hypertension is a complex syndrome influenced by multiple genetic and environmental factors .   in view of some individuals being more susceptible to hypertension than others , although great hope was expressed in genome - wide association studies to unlock the genetic basis of hypertension , the results from such research , however , have told us little . given the limited power of single studies , one practicable strategy is to perform large meta - analyses to reliably evaluate the predetermined candidates in genetic association studies . the gene encoding atrial natriuretic peptide ( anp , chromosome 1p36.21 ) is a logical candidate for involvement in the underlying cause of hypertension . knockout mice deficient in one copy of anp gene was associated with salt - sensitive hypertension . in contrast , overexpression of anp gene via gene therapy in hypertensive mice had lowered systolic blood pressure . these findings therefore encourage the search for human genetic polymorphisms that affect the anp functionality . a large panel of anp gene polymorphisms have been identified ; in particular , an exonic polymorphism t2238c ( rs5065 ) ranks high in association with hypertension ; however , the results in some individually underpowered studies are often irreproducible [ 6 , 7 ] . to derive a more precise estimation , we therefore meta - analyzed the association of anp gene t2238c polymorphism with occurrence of hypertension from both english and chinese literature , while addressing between - study heterogeneity and publication bias . we searched pubmed and embase , as well as china biological medicine ( http://sinomed.imicams.ac.cn/index.jsp ) and wanfang ( http://www.wanfangdata.com.cn ) databases for articles published before 10 february 2011 using the boolean combinations of keywords ( atrial natriuretic peptide or natriuretic peptide precursor a or anp or nppa ) and ( hypertension or blood pressure ) and ( polymorphism or allele or genotype or variant or variation ) . search results were limited to human populations and articles written in both english and chinese languages . the full text of the retrieved articles was scrutinized to decide whether information on the topic of interest was included . reference lists of these retrieved articles and systematic reviews were also checked to determine whether citations of articles were not initially identified . for these articles involving more than one geographic or ethnic heterogeneous groups , each group was treated separately . articles were included in this meta - analysis if they examined the hypothesis that anp gene t2238c polymorphism was associated with hypertension , if they followed a case - control or cross - sectional study design , and if they provided sufficient information on t2238c genotype counts between hypertensive patients and controls for determining an estimate of odds ratio ( or ) and its corresponding 95% confidence interval ( ci ) . hypertension was defined as systolic blood pressure equal to or above 140  mmhg or diastolic blood pressure equal to or above 90  mmhg or previous treatment with antihypertensive drugs . studies evaluating secondary hypertension or other types of monogenic hypertension were excluded . where there were multiple articles from the same study population , the following characteristics were extracted independently and entered into separate databases by wenquan niu and yue qi ( who is an assistant researcher in beijing institute of heart , lung & blood vessel diseases , anzhen hospital ) from each qualified study : first author 's last name , publication date , population ethnicity , study design , diagnostic criteria , baseline characteristics of the study population ( such as age , gender , and body mass index ) , and the t2238c genotype counts in patients and controls . for consistency , quantitative variables expressed as mean  standard error ( se ) were converted to mean  standard deviation ( sd ) . any encountered discrepancies were adjudicated by a discussion , and a consensus was reached . the random - effects model using the dersimonian & laird method was implemented to bring the individual effect - size estimates together , and the estimate of heterogeneity was taken from the mantel - haenszel model . unadjusted or and 95% ci were used to compare genetic contrasts between patients and controls . between - study heterogeneity was assessed by the inconsistency index i statistic ( ranging from 0 to 100% ) , which was documented for the percentage of the observed between - study variability due to heterogeneity rather than chance , with higher values suggesting the existence of heterogeneity [ 14 , 15 ] . in the case of between - study heterogeneity , we examined the study characteristics that can stratify the studies into subgroups with homogeneous effects . in addition , to estimate the extent to which one or more covariates explain heterogeneity , meta - regression , as an extension to random - effects meta - analysis , was employed . cumulative meta - analysis was conducted to identify the influence of the first published study on the subsequent publications , and the evolution of the combined estimates over time according to the ascending date of publication . to identify potentially influential studies , sensitivity analysis was undertaken by removing an individual study each time to check whether any of these estimates can bias the overall estimate . egger 's test can detect funnel plot asymmetry by determining whether the intercept deviates significantly from zero in a regression of the standardized effect estimates against their precision . probability less than 0.05 was judged significant with the exception of the i statistic and egger 's test , where a significance level of less than 0.1 was chosen . based on our search strategy , the primary screening produced 25 potentially relevant articles , of which 7 met the inclusion criteria with an attempt to evaluate the association of anp gene t2238c polymorphism with the occurrence of hypertension [ 612 ] . of these 7 articles , three [ 68 ] were published in english and four in chinese language [ 912 ] . five out of 7 articles were conducted in asians ( four in chinese [ 912 ] and one in japanese ) . overall 2238c allele frequency was 4.19% in patients and 8.14% in controls with the highest frequency noted in an african - american population ( 41.67% and 38.64% )   and the lowest frequency in a chinese han population ( 0.92% and 0.82% ) . genotyping for t2238c polymorphism across all studies , except two using the gene chip technology [ 9 , 12 ] and one using taqman assay , was conducted using polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) followed by enzyme scai digestion . considering the low frequency of 2238cc genotype , we only assessed the association of 2238c allele ( relative to 2238 t allele ) with hypertension risk in this meta - analysis . overall speaking , comparison of allele 2238c with 2238 t generated a 23% reduced , albeit nonsignificant , risk for hypertension ( 95% ci : 0.381.59 ; p = .485 ) ( figure 2 ) . meanwhile , there was strong evidence of between - study heterogeneity ( i = 88.3% , p < .0005 ) and high possibility of publication bias as reflected by the suggestive asymmetry of funnel plot ( figure 3 ) and the egger 's test ( p = .051 ) . in the cumulative meta - analysis , there was no evidence suggesting the first published study that reported a potentially significant result and then trigged the subsequent replication ( data not shown ) . however , the influential analysis revealed that there was one study in a kazakh chinese population   influencing the overall results significantly ( figure 2 ) . when this most influential study was removed , the overall estimate was strongly attenuated with or approaching the unity ( or = 0.97 ; 95% ci : 0.621.51 ; p = .897 ) . likewise , between - study heterogeneity was remarkably reduced ( i = 51.2% , p = .069 ) , and there was a low probability of publication bias ( p = .479 ) ( figure 3 ) . considering the significance of heterogeneity , we considered as a better choice to try investigating its sources by conducting subgroup analysis in characteristic - homogeneous groups . to evaluate the possible effect of study design on the variability of overall estimates , studies were divided into population - based and hospital - based studies , and importantly the magnitude of association in population - based studies was strikingly reinforced with the 2238c allele conferring a significant protective effect on hypertension ( or = 0.33 ; 95% ci : 0.130.80 ; p = .015 ) , whereas this effect was reversed in hospital - based studies with no attainable significance ( or = 1.15 ; 95% ci : 0.791.68 ; p = .454 ) ( figure 4(a ) ) . further subgroup analysis by ethnicity suggested heterogeneous associations of t2238c polymorphism with hypertension , by showing a suggestive risk effect of 2238c allele in blacks ( or = 1.13 ; p = .66 ) , a significant protective effect in whites ( or = 0.53 ; p = .019 ) , and a moderate protective effect in asians ( or = 0.79 ; p = .663 ) ( figure 4(b ) ) . because sample size of study is also a major factor affecting the reliability , we therefore used an additional strategy to estimate the effect by grouping only those large association studies with an arbitrary cutoff of 500 individuals . two out of seven studies had sample sizes greater than 500 and conferred a 53% reduced risk for 2238c allele relative to 2238 t allele ( 95% ci : 0.092.45 ; p = .374 ) , accompanying high between - study heterogeneity ( i = 94.7% , p < .0005 ) ( figure 4(c ) ) . to further account for between - study heterogeneity within a multivariable framework , we performed meta - regression by incorporating various study - level covariates including averaged levels of age , male percent , and body mass index ( bmi ) between patients and controls , as well as study design and ethnicity altogether , and we interestingly and exclusively observed that study design was a significant source of between - study heterogeneity ( p = .042 ) . this study , including 4068 subjects , to our knowledge , is the first meta - analysis examining the relationship between anp gene t2238c polymorphism and hypertension . although some statistical biases could not be eliminated , our results suggested that carriers of 2238c allele were at moderate decreased risk of developing hypertension , whereas study design was identified as a potentially significant source of between - study heterogeneity by both subgroup and meta - regression analyses , indicating the robustness of our results . genetic heterogeneity is an inevitable problem in any disease identification strategy . as shown in this study , we speculated that anp gene t2238c polymorphism might have diverse roles in different ethnic populations . on one hand , striking differences were noted in terms of mutant 2238c allele frequency between african - americans and asians , with the former almost tenfold higher than the latter , suggesting that different genetic backgrounds may cause this discrepancy , or different populations may have different linkage disequilibrium patterns . a polymorphism may be in close linkage with another nearby causal variant in one ethnic population but not in another . the anp gene t2238c polymorphism may be in close linkage with different nearby causal variants in different populations . on the other hand , in our subgroup analysis , t2238c polymorphism showed significant heterogeneous associations with hypertension across different ethnic groups , with 2238c allele in african - americans being completely at odds with that in whites and asians , suggesting that this polymorphism might have a multifunctional role in the pathogenesis of hypertension or interact with other genetic and environmental factors . however , considering the relative small sample sizes in each group , we suggest that confirmation in large , well - designed studies is critical . besides the disturbing influence of ethnicity in this meta - analysis interestingly , we observed that magnitude of association was reversed in population - based studies relative to in hospital - based studies although no significance was identified for the latter . regarding this point , we agree that control for population stratification remains an important consideration in hospital - based studies , because in this meta - analysis , most studies have recruited subjects from only one hospital , and thus there might be a narrow socioeconomic profile for both patients and controls . moreover , in hospital - based studies , poor comparability between cases and controls might exert a confounding effect on the true association in light of a regional specialty for the disease under study and the differential hospitalization rates between cases and controls . in contrast , subjects drawn from community or a fixed group might be representative of the true population , leading us to believe that results from population - based studies might hold the water . considering the wider confidence intervals of estimates and small sample sizes in population - based studies , more studies are required to quantify this effect size reliably . despite the clear strengths of our study including relatively large sample sizes and robustness of statistical analyses , interpretation of our current study , however , should be viewed in light of several technical limitations . because only published studies were retrieved in this meta - analysis and the  grey  literature ( articles in languages other than english and chinese ) was not included , publication bias might be possible , even though our funnel plots and statistical tests did not show it . in addition , most studies in this meta - analysis have recruited subjects aged 50 years , for whom environmental factors are likely to contribute more prominently than the genetic component to the development of hypertension , suggesting that large association studies in a younger population of hypertensive subjects are of added interest . moreover , the single - locus - based nature of meta - analysis precluded the possibility of gene - gene and gene - environment interactions , as well as haplotype - based effects , suggesting that additional studies assessing these aspects will be necessary . furthermore , we only centered on anp gene t2238c polymorphism and did not cover other candidate genes or polymorphisms . it seems likely that the t2238c polymorphism individually makes a moderate contribution to risk prediction in hypertensive subjects , but whether this polymorphism integrated with other risk factors will enhance the prediction requires additional research . thus , the jury must refrain from drawing a conclusion until large , well - performed studies confirm or refuse our results . taken together , we expand previous single studies on hypertension by suggesting that anp gene t2238c polymorphism might contribute to the occurrence of hypertension , especially in population - based studies . also our observations leave open the question regarding the heterogeneous effect of 2238c allele across different ethnic populations . further genetic and functional studies are warranted to elucidate the relationship between t2238c polymorphism and hypertension , and mechanisms of the anp gene and hypertension .