Synergic effect of polymorphisms in ERCC6 5' flanking region and complement factor H on age-related macular degeneration predisposition

Abstract

This study investigates age-related macular degeneration (AMD) genetic risk factors through identification of a functional single-nucleotide polymorphism (SNP) and its disease association. We chose ERCC6 because of its roles in the aging process, DNA repair, and ocular degeneration from the gene disruption. Bioinformatics indicated a putative binding-element alteration on the sequence containing C-6530>G SNP in the 5' flanking region of ERCC6 from Sp1 on the C allele to SP1, GATA-1, and OCT-1 on the G allele. Electrophoretic mobility shift assays displayed distinctive C and G allele-binding patterns to nuclear proteins. Luciferase expression was higher in the vector construct containing the G allele than that containing the C allele. A cohort of 460 advanced AMD cases and 269 age-matched controls was examined along with pathologically diagnosed 57 AMD and 18 age-matched non-AMD archived cases. ERCC6 C-6530>G was associated with AMD susceptibility, both independently and through interaction with an SNP (rs380390) in the complement factor H (CFH) intron reported to be highly associated with AMD. A disease odds ratio of 23 was conferred by homozygozity for risk alleles at both ERCC6 and CFH compared with homozygozity for nonrisk alleles. Enhanced ERCC6 expression was observed in lymphocytes from healthy donors bearing ERCC6 C-6530>G alleles. Intense immunostaining of ERCC6 was also found in AMD eyes from ERCC6 C-6530>G carriers. The strong AMD predisposition conferred by the ERCC6 and CFH SNPs may result from biological epistasis, because ERCC6 functions in universal transcription as a component of RNA pol I transcription complex.

Fig. 1.

EMSA using nuclear extracts from human pigmented epithelium cells (ARPE-19). The oligos containing ERCC6−6530C or ERCC6−6530G were labeled with infrared dye (IRD) and annealed to their complement oligo to generate double-strand DNA. The nuclear extract was incubated with the labeled probes and titrated with different amounts of unlabeled probes. The reaction mixture was subjected to gel electrophoresis, and IRD signal was collected with Li-Cor IR² global system. The nuclear extract was bound to oligo containing ERCC6−6530C, which formed one main complex (complex C); the nuclear extract was bound to oligo containing ERCC6−6530G, which formed three main complexes (complex A, B, and D).

Fig. 2.

Correlation between luciferase activities and an SNP in 5′ flanking region of ERCC6. Transcription activity of the −7,025 to −6,040 nucleotide region of ERCC6. (Upper) Firefly luciferase activities were normalized against the internal control Renilla luciferase activity values. (Lower) The data indicate the mean values with the SDs from four independent experiments. The two cell lines (ARPE-19 and MCF7) show similar patterns.

Fig. 3.

ERCC6 transcript in human lymphocyte (n = 5 in each group) detected by real time RT-PCR. The donors were age-matched. Cells with G allele of ERCC6−6530 express more ERCC6 mRNA than those with the C allele. The fold change was normalized against β-actin. ∗, P < 0.05 vs. CC and CG genotypes.

Fig. 4.

Human ocular ERCC6 transcript detected by RT-PCR. Lane 1, negative control; lane 2, NEI Bank RPE cDNA library; lane 3, human retinal cDNA.

Fig. 5.

Photomicrographs illustrating positive ERCC6 retina cells (arrows) in the retina. (Left) Only few ganglion cells were positive (arrow) in the macula of a normal eye. (Center and Right) The AMD eyes showed replacement of the photoreceptor cells (asterisk) and retinal pigmented epithelia (arrowheads) by a layer of neovascular fibrous tissue. Increased positive staining is observed in the AMD eyes as compared with the normal eye; the AMD eye with ERCC6−6530C/G shows strongest staining. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigmented epithelium. Avidin–biotin–immunoperoxidase staining. (Magnification: ×200).