stroke is the leading cause of disability and third cause of death in developed countries ; an estimated 5.7 million people die of stroke annually worldwide . ischemic stroke ( is ) , the most common type of stroke , accounts for approximately 85% of all strokes . recently , various epidemiologic studies in families and twins have revealed that genetic factors played an important role in the pathogenesis of is , including il1- , hdac9 , tnf- , and acot4 . phosphodiesterase 4d ( pde4d ) is a large gene spanning > 1.5 mb on chromosomal region 5q1 ; pde4d has 22 exons , 8 splice variants , and several hundred snps . pde4d is associated with several processes involved in the occurrence of stroke , including cell proliferation , migration , and inflammation [ 69 ] . recent studies showed that polymorphisms in pde4d gene might be risk factors for is [ 1,1013 ] . first demonstrated that snp56 in pde4d gene was associated with the is risk via whole - genome linkage screens in the icelandic population . to date , the following gwas in different populations that investigated the association between pde4d snp56 and is have produced inconclusive results . for instance , several studies suggested a positive association between snp56 and is susceptibility [ 1517 ] ; nevertheless , some studies could not replicate it [ 1821 ] . these discrepancies may be due to a variety of explanations , including studies with a small sample sizes , inadequate statistical power , different analytical methods , ethnic differences , and different stroke subtypes . meta - analysis is an efficient method to provide more credible evidence by systematically summarizing all eligible data from independent studies . to date , thus , we aimed to perform a meta - analysis to clarify the effect of pde4d snp56 on susceptibility to is . a comprehensive electronic search was conducted of pubmed , embase , and web of science , using the keywords stroke , polymorphism or mutation or variant , snp56 or rs702553 , and pde4d or phosphodiesterase 4d . the reference lists of reviews and retrieved articles were hand - checked for additional potential studies . studies were considered eligible in our meta - analysis if the following criteria were fulfilled : ( 1 ) the study reported the association between pde4d snp56 and risk of ischemia stroke ; ( 2 ) the study provided sufficient information of allele or genotype frequencies ; ( 3 ) the study was a prospective cohort or case - control study . comments , letters , and review articles were excluded , as were studies containing overlapping data with other studies and studies having no control population . a study reporting the results for different subpopulations was treated as separate studies . the following data were extracted independently from each study by 2 authors : first author , year of publication , ethnicity of the individuals ( categorized as europeans or asians ) , numbers of cases and controls , sex and mean age in cases and controls , and genotype frequency . in addition , the corresponding author was contacted for detailed data when there was insufficient information of genotype distributions . the odds ratios ( ors ) and corresponding 95% cis were used to measure the strength of the association between pde4d snp56 and susceptibility to is . the significance of the pooled or was determined by the z test , and p<0.05 was considered significant . moreover , the pooled ors were estimated for t vs. a , tt vs. aa ( homozygote comparison ) , tc vs. aa ( heterozygote comparison ) , tt vs. ta+aa ( recessive model ) , and tt+ta vs. aa ( dominant model ) . finally , the hardy - weinberg equilibrium ( hwe ) in the control group was also assessed , and a p<0.05 was considered significant disequilibrium . heterogeneity across studies was evaluated by using the chi - square  based q test and i test . for the q test i values range between 0% and 100% , with higher values denoting a greater degree of heterogeneity ( considered when i>50% ) . a random - effects model ( dersimonian - laird method ) was used when p<0.05 or i>50% ; otherwise , a fixed - effects model ( mantel - haenszel method ) was used . a metaregression was used to investigate whether any particular covariates could explain the observed between - study heterogeneity . to evaluate the stability of the results , sensitivity analysis was performed by sequentially excluding 1 study each time , to assess the effect of a single study on the pooled ors . moreover , a cumulative analysis was carried out to measure the genetic effects as they accumulated over time . publication bias was assessed using visual inspection of funnel plots and the egger regression test ; p<0.05 was considered significant . all statistical analyses were performed by stata software , version 12 ( statacorp lp , college station , texas , usa ) . a comprehensive electronic search was conducted of pubmed , embase , and web of science , using the keywords stroke , polymorphism or mutation or variant , snp56 or rs702553 , and pde4d or phosphodiesterase 4d . the reference lists of reviews and retrieved articles were hand - checked for additional potential studies . studies were considered eligible in our meta - analysis if the following criteria were fulfilled : ( 1 ) the study reported the association between pde4d snp56 and risk of ischemia stroke ; ( 2 ) the study provided sufficient information of allele or genotype frequencies ; ( 3 ) the study was a prospective cohort or case - control study . comments , letters , and review articles were excluded , as were studies containing overlapping data with other studies and studies having no control population . the following data were extracted independently from each study by 2 authors : first author , year of publication , ethnicity of the individuals ( categorized as europeans or asians ) , numbers of cases and controls , sex and mean age in cases and controls , and genotype frequency . in addition , the corresponding author was contacted for detailed data when there was insufficient information of genotype distributions . the odds ratios ( ors ) and corresponding 95% cis were used to measure the strength of the association between pde4d snp56 and susceptibility to is . the significance of the pooled or was determined by the z test , and p<0.05 was considered significant . moreover , the pooled ors were estimated for t vs. a , tt vs. aa ( homozygote comparison ) , tc vs. aa ( heterozygote comparison ) , tt vs. ta+aa ( recessive model ) , and tt+ta vs. aa ( dominant model ) . finally , the hardy - weinberg equilibrium ( hwe ) in the control group was also assessed , and a p<0.05 was considered significant disequilibrium . heterogeneity across studies was evaluated by using the chi - square  based q test and i test . for the q test , a p value of < 0.05 was considered significant heterogeneity . i values range between 0% and 100% , with higher values denoting a greater degree of heterogeneity ( considered when i>50% ) . a random - effects model ( dersimonian - laird method ) was used when p<0.05 or i>50% ; otherwise , a fixed - effects model ( mantel - haenszel method ) was used . a metaregression was used to investigate whether any particular covariates could explain the observed between - study heterogeneity . to evaluate the stability of the results , sensitivity analysis was performed by sequentially excluding 1 study each time , to assess the effect of a single study on the pooled ors . moreover , a cumulative analysis was carried out to measure the genetic effects as they accumulated over time . publication bias was assessed using visual inspection of funnel plots and the egger regression test ; p<0.05 was considered significant . all statistical analyses were performed by stata software , version 12 ( statacorp lp , college station , texas , usa ) . the systematic literature search identified 68 publications in pubmed , embase , and web of science . after a preliminary screening , 21 articles that met the inclusion criteria and full texts were reviewed and analyzed in detail ; 7 articles reported other variants in pde4d gene [ 2935 ] . finally , 12 articles on pde4d snp56 and is risk were included [ 1421,3841 ] . among them thus , 15 publications were included in the present meta - analysis , and the characteristics of all included studies are summarized in table 1 . a total of 15 studies involving 19,487 subjects ( including 8731 patients and 10,756 control subjects ) were included in the meta - analysis . here , we observed a wide spectrum of the t allele frequency across different ethnicities . briefly , european control subjects carried a higher frequency of pde4d snp56 t allele ( 65.684.13% ) , compared with that in asian controls ( 40.935.68% ; p<0.01 ; figure 2 ) . the meta - analysis results of the association between the allele contrast and genetic models of the pde4d snp56 polymorphism and the susceptibility to is was shown in table 2 . the results of combined analyses comprising 6448 patients and 9700 controls revealed a nonsignificant association between the snp56 variant in pde4d gene and risk of is ( t vs. a : or=1.01 , 95%ci=0.881.15 , p=0.90 ; tt vs. aa : or=1.00 , 95%ci=0.751.34 , p=0.99 ; ta vs. aa : or=1.00 , 95%ci=0.831.21 , p=0.98 ; tt+ta vs. aa : or=0.99 , 95%ci=0.791.24 , p=0.93 ; tt vs. ta+aa : or=1.03 , 95%ci=0.861.25 , p=0.74 ) . in the subgroup analysis stratified by ethnicity , no significant association was observed between pde4d snp56 and is susceptibility in europeans ( t vs. a : or=0.96 , 95%ci=0.861.08 , p=0.54 ) or asians ( t vs. a : or=1.08 , 95%ci=0.801.44 , p=0.62 ; figure 3 ) . the results showed that there was significant heterogeneity in most comparisons in the analysis of is ( t vs. a : p<0.01 for q test , i=84.7% ; tt vs. aa : p<0.01 for q test , i=83.3% ; ta vs. aa : p<0.01 for q test , i=70.0% ; tt+ta vs. aa : p<0.01 for q test , i=81.0% ; tt vs. ta+aa : p<0.01 for q test , i=78.5% ; table 2 ) . we carried out metaregression analysis to assess the source of heterogeneity by ethnicity , sample sizes , and year of publication , which did not indicate any sources that contributed to the substantial heterogeneity . sensitivity analysis showed that no single study qualitatively changed the pooled ors , indicating that the results of this meta - analysis are highly stable ( figure 4 ) . excluding 2 studies that deviated from hwe , the pooled ors did not change at all . [ data not shown , ] cumulative meta - analysis conducted by publication year demonstrated that estimated pooled ors tended to be stable with the accumulation of more data over time ( figure 5 ) . funnel plots and the egger test were performed to assess publication bias . as shown in figure 6 , the shapes of the funnel plots did not show any evidence of obvious asymmetry for pde4d snp56 and is . in addition , the results of the egger test also did not show any evidence of publication bias : t vs. a : p=0.53 , tt vs. aa : p=0.52 , ta vs. aa : p=0.22 , tt+ta vs. aa : p=0.24 , tt vs. ta+aa : p=0.76 . the systematic literature search identified 68 publications in pubmed , embase , and web of science . after a preliminary screening , 21 articles that met the inclusion criteria and full texts were reviewed and analyzed in detail ; 7 articles reported other variants in pde4d gene [ 2935 ] . finally , 12 articles on pde4d snp56 and is risk were included [ 1421,3841 ] . among them thus , 15 publications were included in the present meta - analysis , and the characteristics of all included studies are summarized in table 1 . a total of 15 studies involving 19,487 subjects ( including 8731 patients and 10,756 control subjects ) were included in the meta - analysis . here , we observed a wide spectrum of the t allele frequency across different ethnicities . briefly , european control subjects carried a higher frequency of pde4d snp56 t allele ( 65.684.13% ) , compared with that in asian controls ( 40.935.68% ; p<0.01 ; figure 2 ) . the meta - analysis results of the association between the allele contrast and genetic models of the pde4d snp56 polymorphism and the susceptibility to is was shown in table 2 . the results of combined analyses comprising 6448 patients and 9700 controls revealed a nonsignificant association between the snp56 variant in pde4d gene and risk of is ( t vs. a : or=1.01 , 95%ci=0.881.15 , p=0.90 ; tt vs. aa : or=1.00 , 95%ci=0.751.34 , p=0.99 ; ta vs. aa : or=1.00 , 95%ci=0.831.21 , p=0.98 ; tt+ta vs. aa : or=0.99 , 95%ci=0.791.24 , p=0.93 ; tt vs. ta+aa : or=1.03 , 95%ci=0.861.25 , p=0.74 ) . in the subgroup analysis stratified by ethnicity , no significant association was observed between pde4d snp56 and is susceptibility in europeans ( t vs. a : or=0.96 , 95%ci=0.861.08 , p=0.54 ) or asians ( t vs. a : or=1.08 , 95%ci=0.801.44 , p=0.62 ; figure 3 ) . the results showed that there was significant heterogeneity in most comparisons in the analysis of is ( t vs. a : p<0.01 for q test , i=84.7% ; tt vs. aa : p<0.01 for q test , i=83.3% ; ta vs. aa : p<0.01 for q test , i=70.0% ; tt+ta vs. aa : p<0.01 for q test , i=81.0% ; tt vs. ta+aa : p<0.01 for q test , i=78.5% ; table 2 ) . we carried out metaregression analysis to assess the source of heterogeneity by ethnicity , sample sizes , and year of publication , which did not indicate any sources that contributed to the substantial heterogeneity . sensitivity analysis showed that no single study qualitatively changed the pooled ors , indicating that the results of this meta - analysis are highly stable ( figure 4 ) . excluding 2 studies that deviated from hwe , the pooled ors did not change at all . [ data not shown , ] cumulative meta - analysis conducted by publication year demonstrated that estimated pooled ors tended to be stable with the accumulation of more data over time ( figure 5 ) . funnel plots and the egger test were performed to assess publication bias . as shown in figure 6 , the shapes of the funnel plots did not show any evidence of obvious asymmetry for pde4d snp56 and is . in addition , the results of the egger test also did not show any evidence of publication bias : t vs. a : p=0.53 , tt vs. aa : p=0.52 , ta vs. aa : p=0.22 , tt+ta vs. aa : p=0.24 , tt vs. ta+aa : p=0.76 . despite substantial progress in prevention and treatment , stroke , a multifactorial disease , remains the leading cause of disability and is also responsible for 10% of deaths each year in developed countries . previous studies showed that development of is can be attributed to environmental and genetic factors . in the last few decades , a series of genetic studies have provided evidence supporting an important role for genetics in the pathogenesis of is , such as variants in interleukin-6 , leptin receptor , and angiotensin - converting enzyme [ 4446 ] . one of the widely studied candidate genes was pde4d , which belongs to a large superfamily of phosphodiesterases ( pdes ) . pde genes encode pde enzyme , which can regulate camp levels and is also the key signal transduction molecule in multiple tissues , including kidney , macrophages , b and t lymphocytes , monocytes , as well as vascular smooth muscle cells . moreover , several studies have demonstrated that pde4d was associated with cell proliferation , migration , and inflammation  processes involved in stroke occurrence [ 69 ] . the association between specific pde4d snp56 and is was initially identified via gwas by the decode group . a meta - analysis is a proper method that can overcome the problem of small sample sizes and inadequate statistical power in different genetic studies by combining results from individual studies . to date , accumulated meta - analysis has been performed to clarify the association of genetic polymorphisms with is risk . variants in several genes were identified as risk factors for is , including apolipoprotein b , beta-2 adrenergic receptor , methylenetetrahydrofolate reductase ( mthfr ) , and hdac9 [ 4953 ] . however , some other variants , such as il-6 g572c , pon1 l55 m , and cyp11b2 c-344 t were not associated with susceptibility to is . thus , we saw the need to perform a meta - analysis aimed to investigate the contribution of snp56 in the pde4d gene to is . in this comprehensive meta - analysis involving 8731 patients and 10,756 controls , the results showed a wide spectrum of t allele frequency of pde4d snp56 across different ethnicities . compared with asian controls , european controls carried a higher frequency of t allele ( asians vs. europeans : 40.935.68% and 65.684.13% , respectively ; p<0.01 ) . it is widely accepted that genetic markers in predisposition to is vary across different ethnic populations . for instance , mthfr a1298c polymorphism was strongly associated with is risk in asians but not in europeans [ 5658 ] . , we found that snp56 in the pde4d gene might not be significantly associated with is risk . subsequently , in the subgroup analysis stratified by ethnicity , no significant association between snp56 and susceptibility to is was found in either european or asian population . however , the results might not be conclusive because is is a complex multifactorial disease and environmental factors also play an important role in the development of is . thus , the lack of association might also be attributed to variations in climate and in lifestyle , diet , and economic status of different individuals . the results of metaregression did not show any sources that contribute to the heterogeneity , including year of publication , sample size , and ethnicity . however , subtype information of is might contribute to the significant heterogeneity . for example , the significant association in the first gwas was strongest for carotid and cardiogenic stroke . in this meta - analysis , it is difficult to carry out a subgroup analysis by stroke subtype because of the lack of sufficient data . compared with those with normal blood pressure , patients with hypertension were at a higher risk for diabetes , another stroke risk factor . thus , it might be easier to discern the effect of pde4d snp56 on is risk in the absence of hypertension . moreover , a difference in the proportion of hypertensives might also contribute to the inconsistent results . this meta - analysis significantly increased statistical power by pooling data from different studies ; however , several limitations of this meta - analysis need to be considered for interpretation of our results . first , significant heterogeneity between studies was observed in the current meta - analysis , which might be attributed to different genetic backgrounds , different lifestyles , distinct environments , stroke subtypes , hypertension status , and abnormal physiologic variables . second , we only included studies published in english , which might introduce a language bias . distinct is subtypes had different risk factors and etiologies , thus a subgroup analysis by types of is was necessary in further meta - analysis . finally , the development of is is multifactorial , including genetic and environmental factors . in conclusion , despite these limitations , the current meta - analysis included 15 genetic studies suggested that snp56 in pde4d gene might not be associated with the susceptibility to is both in europeans and asians . further well - designed studies with large sample size are required to validate our results . moreover , gene - gene and gene - environment interactions should also be investigated to clarify possible roles of multiple risk factors in the development of is .
backgroundrecent studies demonstrated that polymorphisms in the pde4d gene were associated with several processes involved in the occurrence of ischemic stroke ( is ) . 
 the association between specific pde4d single - nucleotide polymorphism 56 ( snp56 ) and is risk was initially identified via genome - wide association studies ( gwas ) , although the gwas in different populations produced inconclusive results . 
 thus , we performed a meta - analysis to better explain the association between pde4d snp56 and is risk.material/methodsa literature search was conducted using pubmed , embase , and web of science up to june 1 , 2015 . 
 a fixed - effects or random - effects model was used to calculate the pooled odds ratios ( ors ) based on the results from the heterogeneity tests.resultsfinally , we performed a meta - analysis of 15 studies , involving 8731 is patients and 10,756 controls . 
 the results showed nonsignificant association between pde4d snp56 and is risk ( t vs. a : or=1.01 , 95%ci=0.881.15 , p=0.90 ) . 
 similarly , in the subgroup analysis by ethnicity , no significant association was observed in asian ( t vs. a : or=1.08 , 95%ci=0.801.44 , p=0.62 ) or european ( t vs. a : or=0.96 , 95%ci=0.861.08 , p=0.54 ) population . 
 moreover , funnel plots and egger regression testing showed no evidence of publication bias.conclusionsin summary , current evidence suggested that pde4d snp56 might not be associated with an increased susceptibility to is . 
 however , this conclusion needs further validation by well - designed studies with large sample sizes .