coronary heart disease ( chd ) , a complex disease , is the most important cause of mortality and morbidity worldwide . chd is often initially detected from clinical manifestations such as myocardial infarction , angina , or sudden death due to artery occlusion . the environmental factors , such as tobacco smoking , physical activity and alcohol intake , and dietary fat consumption , in particular , the amount of saturated fatty acids , have been considered as the main risk factors for chd . in recent years , several susceptibility genes have been proposed to manifest an association with chd . among the genetic determinants that have been more widely studied related to chd risk is the common polymorphism in the apolipoprotein e ( apoe ) gene . apoe is located on chromosome 19q13.2 , with 3 common isoforms ( 2 , 3 and 4 ) and 6 genotypes ( e2e2 , e2e3 , e3e3 , e2e4 , e3e4 , and e4e4 ) . the alleles differ at two single - nucleotide polymorphisms , giving rise to different amino acids at protein positions 112 and 158 ; cysteine at 112 and arginine at 158 for 3 , two cysteine for 2 and two arginine for 4 . 3 accounting for approximately 5090% in all population is considered to be the wild type allele whereas alleles 2 and 4 are considered variants which vary between 5 - 35% and 1 - 15% , respectively . apoe is highly polymorphic and plays an imperative role in endogenous lipoprotein metabolism and tissue distribution . it is present in lipoprotein particles and mediates lipoprotein through binding to the low - density lipoprotein ( ldl ) and lipoprotein remnant receptors . defects in the apoe protein could diminish its ability to bind to the receptors , which may lead to an elevated blood cholesterol level . it has been well - established that the presence of the apoe 4 allele is associated with higher levels of total and ldl serum cholesterol , whereas the presence of the apoe 2 allele is associated with the lower effect with reference to cholesterol effects from the 3 allele . these results suggest that apoe is associated with chd . to date , numerous epidemiological studies have recently focused on the association between apoe 2/3/4 polymorphism and chd risk . for example , singh et al . found a significant association of 4 polymorphism , especially e3/e4 genotype with chd , along with high - density lipoprotein ( hdl ) and ldl concentrations . moreover , there was no significant difference between the mean of triglyceride and hdl levels among different apoe genotypes . therefore , the present meta - analysis is designed to derive a more precise estimation . relevant studies were identified from the following electronic databases : pubmed , embase , web of science , cnki , cbm , wanfang , vip and hand searching of relevant journals and the reference lists of retrieved articles , with the last updated search performed before september 2012 . the search was performed using various combinations of keywords including (  apoe  ) and (  polymorphism  or  variant  or  mutation  ) and (  chd  ) without language limitation . the references of the selected papers were also checked by hand - search for other potential articles that possibly have been missed in the initial search . to be eligible for inclusion in this meta - analysis , a study must meet the following criteria : ( 1 ) apoe gene polymorphism in chd ; ( 2 ) sufficient data for calculating an odds ratio ( or ) with its 95% confidence interval ( ci ) . when duplicate articles were published , only the newest or most informative single article was selected . study was conducted according to the preferred reporting items for systematic reviews and meta - analyses statement . the following characteristics were extracted from each study : first author , year of publication , ethnicity of the study population , sample size , genotype method ; source of case and control subjects . the quality of the included studies was assessed using the following criteria modified from the previous report , and studies with four or five adequate items were defined as high quality . description of the case and control groups ( not described , inadequate , adequate).assessment and validation of chd in the patients ( not described , inadequate , adequate ) . adequate validation would include confirmation by coronary angiography ; inadequate validation would include recollection of the patient as the only clinical evidence without examination.description of the laboratory procedures for the genotyping ( not described , inadequate , adequate).confirmation of hardy - weinberg equilibrium ( hwe ) in the genotype distribution ( not described , inadequate , adequate ) . since hwe is a surrogate to assess study quality , and the effect of hwe is associated with problems in the design and conduct of genetic association studies , studies with departures from hwe were defined as low - quality studies.equal assessment for confounding factors in the case and control groups ( not described , inadequate , adequate ) . description of the case and control groups ( not described , inadequate , adequate ) . assessment and validation of chd in the patients ( not described , inadequate , adequate ) . adequate validation would include confirmation by coronary angiography ; inadequate validation would include recollection of the patient as the only clinical evidence without examination . description of the laboratory procedures for the genotyping ( not described , inadequate , adequate ) . confirmation of hardy - weinberg equilibrium ( hwe ) in the genotype distribution ( not described , inadequate , adequate ) . since hwe is a surrogate to assess study quality , and the effect of hwe is associated with problems in the design and conduct of genetic association studies , studies with departures from hwe were defined as low - quality studies . equal assessment for confounding factors in the case and control groups ( not described , inadequate , adequate ) . allele frequencies at genetic polymorphisms from each study were determined by the allele counting method . hwe for the controls in each study was assessed by chi - square interval or fisher exact test . the combined or and 95% ci was used to compare contrasts of genotypes and alleles between cases and controls . statistical heterogeneity was measured using the chi - square based q statistic ( p < 0.10 was considered statistically significant heterogeneity ) . we also quantified the effect of heterogeneity using i statistic : i values of 25% , 50% , and 75% were defined as low , moderate , and high estimates , respectively . either a random - effects model ( dersimonian - laird method ) or fixed - effects model ( mantel - haenszel method ) was used to calculate pooled effect estimates in the presence or absence of heterogeneity , respectively . finally , potential publication bias was evaluated through funnel plot visual analysis and with the begg 's and egger 's tests . a p < 0.05 was considered statistically significant . for the possible publication bias , we used trim and fill method to evaluate the influence to the results . all statistical analyses were performed by stata version 10 ( statacorp lp , college station , tx , usa ) . relevant studies were identified from the following electronic databases : pubmed , embase , web of science , cnki , cbm , wanfang , vip and hand searching of relevant journals and the reference lists of retrieved articles , with the last updated search performed before september 2012 . the search was performed using various combinations of keywords including (  apoe  ) and (  polymorphism  or  variant  or  mutation  ) and (  chd  ) without language limitation . the references of the selected papers were also checked by hand - search for other potential articles that possibly have been missed in the initial search . to be eligible for inclusion in this meta - analysis , a study must meet the following criteria : ( 1 ) apoe gene polymorphism in chd ; ( 2 ) sufficient data for calculating an odds ratio ( or ) with its 95% confidence interval ( ci ) . when duplicate articles were published , only the newest or most informative single article was selected . study was conducted according to the preferred reporting items for systematic reviews and meta - analyses statement . the following characteristics were extracted from each study : first author , year of publication , ethnicity of the study population , sample size , genotype method ; source of case and control subjects . the quality of the included studies was assessed using the following criteria modified from the previous report , and studies with four or five adequate items were defined as high quality . description of the case and control groups ( not described , inadequate , adequate).assessment and validation of chd in the patients ( not described , inadequate , adequate ) . adequate validation would include confirmation by coronary angiography ; inadequate validation would include recollection of the patient as the only clinical evidence without examination.description of the laboratory procedures for the genotyping ( not described , inadequate , adequate).confirmation of hardy - weinberg equilibrium ( hwe ) in the genotype distribution ( not described , inadequate , adequate ) . since hwe is a surrogate to assess study quality , and the effect of hwe is associated with problems in the design and conduct of genetic association studies , studies with departures from hwe were defined as low - quality studies.equal assessment for confounding factors in the case and control groups ( not described , inadequate , adequate ) . description of the case and control groups ( not described , inadequate , adequate ) . assessment and validation of chd in the patients ( not described , inadequate , adequate ) . adequate validation would include confirmation by coronary angiography ; inadequate validation would include recollection of the patient as the only clinical evidence without examination . description of the laboratory procedures for the genotyping ( not described , inadequate , adequate ) . confirmation of hardy - weinberg equilibrium ( hwe ) in the genotype distribution ( not described , inadequate , adequate ) . since hwe is a surrogate to assess study quality , and the effect of hwe is associated with problems in the design and conduct of genetic association studies , studies with departures from hwe were defined as low - quality studies . equal assessment for confounding factors in the case and control groups ( not described , inadequate , adequate ) . allele frequencies at genetic polymorphisms from each study were determined by the allele counting method . hwe for the controls in each study was assessed by chi - square interval or fisher exact test . the combined or and 95% ci was used to compare contrasts of genotypes and alleles between cases and controls . statistical heterogeneity was measured using the chi - square based q statistic ( p < 0.10 was considered statistically significant heterogeneity ) . we also quantified the effect of heterogeneity using i statistic : i values of 25% , 50% , and 75% were defined as low , moderate , and high estimates , respectively . either a random - effects model ( dersimonian - laird method ) or fixed - effects model ( mantel - haenszel method ) was used to calculate pooled effect estimates in the presence or absence of heterogeneity , respectively . finally , potential publication bias was evaluated through funnel plot visual analysis and with the begg 's and egger 's tests . all statistical analyses were performed by stata version 10 ( statacorp lp , college station , tx , usa ) . through screening the title and reading the abstract , 135 papers were excluded . of these , six papers were published in other language ( russian : 2 ; spanish 2 ; portuguese : 1 ; norwegian : 1 ) . of the 26 studies , 2 studies did not have a control group , 5 did not provide enough information on genotype distribution , leaving 19 studies for more detailed assessment . thus , in accordance with the objectives of the present study , we analyzed them independently . as a result , a total of 14 studies from 13 independent articles including 5746 chd cases and 19,120 controls were subjected to the final analysis . of the 14 studies , 6 studies were conducted in mongoloid population , 5 were conducted in caucasians population , and 3 in other population . characteristics of studies included in the current meta - analysis are shown in table 1 . basic information of studies included in the meta - analysis na : not available ; hwe : hardy - weinberg equilibrium ; pcr - rflp : polymerase chain reaction with restriction fragment length polymorphism ; pcr - spm : polymerase chain reaction with solid - phase minisequencing ; bmi : body mass index ; chd : coronary heart disease . according to the quality criteria , there were thirteen studies with high quality , and only one with low quality . a summary of meta - analysis findings regarding the relation between the apoe 2/3/4 polymorphism is presented in table 2 . summary estimates of the association between apoe gene polymorphism and chd : overall analysis and subgroup analysis z test used to determine the significance of the overall or . pz < 0.05 was considered to be significant ; f : fixed - effects model ; r : random - effects model ; chi - square q statistic test used to assess the heterogeneity in each group . pq > 0.1 , fixed - effects model was used , pq < 0.1 , random - effects model was used . or : odds ratio ; ci : confidence interval ; chd : coronary heart disease ; apoe : apolipoprotein e. forest plot on the association between 2 allele ( vs. 3 allele ) and chd . forest plot on the association between 2 carriers ( vs. 3 carriers ) and chd . forest plot on the association between 4 allele ( vs. 3 allele ) and chd . forest plot on the association between 4 carriers ( vs. 3 carriers ) and chd . meta - analysis showed a decreased risk for the 2 allele compared with the 3 allele in overall population ( or = 0.82 , 95% ci : 0.750.90 , p < 0.001 ) , without heterogeneity among studies ( i = 27.1% , p = 0.164 ) . when stratified by race , the pooled or was just statistically significant in caucasian population , not in mongoloid and others . we also compared the model of 2 carriers versus 3 carriers . under a fixed - effect model , the pooled or for 2 carriers versus 3 carriers was 0.81 ( 95% ci : 0.730.89 , p < 0.001 ) . analysis after stratification by race indicated significant association with chd in caucasian population . therefore , carriers of apoe 2 allele had a decreased risk for chd , especially in caucasians . meta - analysis showed an increased risk for the 4 allele compared with the 3 allele in overall population ( or = 1.34 , 95% ci : 1.151.57 , p < 0.001 ) with significant inter - study heterogeneity ( i = 71.3% , p < 0.001 ) . when stratified by race , heterogeneity was not detected . the combined or obtained using a fixed - effect model was significant in mongoloid population . similarly , for genotype contrast of 4 carriers versus 3 carriers , a significant association was noticed in overall population ( or = 1.29 , 95% ci : 1.121.48 , p < 0.001 ) . analysis after stratification by race indicated a significant association with chd risk in mongoloid population ( or = 1.76 , 95% ci : 1.442.15 , p < 0.001 ) . therefore , carriers of apoe 4 allele had an increased risk for chd , especially in mongoloid population . both begg 's test and egger 's test were performed to assess the publication bias of the literature . funnel plots on the distribution of the ors from individual studies in relation to their respective standard deviation are shown in figures 6 and 7 . no evidence of publication bias was observed in the genetic model of overall 2 allele versus 3 allele ( pbegg = 0.583 , pegger = 0.820 ) . however , the shape of the funnel plot for the genetic model of overall 4 allele versus 3 allele seemed to be asymmetry , suggesting potential publication bias ( pbegg = 0.002 ) . this might be a limitation for our analysis because the studies with null findings , especially those with small sample size , were less likely to be published . by using the trim and fill method , we showed that if the publication bias was the only source of the funnel plot asymmetry , it needed seven more studies to balance the funnel plot . the adjusted risk estimate was attenuated but remained significant ( 4 allele vs. 3 allele : or = 1.08 , 95% ci : 1.021.15 , p = 0.009 ; 4 carriers vs. 3 carriers : or = 1.08 , 95% ci : 1.021.16 , p = 0.016 ) , indicating the stability of our results . funnel plot for the results of the meta - analysis of the 2 allele vs. 3 allele . funnel plot for the results of the meta - analysis of the 4 allele vs. 3 allele . through screening the title and reading the abstract , 135 papers were excluded . of these , six papers were published in other language ( russian : 2 ; spanish 2 ; portuguese : 1 ; norwegian : 1 ) . of the 26 studies , 2 studies did not have a control group , 5 did not provide enough information on genotype distribution , leaving 19 studies for more detailed assessment . thus , in accordance with the objectives of the present study , we analyzed them independently . as a result , a total of 14 studies from 13 independent articles including 5746 chd cases and 19,120 controls were subjected to the final analysis . of the 14 studies , 6 studies were conducted in mongoloid population , 5 were conducted in caucasians population , and 3 in other population . characteristics of studies included in the current meta - analysis are shown in table 1 . basic information of studies included in the meta - analysis na : not available ; hwe : hardy - weinberg equilibrium ; pcr - rflp : polymerase chain reaction with restriction fragment length polymorphism ; pcr - spm : polymerase chain reaction with solid - phase minisequencing ; bmi : body mass index ; chd : coronary heart disease . according to the quality criteria , there were thirteen studies with high quality , and only one with low quality . a summary of meta - analysis findings regarding the relation between the apoe 2/3/4 polymorphism is presented in table 2 . summary estimates of the association between apoe gene polymorphism and chd : overall analysis and subgroup analysis z test used to determine the significance of the overall or . pz < 0.05 was considered to be significant ; f : fixed - effects model ; r : random - effects model ; chi - square q statistic test used to assess the heterogeneity in each group . pq > 0.1 , fixed - effects model was used , pq < 0.1 , random - effects model was used . or : odds ratio ; ci : confidence interval ; chd : coronary heart disease ; apoe : apolipoprotein e. forest plot on the association between 2 allele ( vs. 3 allele ) and chd . forest plot on the association between 2 carriers ( vs. 3 carriers ) and chd . forest plot on the association between 4 allele ( vs. 3 allele ) and chd . forest plot on the association between 4 carriers ( vs. 3 carriers ) and chd . meta - analysis showed a decreased risk for the 2 allele compared with the 3 allele in overall population ( or = 0.82 , 95% ci : 0.750.90 , p < 0.001 ) , without heterogeneity among studies ( i = 27.1% , p = 0.164 ) . when stratified by race , the pooled or was just statistically significant in caucasian population , not in mongoloid and others . we also compared the model of 2 carriers versus 3 carriers . under a fixed - effect model , the pooled or for 2 carriers versus 3 carriers was 0.81 ( 95% ci : 0.730.89 , p < 0.001 ) . analysis after stratification by race indicated significant association with chd in caucasian population . therefore , carriers of apoe 2 allele had a decreased risk for chd , especially in caucasians . meta - analysis showed an increased risk for the 4 allele compared with the 3 allele in overall population ( or = 1.34 , 95% ci : 1.151.57 , p < 0.001 ) with significant inter - study heterogeneity ( i = 71.3% , p < 0.001 ) . when stratified by race , heterogeneity was not detected . the combined or obtained using a fixed - effect model was significant in mongoloid population . similarly , for genotype contrast of 4 carriers versus 3 carriers , a significant association was noticed in overall population ( or = 1.29 , 95% ci : 1.121.48 , p < 0.001 ) . analysis after stratification by race indicated a significant association with chd risk in mongoloid population ( or = 1.76 , 95% ci : 1.442.15 , p < 0.001 ) . therefore , carriers of apoe 4 allele had an increased risk for chd , especially in mongoloid population . both begg 's test and egger 's test were performed to assess the publication bias of the literature . funnel plots on the distribution of the ors from individual studies in relation to their respective standard deviation are shown in figures 6 and 7 . no evidence of publication bias was observed in the genetic model of overall 2 allele versus 3 allele ( pbegg = 0.583 , pegger = 0.820 ) . however , the shape of the funnel plot for the genetic model of overall 4 allele versus 3 allele seemed to be asymmetry , suggesting potential publication bias ( pbegg = 0.002 ) . this might be a limitation for our analysis because the studies with null findings , especially those with small sample size , were less likely to be published . by using the trim and fill method , we showed that if the publication bias was the only source of the funnel plot asymmetry , it needed seven more studies to balance the funnel plot . the adjusted risk estimate was attenuated but remained significant ( 4 allele vs. 3 allele : or = 1.08 , 95% ci : 1.021.15 , p = 0.009 ; 4 carriers vs. 3 carriers : or = 1.08 , 95% ci : 1.021.16 , p = 0.016 ) , indicating the stability of our results . funnel plot for the results of the meta - analysis of the 2 allele vs. 3 allele . funnel plot for the results of the meta - analysis of the 4 allele vs. 3 allele . many epidemiological studies have investigated the relationship between the apoe polymorphism and the risk of chd , but because of small sample size and the low statistical power of individual studies , results have been contradictory . the main purpose of the present meta - analysis is to summarize the published literature and attempt to clarify the role of apoe 2/3/4 polymorphism in chd . we quantitatively combined 18 case  control studies including 6342 chd cases and 19,688 controls , and found that carrying one or more 4 allele was associated with an increased risk for chd , whereas individuals with one or more 2 allele had a significantly lower risk of developing chd compared to those with 3 allele . therefore , it is safe to assume that 4 allele is an independent factor for the development of chd , however , 2 allele tends to be a protective factor . as for the caucasian population , significant association was observed in the contrast of the genetic model of 2 allele vs. 3 allele and 2 carriers vs. 3carriers . however , the genetic model 4 allele versus 3 allele and 4 carriers versus 3carriers remained significant difference only in mongoloid population . first reported that the presence of apoe polymorphisms has inspired widespread interest in the genetic associations between these single - nucleotide polymorphisms and many complex diseases including alzheimer 's disease , type 2 diabetes , hypertension , and others . structural defects in apoe could result in an impaired interaction between apoe - containing lipoproteins and their receptors and induce the development of atherogenic dyslipidemias and premature cardiovascular disease , including chd . several former meta - analyses had documented that 4 was associated with increased risk for chd . in our study , we also found that 2 carriers had a lower risk of chd . it is worth mentioning that testing for deviations from hwe in controls is an important requirement in population genetic studies . hwe is based on five basic assumptions : ( 1 ) the population is large ( i.e. , there is no genetic drift ) ; ( 2 ) there is no gene flow between population due to migration or transfer of gametes ; ( 3 ) mutations are negligible ; ( 4 ) individuals are mating randomly ; and ( 5 ) natural selection is not operating on the population . deviation from hwe may point to genotyping error , racial heterogeneity , or selection bias . in our study , four of 18 studies heterogeneity did not exist in overall comparisons of 2 allele versus 3 allele and 2 carriers versus 3carriers , but in the genetic model of 4 allele versus 3 allele and 4 carriers versus 3carriers , which may affect the results of the present meta - analysis . we adopted the random - effects model , as it is more conservative than the fixed - effects model and accounts for additional sources of inter - study variation when heterogeneity exists . this heterogeneity may attribute to the potential confounding due to case definition and sampling , sample size , genotyping method , difference of ethnicity , or it may be due to interaction with other risk factors . in order to understand the sources of heterogeneity , we conducted the subgroup analysis by race . significant bias was observed in genetic model of 4 allele versus 3 allele and 4 carriers versus 3 carriers . first , our meta - analysis took into consideration only fully published data , and the abstract and conference papers were excluded . second , this meta - analysis only focused on papers published in english language , and some eligible studies which were reported in other languages might be missed . third , positive results tend to be accepted by journals while negative results are often rejected or not even submitted . we should point out that the publication bias might partly account for the results , but which were not affected deeply . when we adjusted the results using the trim and fill method , the adjusted risk estimate was attenuated but remained significant , indicating the stability of our results . in addition , heterogeneity and publication bias , some limitations of this meta - analysis should be acknowledged . due to the relative small number of the eligible studies , we were unable to perform further subgroup analysis such as by gender , body mass index . besides , chd is a complex disease with various involved factors including environmental and genetic factors . however , many eligible studies included in this meta - analysis did not consider most of the important environmental factors , moreover , a single gene polymorphism is unlikely to provide the complete explanation of genetic risk for chd . therefore , gene - gene and gene - environment interactions should be considered in future studies . our study suggested a genetic association between apoe gene and chd , 4 increased the risk of chd , whereas 2 decreased the risk of chd . well - designed case - control studies with larger sample sizes regarding the association of apoe polymorphism and chd need to be performed in the future .
background : coronary heart disease ( chd ) is a multifactorial disease and is thought to have a polygenic basis . 
 apolipoprotein e ( apoe ) gene is one such candidate with its common 2/3/4 polymorphism in chd . in recent years 
 , numerous case - control studies have investigated the relationship of apoe polymorphism with chd risk . 
 however , the results are confusing.methods:to clarify this point , we undertook a meta - analysis based on 14 published studies including 5746 chd cases and 19,120 controls . 
 crude odds ratios ( ors ) with 95% confidence intervals ( cis ) were assessed for association using a random - effects or fixed - effects model using stata version 10 ( statacorp lp , college station , tx , usa).results : overall , the analysis showed that carriers of apoe 2 allele decreased risk for chd ( 2 allele vs. 3 allele : or = 0.82 , 95% ci : 0.750.90 , p < 0.001 ; 2 carriers vs. 3 carriers : or = 0.81 , 95% ci : 0.730.89 , p < 0.001 ) , compared with those carrying 3 allele , especially in caucasian population . 
 however , those with 4 allele had a significant increased risk for chd ( 4 allele vs. 3 allele : or = 1.34 , 95% ci : 1.151.57 , p < 0.001 ) , especially in mongoloid population . 
 potential publication bias was observed in the genetic model of 4 versus 3 , but the results might not be affected deeply by the publication bias . when we accounted for publication bias using the trim and fill method , the results were not materially alerted , suggesting the stability of our results.conclusions:taken together , our meta - analysis supported a genetic association between apoe gene and chd . 
 4 increased the risk of chd , whereas 2 decreased the risk of chd .