Association of rs6265 and rs2030324 Polymorphisms in Brain-Derived Neurotrophic Factor Gene with Alzheimer’s Disease: A Meta-Analysis

Background The association between polymorphisms rs6265 and rs2030324 in brain-derived neurotrophic factor (BDNF) and Alzheimer’s disease (AD) has been widely reported, but the results remain controversial. Methods A comprehensive search of Pubmed, Web of Science, China National Knowledge Infrastructure (CNKI), Wanfang Med Online and China Biology Medical literature database (CBM) was performed. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using fixed or random-effects models. We excluded the studies with OR>3.0 or OR<0.3 for sensitive analysis. Subgroup analysis by ethnicity, form of AD and gender was carried out. Meta-regression was conducted to explore the potential sources of between-study heterogeneity. Results 29 articles with 7548 cases and 7334 controls concerning rs6265 and 22 articles with 5796 cases and 5706 controls concerning rs2030324 were included in this meta-analysis. The combined evidence suggested rs6265 contributing significantly to the increased risk of AD in females (codominant: fixed-effects model (FEM): OR = 1.13, 95% CI = 1.04–1.23; dominant: FEM: OR = 1.17, 95% CI = 1.05–1.31), especially for Caucasian females (codominant: FEM: OR = 1.18, 95% CI = 1.03–1.34; dominant: FEM: OR = 1.18, 95% CI = 1.01–1.37) and female late-onset Alzheimer’s disease (LOAD) patients (codominant: FEM: OR = 1.22, 95% CI = 1.05–1.41; dominant: FEM: OR = 1.23, 95% CI = 1.03–1.46). No evidence indicated an association between rs2030324 with AD in codominant (random-effects model (REM): OR = 1.06, 95% CI = 0.89–1.26) and dominant (REM: OR = 1.05, 95% CI = 0.86–1.27) models. Conclusion This meta-analysis suggested A allele of rs6265 might increase the risk of AD in Caucasian females and female LOAD patients. In addition, no evidence indicated an association between rs2030324 with AD. Further studies are needed to confirm these results.


Introduction
Alzheimer's disease (AD) is an age-associated neurodegenerative disorder characterized by progressive decline in cognitive function, which typically begins with deterioration in memory [1]. The number of people with dementia worldwide in 2010 is estimated at 35.6 million and is projected to nearly double every 20 years to 65.7 million in 2030 and 115.4 million in 2050. AD is the most common form of dementia and possibly contributes to 60-70% of cases [2]. In the US alone, AD is related with an estimated healthcare cost of US$172 billion per year [3]. The overwhelming number of AD patients, combined with the staggering economic burden, makes AD a public health problem.
The key pathological changes that observed in AD brain tissue are the accumulation of neuritic extracellular amyloid plaques and intracellular neurofibrillary tangles [4]. However, the neuropathological etiology of AD remains unclear, but are probably related with the combined interaction between gene variants and environmental factors [5]. There have been many genetic polymorphisms reported to be associated with AD, such as amyloid precursor protein (APP), presenilin-1(PSEN1), presenilin-2(PSEN2) [6], apolipoprotein E (APOE) [7,8] and sortilin-related receptor 1 (SORL1) [9]. However, as a complex disorder, the genes mentioned-above can not explain the overall genetic susceptibility and it is supposed that some other genes may participate in the development of AD.
Brain-derived neurotrophic factor (BDNF), as a member of the neurotrophic family, plays an important role in the growth, development, differentiation and regeneration of various types of neurons in the central nervous system [10]. Autopsy studies found reduced mRNA expression of BDNF in the hippocampus of patients with AD [11], which implicates the possible participation of BDNF in the pathogenesis of AD. There have been many polymorphisms studied in BDNF gene, such as rs11030104, rs16917204, rs7103411, rs6265 and rs2030324. However, only the last two polymorphisms have been widely studied, with no linkage disequilibrium (LD) between them. What's more, these results are inconsistent and individual studies have relatively small power to confirm this association. For example, the G allele of rs6265 confers risk effect for AD in subjects of Japanese (OR = 1.23, 95% CI = 1.02-1.47) [12], but no significant association was found in Italians [13]. Therefore, we performed a metaanalysis to identify the association of the two polymorphisms in BDNF and AD susceptibility. The search used the following keywords: ''Alzheimer's disease'' or ''AD'' and ''brain-derived neurotrophic factor'' or ''BDNF'' and ''polymorphism'' or ''mutation'' or ''variant''. We also reviewed the references of included articles to identify additional articles not captured by our database searches.

Inclusion Criteria
Two investigators reviewed all relevant studies independently to determine whether an individual study was eligible for inclusion. If the two investigators disagreed about the eligibility of a study, a senior researcher was invited to the discussion. The inclusion criteria were as follows: (1) case-control or cohort study published as an original study to evaluate the association between rs6265 and rs2030324 polymorphisms in BDNF gene and AD susceptibility; (2) AD were diagnosed according to NINCDS-ADRDA criteria, DSM-IV criteria or CERAD criteria; (3) genotype frequencies were reported in the articles or could be calculated or ORs and 95% CIs can be obtained; (4) the genotype frequencies of controls are consistent with Hardy-Weinberg equilibrium (HWE); (5) if one study from the same population had been published more than once, we choose the most recent or complete one; (6) English or Chinese language articles were included.

Data Extraction
Two investigators extracted the data independently and reached a consensus on all items. Information extracted from each study was as follows: first author, publication year, country, ethnicity of studied population, diagnostic criteria for cases, sample size, genotype distributions, mean age and the percentage of male.

Statistical Analysis
The chi-square (x 2 ) analysis was used to test deviation from Hardy-Weinberg equilibrium (HWE) for the rs6265 and rs2030324 genotype distribution of BDNF gene in control groups, and P,0.05 was considered as departure from HWE. Pooled measure was used as the inverse variance weighted mean of the logarithm of odds ratio (OR) with 95% confidence intervals (CI) to evaluate the strength of the association of rs6265 and rs2030324 in BDNF gene with risk of AD. We conducted analysis for each polymorphism considering dominant (AA+GA vs. GG for rs6265, TT+CT vs. CC for rs2030324), recessive (AA vs. GA+GG for rs6265, TT vs. CT+CC for rs2030324) and codominant (A vs. G for rs6265, T vs. C for rs2030324) models, respectively. I 2 of Higgins and Thompson was used to describe heterogeneity among studies [14]. The random-effects model (REM) was adopted if significant heterogeneity (I 2 .50%) was found; otherwise, the fixed-effects model (FEM) was adopted, and Mantel-Haenszel was used to assess the fixed effects. For rs6265 and rs2030324, metaregression with restricted maximum likelihood estimation was performed to explore the potentially important covariates that might exert substantial impacts on between-study heterogeneity. Specific genetic variants causally associated with common diseases would have small effects (risk ratios mostly ,2.0) [15,16]. Therefore, for sensitive analysis, we excluded the studies with OR.3.0 or OR,0.3 for both of the two polymorphisms (rs6265 and rs2030324) to control the impact of outlier values resulting from low cell counts within each single study on the pooled effect. Moreover, the 'leave one out' sensitive analysis was performed using I 2 .50% as the criteria to evaluate the key studies with substantial impact on between-study heterogeneity [17]. When heterogeneity was observed, subgroup analysis was also carried out. Publication bias was evaluated by Harbord's test. An influence analysis was performed to describe how robust the pooled estimator is to removal of individual studies. If the point estimate of its omitted analysis lies outside the 95% CI of the combined analysis, the individual study is suspected of excessive influence [18]. All the statistical analyses were conducted using the STATA version 10 (Stata Corporation, College Station, TX, USA). Two-tailed P#0.05 was considered as statistically significant.

Literature Search and Study Characteristics
The search strategy identified 309 articles from English databases (177 articles from Web of Science, 132 articles from Pubmed), 176 articles from Chinese databases (161 articles from China National Knowledge Infrastructure, 9 articles from Wanfang Med Online, 6 articles from China Biology Medical literature database). 64 articles were reviewed in full-text. Furthermore, we excluded 30 articles according to the inclusion criteria, and obtained 1 additional article through references review. Finally, 35 articles were included in this meta-analysis. Figure 1 showed the flow diagram of literature search.

Influence Analysis and Publication Bias
For both of the two polymorphisms, no individual study has excessive influence on the pooled effect in any of dominant, recessive and codominant models (data not shown). Harbord's test showed no publication bias for both two polymorphisms.

Quantitative Synthesis
Overall analysis for rs6265 and rs2030324. For overall analysis, no significant association was found between A allele and risk of AD in codominant (REM: OR = 1.03, 95% CI = 0.95-1.  Table 4. Figure 2 presented the forest plot of ORs in codominant model (A vs. G) in overall analysis for rs6265.
Evidence for heterogeneity (I 2 .50%) was found in codominant and dominant models considering the association of rs6265 and rs2030324 polymorphisms with AD. Univariate meta-regression with the covariates of publication year, ethnicity, diagnostic criteria, form of AD, age (ratio of mean age in case group to that in control group) and gender (ratio of male percent in case group to that in control group) for the above-mentioned polymorphisms showed that no covariates have significant effect on between-study heterogeneity.     44). However, no association was found in male LOAD patients in any of the above-mentioned models. Figure Table 5 and Table S2 and  Table S3 in File S1.

Sensitivity Analysis
After excluding articles with OR.3.0 or OR,0.3, low heterogeneity (I 2 ,50%) was found in the codominant, dominant and recessive models, and the results for both two polymorphisms are consistent with the ones without sensitive analysis. Furthermore, the ''leave one out'' analysis was carried out when significant heterogeneity (I 2 .50%) was observed. After excluded key articles with substantial impact on between-study heterogeneity, the data showed rs6265 confers a risk effect for Caucasians in codominant model (FEM: OR = 1.08, 95% CI = 1.00-1.17, P = 0.042). With respect to rs2030324, the result indicated T allele was associated with EOAD patients in codominant (FEM: OR = 2.02, 95% CI = 1.18-3.47) and dominant model (FEM: OR = 2.01, 95% CI = 1.15-3.52).

Discussion
To our knowledge, many case-control studies have been carried out to investigate the role of BDNF gene in the development of AD. However, these results remained controversial. 29 articles concerning rs6265 and 22 articles concerning rs2030324 were included in our meta-analysis. With respect to rs2030324, there was no evidence for an association with AD. What's more, the combined evidence suggested that rs6265 was not associated with AD for overall analysis. After stratified by ethnicity in females, our Table 4. The overall pooled measure on the relation of BDNF gene rs6265 and rs2030324 polymorphism with AD. data indicated rs6265 lead to the increased risk of AD in Caucasian females, but not for Asians. When stratified by gender in LOAD patients, the A allele was found contributing significantly to the increased risk of AD in female LOAD patients. The results of our study were not consistent with the previous meta-analysis conducted by Fukumoto et al. [22] in 2010 including 16 studies revealing a gender-related association between rs6265 polymorphism and AD susceptibility. The reason might be that the quantitative assessments in our study were based on a larger sample size and we have performed a detailed subgroup analysis. The Met66-BDNF protein has been shown to be associated with reduced transport of BDNF from the Golgi region to appropriate secretory granules in neurons, compared with the Val66-BDNF protein [52]. Moreover, the A allele of rs6265 was related with poorer episodic memory, abnormal hippocampal activation, and lower hippocampal n-acetyl aspartate (NAA) in human subjects [53]. Epidemiological studies showed higher incidence and prevalence of AD in women than in men [3,5]. Molecular mechanisms underlying this correlation had been considered as colocalization estrogen receptors with BDNFsynthesizing neurons in the forebrain [54] and induction BDNF expression by estrogen through the estrogen response element [55]. In addition, though the two forms of AD (EOAD and LOAD) have different patterns of genetic epidemiology [4], the results of our study revealed no difference between them. Between-study heterogeneity is common in meta-analysis for genetic association studies [56] and it is essential to explore the potential sources of between-study heterogeneity [57]. This metaanalysis also showed significant between-study heterogeneity in dominant model and codominant model for both two polymorphisms. An indeterminate number of characteristics that varied among studies could be the sources of between-study heterogeneity, such as publication year, ethnicity, diagnostic criteria, form of AD, age, and gender etc. Therefore, in order to explore the potential sources of between-study heterogeneity for rs6265 and rs2030324, meta-regression was adopted. However, the aforementioned covariates were not important contributors to this disease-effect heterogeneity. Considering that our meta-analysis showed significant heterogeneity, subgroup analyses by ethnicity (Caucasian and Asian), form of AD (EOAD and LOAD) and gender (female and male) etc. were performed to explore the sources of heterogeneity. However, between-study heterogeneity still existed in subgroups, suggesting the presence of other unknown confounders. AD is a complex multi-factorial disease and is related with the combined effects between gene variants and environmental factors. Therefore, other genetic and environment variables, as well as their possible interaction, may be potential contributors to this disease-effect unconformity. We further conducted a sensitivity analysis excluding articles with OR.3 or OR,0.3. After sensitivity analysis, low heterogeneity (I 2 ,50%) was found in the codominant, dominant and recessive models, and the results are consistent with the one before heterogeneity analysis for rs6265 and rs2030324, strongly identified the stability of our results. Moreover, no publication bias was found in any of the above-mentioned models for both two polymorphisms.
The major strength of our meta-analysis is that the results were based on the large number of participators, allowing a much greater possibility of reaching definitive conclusions. Additionally, we conducted subgroup analyses to explore the potential sources of heterogeneity, and sensitivity analysis was carried out to ensure the stability of our results. However, our meta-analysis also had some limitations. Firstly, lack of the original data of included articles made it impracticable to excluded potential confounders completely, especially the confounding of age. Secondly, different diagnostic criteria may have possible influence on the diagnosis of AD.     In conclusion, this meta-analysis suggested A allele of rs6265 might increase the risk of AD in Caucasian females and female LOAD patients. In addition, no evidence indicated an association between rs2030324 with AD. Since potential biases and confounders could not be ruled out completely in this study, further studies are needed to confirm these results.

Supporting Information
File S1 This includes the files Search Strategy S1 and Tables S1 to S3. Search Strategy S1. Keywords of literature search for different database. Table S1. Genotype and allele distribution for rs2030324 polymorphism in female and other subgroups. Table S2. Subgroup analysis on the relation of BDNF gene rs6265 polymorphism with AD in dominant and recessive model. Table S3. Subgroup analysis on the relation of BDNF gene rs2030324 polymorphism with AD in dominant and recessive model.

Author Contributions
Conceived and designed the experiments: YL. Performed the experiments: YL SC ZTX DFZ. Analyzed the data: YL SC ZTX DFZ. Contributed reagents/materials/analysis tools: YL SC ZTX DFZ. Wrote the paper: YL SC.