Alzheimer\'s disease (AD) is a progressive and fatal brain disorder that causes memory loss, steady deterioration of cognition, and dementia[@b1].  It is the sixth leading cause of all deaths in the United States, and deaths increased by 66% between 2000 and 2008[@b2].  Approximately 13% of people over the age of 65 years and 45% over the age of 85 years are estimated to have AD[@b3].  Therefore, discussing the risk factors and pathogenesis of AD, is of great significance for early detection, prevention and control of the susceptible population. 

During the last two decades, researchers have found that Apolipoprotein E (ApoE) gene, located on chromosome 19, is closely associated with the onset of AD[@b4][@b5].  ApoE is a 299-aminoacid protein encoded by the ApoE gene and has a molecular mass of \~34 kDa[@b6].  It is a major cholesterol carrier that supports lipid transport and injury repair in the brain[@b7].  Three common polymorphisms in the ApoE gene, ε2, ε3, and ε4, including 6 genotypes, three homozygote (ε2/ε2, ε3/ε3, ε4/ε4) and three heterozygote (ε3/ε2, ε4/ε2, ε4/ε3)[@b8], result in a single amino acid change in the ApoE protein.  Differences between the three ApoE isoforms are limited to amino acid residues 112 and 158[@b9].  Of these, the ε3 allele is the most common, followed by ε4 and ε2, although these frequencies vary between populations.  ApoE polymorphic alleles are the main genetic determinants of AD risk: individuals carrying the ε4 allele are at increased risk of AD compared with those carrying the more common ε3 allele, whereas the ε2 allele decreases risk[@b10].  Moreover, the ε4 allele is most highly associated with AD at a large range of ages and in all ethnic groups; its presence is related with increased risk of cerebral amyloid angiopathy and age-associated cognitive decline during normal ageing[@b11][@b12]; It is also associated with hyperlipidaemia and hypercholesterolaemia, which lead to atherosclerosis, coronary heart disease and stroke[@b13].  The effects of ApoE genotype on risk of these diseases are likely to be mediated by differential effects of ApoE on amyloid-β accumulation in the brain and its vasculature[@b14][@b15].  Response to treatment for AD might differ according to ApoE genotype[@b16]. 

Although numerous studies have demonstrated the association, inconsistency was presented for different allele frequencies among study populations, particularly in different ethnic and geographical groups.  The purpose of conducting this meta-analysis is to reduce heterogeneity and summarize the published evidence on the prevalence of the ApoE polymorphism among patients diagnosed with AD in Chinese population. 

Results
=======

Study selection and characteristics
-----------------------------------

The electronic database search identified 312 references.  Of those, 218 records excluded after title review and 103 articles were judged potentially relevant.  Following abstracts screened for relevance, 48 full-text articles comprehensively assessed against inclusion criteria.  Overall, the initial search with the keywords and the subject terms identified 20 publications, including 1576 cases and 1741 controls that met the inclusion criteria and were eligible for review. [ Figure 1](#f1){ref-type="fig"} shows the study flow. 

Of the 20 reports focusing on the relationship between ApoE polymorphism and AD, 18 were from mainland[@b17][@b18][@b19][@b20][@b21][@b22][@b23][@b24][@b25][@b26][@b27][@b28][@b29][@b30][@b31][@b32][@b33][@b34], two were from Taiwan[@b35][@b36].  The distribution of the genotypes in the control group was consistent with Hardy-Weinberg equilibrium (HWE).  The detailed characteristics of the included studies were shown in [Table 1](#t1){ref-type="table"}.  The distributions of genotypes in the individual studies were presented in [Table 2](#t2){ref-type="table"}. 

Association between ApoE allele and AD
--------------------------------------

The results of each allele and genotypes of ApoE in this meta-analysis were listed in [Table 3](#t3){ref-type="table"}.  The heterogeneity between studies was not significant excepting the allele ε2.  The fixed effect model or the random effect model was employed for calculating the pooled OR.  Overall, this meta-analysis showed that the frequency of ApoE ε4 allele is higher in AD than that in the health controls, and demonstrated statistically significant positive association between risk factor ε4 allele carriers and AD in Chinese population (OR = 3.93, 95% CI = 3.37--4.58, P \< 0.00001), as shown in [Figure 2](#f2){ref-type="fig"}.  The frequency of the ApoE ε3 is lower in AD than that in the health controls and the difference is also statistically significant (OR = 0.42, 95% CI = 0.37--0.47, P \< 0.00001), implying the protection effect of ε3 allele in developing AD.  No significant association was found between ApoE ε2 allele and AD (OR = 0.93, 95% CI = 0.66--1.29, P = 0.65) in a random-effect model. 

Association between ApoE genotype and AD
----------------------------------------

As shown in [table 3](#t3){ref-type="table"}, the heterogeneity between studies was not significant (I^2^ \< 50%) and the fixed effect model was used for calculating the pooled OR.  ApoE ε4/ε4 and ApoE ε4/ε3 have statistically significant association with AD (ε4/ε4: OR = 11.76, 95% CI = 6.38--21.47, P \< 0.00001; ε4/ε3: OR = 3.08, 95% CI = 2.57--3.69, P \< 0.00001). [ Figure 3](#f3){ref-type="fig"} showed the association between ε4/ε4 and AD in Chinese population.  Genotype ε3/ε3 also has significant association with AD (OR = 0.39, 95% CI = 0.33--0.45, P \< 0.00001).  ApoE ε3/ε2 and ApoE ε4/ε2 have slight association with AD.  There is no association between ε2/ε2 and AD. 

Sensitivity analysis and publication bias
-----------------------------------------

For this meta-analysis, the influence of a single study on the overall meta-analysis estimate was investigated by omitting one study at a time, respectively.  The risk ratio was not significantly influenced by omitting any single study. 

The distribution of the ORs from individual studies in relation to their respective standard deviation in funnel plot, as shown in [Figure 4](#f4){ref-type="fig"} and [Figure 5](#f5){ref-type="fig"}.  The funnel revealed no evidence of asymmetry.  Thus, there was no possibility of publication bias risk in the meta-analysis. 

Discussion
==========

ApoE gene, known to mediate the regulation of cholesterol and triglyceride metabolism, is immunochemically localized to the senile plaques, vascular amyloid, and neurofibrillary tangles of AD[@b37][@b38].  In 1993, Strittmatter et al.  for the first time demonstrated that there was a highly significant association of ApoE ε4 allele and late-onset familial AD[@b39].  Subsequently, most studies reported gene frequency of ApoE ε4 was significantly increased in sporadic AD than in controls.  Sando et al.  proved that ApoE ε4 is a very strong risk factor for AD in the population of central Norway, and lowers age at onset of late onset AD (LOAD) significantly[@b40]; Rhinn et al.  identified an ApoE ε4 associated molecular pathway that promotes LOAD[@b41]; Genin et al.  demonstrated that ApoE ε4 is a risk factor not only for late-onset but for early-onset AD as well[@b42].  Together, these results urge a reappraisal of the impact of ApoE in AD. 

Previous meta-analysis demonstrated that ApoE ε4 genotype prevalence varies among AD patients by region and within each country[@b43]: the highest estimates were in Northern Europe; the lowest estimates were in Asia and Southern Europe.  To further explore and examine the association of prevalence district of ApoE genotype and AD risk, we conducted this meta-analysis only in Chinese population.  Overall, our results showed that the risk of developing AD in ε4 allele carriers was 3.93-fold higher than individuals without ε4 allele.  The risk of developing AD in individuals with ε4/ε4 genotype was 11.76-fold higher than individuals without ε4/ε4 genotype.  There probably exists a dose-dependent association between the number of ApoE ε4/ε4 allele and the risk of AD in Chinese population.  Furthermore, the frequency of the ApoE ε3 is lower in AD than that in the health controls and the difference is also statistically significant, implying the protection effect of ε3 allele in developing AD.  No association was found between ε2 allele and AD risk. 

The ε4 allele of ApoE is the "risk" variant for several phenotypes compared with ε3 ("neutral"), and ε2 (generally considered "protective", although less consistently).  The ApoE gene confers differential susceptibility to AD etiology depending on the combination of the 3 alleles as well as the age and ethnicity of the person.  The ε4 allele of ApoE is the strongest genetic risk factor for the development of AD.  Although multiple genetic and environmental risk factors are involved in LOAD pathogenesis, overall impairment in Aβ clearance is probably a major contributor to disease development[@b44].  Accumulation of amyloid-β (Aβ) is hypothesized to initiate synaptic and neuronal dysfunction that ultimately lead to neuronal cell death in AD, and several lines of evidence strongly suggest that the differential effects of ApoE isoforms on Aβ aggregation and/or clearance plays a major role in AD pathogenesis[@b45][@b46].  The ApoE isoforms could influence the risk for AD via other mechanisms as well[@b47].  Relative to the common ε3 allele, possession of ε4 increases disease risk and decreases age at onset in a dose-dependent manner[@b48].  In contrast, possession of the ε2 allele may confer protection against AD, as carriers of this allele are less likely to develop the disease than ε3 homozygotes[@b49].  The ApoE ε4 allele is associated with greater accumulation of both Aβ plaques and neurofibrillary tangles than the ε3 allele[@b50], while carriers of the ε2 allele typically develop less AD-related pathology than both ε4 carriers and ε3 homozygotes.  Pomara et al.  showed a potential effect of the ApoE ε2 allele and of family history of Alzheimer\'s disease on brain amyloid-β in normal elderly[@b51].  Hostage et al.  confirmed and extended prior data on the opposing effects of the ApoE ε4 and ε2 alleles on hippocampal morphology across the spectrum of cognitive aging[@b48].  This general pattern holds for many physiological phenomena influenced by ApoE genotype, and potentially related to AD, including measures of synaptic plasticity and repair[@b52], antioxidant properties, and certain immune responses[@b53], and cholesterol levels[@b54]. 

Currently, genetic testing of ApoE ε4 carrier status is not routinely considered in clinical practice.  As ApoE genotype determines AD risk, and ApoE has crucial roles in cognition[@b55][@b56], ApoE might offer an attractive alternative target for AD therapy.  ApoE genotype status could be included in clinical trial enrolment criteria, as some therapies might be effective only in specific ApoE genotypes.  In addition, ApoE is a crucial regulator of the innate immune system, with ApoE ε4 promoting pro-inflammatory responses that could exacerbate AD pathogenesis. 

Several limitations were presented in this meta-analysis.  Firstly, in AD group of retrieved case-control studies, there may exist mixed dementia.  It might increase the apparent association of ApoE with AD, since ApoE ε4 allele is associated with dementia.  Secondly, ApoE polymorphism may interact with other known and unknown risk factors which should be considered.  Thirdly, the selected studies may have more subject to bias and artifact than prospective studies. 

In conclusion, our meta-analysis suggests that ApoE ε4 carrier is associated with AD and provide a support for the protection effect of ApoE ε3 allele in developing AD in Chinese population.  Although ApoE polymorphism is a well-studied genetic risk factor for developing AD, in some regions most patients do not carry this genotype.  Therefore, additional research with well-designed and large sample sizes is needed to be able to understand both other genetic and environmental risk factors in the future. 

Methods
=======

Identification and eligibility of relevant studies
--------------------------------------------------

We conducted a comprehensive literature search using the electronic database of PubMed, Wanfang database and CNKI (China National Knowledge Infrastructure) for relevant articles assessing the association of ApoE polymorphism and AD in Chinese population from January 2000 to November 2013.  The Medical Subject Heading (MeSH) terms "Alzheimer\'s disease", "AD", "ApoE", "Apolipoprotein E", and "polymorphism" were employed as the searching words.  The equivalent Chinese terms were used in the Chinese databases.  All studies matching the eligibility criteria were retrieved, and references were checked for other relevant publications. 

Criteria for article screening
------------------------------

Studies eligible for inclusion in this meta-analysis must meet the following criteria: 1) case-control or cohort study; 2) measure the relationship between ApoE polymorphism and AD; 3) clinical diagnosis of AD based on standards of the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) and the Alzheimer\'s Disease and Related Disorders Association (NINCDS/ADRDA) work group[@b57]; 4) the results were expressed as odds ratio (OR) and corresponding 95 percent confidence interval (95% CI); 5) genotype distribution of control for a certain polymorphism must be in HWE; and 6) when the same authors reported two or more publications on possibly the same patient populations, only the most recent or complete study was included into this meta-analysis. 

Quality assessment and data extraction
--------------------------------------

Two investigators independently extracted data and reached a consensus on all of the items.  Any disagreement was resolved by discussing with the third expert.  Data retrieved from the reports included first author, publication year, demographics, number of cases and controls, distribution of genotypes, and the diagnosis criteria of AD. 

Statistical analysis
--------------------

The odd ratio (OR) with 95% confidence intervals (95% CI) was used to assess the relationship of ApoE polymorphism and AD.  The significance of the pooled OR was determined by the Z test, and a P value less than 0.05 was considered significant.  The heterogeneity for the included articles was evaluated using Cochran\'s Q test and I^2^ statistics.  P-value less than 0.05 and I^2^ less than 50% were considered to be statistically significant.  When a significant heterogeneity existed across the included studies (I^2^ \> 50%), the random effect model was used[@b58]; When there was no significant heterogeneity across the included studies (I^2^ \< 50%), the fixed effect model was used[@b58].  To assess whether our results were substantially influenced by the presence of any individual study, we conducted a sensitivity analysis by systematically removing each study and recalculating the significance of the result.  Begg\'s funnel plot was performed to examine the publication bias.  Analyses were carried out using the Review manager 5.2 (The Cochrane Collaboration).  All tests were two sided. 

Author Contributions
====================

Conceived and designed the study: J.Q.Z. and F.W.; Performed the experiments: M.Y.L., C.B., J.Q.Z. and F.W.; Contributed material/analysis tools: M.Y.L., J.Q.Z. and F.W.; Statistical analyses and paper writing, revising: M.Y.L., C.B., J.Q.Z. and F.W.

This study was supported by the National Science Foundation of China (NSFC, No.  81171035). 

![ Flow chart of literature screening.](srep04383-f1){#f1}

![ Forest plot on the association between ε4 allele carriers and AD in Chinese population.](srep04383-f2){#f2}

![ Forest plot on the association between ε4/ε4 genotype and AD in Chinese population.](srep04383-f3){#f3}

![ Funnel plot on the association between ε4 allele carriers and AD.](srep04383-f4){#f4}

![ Funnel plot on the association between ε4/ε4 genotype and AD.](srep04383-f5){#f5}

###### Main characteristics of the eligible studies

                                   Total no.  of           Mean-age                   
  -------------- ---------------- -------------- ----- -------------- -------------- --------------
  Zhu-2000           Shandong           36        36    NINCDS-ADRDA        \-             \-
  Huang-2001        Guangzhou           41        85     DSM-III-R         80.8           59.6
  Jia-2002           Beijing            58        60    NINCDS-ADRDA    68.6 ± 7.6     65.9 ± 8.5
  Huang H-2002        Taiwan            99        96    NINCDS-ADRDA    76.3 ± 6.9     72.2 ± 6.9
  Bi-2002            Haerbin            42        40    NINCDS-ADRDA    70.4 ± 6.6     68.1 ± 4.3
  Zhou-2003          Wulumuqi           51        52    NINCDS-ADRDA    74.1 ± 9.7    69.4 ± 11.4
  Chen-2003       Beijing/Shanxi       160        195   NINCDS-ADRDA    69.4 ± 9.5     69.8 ± 7.8
  Zhang-2004         Shandong           32        40      CCMD-2-R      74.1 ± 7.3     63.9 ± 7.3
  He-2005            Beijing            27        67    NINCDS-ADRDA    82.9 ± 7.9         \-
  Wang-2006           Taiwan           151        161      DSM-IV       74.8 ± 7.9     62.5 ± 8.7
  Li-2006            Guizhou            30        30       DSM-IV      70.2 ± 11.6    71.4 ± 10.5
  Yang J-2008         Yunnan            58        96    NINCDS-ADRDA   74.0 ± 8.48    74.2 ± 4.72
  Yang L-2008         Henan            102        98    NINCDS-ADRDA    78.5 ± 7.3     76.5 ± 9.3
  Wu-2009            Shanghai          262        118   NINCDS-ADRDA   76.91 ± 5.10   60.72 ± 4.88
  Duan-2009           Henan             32        76      DSM-IV-R      70.6 ± 9.8     63.5 ± 6.7
  Mai-2010          Guangdong           88        97      DSM-IV-R      79.6 ± 9.2     79.7 ± 8.6
  Jiang-2010         Guangxi            79        156   NINCDS-ADRDA   72.8 ± 9.  5    71.2 ± 9.  3
  Zhou C-2012       Chongqing           68        72     DSM-IV-TR     70.6 ± 6.  8    71.2 ± 6.  6
  Lv-2012            Shanghai          100        106   NINCDS-ADRDA   77.0 ± 4.  8    79.5 ± 5.  0
  Dong-2013          Liaoning           60        60    NINCDS-ADRDA        \-             \-

###### Distribution of genotypes in the individual studies

  First author    cases   controls                                                   
  -------------- ------- ---------- ----- --- ----- ---- --- --- ----- --- ---- ---- ---
  Zhu             2000       3       16    0   12    5    0   0   28    0   4    4    0
  Huang           2001       4       20    1    6    7    3   0   62    0   9    0    4
  Jia             2002       1       29    0   15    7    6   1   47    0   4    8    0
  Huang           2002       6       53    5   23    9    3   0   66    3   15   12   0
  Bi              2002       2       20    0   15    2    3   0   28    1   6    3    2
  Zhou            2003       1       26    3   19    2    0   0   35    4   4    9    0
  Chen            2003       10      85    0   47    11   7   0   137   0   26   29   3
  Zhang           2004       1       18    0    9    2    2   0   32    0   3    4    1
  He              2005       0       13    0    9    4    1   0   55    0   5    5    0
  Wang            2006       13      75    0   54    7    2   0   120   1   27   11   2
  Li              2006       2       17    0    9    1    1   0   23    0   1    2    1
  Yang L          2008       10      44    1   35    5    7   1   82    0   8    6    1
  Yang J          2008       1       40    0   14    3    0   0   75    0   8    11   2
  Wu              2009       30      118   0   102   12   1   1   77    2   23   13   2
  Duan            2009       5        8    0   12    4    3   1   53    1   9    10   2
  Mai             2010       6       45    2   18    14   3   0   65    0   12   16   4
  Jiang           2010       2       47    0   20    6    4   0   107   0   17   31   1
  Zhou C          2012       5       37    0   17    7    2   0   49    0   9    11   3
  Lv              2012       11      44    2   33    10   0   0   76    2   15   12   1
  Dong            2013       4       27    0   23    3    2   1   44    0   9    6    0

###### Meta-analysis of Apolipoprotein E gene polymorphism in Alzheimer\'s disease

                   Fix-effect model     Random-effect model                                                           
  -------------- --------------------- --------------------- -------- ----- -------------------- ----------- -------- -----
  **Allele**                                                                                                             
  ApoE ε2          0.88 (0.73, 1.07)           0.20           0.0002   61%   0.93 (0.66, 1.29)      0.65      0.0002   61%
  ApoE ε3          0.42 (0.37, 0.47)         \<0.00001         0.07    34%   0.42 (0.36, 0.49)    \<0.00001    0.07    34%
  ApoE ε4          3.93 (3.37, 4.58)         \<0.00001         0.81    0%    3.89 (3.33, 4.54)    \<0.00001    0.81    0%
  **Genotype**                                                                                                           
  ApoE ε4/ε4      11.71 (6.38, 21.47)        \<0.00001         0.98    0%    9.84 (5.28, 18.34)   \<0.00001    0.98    0%
  ApoE ε3/ε3       0.39 (0.33, 0.45)         \<0.00001         0.25    16%   0.38 (0.33, 0.45)    \<0.00001    0.25    16%
  ApoE ε2/ε2       1.06 (0.55, 2.06)           0.86            0.60    0%    1.09 (0.52, 2.27)      0.83       0.60    0%
  ApoE ε4/ε3       3.08 (2.57, 3.69)         \<0.00001         0.06    35%   3.16 (2.48, 4.02)    \<0.00001    0.06    35%
  ApoE ε3/ε2       0.67 (0.53, 0.85)          0.0008           0.18    23%   0.66 (0.49, 0.88)      0.005      0.18    23%
  ApoE ε4/ε2       2.10 (1.35, 3.26)           0.001           0.32    11%   1.92 (1.13, 3.28)      0.02       0.32    11%

P~h~, P~heterogeneity~.
