Two DNA Variants Associated With Age–Related Macular Degeneraton in a Case–Control Study

Abstract

Abstract: : Purpose: Both genetic and environmental factors are implicated in age–related macular degeneration (AMD) pathogenesis. Candidate gene association studies provide evidence that single nucleotide polymorphisms (SNPs) may play a role in AMD. We have reported an association between AMD and two CX3CR1 SNPs (V249I and T280M) (FASEBS J, 2004). Here we investigate two additional SNPs associated with AMD in a case–control study. Methods: Genomic DNA was subjected to PCR amplification following extraction from peripheral blood collected from 192 age–matched controls with no clinical signs of AMD (AC), 194 random unscreened healthy volunteers with a younger mean age (RC), and 128 advanced AMD patients (CP). Genomic DNA was also extracted, following microdissection and whole genome amplification, from 40 archived paraffin–embedded slides of pathologically diagnosed AMD eyes (PP). The selected candidate gene SNPs were A2518G in the nonsynonymous coding region of chemokine CCL2 and 530A–>G in the promoter region of the DNA repair related CSB (Cockayne syndrome) gene. These SNPs were screened using PCR amplification followed by restriction fragment length polymorphism (RFLP). Gel shift assay was also conducted. Results: The average age of the 4 groups of CP, AC, RC, and PP were 78.8, 65.7, 45.2, and 82.8 years, respectively. CCL2 2518G allele frequency was 25.5% in the CP as compared to 32.2% in the AC. For the CSB 530A–>G allele frequencies: 38.2% in CP, 26.8% in AC and 26.8% in RC were found. The PP had the highest allele frequency of 46.1%. This association is in a gene dosage effect manner. The highest odds ratio of CSB–530 G allele carrier was 2.36 (PP), p=0.0006. Gel shift assay showed lower nuclear protein binding of the G allele as compared to the A allele. Conclusions: The data of this case–control study suggest that a gene dose affected association exists between the CSB–530 G allele variant and increased risk of AMD development. Efficient DNA repair mechanisms are required to maintain cellular integrity after DNA damage. A CSB SNP in the promoter region may result in regulatory functional alterations of the CSB activator element thereby impairing DNA repair. Although there is a trend towards AMD association with the CCL2 A2518G SNP, larger samples and further studies are needed to confirm this potential association.

Keywords: age-related macular degeneration • clinical (human) or epidemiologic studies: risk factor assessment • retina