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Sequence variants of interleukin 6 (IL-6) are significantly associated with a decreased risk of late-onset Alzheimer's disease

Background Interleukin 6 (IL-6) has been related to beta-amyloid aggregation and the appearance of hyperphosphorylated tau in Alzheimer's disease (AD) brain. However, previous studies relating IL-6 genetic polymorphisms to AD included few and unrepresentative single nucleotide polymorphisms (SNPs) and the results were inconsistent. Methods This is a case-control study. A total of 266 patients with AD, aged≧65, were recruited from three hospitals in Taiwan (2007-2010). Controls (n = 444) were recruited from routine health checkups and volunteers of the hospital during the same period of time. Three common IL-6 haplotype-tagging SNPs were selected to assess the association between IL-6 polymorphisms and the risk of late-onset AD (LOAD). Results Variant carriers of IL-6 rs1800796 and rs1524107 were significantly associated with a reduced risk of LOAD [(GG + GC vs. CC): adjusted odds ratio (AOR) = 0.64 and (CC + CT vs. TT): AOR = 0.60, respectively]. Haplotype CAT was associated with a decreased risk of LOAD (0 and 1 copy vs. 2 copies: AOR = 0.65, 95% CI = 0.44-0.95). These associations remained significant in ApoE e4 non-carriers only. Hypertension significantly modified the association between rs2069837 polymorphisms and the risk of LOAD (pinteraction = 0.03). Conclusions IL-6 polymorphisms are associated with reduced risk of LOAD, especially in ApoE e4 non-carriers. This study identified genetic markers for predicting LOAD in ApoE e4 non-carriers.


Background
Dementia is a neurodegenerative disease characterized by decline or loss in cognitive function. Alzheimer's disease (AD) is the most common cause of dementia. In the United States in 2006, AD was the fifth leading cause of death in the elderly (age 65 or older) [1]. In Taiwan, the prevalence of dementia is around 1.7-4.3% among the elderly [2] and the number of dementia patients keeps increasing. Therefore, dementia has become an important health issue in aging populations.
Interleukin-6 (IL-6), an inflammatory cytokine, plays an important role in the development and differentiation of neurons in both peripheral and central nervous system [3]. IL-6 promotes the activation of microglia [4] and then induces the synthesis of acute phase proteins [5] and phosphorylation of tau protein in neurons [6]. In AD brain, microglia and astrocytes are stimulated by IL-6 and are recruited to release proinflammatory cytokines and acute-phase proteins, such as C-reactive protein (CRP) [7]. Therefore, IL-6 plays a pivotal role in brain inflammation that maybe important in AD pathogenesis.
Previous studies relating IL-6 polymorphisms to AD risk have been inconsistent. A Caucasian study found that the CC genotype of IL-6 promoter SNP rs1800795 was significantly associated with an increased risk for AD [8]. However, this association has not been observed in other Caucasian studies [9][10][11]. In addition, the GG genotype of promoter SNP rs1800796 has been associated with a decreased risk for AD in two Chinese populations [12,13]. For the latter study [13], a significant association was observed only for Apolipoprotein E (ApoE) e4 carriers. In contrast, no significant association was observed in a Japanese population [14]. Furthermore, the variant rs13447446 was very rare in a Korean study [minor allele frequency (MAF) = 0.006] [15] and a Chinese study (MAF = 0) [12] and thus no association analysis was performed. In summary, previous studies (see Table 1) have been inconsistent in relating IL-6 polymorphisms to AD risk. This may be attributable to different ethnic groups, SNPs selected, study period, or sample size.
IL-6 plays an important role in inflammation. Past studies relating IL-6 polymorphisms to AD risk have been limited to few and unrepresentative SNPs in IL-6. Some SNPs (e.g., rs13447446) even showed no variations in Asians. Therefore, this study used a systematic approach to select haplotype-tagging SNPs (htSNPs) in IL-6 to explore their association with AD risk. Because ApoE e4 status and some vascular risk factors [e.g., hypertension, hyperlipidemia, and type 2 diabetes mellitus (DM)] may affect the pathogenesis of dementia [16][17][18], this study further explored the effect of modification by these factors.

Study population
This was a case-control study. A total of 294 late-onset AD (LOAD) cases were recruited from the neurology clinics of three teaching hospitals in northern Taiwan from 2007 to 2010. Healthy controls (n = 503) were recruited from elderly health checkup and volunteers of the hospital during the same period of time. All participants were Taiwanese aged 65 years or older. Participants with a history of depression, dementia subtypes other than AD, Parkinson's disease, hemorrhagic stroke, cerebral infarction, or organic brain tumors were excluded. After further exclusion of participants without blood samples, a total of 266 LOAD and 444 controls were included for data analyses. The study protocol has been approved by the Institutional Review Boards of National Taiwan University Hospital, En Chu Kong Hospital, and Cardinal Tien's Hospital. Written informed consent was obtained from each study participant. The consent from the legal guardian/next of kin was obtained when patients had serious cognitive impairment. A self-reported questionnaire was administered to collect information on demography, comorbidity (e.g., hypertension, hyperlipidemia, and type 2 DM), and lifestyle.  [22,23]. Two htSNPs were selected among four common SNPs using tagSNP program [24]. For comparison purpose, SNP rs1800796 was further included because it has been related to AD risk [12,13]. Blood samples were collected in tubes containing sodium EDTA from each participant for genotyping. After centrifugation, genomic DNA was extracted from buffy coats using a QuickGene-Mini80 system (Fujifilm, Tokyo, Japan) and then stored in a -80°C freezer. Genotypes of ApoE SNPs were determined using the assay developed by Chapman et al. [25]. Genotypes of IL-6 htSNPs were determined by Taqman Assay (Applied Biosystems Inc., CA, USA) with genotyping success rate greater than 95%. Five percent of internal samples were selected and replicated for quality control, and the concordance rate was 100%.
A likelihood ratio test was used to evaluate how ApoE e4 status and vascular risk factors (e.g., type 2 DM, hypertension, and hyperlipidemia) modified the association between IL-6 polymorphisms and risk of LOAD. Stratified analyses were performed subsequently to assess the association between IL-6 polymorphisms and risk of LOAD by these vascular risk factors. SAS version 9.2 (SAS Institute, Cary, NC) was used for statistical analyses and all statistical tests were two-sided.

IL-6 haplotypes and AD risk
Three common (frequency ≥ 5%) htSNPs spanning IL-6 formed one haplotype block, which was determined by the modified Gabriel et al. algorithm [22,23] (Figure 1). Three common haplotypes (CAT, GGC, and GAC) with a cumulative frequency of 99% in controls were identified in IL-6 ( Table 5). Because HAP1 CAT is a "major" haplotype (haplotype frequency > 0.50), 2 copies of HAP1 were used as the reference group to be consistent with the reference groups used for HAP2 and HAP3, which are "minor" haplotypes (haplotype frequency < 0.50). Participants carrying 0 or 1 copies of major HAP1 CAT had a decreased risk of LOAD (AOR = 0.65, 95% CI = 0.44-0.95) compared to those carrying 2 copies of major HAP1.
For IL-6 haplotypes, vascular risk factors or ApoE e4 status did not significantly modify the association between IL-6 haplotypes and the risk of LOAD (p interaction > 0.05). After stratification by hypertension history, hypertensive patients carrying 0 or 1 copy of HAP1 had a decreased risk of LOAD (AOR = 0.52, 95% CI = 0.29-0.95, Table 7). After stratification by type 2 DM, non-DM patients carrying minor HAP1 were associated with a decreased LOAD risk (AOR = 0.59, 95% CI = 0.39-0.92, Table 7).

Discussion
In addition to replicating findings for rs1800796 explored in previous Asian studies [12][13][14], this study for the first time identifies two IL-6 htSNPs (rs2069837 and rs1524107) representative for the Chinese population, and rs1524107 showed a significant association with LOAD. We found that a variant of SNP rs1800796 is significantly associated with decreased risk of LOAD, which is consistent with the findings from a Chinese study [12]. In addition, we found that the intronic SNP rs1524107 and a haplotype CAT have a significant protective effect on LOAD risk, which has not been reported previously. Unlike rs1800796 and rs1524107, variant rs2068937 was not significantly associated with LOAD (Table 4). Although they are located in one block [i.e., highly linkage disequilibrium (LD) with dark gray in Figure 1], rs2068937 shows a low correlation with rs1800796 (r 2 = 0.58) and rs1524107 (r 2 = 0.62) which explains its lack of a significant association with LOAD. rs1800796 is a promoter SNP, which may enhance transcription efficiency [28] and affect plasma CRP levels [29] and subsequent inflammatory responses. These results indicate that the promoter SNP rs1800796 might be involved in the course of inflammation, and the variant rs1800796 could alter IL-6 production in the development of AD. Sequence variants of rs1524107 may affect disease risk via their effect on alternative splicing, e.g., altering mRNA folding or the stability of mRNA structure, and subsequent protein production. Besides, the most common IL-6 haplotype CAT, composed of three major alleles, was associated with decreased risk of LOAD. Therefore, carrying 0 or 1 copies of CAT showed consistent protective effects on LOAD risk, as did the variant IL-6 SNPs. These sequence changes may either block or attenuate inflammation signaling which leads to reduced risk of AD. This supports the finding of a protective effect from IL-  Figure 1 IL-6 linkage disequilibrium (LD) plot. This plot was generated by using a Haploview program. Three SNPs form one block. The SNP name, e.g., SNP1, SNP2, and SNP3; indicates the three htSNPs genotyped in this study. Three common haplotypes (frequency ≥ 0.05) were identified. The level of pair wise r 2 , which indicates the association degree between two SNPs in LD block, is shown numerically in the cell of the LD structure. The level of pairwise D', which indicates the strength of LD between two SNPs, is shown in the LD structure in gray scale.
6 polymorphisms on LOAD. The postulated mechanism of IL-6 in LOAD is demonstrated in Figure 2.
Although ApoE e4 status is a well-known predictor for AD risk, over half of AD cases (61% in this study, Table  2) do not carry the ApoE e4 allele. Therefore, prediction of LOAD risk among ApoE e4 non-carriers becomes an important task. This study found significant associations between rs1800796 or rs1524107 and LOAD in ApoE e4 non-carriers, which remained statistically significant after correction for type I error by using FDR. A Chinese study [13] found that the variant rs1800796 was associated with AD risk in ApoE e4 carriers but not in the wider population. This may be a chance finding because a significant protective effect of rs1800796 variants on AD risk was observed only after stratification by ApoE e4 status. In contrast, both He et al. [12] and our study found a significant association before stratification. A previous study showed that elevated serum IL-6 induces hypertriglyceridemia [30], which manifests before the deposition of beta amyloid (Aβ) in AD [31]. Therefore, IL-6 polymorphisms may be important in predicting LOAD in ApoE e4 non-carriers.
Genotyping data from public domains (HapMap and dbSNP) show that IL-6 and ApoE SNPs show geographic variations ( Table 3). MAFs of three IL-6 SNPs (rs1800796, rs2069837, and rs1524107) are similar for a Two copies of HAP1 was used as the reference group because it is a "major" haplotype (haplotype frequency > 0.5).
All models were adjusted for age, gender, education, and ApoE e4 status.  Abbreviation: AOR, adjusted odds ratio; DM, diabetes mellitus; LOAD, lateonset Alzheimer's disease. All models were adjusted for age, gender, education, and ApoE e4 status. a 2 copies of haplotype CAT was used as the reference group because it is a major haplotype. b p interaction value was obtained by using the dominant model. controls of this Chinese population (0.19-0.27) and CHB of the HapMap project (0.17-0.29). In comparison with these two Chinese populations, for the same IL-6 SNPs, Japanese populations have lower MAFs (0.10-0.18), and the MAFs are even lower in Caucasians (0.04-0.08). For ApoE SNPs, the MAF of AopE112 (rs429358) is highest among Caucasians (dbSNP: 0.15), followed by controls in this Chinese population (0.08), and by Japanese (0.01) and CHB (0.00) from the HapMap Project. The inconsistency between the two Chinese populations for ApoE112 may be attributable to the small number of samples genotyped in HapMap study (n = 45 for CHB and n = 43 for JPN). For ApoE158 (rs7412), the MAFs were similar between Chinese (0.08-0.11) and Caucasians (0.08) across ethnic groups with a lower value observed in Japanese (0.05).
Hypertension significantly modified the association of IL-6 SNPs with LOAD risk. The protective effect of three IL-6 SNPs for LOAD was especially evident among participants with hypertension (Table 6), which may be a result of medication for treating hypertension that can lower inflammatory responses ( Figure 2). For example, angiotensin receptor blockers or angiotensinconverting enzyme inhibitors have been used to lower blood pressure. Their accompanying neuroprotective effects can reduce neuronal damage and lead to slowed progression of AD [32,33]. Sequence variants of IL-6 may lower blood pressure, and subsequent brain hypoperfusion and neural degeneration, which eventually decreases LOAD risk ( Figure 2). However, experimental studies are needed to clarify the underlying mechanism.
This study had some strengths. First, the htSNPs in IL-6 were identified to explore LOAD risk for the first time. Second, the selection of two representative htSNPs captured abundant genetic information regarding the IL-6 gene (r 2 = 1.00) as compared to the genetic information captured by the single promoter SNP (rs1800796, r 2 = 0.81) in previous candidate-gene studies [12,13]. Third, the associations between IL-6 SNPs and LOAD risk remained significant after correction for type I error using FDR, which indicates that these findings are not chance findings. In addition, high false positive rate in genomewide association studies prevent identifying SNPs associated with disease outcome but with moderate p values in the exploratory stage [34]. This study, which selected htSNPs that are representative for Chinese and captured abundant genetic information for IL-6, may solve the above issue. Furthermore, brain imaging was used to exclude other diseases with similar presentation as LOAD.
Our study had some limitations. A self-report questionnaire was used to collect information on vascular risk factors (e.g., hypertension, hyperlipidemia, and type 2 DM). Because these diseases/conditions are major health issues, participants' recall of disease/condition diagnosis and their awareness of these diseases/conditions tend to be relatively accurate [35][36][37]. Therefore, information bias should not be a concern.
Conclusions In addition to a significant association of rs1800796 with LOAD, this study for the first time found that an IL-6 htSNP (rs1524107) and a haplotype CAT were significantly associated with LOAD risk after correction for multiple tests. These associations remained significant in ApoE e4 non-carriers only. Because the majority of AD patients are ApoE e4 non-carriers (61% in this study and another Chinese study [13]), IL-6 SNPs may be important markers in predicting LOAD risk in ApoE e4 noncarriers. In addition, hypertension significantly modified the association of IL-6 polymorphisms with LOAD risk. Future large studies are warranted to explore the role of IL-6 in risk of LOAD.