Toll-like receptor 2 -196 to -174 del polymorphism influences the susceptibility of Han Chinese people to Alzheimer's disease

Toll-like receptor 2 (TLR2) represents a reasonable functional and positional candidate gene for Alzheimer's disease (AD) as it is located under the linkage region of AD on chromosome 4q [1], and is functionally involved in the microglia-mediated inflammatory response and amyloid β (Aβ) clearance [2‐6]. Genetic studies on the TLR2 gene have identified a number of polymorphisms which have been shown to affect host defense, disease progression and be linked to differential disease susceptibilities [7]. We have assessed the involvement of 7 single nucleotide polymorphisms (SNPs) (Arg 677Trp, Arg753Gln, rs1898830, rs11938228, rs3804099, rs3804100, and rs7656411) as well as a short tandem GT repeat polymorphism in intron 2 of the TLR2 gene in the risk of developing late-onset AD (LOAD) [8, 9], and found an association of GT repeat polymorphism with an increased risk for LOAD in our previous studies. A 22-bp nucleotide deletion at position -196 to -174 of the untranslated 5'-region in TLR2 gene is associated with reduced transcriptional activity compared to the wild type allele in luciferase reporter assays [10]. This polymorphism has already been shown to be associated with an increased risk of noncardiac gastric cancer, susceptibility to cervical cancer, hepatitis C viral loads and susceptibility to hepatocellular carcinoma [11‐13]. In light of the important role that TLR2 plays with respect to the immune response in the pathogenesis of AD [2‐6], we hypothesized that the -196 to -174 del/ins polymorphism in the TLR2 gene might be associated with AD.

The alleles and genotypes frequencies of LOAD patients and controls in the total sample and after stratification for ApoE ε4 allele are given in Table 1. The distribution of genotypes of TLR2 polymorphisms were within the range of Hardy-Weinberg equilibrium (P = 0.21). There were significant differences in genotype and allele frequencies between LOAD and control groups (genotype P = 0.026, allele P = 0.009). The -196 to -174 del allele significantly raised the risk of developing LOAD (OR = 1.31, 95%CI = 1.07-1.60, Power = 84.9%). In subjects without ApoE ε4 allele, the allele and genotype distributions between LOAD patients and controls remain significantly different (genotype P = 0.027, allele P = 0.009). However, in subjects with the ApoE ε4 allele, there were no significant differences. In order to rule out confounding in our crude association analyses, we reevaluated the polymorphism effect under 3 different models using logistic regression adjusting for age and ApoE ε4 status (Table 2). The -196 to -174 del polymorphism was still found to increase the risk of LOAD via a recessive model (OR = 1.64, 95% CI = 1.13-2.39, P = 0.01, Bonferroni corrected P = 0.03).

Many experimental and clinical studies have suggested that TLR2 might play an important role in the pathogenesis of AD [2‐6]. TLR2 is a member of pattern-recognition receptors in the innate immune system [7]. Increased levels of TLR2 mRNA have been found in microglia isolated from AD patients [2]. Jana et al. have supplied several lines of evidence supporting the opinion that Aβ peptides activate microglia via TLR2 as inhibition of TLR2 through function-blocking antibodies or siRNA knockdown prevents fibrillar forms of Aβ from inducing nitrite, interleukin-6 (IL-6), or tumor necrosis factor-alpha (TNF-α) production [3]. Although an increasing volume of data favors TLR2 -mediated neurotoxicity, TLR2 may also be essential for Aβ clearance and in that way provide neuroprotection in AD [4]. Reed-Geaghan et al. reported that CD14, TLR4, and TLR2 are necessary for binding fibrillar Aβ (fAβ) to the cell surface, and are required for phenotypic activation of microglia and induction of phagocytosis [5]. Richard et al. demonstrated that TLR2 deficiency in transgenic AD mice could increase Aβ deposition and accelerate cognitive decline [6].

The -196 to -174 del polymorphism in the TLR2 gene, located on chromosome 4, causes a 22 bp nucleotide deletion that alters the promoter activity of TLR2. The TLR2 del/del genotype is reported to show decreased transactivation of responsive promoters [10]. Consequently, it might be speculated that expression of TLR2 in microglial cells might exhibit low levels with the del/del genotype. Further, it can be presumed that the del/del genotype is more conducive to the occurrence of AD. Our results suggest a significant association between the -196 to -174 del allele of TLR2 and the risk of developing LOAD in the Han Chinese population. Interestingly, this association was restricted to non-ApoE ε4 carriers, as no association was found for ApoE ε4 carriers. One possible interpretation is that the genetic effect of TLR2 is relevant in predisposing to AD only in the absence of the ApoE ε4 allele, while in ε4 carriers the genetic effect is determined by this strong susceptibility factor. There is overlap in the study populations used in present study and in our previous study [9]. Linkage disequilibrium (LD) between the GT repeat polymorphism and the -196 to -174 del polymorphism within the TLR2 gene in the overlap study population was measured by calculating the D' and r2 statistics. These were found to be in weak linkage disequilibrium (D' = 0.376 and r² = 0.009). Hence, haplotype frequencies were not estimated, and both the GT repeat and the -196 to -174 del polymorphisms might independently influence the risk of LOAD.