Synergetic Effect of a Functional Variation in ERCC6 5’–Flanking Region and a Complement Factor H Variation Confers High Risk of Age–Related Macular Degeneration

Abstract

Purpose: : ERCC6 gene is involved in the aging process through its function in DNA metabolism. Disruption of this gene causes degeneration of multiple organs including retina. This study aims to identify single nucleotide polymorphism (SNP) of ERCC6 in association with age–related macular degeneration and the SNP–SNP interaction with SNPs in known AMD genes, such as complement factor H (CFH). The function of the ERCC6 SNP was also characterized.

Methods: : A cohort of 460 advanced AMD and 269 age–matched controls were included. Another set of 40 pathologically diagnosed AMD cases were used for the purpose of replication. The typing of ERCC6–6530C/G (rs3793784) and CFH intron GC (rs380390) was performed using PCR–RFLP. In silico analysis, electrophoretic mobility shift assay (EMSA) and luciferase reporter assays were performed to determine the regulatory function of ERCC6–6530C/G.

Results: : Both ERCC6–C6530G and CFH intron GC were associated with AMD. The disease odds ratios were 1.60 in ERCC6–6530 GG and 6.77 in CFH intron CC. However, individuals homozygous for both the ERCC6–6530 and the CFH intron risk alleles (GG/CC) conferred a 23.1–fold increased disease risk as compared with the CC/GG homozygocity. The archived AMD cases replicated the ERCC6 SNP association. Bioinformatics analysis indicates a putative binding element shift on the sequence flanking the ERCC6 SNP from Sp1 on the C allele to SP1, GATA–1 and OCT–1 on the G allele. EMSA displayed distinctive C and G allele binding patterns to retinal pigment epithelial nuclear protein. Luciferase expression was also higher in the vector construct containing the ERCC6–6530 G allele than that containing the C allele.

Conclusions: : A C/G SNP in the 5’–flanking region of ERCC6 is associated with AMD, both independently and via interaction in a synergistic mode with a SNP within CFH . The ERCC6–C6530G is located in a regulatory region and the G allele of ERCC6–6530 elevates the gene expression. ERCC6 is known to be a transcriptional factor by functioning as a component of RNA pol I transcription complex. Due to a linkage disequilibrium of CFH intron SNP and CFH tyr402his, overexpression of a malfunctioning variant protein could contribute in the combined risk of two SNPs for AMD.

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