Alzheimer's risk variants in the clusterin gene are associated with alternative splicing

Abstract

Genetic variation in CLU encoding clusterin has been associated with Alzheimer's disease (AD) through replicated genome-wide studies, but the underlying mechanisms remain unknown. Following earlier reports that tightly regulated CLU alternative transcripts have different functions, we tested CLU single nucleotide polymorphisms (SNPs) including those associated with AD for quantitative effects on individual alternative transcripts. In 190 temporal lobe samples without pathology we found that the risk allele of the AD associated SNP rs9331888 increases the relative abundance of transcript NM_203339 (P=4.3×10(-12)). Using an independent set of 115 AD and control samples, we replicated this result (p=0.0014) and further observed that multiple CLU transcripts are at higher levels in AD compared to controls. The AD SNP rs9331888 is located in the first exon of NM_203339 and therefore, it is a functional candidate for the observed effects. We tested this hypothesis by in vitro dual luciferase assays using SK-N-SH cells and mouse primary cortical neurons and found allelic effects on enhancer function, consistent with our results on post-mortem human brain. These results suggest a biological mechanism for the genetic association of CLU with AD risk and indicate that rs9331888 is one of the functional DNA variants underlying this association.

Keywords: Alzheimer's dementia; CLU; clusterin; gene regulation; splicing; transcription.

Figure 1

(a) CLU alternative transcripts from reference sequence (RefSeq) and UCSC examined in this study. Single-nucleotide polymorphisms (SNPs) genotyped within the gene are shown and SNPs reported in recent genome-wide association studies (GWAS) are marked with an asterisk. (b) Enlargement of the region around rs9331888. The SNP location is marked by a vertical red line. Selected tracks from the UCSC genome browser ENCODE data, as well as phylogenetic conservation data are shown. For clarifications, see the UCSC genome database website. (c) The sequences inserted into reporter constructs are shown named U (upstream) S (short) and D (downstream). Each sequence was made to carry either a reference or a risk allele at rs9331888, shown here by a white asterisk and found to influence Alzheimer's disease (AD) risk in the Lambert et al. GWAS.

Figure 2

Dual luciferase reporter assays comparing constructs carrying reference (ref) or risk alleles in the three different constructs shown in Figure 1c in front of an SV40 promoter. Firefly, relative to renilla luciferase levels, is shown with s.e. bars based on four replicates. All constructs show significant differences between the two rs9331888 alleles in primary neuron culture and two of three also show differences in SK-N-SH cells. As expected, the risk allele shows higher activity. RLU, relative luciferase units.