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

Background 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, and functionally is involved in the microglia-mediated inflammatory response and amyloid-β clearance. The -196 to -174 del polymorphism affects the TLR2 gene and alters its promoter activity. Methods We recruited 800 unrelated Northern Han Chinese individuals comprising 400 late-onset AD (LOAD) patients and 400 healthy controls matched for gender and age. The -196 to -174 del polymorphism in the TLR2 gene was genotyped using the polymerase chain reaction (PCR) method. Results There were significant differences in genotype (P = 0.026) and allele (P = 0.009) frequencies of the -196 to -174 del polymorphism between LOAD patients and controls. The del allele was associated with an increased risk of LOAD (OR = 1.31, 95% CI = 1.07-1.60, Power = 84.9%). When these data were stratified by apolipoprotein E (ApoE) ε4 status, the observed association was confined to ApoE ε4 non-carriers. Logistic regression analysis suggested an association of LOAD with the polymorphism in a recessive model (OR = 1.64, 95% CI = 1.13-2.39, Bonferroni corrected P = 0.03). Conclusions Our data suggest that the -196 to -174 del/del genotype of TLR2 may increase risk of LOAD in a Northern Han Chinese population.


Background
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][3][4][5][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][12][13]. In light of the important role that TLR2 plays with respect to the immune response in the pathogenesis of AD [2][3][4][5][6], we hypothesized that the -196 to -174 del/ins polymorphism in the TLR2 gene might be associated with AD.

Subjects
Our study was comprised of 400 sporadic LOAD cases (189 female and 211 male; age > 65 years; mean age = 82.8 ± 7.1 years; age at onset = 75.4 ± 5.9 years) and 400 healthy controls subjects matched for sex and age (189 female and 211 male; mean age = 81.4 ± 5.4 years). All participants originated from Northern Han Chinese populations. A clinical diagnosis of probable AD was established according to the criteria of National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's disease and Related Disorders Association (NINCDS-ADRDA) [14]. All AD cases were defined as sporadic because family histories showed no mention of first-degree relatives with dementia. Control subjects were unrelated individuals selected from the Health Examination Center of the Qingdao Municipal Hospital. These subjects were confirmed healthy and neurologically normal by complete neurological and medical examinations comprised of medical history and laboratory examinations. Written informed consent was obtained from all individuals and the study was approved by the Institute Ethical Committee.

Genotyping
Genomic DNA was extracted from peripheral blood leukocytes using the standard method [15]. Polymorphisms at TLR2 -196 to -174del were investigated using the polymerase chain reaction (PCR) method, following the procedures described by Tahara et al [11]. The apolipoprotein E (ApoE) genotype was determined according to the method described by Donohoe et al [16]. Two investigators independently reviewed all results.

Statistical analysis
Hardy-Weinberg equilibrium was assessed using the Chi-square test. Genotype and allele distributions were compared using the Chi-square test. Differences in allele and genotype distribution between cases and controls were analyzed using logistic regression adjusted for age and ApoE ε4 status under various genetic models. The Wald P value was multiplied by 3 as a Bonferroni adjustment for the 3 genetic models tested. The P value, odds ratios (OR) and 95% confidence intervals (CI) were calculated. Estimation of the statistical power was performed with the STPLAN 4.3 software. Data were analyzed using a commercially available statistical package (SPSS Version 13.0, SPSS Inc., Chicago, IL). The criterion used for significant differences is P < 0.05.

Results
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). pathogenesis of AD [2][3][4][5][6]. TLR2 is a member of patternrecognition 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].

Many experimental and clinical studies have suggested that TLR2 might play an important role in the
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 r 2 = 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.

Conclusions
Our data suggest that the -196 to -174 del/del genotype of TLR2 may increase the risk of LOAD in a Northern Han Chinese population. Additional independent replications and functional genetic analyses are warranted to elucidate the potential mechanisms and the epidemiologic relevance of these associations.  Adjusted for age and for carriage of at least one ApoE ε4 allele. Dom, dominant model: 1 (del/del+del/ins) versus 0 (ins/ins); Rec, recessive model: 1 (del/del) versus 0 (del/ins + ins/ins); Add, additive model: 0 (ins/ins) versus 1 (del/ins) versus 2 (del/del); OR, odds ratio; CI, confidence interval. Pc, corrected P for multiple testing by Bonferroni correction (P value was multiplied by 3 as a Bonferroni adjustment for the 3 genetic models tested). NC, not calculated.