Concordant association of insulin degrading enzyme gene (IDE) variants with IDE mRNA, Abeta, and Alzheimer's disease

Abstract

Background: The insulin-degrading enzyme gene (IDE) is a strong functional and positional candidate for late onset Alzheimer's disease (LOAD).

Methodology/principal findings: We examined conserved regions of IDE and its 10 kb flanks in 269 AD cases and 252 controls thereby identifying 17 putative functional polymorphisms. These variants formed eleven haplotypes that were tagged with ten variants. Four of these showed significant association with IDE transcript levels in samples from 194 LOAD cerebella. The strongest, rs6583817, which has not previously been reported, showed unequivocal association (p = 1.5x10(-8), fold-increase = 2.12,); the eleven haplotypes were also significantly associated with transcript levels (global p = 0.003). Using an in vitro dual luciferase reporter assay, we found that rs6583817 increases reporter gene expression in Be(2)-C (p = 0.006) and HepG2 (p = 0.02) cell lines. Furthermore, using data from a recent genome-wide association study of two Croatian isolated populations (n = 1,879), we identified a proxy for rs6583817 that associated significantly with decreased plasma Abeta40 levels (ss = -0.124, p = 0.011) and total measured plasma Abeta levels (b = -0.130, p = 0.009). Finally, rs6583817 was associated with decreased risk of LOAD in 3,891 AD cases and 3,605 controls. (OR = 0.87, p = 0.03), and the eleven IDE haplotypes (global p = 0.02) also showed significant association.

Conclusions: Thus, a previously unreported variant unequivocally associated with increased IDE expression was also associated with reduced plasma Abeta40 and decreased LOAD susceptibility. Genetic association between LOAD and IDE has been difficult to replicate. Our findings suggest that targeted testing of expression SNPs (eSNPs) strongly associated with altered transcript levels in autopsy brain samples may be a powerful way to identify genetic associations with LOAD that would otherwise be difficult to detect.

Figure 1. Association of variants 3 (rs5786996) and 311 (rs6583817) with LOAD and IDE mRNA levels.

(A) Variants were analyzed for association with LOAD using an additive logistic regression model with age at diagnosis/entry, sex and APOE ε4 dosage as covariates. N; total genotype count, MAF; minor allele frequency. For logistic regression of the combined series, individual series were included as covariates. P values demonstrating that there were no series-related effects on association (p*) were obtained by comparing this model to one that adjusts for series x genotype interactions in addition to age at diagnosis/entry, sex, APOE ε4 dosage, and series effects. (B) OR and 95% CI forest plots were generated from the data tabulated in panel a. (C) Association of the two variants with IDE mRNA levels in cerebellum. The horizontal black line and dots represent median relative IDE mRNA level expressed as 2^−ΔΔCt for each variant. Boxes represent the 25th and 75th percentiles and whiskers represent the full data range.11 = homozygotes for the major allele, 12 = heterozygotes, 22 = minor allele homozygotes. Samples with 12 and 22 genotypes for rs6583817 were combined because only two samples were 22. Variants were analyzed for association with the level of IDE transcript (ΔCT) using an additive linear regression model with age at diagnosis/entry, sex and APOE ε4 dosage as covariates.

Figure 2. In vitro functional effects of variant 311 (rs6583817) on reporter gene expression.

pGL3P constructs containing v311 sequence were transfected into (A) Be(2)-C and (B) HepG2 cell lines. 5′ and 3′ refer to the position of the cloned sequences relative to the luciferase gene and promoter in the pGL3P vector. G refers to vectors containing v311 major allele sequence and A refers to vectors containing v311 minor allele sequence. P refers to the pGL3P positive control vector (containing SV40 promoter) and B refers to the pGL3B negative control vector (containing no promoter). Error bars represent SEM (standard error of the mean). Note that the major allele sequence encompassing v311 had a repressor effect in Be(2)-C cells that achieved significance when the 3′G construct was compared to the pGL3P positive control vector (P) alone (fold-change = 0.48, p = 0.04). In contrast, the wild type sequence had a significant enhancer effect when the 3′G construct was compared to pGL3P in HepG2 cells (5′G fold-change = 2.4, p = 0.02, 3′G fold-change = 1.5). This difference probably reflects differential regulation of IDE expression by the transcription factors expressed in each cell line.

Figure 3. In vitro functional effects of variant 3 (rs5786996) on reporter gene expression.

Note that variant 3 (-/C) has a single C insertion. Thus the major allele is indicated by a – and the minor allele by a C. pGL3P constructs containing v3 sequence were transfected into (A) Be(2)-C and (B) HepG2 cell lines. 5′ and 3′ refer to the position of the cloned sequences relative to the luciferase gene and promoter in the pGL3P vector. “-” = vector containing v3 major allele sequence, “C” = vector containing v3 minor allele insertion sequence, “P” = pGL3P positive control vector (containing SV40 promoter), “B” = pGL3B negative control vector (containing no promoter). Error bars represent standard error of the mean. Note that in Be(2)-C cells, the v3 sequence had a repressor effect when compared to the pGL3P positive control vector (P) alone that achieved significance (5′C; fold-change = 0.82, p = 0.03, 3′-; fold-change = 0.65, p = 0.001; 3′C; fold-change = 0.19, p<0.0001) for all except the 5′- major allele sequence that showed no significant change in expression. In HepG2 cells, the 5′major allele sequence had an enhancer effect (5′-; fold-change = 1.62, p = 0.008) while the 5′C vector showed no significant change in expression compared to P. Both 3′ constructs had repressor activity (3′- fold-change = 0.56, p = 0.0008, 3′C fold-change = 0.14, p<0.0001).