| Abstract: | Age-related macular degeneration (AMD) is a progressive disease and major cause of severe visual loss. Toward the discovery of tools for early identification of AMD susceptibility, we evaluated the combined predictive capability of proteomic and genomic AMD biomarkers. We quantified plasma carboxyethylpyrrole (CEP) oxidative protein modifications and CEP autoantibodies by ELISA in 916 AMD and 488 control donors. CEP adducts are uniquely generated from oxidation of docosahexaenoate-containing lipids that are abundant in the retina. Mean CEP adduct and autoantibody levels were found to be elevated in AMD plasma by ～60 and ～30%, respectively. The odds ratio for both CEP markers elevated was 3-fold greater or more in AMD than in control patients. Genotyping was performed for AMD risk polymorphisms associated with age-related maculopathy susceptibility 2 (ARMS2), high temperature requirement factor A1 (HTRA1), complement factor H, and complement C3, and the risk of AMD was predicted based on genotype alone or in combination with the CEP markers. The AMD risk predicted for those exhibiting elevated CEP markers and risk genotypes was 2–3-fold greater than the risk based on genotype alone. AMD donors carrying the ARMS2 and HTRA1 risk alleles were the most likely to exhibit elevated CEP markers. The results compellingly demonstrate higher mean CEP marker levels in AMD plasma over a broad age range. Receiver operating characteristic curves suggest that CEP markers alone can discriminate between AMD and control plasma donors with ～76% accuracy and in combination with genomic markers provide up to ～80% discrimination accuracy. Plasma CEP marker levels were altered slightly by several demographic and health factors that warrant further study. We conclude that CEP plasma biomarkers, particularly in combination with genomic markers, offer a potential early warning system for assessing susceptibility to this blinding, multifactorial disease.Age-related macular degeneration (AMD)1 is the most common cause of legal blindness in the elderly in developed countries (1). It is a complex, progressive disease involving multiple genetic and environmental factors that can result in severe visual loss. Early risk factors include the macular deposition of debris (drusen) on Bruch membrane, the extracellular matrix separating the choriocapillaris from the retinal pigment epithelium (RPE). Later stages of “dry” AMD involve the degeneration of photoreceptor and RPE cells resulting in geographic atrophy. In “wet” AMD, abnormal blood vessels grow from the choriocapillaris through Bruch membrane (choroidal neovascularization (CNV)). CNV occurs in 10–15% of AMD cases yet accounts for over 80% of debilitating visual loss in AMD. Anti-vascular endothelial growth factor treatments can effectively inhibit the progression of CNV (1), and antioxidant vitamins and zinc can slow dry AMD progression for select individuals (2). However, there are no universally effective therapies for the prevention of dry AMD or the progression from dry to wet AMD nor are there therapies to repair retinal damage in advanced AMD. The prevalence of advanced AMD in the United States is projected to increase by 50% to ～3 million by the year 2020 largely because of the rapidly growing elderly population (3). Accordingly early identification of AMD susceptibility and implementation of preventive measures are important therapeutic strategies (1).The molecular mechanisms causing AMD remain unknown, although inflammatory processes have been implicated by the identification of AMD susceptibility genes encoding complement factors (4–10) and the presence of complement proteins in drusen (11–13). Oxidative stress has long been associated with AMD pathology as shown by the finding that smoking significantly increases the risk of AMD (14) and that antioxidant vitamins can selectively slow AMD progression (2). A direct molecular link between oxidative damage and AMD was established by the finding that carboxyethylpyrrole (CEP), an oxidative protein modification generated from docosahexaenoate (DHA)-containing phospholipids, was elevated in Bruch membrane and drusen from AMD patients (11). Subsequently CEP adducts as well as CEP autoantibodies were found to be elevated in plasma from AMD donors (15), and CEP adducts were found to stimulate neovascularization in vivo, suggesting a role in the induction of CNV (16). From such observations, oxidative protein modifications were hypothesized to serve as catalysts of AMD pathology (11, 15, 17). In support of this hypothesis, mice immunized with CEP-adducted mouse albumin develop a dry AMD-like phenotype that includes sub-RPE deposits resembling drusen and RPE lesions mimicking geographic atrophy (18).Although identified AMD susceptibility genes account for over half of AMD cases (19), many individuals carrying AMD risk genotypes may never develop the disease. Likewise only a fraction of those diagnosed with early AMD progress to advanced stage disease with severe visual loss (2). Toward the discovery of better methods to predict susceptibility to advanced AMD, we quantified CEP adducts and autoantibodies in over 1400 plasma donors and also genotyped many of these donors for AMD risk polymorphisms in complement factor H (CFH) (4–7), complement C3 (9, 10), age-related maculopathy susceptibility 2 (ARMS2; also known as LOC387715) (19–22), and high temperature requirement factor A1 (HTRA1) (23, 24). The results demonstrate that combined CEP proteomic and genomic biomarker measurements are more effective in assessing AMD risk than either method alone. |
