Genome-wide association analyses of genetic, phenotypic, and environmental risks in the age-related eye disease study

Abstract

Purpose: To present genome-wide association analyses of genotypic and environmental risks on age-related macular degeneration (AMD) using 593 subjects from the age-related eye disease study (AREDS), after adjusting for population stratification and including questionable controls.

Methods: Single nucleotide polymorphism (SNP) associations with AMD for the non-Hispanic white population were investigated using a log-additive model after adjusting for population stratification. Replication of possible SNP-disease association was performed by genotyping an independent group of 444 AMD case and 300 control subjects. Logistic regression models were used to assess interaction effects between smoking and SNPs associated with AMD. Independent genetic risk effects among the disease-associated SNPs were also investigated using multiple logistic regression models.

Results: Population stratification was observed among the individuals having a self-reported race of non-Hispanic white. Risk allele frequencies at established AMD loci demonstrated that questionable control subjects were similar to control subjects in the AREDS, suggesting that they could be used as true controls in the analyses. Genetic loci (complement factor H [CFH], complement factor B [CFB], the age-related maculopathy susceptibility 2 locus containing the hypothetical gene [LOC387715]/the high-temperature requirement A-1 [HTRA1], and complement component 3 [C3]) that were already known to be associated with AMD were identified. An additional 26 novel SNPs potentially associated with AMD were identified, but none were definitely replicated in a second independent group of subjects. Smoking did not interact with known AMD loci, but was associated with late AMD. Statistically independent genetic signals were observed within the Pleckstrin homology domain-containing family A member 1 (PLEKHA1) region near LOC387715/HTRA1 and within a haplotype spanning exon 19 of the C3 gene.

Conclusions: Population stratification among Caucasian subjects from the multicentered AREDS was observed, suggesting that it should be adjusted for in future studies. The AREDS questionable control subjects can be used as control subjects in the AREDS genome-wide association study (GWAS). Smoking was an independent risk factor for advanced AMD in the AREDS subjects. There continues to be evidence that the 10q26 (age-related maculopathy susceptibility 2 gene [ARMS2]) locus spanning PLEKHA1-LOC387715-HTRA1 and the C3 gene may contain multiple independent genetic risks contributing to AMD.

Figure 1

Comparison of the frequency of the risk allele for four single nucleotide polymorphisms (SNPs) known to contribute to age-related macular degeneration (AMD) in the age-related eye disease study (AREDS) “control” and “questionable control” subjects. Each of these SNPs has been consistently associated with AMD in multiple studies. The figure shows that the questionable control category was similar to the control category in terms of the frequency of the risk allele. Questionable control subjects can be used as controls in analyses using the AREDS data set. Abbreviations: RCA is the regulation of complement activation locus containing the gene encoding CFH; please see introduction for other gene and locus names.

Figure 2

Population stratification in the age-related eye disease study (AREDS) subjects. Three race categories are presented in pink circle for white, non-Hispanics (White), in black triangle for blacks (Black), and in green cross for subjects who are neither white, non-Hispanics nor blacks. A plot of the first two principal components using 103,895 single nucleotide polymorphisms (SNPs) passing quality-control filters. Of the total 590 subjects, the self-reported race was white, non-Hispanic for 576, black for 12, and “other” for 2. The figure shows that one black subject and two other subjects were similar to the white subjects. There was also stratification among the white subjects, for which we corrected in subsequent analyses. The x-axis shows the first principal component axis (PC1), while the y-axis shows the second component (PC2).

Figure 3

Quantile-quantile plots of the age-related eye disease study (AREDS) genome-wide association study (GWAS). A: Scatter plot of the −log10 (p values) expected under the null hypothesis of no genetic association versus the observed −log10 (p values) are shown for all subjects. B: This is a scatter plot as in A, but for white subjects only. C: This is a scatter plot of white subjects corrected for population stratification using principal components methods without exclusion of known loci. D: This is a scatter plot of white subjects corrected for population stratification using principal components methods with exclusion of known loci. A–C: These plots show that correction for population stratification reduced the genomic inflation factor from 1.23 to 1.01. D: This plot suggests that there is evidence for additional single nucleotide polymorphisms (SNPs) contributing to age-related macular degeneration (AMD) after statistically accounting for known loci.

Figure 4

The association of single single nucleotide polymorphisms (SNPs) with age-related macular degeneration (AMD) across the genome. Significance across the genome is illustrated by plotting −log 10 (p values) from the log-additive genetic model corrected for population stratification. The loci with p values less than 0.00001 are labeled and the SNPs are provided in Table 2.