Background Age-related macular degeneration (AMD) is a heritable, multi-factorial, progressive neurodegenerative disease that leads to loss of central vision through RPE dysfunction and death of photoreceptors. In developed countries, AMD is the leading cause of blindness in those >65 years of age. Genes in the complement pathway and a region of chromosome 10 have been implicated as the major genetic contributors to disease. However, association has also been shown with at least 17 additional loci that could be entry points into the understanding of AMD biology and potential therapeutic targets. Results To accelerate the pace of discovery in AMD genetics, we joined an international group of 18 laboratories in the AMD Gene Consortium supported by NEI. We performed a meta-analysis of genome-wide association studies (GWAS), combining data for >7,600 cases with advanced disease (with geographic atrophy, neovascularization or both) and >50,000 controls. We combined results through meta-analysis and after genotyping additional 9,500+ cases and 8,200+ controls for 32 variants, representing loci with promising evidence of association. We identified 19 significantly associated loci that show enrichment for genes involved in the regulation of complement activity, lipid metabolism, extracellular matrix remodeling and angiogenesis (1). Our results include seven loci with associations reaching P <5 x 10e-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. The pursuit of investigations of these AMD-associated loci will provide better genetic and biological understanding of AMD and the opportunity to develop novel therapeutic approaches. Of the list generated from the meta-analysis of AMD patients, we have selected 4 genes for functional follow up in zebrafish, based on their expression pattern in mouse and human. Our experimental plan includes the use of morpholinos to knock down the expression of these genes and by overexpression using mRNA. Phenotypic analysis focuses on changes in morphology and in the development of retinal cell types by immunohistochemistry (IHC). Our preliminary data indicate that knock down of two of the genes results in a reproducible small eye phenotype. However, further validation by histology, IHC and additional methods is necessary before making any definitive conclusions. Zebrafish studies serve as a prelude to work in mice being planned for the upcoming year. To identify rare coding variants in associated AMD susceptibility genes, we undertook targeted re-sequencing approach in collaboration with colleagues at the University of Michigan and Washington University Genome Center. In addition to validating reported rare alleles, we identified a rare variant in complement C3 (6). We are also sequencing exomes in as many as 18 AMD families to identify rare to low frequency functional variants that segregate with the disease. Although AMD is a complex disease, it might be possible to identify high-penetrance causative allele(s) in large multigenerational families. We can test whether the genes with rare variants overlap with the genes for common AMD risk loci or converge on specific pathways. Even within the same family, AMD phenotypes can vary from mild (only drusen) to more severe geographic atrophy or choroidal neovascularization. By whole exome analysis, we aim to identify subsets of genetic variants that are associated with severity. This work has been completed, and the data analysis is in progress. In addition, we performed exome sequencing in three affected and one unaffected member of a two-generation family with autosomal dominant form of early onset macular degeneration to identify the causative gene. Our analysis revealed a rare non-synonymous variant p.Glu1144Lys in FBN2, a glycoprotein of the elastin-rich extracellular matrix (ECM). We localized FBN2 to Bruchs membrane in human and monkey eyes. Expression is reduced in aging and AMD eyes. We screened FBN2 gene for rare variants in additional maculopathy patients and identified four rare non-synonymous variants that are predicted to alter FBN2 structure or function. We also examined the association of a non-synonymous FBN2 variant rs154001 (p. Val965Ile) with AMD in over 10,000 cases and almost an equal number of controls. Our analysis detected a significant association (OR= 1.10;p-value= 3.7910 x 10e-5) of a common FBN2 coding variant, rs154001 (p.Val965Ile), with AMD. Thus, our results show that rare and common variants in a single gene can contribute to Mendelian and complex forms of macular degeneration and highlighted the importance of studying orphan diseases for understanding more common clinical phenotypes. Furthermore, we provided strong evidence for a key role of Bruchs membrane in maintaining blood-retina homeostasis. Our collaborators have used the GWAS data from previous studies for examining association with intraocular pressure (2).
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