The most significantly associated SNP in the experiment, rs10033900 (P
=9.11 × 10−8
), resides in the chromosome 4 linkage peak region according to Fisher et al1
and was very close to genome-wide significance levels as put forth by Dudbridge and Gusnanto and Pe'er et al19,20
(Supplementary Table 1 shows the full results of the screen). Several nearby SNPs were also associated with P
<0.0005, suggesting this association was not because of a sporadic genotyping artifact. This SNP is 2781
bp upstream of the 3′ UTR of CFI.
Given this compelling result, we decided to genotype a much higher density of SNPs in this region. Our originally associated SNP (rs10033900) remained the most highly associated SNP with a P-value of 6.46 × 10−8 (OR=0.7056 referring to lower-risk C allele) (). This SNP showed a very high level of genotyping concordance between the Illumina assay and the iPLEX assay designs.
29 SNPs tested across PLA2G12A and CFI on chromosome 4
We tested 29 SNPs across this region for association; conditioning on our most associated SNP, rs10033900, and observed no significant independent associations. We did, however, observe modest residual association at two neighboring, highly correlated SNPs (). This result suggests that rs10033900 may not be the causal variant but may be highly correlated with said variant. Therefore, we applied multimarker haplotype tests in an attempt to refine and isolate the association signal. We tested the two-marker haplotype of the two closest SNPs to rs10033900, both 5′ (rs13117504) and 3′ (rs11726949). The two-marker haplotype between rs13117504 and rs10033900 shows a somewhat stronger association to AMD than either SNP alone with a P-value of 1.18 × 10−8 (). None of these three SNPs appear to be functional, although rs11726949 is in intron 11 of CFI. We also tested for differences in association between the neovascular (‘wet') and geographic atrophy (‘dry') forms of advanced AMD and found only a 0.2% difference in MAF (46%) between the two groups.
Two-marker haplotype association results for most significant SNPs in CFI region
We conservatively defined the span of LD that encompassed the SNPs of interest. All other HapMap SNPs in this region are correlated to rs10033900 and rs13117504 with an r2>0.35. Multimarker haplotype testing across this region did not show any further association above the levels observed by the two-marker haplotype created between rs13117504 and rs10033900. We then sequenced all of the exons in this region to determine whether an obvious functional variant exists that explains this association. This block of LD spans the last two exonic regions of the 3′ end of CFI and all four exons of phospholipase A(2) group 12A (PLA2G12A). We found no SNPs in either gene transcript that could statistically explain the association observed at rs10033900. We did find a novel SNP just 5′ of exon 12 in CFI, but this SNP does not appear to be in high r2 with our associated SNP or haplotype and is, therefore, not the biological source of association.
We next evaluated the role of epistasis between rs10033900 and rs13117504 and the six variants previously established to be associated with AMD.2, 3, 4, 5, 6, 7, 8, 9
Specifically, two variants at CFH, two variants at the CFB/C2 locus, one at the LOC387715/HTRA1 locus, and one at the C3 locus were established as unequivocally associated to AMD risk in this cohort – the typing and analysis of which was described in Maller et al.8, 9
Using logistic regression, we observed no statistically significant interaction terms between any pair of these SNPs. Although weak interactions cannot be excluded, this result suggests that despite targeting the same pathway, these variants largely confer risk in an independent, log-additive fashion.
Given the independent action of this new variant, we were able to add it to the multilocus model from Maller et al.9
We estimate that this variant accounts for approximately 1% of the population variance of liability.21