Background We recently completed a meta-analysis study in AMD. We are employing comprehensive and collaborative approaches and using human genetics along with mouse and zebrafish animal models. Results 1. Age-related macular degeneration (AMD) AMD is the most common cause of incurable visual impairment in high-income countries. We participate in several large multi-center, national and international studies to discern the genetic component of this disease and its superimposition to environmental factors. We are participating in the NEI-sponsored AMD Gene Consortium. Meta-analysis of the GWAS data (from 18 groups worldwide) including >17,100 cases with advanced disease and >60,000 controls has revealed 19 AMD susceptibility loci including seven novel loci. In most, the strongly associated marker is close to more than one gene. These loci are enriched for genes involved in regulation of complement activity, lipid metabolism, extracellular matrix (ECM) remodeling and angiogenesis, providing insights into the pathophysiology of AMD. To catalog the complete spectrum of functional alleles, we have initiated targeted re-sequencing of 57 candidate AMD-associated genes in 3000 cases and controls (collaboration with Wash U Genome Center and Goncalo Abecasis). Our studies are expected to unravel rare coding variants in the associated genes, thus opening new avenues for biological understanding of AMD pathogenesis. In another consortium approach to catalogue the rare variants (MAF >0.1), we are involved in exome chip genotyping arrays of 38,000 AMD patients, which should provide enough power to establish the effect of rare variants and explain additional unresolved heritability (P.I. Goncalo Abecasis). Disease-associated SNPs are often located near more than one gene, and a genetic approach cannot pinpoint the pathogenic allele(s). We are using zebrafish as a model to knockdown or overexpress the associated genes and examine the eye/retina phenotype. To date, we have injected splice-site morpholinos to five genes. We hope to establish a quick functional screening tool to select genes for further biological characterization. We are also sequencing exomes in AMD families to identify rare to low frequency functional variants that segregate with the disease. Though AMD is a complex disease, it might be possible to identify high-penetrant 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 vary from mild (only drusen) to more severe geographic atrophy or choroidal neovascularization. Whole exome analysis may allow us to identify a subset of genetic variants that are associated with severity Complement factor H shows very strong association with 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. We conclude that causal variation in this region potentially encompasses both regulatory effects at single markers and copy number (3). Previous case-control studies in populations of European ancestry had provided little differentiation in AMD subtype, and insufficient power to confirm or refute effect modification by smoking. In a collaborative study, we found that the Y402H variant confers a 2-fold higher risk of late-AMD per copy in individuals of European descent. This is stable to stratification by study design and AMD classification and is not modified by smoking. The lack of association in non-Europeans requires further verification (5). We also examined the role of complement factor D (CFD) in AMD by analysis of genetic association, copy number variation, and plasma CFD concentrations. CFD regulates activation of the alternative complement pathway, which is implicated in AMD pathogenesis. We found evidence for genetic association between a CFD gene SNP and AMD and a significant increase in plasma CFD concentration in AMD cases compared with controls, consistent with a role for CFD in AMD pathogenesis (6). 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. Our pooled analysis (n = 21,160) demonstrates associations between late AMD and both APOepsylon4 and APOepsylon2, following adjustment for age group and sex within each study and smoking status. We did not identify additional risks beyond epsylon2 and epsylon4 haplotypes. Our data provide evidence supporting the role of cholesterol modulation, and low-density cholesterol specifically, in AMD disease etiology (2). 2. Diabetic Retinopathy We collaborated with the Candidate gene Association Resource (CARe) group to investigate genetic associations using candidate genes that had been previously associated with type 2 diabetes (T2D), DR, and diabetic nephropathy (DN). Although we found little evidence of a major DR gene, we identified variants in P-selectin (SELP) and alpha-L-iduronidase (IDUA) that warrant further investigation (4). Collaborating with Raj Kandpal (Western University of Health Sciences), we identified transcriptional and splicing changes in the retina of diabetic rats, some of which are inhibited by therapies that prevent vascular pathology, pointing to possible molecular mechanisms for drug effects (1).
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