Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease

We undertook a two-stage genome-wide association study of Alzheimer's disease involving over 16,000 individuals. In stage 1 (3,941 cases and 7,848 controls), we replicated the established association with the APOE locus (most significant SNP: rs2075650, p= 1.8×10−157) and observed genome-wide significant association with SNPs at two novel loci: rs11136000 in the CLU or APOJ gene (p= 1.4×10−9) and rs3851179, a SNP 5′ to the PICALM gene (p= 1.9×10−8). Both novel associations were supported in stage 2 (2,023 cases and 2,340 controls), producing compelling evidence for association with AD in the combined dataset (rs11136000: p= 8.5×10−10, odds ratio= 0.86; rs3851179: p= 1.3×10−9, odds ratio= 0.86). We also observed more variants associated at p< 1×10−5 than expected by chance (p=7.5×10−6), including polymorphisms at the BIN1, DAB1 and CR1 loci. Alzheimer's disease (AD) is the most common form of dementia, is highly heritable (heritability of up to 76%) but genetically complex1. Neuropathologically, the disease is characterized by extracellular senile plaques containing β-amyloid (Aβ) and intracellular neurofibrillary tangles containing hyperphosphorylated τ protein1. Four genes have been definitively implicated in its etiology. Mutations of the amyloid precursor protein (APP) gene and the presenilin 1 and 2 genes (PSEN1, PSEN2) cause rare, Mendelian forms of the disease usually with an early-onset. However, in the more common form of AD, only apolipoprotein E (APOE) has been established unequivocally as a susceptibility gene1. Aiming to identify novel AD loci, several genome-wide association studies (GWAS) have been conducted prior to the present study. All have identified strong evidence for association to APOE, but less convincing evidence implicating other genes2-9. This outcome is consistent with the majority of findings from GWAS of other common phenotypes, where susceptibility alleles typically have effect sizes with odds ratios (OR) of 1.5 or less, rather than of the magnitude for APOE (OR~3). Detecting such modest effects requires much larger samples than those that have been applied in the GWAS of AD to date10, which have all included fewer than 1,100 cases. Based upon the hypothesis that risk alleles for AD are likely to confer ORs in the range seen in other common diseases, we undertook a more powerful GWAS than has been undertaken to date. We established a collaborative consortium from Europe and the USA from which we were able to draw upon a combined sample of up to 19,000 subjects (before quality control) and conducted a two-stage study. In Stage 1, 14,639 subjects were genotyped on Illumina platforms. 5,715 samples were genotyped for the present study using the Illumina 610-quadchip; genotypes for the remaining subjects were either made available to us from population control datasets or through collaboration and were genotyped on the Illumina HumanHap550 or the HumanHap300 BeadChips. Prior to association analysis, all samples and genotypes underwent stringent quality control, which resulted in the elimination of 53,383 autosomal SNPs and 2,850 subjects. Thus, in Stage 1, we tested 529,205 autosomal SNPs for association in up to 11,789 subjects (3,941 AD cases, 7,848 controls of which 2,078 were elderly screened controls, see Supplementary Table 1). The genomic control inflation factor (λ)11 was 1.037, suggesting little evidence for residual stratification. In addition to the known association with the APOE locus, GWA analysis identified two novel loci at a genome-wide level of significance (see Fig. 1). Table 1 shows SNPs which were genome-wide significant (GWS) in stage 1; 13 GWS SNPS map within or close to the APOE locus on chromosome 19 (p< 3×10−8 − 2×10−157) and the top 5 are shown in Table 1 (see Supplementary Table 2 for the complete list). The other two SNPs represent novel associations. One of the novel SNPs (rs11136000) is located within an intron of clusterin (CLU, also known as APOJ) on chromosome 8 (p= 1.4×10−9, OR=0.840); the other SNP (rs3851179) is 88.5 kb 5′ to PICALM on chromosome 11 (p = 1.9×10−8, OR=0.849). Note that there was no significant difference in allele frequencies between elderly, screened controls and Harold et al. Page 3 Nat Genet. Author manuscript; available in PMC 2010 April 1. U PM C Fders G rup Athor M anscript U PM C Fders G rup Athor M anscript population controls for these two SNPs. In stage 2, the two novel GWS SNPs were genotyped in an independent sample comprising 2,023 AD cases and 2,340 age-matched, cognitively screened controls (Supplementary Table 3). Both were independently associated in this sample (rs11136000: one-tailed p= 0.017, OR= 0.905; rs3851179: one-tailed p= 0.014, OR= 0.897). Meta-analysis of the stage 1 and 2 datasets also produced highly significant evidence of association (rs11136000: p= 8.5×10−10, OR = 0.861 and rs3851179: p= 1.3×10−9, OR= 0.859, two-tailed, Table 1) for CLU and PICALM loci respectively. We sought further evidence from the Translational Genomics Research Institute (TGEN) study9 and the Li et al. study8, two publicly available AD GWAS datasets, but neither of the novel GWS SNPs had been genotyped or could be imputed. As secondary analyses, we tested each novel finding for interaction with APOE status and for association with age at onset. No significant interactions of the novel SNPs with APOE status were observed influencing AD risk (rs11136000xAPOE-ε4 interaction p= 0.674; rs3851179xAPOE-ε4 interaction p=0.735). Although we observed significant effects of the GWS SNPs on age at onset, these were limited to SNPs at the APOE locus (data not shown). In a preliminary attempt to attribute the source of the association to a functional variant, we used publicly available data to identify additional SNPs at each locus that were correlated through linkage disequilibrium (LD) with either novel GWS SNP or that might plausibly have functional effects (see Supplementary Table 4). A synonymous SNP (rs7982) in the CLU gene was in strong LD (r2=0.95 in our extension sample) with the GWS SNP and showed a similar level of evidence for association with AD in the whole sample (meta-p =8×10−10; stage 1 genotypes were imputed). This SNP is in exon 5 of the CLU gene, which codes for part of the beta chain of the protein and may influence a predicted exon splicing enhancer. We note that Tycko and colleagues12 previously published a negative association study on the CLU gene, analyzing 4 SNPs in an AD case-control sample of African-American, Hispanic and Caucasian/ non-Hispanic individuals. Although they identified rs7982 through mutation screening (referred to as VB in their study), the SNP was not tested for association in their sample. The 4 SNPs that were analyzed were rare in Caucasians (minor allele frequency <2%) and there was very limited power to detect association in their Caucasian sub-sample of 53 AD cases and 43 controls. As these 4 SNPs were not genotyped in the present GWAS, there is no overlap between the two studies. Several potentially functional SNPs were identified at the PICALM locus. Of these, two showed good evidence for association; rs561655, which is within a putative transcription factor binding site and rs592297, which is a synonymous SNP in exon 5 of the gene that may influence a predicted exon splicing enhancer. However, neither of these SNPs showed the strength of evidence for association observed for rs3851179, the GWS SNP at the PICALM locus (i.e. rs561655: meta-p= 1×10−7 and rs592297: meta-p= 2×10−7). A number of SNPs in LD with rs3851179 and showing moderate evidence of association in the GWAS (p<1×10−4) were also followed up, most notably rs541458. This SNP is 8kb 5′ to the PICALM gene and was directly genotyped in the extension sample, the TGEN study and the Li et al. study, with p<0.05 in each. Following meta-analysis, this is one of our most significant SNPs (meta-p= 8×10−10) and is also supported by the study of Amouyel et al. published in this issue (p=3×10−3). Further genetic analyses will be required to characterize the true nature of the associations observed at these loci, which is beyond the scope of this paper. We also tested whether the observed number of significant associations observed in the GWAS exceeded what would be expected by chance. Having removed SNPs within the APOE, CLU and PICALM loci (see Methods) we focused on those which showed most evidence for association (p< 1×10−5). Approximately 13 independent signals were observed; less than 4 would be expected by chance (p= 7.5×10−6). Table 2 shows the loci implicated and provides strong evidence for association with the complement receptor 1 (CR1) gene, particularly in the Harold et al. Page 4 Nat Genet. Author manuscript; available in PMC 2010 April 1. U PM C Fders G rup Athor M anscript U PM C Fders G rup Athor M anscript context of the results of the Amouyel et al. study published in this issue. Also noteworthy are the bridging integrator 1 (BIN1) gene, which produces a protein involved in synaptic vesicle endoctyosis13, and the disabled homolog 1 (DAB1) gene, whose product is involved with tyrosine phosphorylation and microtubule function in neurons14. These data thus provide strong evidence that there are several genes associated with AD which remain to be identified. We have also tested over 100 variants highlighted by previous AD GWA studies for association in our sample (see Supplementary note for full discussion). These are summarized in Supplementary Table 5. Until now, the APOE-ε4 allele was the only consistently replicated genetic risk factor for AD. It is therefore intriguing that we find compelling evidence for association with CLU, a gene that encodes another major brain apolipoprotein15, suggesting that susceptibility genes are not randomly distributed through functional pathways. The predominant form of clusterin is a secreted heterodimeric glycoprotein of 75-80kDa. The single copy gene spans about 16kb on chromosome 8p21-