg/kg or less and 4 were taking greater than this recommended dosage. However, if lean body weight was used, 3 patients were taking 6.5 mg/kg or less and 9 were taking greater than this daily dosage. The most common (n = 10) presenting symptom was difficulty with reading; 4 women were asymptomatic. Two patients had preexisting retinal disease, 2 were obese, and none had renal or liver dysfunction. Fundus findings ranged from mild retinal pigment epithelial changes to bull's-eye maculopathy; 3 patients had a normal-appearing macula. Two patients had full-field electroretinograms that showed no abnormalities and 6 showed evidence of generalized retinal dysfunction with reduced rod and cone responses. All 15 patients who underwent multifocal electroretinography testing had evidence of bilateral macular cone dysfunction. Four patterns of visual field abnormality were observed in the 15 patients with abnormal visual fields, the most common (n = 10) being isolated central loss. Repeat electrophysiologic and visual field assessment provided evidence of disease progression despite cessation of medication in 6 patients, with documented progression for 7 years in 1 woman., Conclusions: Sustained visual improvement following cessation of drug therapy was not observed in any patient in this series, and our identification of 6 patients with objective evidence of progression serves to remind physicians of the potentially devastating visual consequences of antimalarial-related retinal toxicity. It is also of note that profound abnormalities detected with visual field and multifocal electroretinography testing can be observed in the presence of a normal macular appearance, and our findings suggest that lean body weight should be used for all patients when calculating daily dosage.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. We conducted a genome-wide association study in a series of families enriched for AMD and completed a meta-analysis of this new data with results from reanalysis of an existing study of a late-stage case-control cohort. We tested the top findings for replication in 1896 cases and 1866 controls and identified two novel genetic protective factors for AMD. In addition to the complement factor H (CFH) (P=2.3 × 10⁻⁶⁴) and age-related maculopathy susceptibility 2 (ARMS2) (P=1.2 × 10⁻⁶⁰) loci, we observed a protective effect at rs429608, an intronic SNP in SKIV2L (P=5.3 × 10⁻¹⁵), a gene near the complement component 2 (C2)/complement factor B (BF) locus, that indicates the protective effect may be mediated by variants other than the C2/BF variants previously studied. Haplotype analysis at this locus identified three protective haplotypes defined by the rs429608 protective allele. We also identified a new potentially protective effect at rs2679798 in MYRIP (P=2.9 × 10⁻⁴), a gene involved in retinal pigment epithelium melanosome trafficking. Interestingly, MYRIP was initially identified in the family-based scan and was confirmed in the case-control set. From these efforts, we report the identification of two novel protective factors for AMD and confirm the previously known associations at CFH, ARMS2 and C3.

Purpose: We sought to determine whether genotype is associated with rate of growth of geographic atrophy (GA) in eyes with age-related macular degeneration (AMD)., Design: Prospective analysis of participants in a randomized controlled clinical trial., Participants: We included 114 eyes of 114 participants in the Age-Related Eye Disease Study (AREDS)., Methods: Fundus photographs from AREDS participants with GA from whom a DNA specimen had been obtained and serial photographs had been taken over a minimum of 2 years were evaluated for progression as determined by change in cumulative area of GA. All fundus photographs were scanned, digitized, and centrally graded longitudinally for area of GA. The relationship of GA progression with previously identified genetic variants associated with AMD was assessed., Main Outcome Measures: Genotype frequencies and change in cumulative area of GA., Results: The mean growth rate of GA for the 114 eyes was 1.79 mm(2)/year (range, 0.17-4.76). No association between growth rate and genotype was present for variants in the CFH, C2, C3, APOE, and TLR3 genes. For the single nucleotide polymorphism rs10490924 in LOC387715/ARMS2, there was a significant association of GA growth rate, both adjusted and unadjusted for initial lesion size, with the homozygous risk genotype as compared with the homozygous nonrisk genotype (unadjusted P = 0.002; Bonferroni-corrected P = 0.014) and for allelic association (Bonferroni-corrected P value = 0.011). Analyses of other measures of GA progression (progression to central GA from extrafoveal GA and development of bilateral GA in those initially with unilateral GA) showed no statistically significant association between progression and the LOC387715/ARMS2/HTRA1 genotype., Conclusions: Growth rates of GA calculated from digitized serial fundus photographs showed no association with variants in the CFH, C2, C3, APOE, or TLR3 genes. There was a nominally significant association with the LOC387715/ARMS2/HTRA1 genotype, although this finding was not supported by analyses of secondary measures of GA progression. Replication in other populations is needed to establish the existence of an association., (Copyright 2010 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

We executed a genome-wide association scan for age-related macular degeneration (AMD) in 2,157 cases and 1,150 controls. Our results validate AMD susceptibility loci near CFH (P < 10(-75)), ARMS2 (P < 10(-59)), C2/CFB (P < 10(-20)), C3 (P < 10(-9)), and CFI (P < 10(-6)). We compared our top findings with the Tufts/Massachusetts General Hospital genome-wide association study of advanced AMD (821 cases, 1,709 controls) and genotyped 30 promising markers in additional individuals (up to 7,749 cases and 4,625 controls). With these data, we identified a susceptibility locus near TIMP3 (overall P = 1.1 x 10(-11)), a metalloproteinase involved in degradation of the extracellular matrix and previously implicated in early-onset maculopathy. In addition, our data revealed strong association signals with alleles at two loci (LIPC, P = 1.3 x 10(-7); CETP, P = 7.4 x 10(-7)) that were previously associated with high-density lipoprotein cholesterol (HDL-c) levels in blood. Consistent with the hypothesis that HDL metabolism is associated with AMD pathogenesis, we also observed association with AMD of HDL-c-associated alleles near LPL (P = 3.0 x 10(-3)) and ABCA1 (P = 5.6 x 10(-4)). Multilocus analysis including all susceptibility loci showed that 329 of 331 individuals (99%) with the highest-risk genotypes were cases, and 85% of these had advanced AMD. Our studies extend the catalog of AMD associated loci, help identify individuals at high risk of disease, and provide clues about underlying cellular pathways that should eventually lead to new therapies.

Purpose. Usher's syndrome is a combined deafness and blindness disorder caused by mutations in several genes with functions in both the retina and the ear. Here the authors studied morphologic and functional changes in an animal model, the Ush2a mouse, and explored whether transplantation of forebrain-derived progenitor cells might affect the progress of morphologic and functional deterioration. Methods. Ush2a mice were tested at postnatal days (P) 70 to P727 using an optomotor test, which provides a repeatable method of estimating rodent visual acuity and contrast sensitivity. A group of mice that received grafts of forebrain-derived progenitor cells at P80 was tested for up to 10 weeks after grafting. At the end of testing, animals were killed, and eyes were processed for histology. Results. The optomotor test showed that both acuity and contrast sensitivity deteriorated over time; contrast sensitivity showed a deficit even at P70. By contrast, photoreceptor loss was only evident later than 1 year of age, though changes in the intracellular distribution of red/green cone opsin were observed as early as P80. Mice that received transplanted cells performed significantly better than control mice and no longer demonstrated abnormal distribution of red/green opsin where the donor cells were distributed. Conclusions. This study showed that vision impairment was detected well before significant photoreceptor loss and was correlated with abnormal distribution of a cone pigment. Cell transplantation prevented functional deterioration for at least 10 weeks and reversed the mislocalization of cone pigment.

Nance-Horan syndrome (NHS) is an X-linked developmental disorder characterized by congenital cataract, dental anomalies, facial dysmorphism and, in some cases, mental retardation. Protein truncation mutations in a novel gene (NHS) have been identified in patients with this syndrome. We previously mapped X-linked congenital cataract (CXN) in one family to an interval on chromosome Xp22.13 which encompasses the NHS locus; however, no mutations were identified in the NHS gene. In this study, we show that NHS and X-linked cataract are allelic diseases. Two CXN families, which were negative for mutations in the NHS gene, were further analysed using array comparative genomic hybridization. CXN was found to be caused by novel copy number variations: a complex duplication-triplication re-arrangement and an intragenic deletion, predicted to result in altered transcriptional regulation of the NHS gene. Furthermore, we also describe the clinical and molecular analysis of seven families diagnosed with NHS, identifying four novel protein truncation mutations and a novel large deletion encompassing the majority of the NHS gene, all leading to no functional protein. We therefore show that different mechanisms, aberrant transcription of the NHS gene or no functional NHS protein, lead to different diseases. Our data highlight the importance of copy number variation and non-recurrent re-arrangements leading to different severity of disease and describe the potential mechanisms involved.

Objective: To study the phenotypic characteristics of patients with a novel p.E292K mutation in BEST1., Methods: Affected individuals underwent ophthalmic examination and testing that included photography, autofluorescence, optical coherence tomography, and electrophysiological testing. Their DNA was analyzed for BEST1 mutations., Results: Five patients aged 5 to 59 years who expressed the p.E292K mutation in BEST1 were identified in 3 families. Electro-oculographic light-rise was subnormal in all probands and carriers. Carriers had normal findings from fundus examination, multifocal electroretinography, and visual acuity, and were emmetropic or myopic. Only probands had hyperopia and fundus findings typical of Best macular dystrophy. Optical coherence tomography of vitelliform lesions demonstrated retinal pigment epithelium elevation without subretinal fluid; atrophic lesions exhibited disruption of the hyperreflective outer retina-retinal pigment epithelium complex. Intense hyperautofluorescence correlated with the vitelliform lesion., Conclusions: Patients with the Glu292Lys variation in BEST1 exhibit intrafamilial and interfamilial phenotypic variability. A disproportionate fraction (26%) of Best disease-causing mutations occurs in exon 8, suggesting that the portion of protein encoded by this exon (amino acids 290-316) may be especially important to bestrophin's function. Relatively good visual acuity with vitelliform lesions can be explained by preservation of the outer retina, demonstrated by optical coherence tomography. Clinical Relevance A novel mutation in this region of BEST1 carries implications for disease pathogenesis.

Purpose: Cell-based therapy rescues retinal structure and function in rodent models of retinal disease, but translation to clinical practice will require more information about the consequences of transplantation in an eye closely resembling the human eye. The authors explored donor cell behavior using human cortical neural progenitor cells (hNPC(ctx)) introduced into the subretinal space of normal rhesus macaques., Methods: hNPC(ctx) transduced with green fluorescent protein (hNPC(ctx)-GFP) were delivered bilaterally into the subretinal space of six normal adult rhesus macaques under conditions paralleling those of the human operating room. Outcome measures included clinical parameters of surgical success, multifocal electroretinogram (mfERG), and histopathologic analyses performed between 3 and 39 days after engraftment. To test the effects of GFP transduction on cell bioactivity, hNPC(ctx)-GFP from the same batch were also injected into Royal College of Surgeons (RCS) rats and compared with nonlabeled hNPC(ctx)., Results: Studies using RCS rats indicated that GFP transduction did not alter the ability of the cells to rescue vision. After cells were introduced into the monkey subretinal space by a pars plana transvitreal approach, the resultant detachment was rapidly resolved, and retinal function showed little or no disturbance in mfERG recordings. Retinal structure was unaffected and no signs of inflammation or rejection were seen. Donor cells survived as a single layer in the subretinal space, and no cells migrated into the inner retina., Conclusions: Human neural progenitor cells can be introduced into a primate eye without complication using an approach that would be suitable for extrapolation to human patients.

Background: Age related macular degeneration (AMD) is a leading cause of blindness. AMD is a complex disorder caused by genetic and environmental factors in which single nucleotide polymorphisms (SNPs) in the genes CFH and LOC387715/HTRA1/ARMS2 have prognostic importance for progression to advanced AMD (with visual loss). CFH may also have a pharmacogenetic role by affecting treatment response to widely used nutritional supplements. This paper examines other AMD susceptibility genes to determine if these genotypes influenced disease progression and treatment response., Methods: Three cohorts, totalling 3137 individuals, were genotyped for SNPs in 13 genes previously published to be associated with advanced AMD (other than CFH and LOC387715/ARMS2/HTRA1). Those genes found associated were then evaluated for their involvement in disease progression. Interactions between the genes and with AREDS (Age-Related Eye Disease Study) nutritional supplements were investigated., Results: Positive independent associations were noted in SNPs in the genes C2 (p = 0.0001, odds ratio (OR) 0.35, 95% confidence interval (CI) 0.2 to 0.6), CFB (p = 0.0001, OR 0.35, 95% CI 0.2 to 0.6), C3 (p = 0.0001, OR 3.91, 95% CI 1.94 to 7.88), APOE (epsilon4, p = 0.01, OR 0.50, 95% CI 0.29 to 0.86) and VEGFA (p = 0.01, OR 2.23, 95% CI 1.06 to 4.68). C2/CFB and C3 were independently related to progression from early/intermediate to advanced AMD with OR 0.32 (95% CI 0.14 to 0.73) and 3.32 (95% CI 1.46 to 7.59), respectively. Gene-gene and pharmacogenetic interactions were not observed. No preferential associations were observed with geographic atrophy or choroidal neovascularisation., Conclusion: This study provides insights into the genetic pathogenesis of AMD. Five genes have now been shown to be independently involved in progression from intermediate disease (before vision loss has occurred) to advanced disease in which blindness is frequent.

Background: Age-related macular degeneration (AMD), a chronic neurodegenerative and neovascular retinal disease, is the leading cause of blindness in elderly people of western European origin. While structural and functional alterations in mitochondria (mt) and their metabolites have been implicated in the pathogenesis of chronic neurodegenerative and vascular diseases, the relationship of inherited variants in the mitochondrial genome and mt haplogroup subtypes with advanced AMD has not been reported in large prospective cohorts., Methodology/prinicipal Findings: We examined the relationship of inherited mtDNA variants with advanced AMD in 1168 people using a three-stage design on samples from 12-year and 10-year prospective studies on the natural history of age-related eye disease. In Stage I we resequenced the entire genome in 99 elderly AMD-free controls and 215 people with advanced AMD from the 12-year study. A consistent association with AMD in 14 of 17 SNPs characterizing the mtDNA T haplogroup emerged. Further analysis revealed these associations were driven entirely by the T2 haplogroup, and characterized by two variants in Complex I genes (A11812G of MT-ND4 and A14233G of MT-ND6). We genotyped T haplogroups in an independent sample of 490 cases and 61 controls from the same study (Stage II) and in 56 cases and 246 controls from the 10-year study (Stage III). People in the T2 haplogroup were approximately 2.5 times more likely to have advanced AMD than their peers (odds ratio [OR] = 2.54, 95%CI 1.36-4.80, P

Background: Age-related macular degeneration is the most common cause of irreversible visual impairment in the developed world. Advanced age-related macular degeneration consists of geographic atrophy and choroidal neovascularization. The specific genetic variants that predispose patients to geographic atrophy are largely unknown., Methods: We tested for an association between the functional toll-like receptor 3 gene (TLR3) variant rs3775291 (involving the substitution of phenylalanine for leucine at amino acid 412) and age-related macular degeneration in Americans of European descent. We also tested for the effect of TLR3 Leu and Phe variants on the viability of human retinal pigment epithelial cells in vitro and on apoptosis of retinal pigment epithelial cells from wild-type mice and Tlr3-knockout (Tlr3(-/-)) mice., Results: The Phe variant (encoded by the T allele at rs3775291) was associated with protection against geographic atrophy (P=0.005). This association was replicated in two independent case-control series of geographic atrophy (P=5.43x10(-4) and P=0.002). No association was found between TLR3 variants and choroidal neovascularization. A prototypic TLR3 ligand induced apoptosis in a greater fraction of human retinal pigment epithelial cells with the Leu-Leu genotype than those with the Leu-Phe genotype and in a greater fraction of wild-type mice than Tlr3(-/-) mice., Conclusions: The TLR3 412Phe variant confers protection against geographic atrophy, probably by suppressing the death of retinal pigment epithelial cells. Since double-stranded RNA (dsRNA) can activate TLR3-mediated apoptosis, our results suggest a role of viral dsRNA in the development of geographic atrophy and point to the potential toxic effects of short-interfering-RNA therapies in the eye., (2008 Massachusetts Medical Society)