QTL mapping of human retina DNA methylation identifies 87 gene-epigenome interactions in age-related macular degeneration.

DNA methylation provides a crucial epigenetic mark linking genetic variations to environmental influence. We have analyzed array-based DNA methylation profiles of 160 human retinas with co-measured RNA-seq and >8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 methylation quantitative trait loci and 12,505 expression quantitative trait loci) and 13,747 DNA methylation loci affecting gene expression, with over one-third specific to the retina. Methylation and expression quantitative trait loci show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration. Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of macular degeneration pathology by genotype-environment interaction in retina.

Whole genome sequencing of 4,787 individuals identifies gene-based rare variants in age-related macular degeneration.

Genome-wide association studies have contributed extensively to the discovery of disease-associated common variants. However, the genetic contribution to complex traits is still largely difficult to interpret. We report a genome-wide association study of 2394 cases and 2393 controls for age-related macular degeneration (AMD) via whole-genome sequencing, with 46.9 million genetic variants. Our study reveals significant single-variant association signals at four loci and independent gene-based signals in CFH, C2, C3, and NRTN. Using data from the Exome Aggregation Consortium (ExAC) for a gene-based test, we demonstrate an enrichment of predicted rare loss-of-function variants in CFH, CFI, and an as-yet unreported gene in AMD, ORMDL2. Our method of using a large variant list without individual-level genotypes as an external reference provides a flexible and convenient approach to leverage the publicly available variant datasets to augment the search for rare variant associations, which can explain additional disease risk in AMD.

The Role of Gene Expression Regulation on Genetic Risk of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a major cause of irreversible vision loss in the elderly. It is a complex multifactorial disease that is caused by the cumulative impact of genetic predisposition, environmental stress, and advanced aging. Knowledge of genetic risk factors underlying AMD susceptibility has advanced rapidly in the past decade that can be largely credited to genome-wide association studies (GWAS) and next-generation sequencing (NGS) efforts. GWAS have identified 34 genetic risk loci for AMD; the majority of which are in the noncoding genome. Several lines of evidence suggest that a complex trait-associated variant is likely to regulate the gene expression (acting as expression quantitative trait loci (eQTLs)), and there is a significant enrichment of GWAS-associated variants within eQTLs. In the last two years, eQTL studies in AMD-relevant tissues have provided functional interpretation of several AMD-GWAS loci. This review highlights the knowledge gained to date and discusses future directions to bridge the gap between genetic predisposition and biological mechanisms to reap the full benefits of GWAS findings.

QTL mapping of human retina DNA methylation identifies 87 gene-epigenome interactions in age-related macular degeneration.

DNA methylation (DNAm) provides a crucial epigenetic mark linking genetic variations to environmental influence. We analyzed array-based DNAm profiles of 160 human retinas with co-measured RNA-seq and > 8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 mQTLs and 12,505 eQTLs) and 13,747 eQTMs (DNAm loci affecting gene expression), with over one-third specific to the retina. mQTLs and eQTMs show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration (AMD). Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of AMD pathology by genotype-environment interaction in retina.

IL-17 Producing Lymphocytes Cause Dry Eye and Corneal Disease With Aging in RXRα Mutant Mouse.

Purpose: To investigate IL-17 related mechanisms for developing dry eye disease in the Pinkie mouse strain with a loss of function RXRα mutation. Methods: Measures of dry eye disease were assessed in the cornea and conjunctiva. Expression profiling was performed by single-cell RNA sequencing (scRNA-seq) to compare gene expression in conjunctival immune cells. Conjunctival immune cells were immunophenotyped by flow cytometry and confocal microscopy. The activity of RXRα ligand 9-cis retinoic acid (RA) was evaluated in cultured monocytes and γδ T cells. Results: Compared to wild type (WT) C57BL/6, Pinkie has increased signs of dry eye disease, including decreased tear volume, corneal barrier disruption, corneal/conjunctival cornification and goblet cell loss, and corneal vascularization, opacification, and ulceration with aging. ScRNA-seq of conjunctival immune cells identified γδ T cells as the predominant IL-17 expressing population in both strains and there is a 4-fold increased percentage of γδ T cells in Pinkie. Compared to WT, IL-17a, and IL-17f significantly increased in Pinkie with conventional T cells and γδ T cells as the major producers. Flow cytometry revealed an increased number of IL-17+ γδ T cells in Pinkie. Tear concentration of the IL-17 inducer IL-23 is significantly higher in Pinkie. 9-cis RA treatment suppresses stimulated IL-17 production by γδ T and stimulatory activity of monocyte supernatant on γδ T cell IL-17 production. Compared to WT bone marrow chimeras, Pinkie chimeras have increased IL-17+ γδ T cells in the conjunctiva after desiccating stress and anti-IL-17 treatment suppresses dry eye induced corneal MMP-9 production/activity and conjunctival goblet cell loss. Conclusion: These findings indicate that RXRα suppresses generation of dry eye disease-inducing IL-17 producing lymphocytes s in the conjunctiva and identifies RXRα as a potential therapeutic target in dry eye.

Single-cell transcriptional profiling of murine conjunctival immune cells reveals distinct populations expressing homeostatic and regulatory genes.

Immune cells in the exposed conjunctiva mucosa defend against environmental and microbial stresses. Expression profiling by single-cell RNA sequencing was performed to identify conjunctival immune cell populations expressing homeostatic and regulatory genes. Fourteen distinct clusters were identified, including myeloid cells (neutrophils, monocytes, macrophages), dendritic cells (DC), and lymphoid cells (B, T, γδT, ILC2, and NK) lineages. Novel neutrophil [lipocalin (Lcn2) high and low), and MHCIIlo macrophage (MP) clusters were identified. More than half of the cells map to myeloid and dendritic cell populations with differential expression profiles that include genes with homeostatic and regulatory functions: Serpinb2 (MHCIIlo macrophage), Apoe (monocyte), Cd209a (macrophage), Cst3 (cDC1), and IL4i1 in migratory DC (mDC). ILC2 expresses the goblet cell trophic factor IL-13. Suppressed inflammatory and activated anti-inflammatory/regulatory pathways were observed in certain myeloid and DC populations. Confocal immunolocalization of identity markers showed mDC (CCR7, FASCIN1) located on or within the conjunctival epithelium. Monocyte, macrophage, cDC1 and IL-13/IL-5+ ILC2 were located below the conjunctival epithelium and goblet cells. This study found distinct immune cell populations in the conjunctiva and identified cells expressing genes with known homeostatic and immunoregulatory functions.

A Novel ARL3 Gene Mutation Associated With Autosomal Dominant Retinal Degeneration.

Despite major progress in the discovery of causative genes, many individuals and families with inherited retinal degenerations (IRDs) remain without a molecular diagnosis. We applied whole exome sequencing to identify the genetic cause in a family with an autosomal dominant IRD. Eye examinations were performed and affected patients were studied with electroretinography and kinetic and chromatic static perimetry. Sequence variants were analyzed in genes (n = 271) associated with IRDs listed on the RetNet database. We applied a stepwise filtering process involving the allele frequency in the control population, in silico prediction tools for pathogenicity, and evolutionary conservation to prioritize the potential causal variant(s). Sanger sequencing and segregation analysis were performed on the proband and other family members. The IRD in this family is expressed as a widespread progressive retinal degeneration with maculopathy. A novel heterozygous variant (c.200A > T) was identified in the ARL3 gene, leading to the substitution of aspartic acid to valine at position 67. The Asp67 residue is evolutionary conserved, and the change p.Asp67Val is predicted to be pathogenic. This variant was segregated in affected members of the family and was absent from an unaffected individual. Two previous reports of a de novo missense mutation in the ARL3 gene, each describing a family with two affected generations, are the only examples to date of autosomal dominant IRD associated with this photoreceptor gene. Our results, identifying a novel pathogenic variant in ARL3 in a four-generation family with a dominant IRD, augment the evidence that the ARL3 gene is another cause of non-syndromic retinal degeneration.

Making Biological Sense of Genetic Studies of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a major cause of blindness in older individuals worldwide. The disease is characterized by deposition of drusen between the retinal pigment epithelium (RPE) and Bruch's membrane, RPE atrophy and death of photoreceptors. AMD is a complex disease with multiple genetic and non-genetic risk factors. Genome-wide association studies (GWAS) have identified 52 variants at 34 genetic loci associated with AMD. A majority of the AMD-GWAS variants are present in non-coding region of the genome and could quantitatively impact distinct human traits [called quantitative trait loci (QTLs)] by affecting regulation of gene expression. The integration of different regulatory features, such as open-chromatin regions, histone marks, transcription factor binding sites, with AMD-GWAS can provide meaningful insights into variant's function. However, functional interpretation of variant-gene relationship in AMD is challenging because of inadequate understanding of cell-type specific and context-dependent information in disease-relevant tissues. Here we focus on the role of sequencing-based omic studies in assigning biological meaning to disease-associated variants and genes. We also discuss the methods and model systems that can be utilized to unravel molecular mechanisms of a complex disorder like AMD.

A mega-analysis of expression quantitative trait loci in retinal tissue.

Significant association signals from genome-wide association studies (GWAS) point to genomic regions of interest. However, for most loci the causative genetic variant remains undefined. Determining expression quantitative trait loci (eQTL) in a disease relevant tissue is an excellent approach to zoom in on disease- or trait-associated association signals and hitherto on relevant disease mechanisms. To this end, we explored regulation of gene expression in healthy retina (n = 311) and generated the largest cis-eQTL data set available to date. Genotype- and RNA-Seq data underwent rigorous quality control protocols before FastQTL was applied to assess the influence of genetic markers on local (cis) gene expression. Our analysis identified 403,151 significant eQTL variants (eVariants) that regulate 3,007 genes (eGenes) (Q-Value < 0.05). A conditional analysis revealed 744 independent secondary eQTL signals for 598 of the 3,007 eGenes. Interestingly, 99,165 (24.71%) of all unique eVariants regulate the expression of more than one eGene. Filtering the dataset for eVariants regulating three or more eGenes revealed 96 potential regulatory clusters. Of these, 31 harbour 130 genes which are partially regulated by the same genetic signal. To correlate eQTL and association signals, GWAS data from twelve complex eye diseases or traits were included and resulted in identification of 80 eGenes with potential association. Remarkably, expression of 10 genes is regulated by eVariants associated with multiple eye diseases or traits. In conclusion, we generated a unique catalogue of gene expression regulation in healthy retinal tissue and applied this resource to identify potentially pleiotropic effects in highly prevalent human eye diseases. Our study provides an excellent basis to further explore mechanisms of various retinal disease etiologies.

A unique PRDM13-associated variant in a Georgian Jewish family with probable North Carolina macular dystrophy and the possible contribution of a unique CFH variant.

Purpose: North Carolina macular dystrophy (NCMD) is an autosomal dominant maculopathy that is considered a non-progressive developmental disorder with variable expressivity. Our study aimed to clinically and genetically characterize macular dystrophy in a family (MOL1154) consisting of six affected subjects with a highly variable maculopathy phenotype in which no correlation between age and severity exists. Methods: Clinical characterization included visual acuity testing and electroretinography. Genetic analysis included Sanger sequencing and whole exome sequencing (WES). Results: WES analysis performed on DNA samples from two individuals revealed a heterozygous deletion of six nucleotides [c.2247_2252del; p.(Leu750_Lys751del)] in the CFH gene. Co-segregation analysis revealed that five of the six NCMD affected subjects carried this deletion, while one individual who had a relatively mild phenotype compatible with dry age-related macular degeneration (AMD) did not carry it. We subsequently analyzed the upstream region of PRDM13 that has previously been reported to be associated with NCMD and identified a unique heterozygous transversion (chr6:100040974A>C) located within the previously described suspected control region in all six affected individuals. This transversion is likely to cause NCMD. Conclusions: NCMD has a wide spectrum of clinical phenotypes that can overlap with AMD, making it challenging to correctly diagnose affected individuals and family members. The DNA sequence variant we found in the CFH gene of some of the affected family members may suggest some role as a modifier gene. However, this variant still does not explain the huge phenotypic variability of NCMD and needs to be studied in other and larger populations.

Family-based exome sequencing identifies rare coding variants in age-related macular degeneration.

Genome-wide association studies (GWAS) have identified 52 independent variants at 34 genetic loci that are associated with age-related macular degeneration (AMD), the most common cause of incurable vision loss in the elderly worldwide. However, causal genes at the majority of these loci remain unknown. In this study, we performed whole exome sequencing of 264 individuals from 63 multiplex families with AMD and analyzed the data for rare protein-altering variants in candidate target genes at AMD-associated loci. Rare coding variants were identified in the CFH, PUS7, RXFP2, PHF12 and TACC2 genes in three or more families. In addition, we detected rare coding variants in the C9, SPEF2 and BCAR1 genes, which were previously suggested as likely causative genes at respective AMD susceptibility loci. Identification of rare variants in the CFH and C9 genes in our study validated previous reports of rare variants in complement pathway genes in AMD. We then extended our exome-wide analysis and identified rare protein-altering variants in 13 genes outside the AMD-GWAS loci in three or more families. Two of these genes, SCN10A and KIR2DL4, are of interest because variants in these genes also showed association with AMD in case-control cohorts, albeit not at the level of genome-wide significance. Our study presents the first large-scale, exome-wide analysis of rare variants in AMD. Further independent replications and molecular investigation of candidate target genes, reported here, would assist in gaining novel insights into mechanisms underlying AMD pathogenesis.

Applications of Genomic Technologies in Retinal Degenerative Diseases.

Next-generation sequencing (NGS)-based technologies are ideal for genomic analyses owing to their cost-effectiveness, unprecedented speed, and accuracy. Acceleration in examining genome, transcriptome, and epigenome has made significant impact in biomedical sciences. This review highlights the applications of high-throughput NGS technologies in improving the molecular understanding of retinal degenerative diseases (RDDs). I focus on NGS-based methods and strategies that are allowing expedited disease gene identifications, improved diagnosis, and deeper understanding of the mechanisms through which genetic variations lead to diseases.

No CFH or ARMS2 Interaction with Omega-3 Fatty Acids, Low versus High Zinc, or ß-Carotene versus Lutein and Zeaxanthin on Progression of Age-Related Macular Degeneration in the Age-Related Eye Disease Study 2: Age-Related Eye Disease Study 2 Report No. 18.

PURPOSE: To assess whether genotypes at 2 major loci associated with age-related macular degeneration (AMD), complement factor H (CFH), or age-related maculopathy susceptibility 2 (ARMS2), modify the response to oral nutrients for the treatment of AMD in the Age-Related Eye Disease Study 2 (AREDS2). DESIGN: Post hoc analysis of a randomized trial. PARTICIPANTS: White AREDS2 participants. METHODS: AREDS2 participants (n = 4203) with bilateral large drusen or late AMD in 1 eye were assigned randomly to lutein and zeaxanthin, omega-3 fatty acids, both, or placebo, and most also received the AREDS supplements. A secondary randomization assessed modified AREDS supplements in 4 treatment arms: lower zinc dosage, omission of ß-carotene, both, or no modification. To evaluate the progression to late AMD, fundus photographs were obtained at baseline and annual study visits, and history of treatment for late AMD was obtained at study visits and 6-month interim telephone calls. Participants were genotyped for the single-nucleotide polymorphisms rs1061170 in CFH and rs10490924 in ARMS2. Bivariate frailty models using both eyes were conducted, including a gene-supplement interaction term and adjusting for age, gender, level of education, and smoking status. The main treatment effects, as well as the direct comparison between lutein plus zeaxanthin and ß-carotene, were assessed for genotype interaction. MAIN OUTCOME MEASURES: The interaction between genotype and the response to AREDS2 supplements regarding progression to late AMD, any geographic atrophy (GA), and neovascular AMD. RESULTS: Complete data were available for 2775 eyes without baseline late AMD (1684 participants). The participants (mean age ± standard deviation, 72.1±7.7 years; 58.5% female) were followed up for a median of 5 years. The ARMS2 risk allele was associated significantly with progression to late AMD and neovascular AMD (P = 2.40 × 10-5 and P = 0.002, respectively), but not any GA (P = 0.097). The CFH risk allele was not associated with AMD progression. Genotype did not modify significantly the response to any of the AREDS2 supplements. CONCLUSIONS: CFH and ARMS2 risk alleles do not modify the response to the AREDS2 nutrient supplements with respect to the progression to late AMD (GA and neovascular AMD).

Assessment of Novel Genome-Wide Significant Gene Loci and Lesion Growth in Geographic Atrophy Secondary to Age-Related Macular Degeneration.

IMPORTANCE: Age-related macular degeneration (AMD) is a common threat to vision loss in individuals older than 50 years. While neovascular complications in AMD are treatable, there is currently no therapy for geographic atrophy secondary to AMD. Geographic atrophy lesion progression over time shows considerable interindividual variability, but little is known about prognostic factors. OBJECTIVE: To elucidate the contribution of common genetic variants to geographic atrophy lesion growth. DESIGN, SETTING, AND PARTICIPANTS: This pooled analysis combined 4 independent studies: the Fundus Autofluorescence Imaging in Age-Related Macular Degeneration (FAM) study, the Directional Spread in Geographic Atrophy (DSGA) study, the Age-Related Eye Disease Study (AREDS), and the Geographic Atrophy Treatment Evaluation (GATE) study. Each provided data for geographic atrophy lesion growth in specific designs. Patients with geographic atrophy secondary to AMD were recruited to these studies. Genotypes were retrieved through the database of Genotypes and Phenotypes (for AREDS) or generated at the Cologne Center for Genomics (for FAM, DSGA, and GATE). MAIN OUTCOMES: The correlation between square root-transformed geographic atrophy growth rate and 7 596 219 genetic variants passing quality control was estimated using linear regression. The calculations were adjusted for known factors influencing geographic atrophy growth, such as the presence of bilateral geographic atrophy as well as the number of lesion spots and follow-up times. MAIN OUTCOMES AND MEASURES: Slopes per allele, 95% CIs, and P values of genetic variants correlated with geographic atrophy lesion growth. RESULTS: A total of 935 patients (mean [SD] age, 74.7 [7.8] years; 547 female participants [59.0%]) were included. Two gene loci with conservative genome-wide significance were identified. Each minor allele of the genome-wide associated variants increased the geographic atrophy growth rate by a mean of about 15% or 0.05 mm per year. Gene prioritization within each locus suggests the protein arginine methyltransferase 6 gene (PRMT6; chromosome 1; slope, 0.046 [95% CI, 0.026-0.066]; P = 4.09 × 10-8) and the lanosterol synthase gene (LSS; chromosome 21; slope, 0.105 [95% CI, 0.068-0.143]; P = 4.07 × 10-7) as the most likely progression-associated genes. CONCLUSIONS AND RELEVANCE: These data provide further insight into the genetic architecture of geographic atrophy lesion growth. Geographic atrophy is a clinical outcome with a high medical need for effective therapy. The genes PRMT6 and LSS are promising candidates for future studies aimed at understanding functional aspects of geographic atrophy progression and also for designing novel and targeted treatment options.

Author Correction: Retinal transcriptome and eQTL analyses identify genes associated with age-related macular degeneration.

In the version of this article initially published, in Supplementary Data 5, the logFC, FC, P value and adjusted P value for advanced AMD versus control (DE 4/1) without age correction did not correspond to the correct gene IDs. The errors have been corrected in the HTML version of the article.

Association of age-related macular degeneration with complement activation products, smoking, and single nucleotide polymorphisms in South Carolinians of European and African descent.

Purpose: Smoking and the incidence of age-related macular degeneration (AMD) have been linked to an overactive complement system. Here, we examined in a retrospective cohort study whether AMD-associated single nucleotide polymorphisms (SNPs), smoking, ethnicity, and disease status are correlated with blood complement levels. Methods: Population: The study involved 91 AMD patients and 133 controls, which included 73% Americans of European descent (EUR) and 27% Americans of African descent (AFR) in South Carolina. Readouts: Participants were genotyped for 10 SNPs and systemic levels of complement factor H (CFH) activity, and the complement activation products C3a, C5a, and Bb were assessed. Main Outcome Measures: Univariate and multivariable logistic regression models were used to examine associations between AMD status and distinct readouts. Results: AMD affects EUR individuals more than AFRs. EUR but not AFR AMD subjects revealed higher levels of Factors C3a and Bb. In all subjects, a 10-unit increase in C3a levels was associated with an approximately 10% increase in the odds of being AMD-positive, and C3a and Bb were associated with smoking. While CFH activity levels were not correlated with AMD, a significant interaction was evident between patient age and CFH activity. Finally, EURs had lower odds of AMD with enhanced copies of rs1536304 (VEGFA) and higher odds with more copy numbers of rs3766404 (CFH). Conclusions: Our results support previous studies of systemic complement components being potential biomarkers for AMD, but they suggest that smoking and disease do not synergistically affect complement levels. We also suggest a novel susceptibility and protective haplotypes in the South Carolinian AMD population. Our studies indicate that augmented complement activation associated with advanced AMD could be attributed to a decrease in CFH activity in younger patients.

Retinal transcriptome and eQTL analyses identify genes associated with age-related macular degeneration.

Genome-wide association studies (GWAS) have identified genetic variants at 34 loci contributing to age-related macular degeneration (AMD)1-3. We generated transcriptional profiles of postmortem retinas from 453 controls and cases at distinct stages of AMD and integrated retinal transcriptomes, covering 13,662 protein-coding and 1,462 noncoding genes, with genotypes at more than 9 million common SNPs for expression quantitative trait loci (eQTL) analysis of a tissue not included in Genotype-Tissue Expression (GTEx) and other large datasets4,5. Cis-eQTL analysis identified 10,474 genes under genetic regulation, including 4,541 eQTLs detected only in the retina. Integrated analysis of AMD-GWAS with eQTLs ascertained likely target genes at six reported loci. Using transcriptome-wide association analysis (TWAS), we identified three additional genes, RLBP1, HIC1 and PARP12, after Bonferroni correction. Our studies expand the genetic landscape of AMD and establish the Eye Genotype Expression (EyeGEx) database as a resource for post-GWAS interpretation of multifactorial ocular traits.

Progression of Geographic Atrophy in Age-related Macular Degeneration: AREDS2 Report Number 16.

PURPOSE: To analyze the prevalence, incidence, and clinical characteristics of eyes with geographic atrophy (GA) in age-related macular degeneration (AMD), including clinical and genetic factors affecting enlargement. DESIGN: Prospective cohort study within a controlled clinical trial. PARTICIPANTS: Age-Related Eye Disease Study 2 (AREDS2) participants, aged 50-85 years. METHODS: Baseline and annual stereoscopic color fundus photographs were evaluated for GA presence and area. Analyses included GA prevalence and incidence rates, Kaplan-Meier rates, mixed-model regression, and multivariable analysis of the square root of GA, area adjusted for covariates, including clinical/imaging characteristics and genotype. MAIN OUTCOME MEASURES: (1) Presence or development of GA; (2) change in the square root of GA area over time. RESULTS: At baseline, 517 eyes (6.2%) of 411 participants (9.8%) had pre-existing GA (without neovascular AMD), with the following characteristics: 33% central, 67% noncentral; and the following configurations: 36% small, 26% solid/unifocal, 24% multifocal, 9% horseshoe/ring, and 6% indeterminate. Of the remaining 6530 eyes at risk, 1099 eyes (17.3%) of 883 participants developed incident GA without prior neovascular disease during mean follow-up of 4.4 years. The Kaplan-Meier rate of incident GA was 19% of eyes at 5 years. In eyes with incident GA, 4-year risk of subsequent neovascular AMD was 29%. In eyes with incident noncentral GA, 4-year risk of central involvement was 57%. GA enlargement rate (following square root transformation) was similar in eyes with pre-existing GA (0.29 mm/year; 95% confidence interval 0.27-0.30) and incident GA (0.28 mm/year; 0.27-0.30). In the combined group, GA enlargement was significantly faster with noncentrality, multifocality, intermediate baseline size, and bilateral GA (P < 0.0001 for interaction in each case) but not with AREDS2 treatment assignment (P = 0.33) or smoking status (P = 0.05). Enlargement was significantly faster with ARMS2 risk (P < 0.0001), C3 non-risk (P = 0.0002), and APOE non-risk (P = 0.001) genotypes. CONCLUSIONS: Analyses of AREDS2 data on natural history of GA provide representative data on GA evolution and enlargement. GA enlargement, which was influenced by lesion features, was relentless, resulting in rapid central vision loss. The genetic variants associated with faster enlargement were partially distinct from those associated with risk of incident GA. These findings are relevant to further investigations of GA pathogenesis and clinical trial planning.

Genome-wide analysis of disease progression in age-related macular degeneration.

Family- and population-based genetic studies have successfully identified multiple disease-susceptibility loci for Age-related macular degeneration (AMD), one of the first batch and most successful examples of genome-wide association study. However, most genetic studies to date have focused on case-control studies of late AMD (choroidal neovascularization or geographic atrophy). The genetic influences on disease progression are largely unexplored. We assembled unique resources to perform a genome-wide bivariate time-to-event analysis to test for association of time-to-late-AMD with ∼9 million variants on 2721 Caucasians from a large multi-center randomized clinical trial, the Age-Related Eye Disease Study. To our knowledge, this is the first genome-wide association study of disease progression (bivariate survival outcome) in AMD genetic studies, thus providing novel insights to AMD genetics. We used a robust Cox proportional hazards model to appropriately account for between-eye correlation when analyzing the progression time in the two eyes of each participant. We identified four previously reported susceptibility loci showing genome-wide significant association with AMD progression: ARMS2-HTRA1 (P = 8.1 × 10-43), CFH (P = 3.5 × 10-37), C2-CFB-SKIV2L (P = 8.1 × 10-10) and C3 (P = 1.2 × 10-9). Furthermore, we detected association of rs58978565 near TNR (P = 2.3 × 10-8), rs28368872 near ATF7IP2 (P = 2.9 × 10-8) and rs142450006 near MMP9 (P = 0.0006) with progression to choroidal neovascularization but not geographic atrophy. Secondary analysis limited to 34 reported risk variants revealed that LIPC and CTRB2-CTRB1 were also associated with AMD progression (P < 0.0015). Our genome-wide analysis thus expands the genetics in both development and progression of AMD and should assist in early identification of high risk individuals.