Association of T174M polymorphism of angiotensinogen gene with essential hypertension: A meta-analysis

The association between T174M polymorphism of angiotensinogen gene and essential hypertension risk remains controversial. We herein performed a meta-analysis to achieve a reliable estimation of their relationship. All the studies published up to May 2013 on the association between T174M polymorphism and essential hypertension risk were identified by searching the electronic repositories PubMed, MEDLINE and EMBASE, Springer, Elsevier Science Direct, Cochrane Library and Google Scholar. Data were extracted and pooled odds ratios (ORs) with 95% confidence intervals (95% CIs) were calculated. Ultimately, nine eligible studies, including 2188 essential hypertension cases and 2459 controls, were enrolled in this meta-analysis. No significant associations were found under the overall ORs for M-allele comparison (M vs. T, pooled OR 0.92, 95% CI 0.62–1.37), MM vs. TT (pooled OR 0.86, 95% CI 0.29–2.51), TM vs. TT n (pooled OR 0.91, 95% CI 0.63–1.32), recessive model (MM vs. TT+TM, pooled OR 0.89, 95% CI 0.35–2.30), dominant model (MM+TM vs. TT, pooled OR 0.91, 95% CI 0.60–1.38) between T174M polymorphism and risk for essential hypertension. This meta-analysis suggested that the T174M polymorphism of the angiotensinogen gene might not be associated with the susceptibility of essential hypertension in Asian or European populations.


Introduction
Hypertension affects approximately 30% of adults in industrialized countries and is the major risk factor for cardiovascular disease (Nguyen et al., 2013). Hypertension is classified into essential hypertension and secondary hypertension according to the etiology. Secondary hypertension is a type of hypertension caused by an identifiable underlying secondary cause, such as renovascular disease, renal failure, pheochromocytoma, aldosteronism and others, while essential hypertension is defined as hypertension in which secondary causes are not present (Carretero and Oparil, 2000). More than 90% of all hypertensive persons are reported to have essential hypertension (Ghosh et al., 2013). Essential hypertension is associated with large and small vascular remodeling that impacts cardiovascular prognosis (Briet and Schiffrin, 2013). It is generally considered as a paradigmatic multi-factors disease which involves a combination of genetic factors, environmental stimuli and their interaction (O'Shaughnessy, 2001). It has been reported that approximately 20-60% of the inter-individual variation in blood pressure is genetically controlled (Kurtz and Spence, 1993). Thus, we hypothesized that exploring potential hypertension susceptibility genes would help us to better understand the etiology of the disease and, eventually, to better control this disease.
Angiotensinogen (AGT) is a liver protein that interacts with renin to produce angiotensin I, the prohormone of angiotensin II, which is the major effector molecule of the renin-angiotensin-aldosterone system (Gu et al., 2011). It is a promising candidate gene for evaluating susceptibility to essential hypertension (Mohana et al., 2012). T174M polymorphism refers to the substitution of threonine to methionine amino acid at position 174 in exon 2 of the AGT gene. A significant association between T174M polymorphism and the risk of essential hypertension has been reported in several studies (Iso et al., 2000;Jiang et al., 2009). However, several other studies did not detect such an association Wang et al., 2002;Nejatizadeh et al., 2008). Therefore, the T174M polymorphism was conflictingly associated with essential hypertension.
In order to achieve an integrative understanding of the association between the T174M polymorphism and the risk of essential hypertension it is necessary to consider the findings as a whole, paying special attention to methodological characteristics of each of the studies. Accordingly, we conducted a systematic review of published findings and used meta-analysis techniques to quantitatively combine the results. This allowed us comprehensively investigate the association between T174M polymorphism and the risk of essential hypertension.

Materials and Methods
Sources for the literature search A literature search was conducted for studies that reported the effect of T174M polymorphism on the risk of essential hypertension. We systematically searched the PubMed, MEDLINE and EMBASE, Springer, Elsevier Science Direct, Cochrane Library and Google Scholar electronic databases for articles published up to May 2013. The following key words were used for searching: "T174M", "hypertension", "angiotensinogen gene", "polymorphism", "variants", "study" or "trial". Furthermore, complimentary searches in the references lists of retrieved papers were performed for any additional studies. The research was restricted to full-text English-language articles of studies in humans.

Search methods
Six investigators (XY. L, ZY. Y, DQ. P, H. D, Y. L and Q. Z) independently searched the electronic databases. The abstracts were first reviewed to obtain potentially eligible articles according to the inclusion criteria. To avoid a possible loss of any relevant article, an additional search was performed through the references cited in identified articles.

Included and excluded criteria of studies
Inclusion criteria of studies Studies were included in the current meta-analysis if they met the following criteria: (1) the investigated patients suffered from essential hypertension (prospective studies, retrospective studies, or cross-sectional studies, etc.); (2) they evaluated the relationship between T174M polymorphism and essential hypertension risk; (3) they had an odds ratio (OR) with an 95% confidence interval (CI), or other available information for estimating the OR (95% CI); (4) they provided available genotype data for the T174M polymorphism.

Exclusion criteria of studies
Studies were excluded if they met the following criteria: (1) they were designed based on family or sibling pairs; (2) the genotype frequency of the T174M polymorphism was not reported; (3) they did not detect an association between T174M polymorphism and susceptibility of essential hypertension; (4) there was insufficient information for extraction of data.

Evaluation of quality and extraction of data
The quality of the included studies was assessed using a ten-point scoring sheet, as previously described (Clark and Baudouin, 2006). The factors including control group, Hardy-Weinberg equilibrium, case group, primer, reproducibility, blinding, power calculation, statistics, corrected statistics and independent replication were evaluated and the score was recorded as 1 if present or 0 if absent. A final quality score was obtained by summation of each component. Two investigators completed the evaluation independently, and any difference was settled by discussion to reach an agreement between these two investigators.
Two investigators (XY. L and H. Y) independently extracted data according to the inclusion criteria listed above. The extracted data included the first author's name, year of publication, country, sample size, participants, study design, genotyping methods, source of control group, and distribution of T174M polymorphism within the participants of the case vs. control group in all studies. Some of these data items were obtained from the author of the primary study. Disagreements were resolved by discussion and reaching an agreement among all investigators, or by contacting the original investigators.

Meta-analysis methods
The strength of the association between T174M polymorphisms and essential hypertension risk was determined by an OR with 95% CI. A chi-square test was used to determine whether or not the observed frequencies of genotypes in the controls conformed to HWE (Hardy-Weinberg expectations), and a p-value < 0.05 was considered as significant disequilib-rium. Pooled ORs were calculated to examine the contrast of A-allele comparison (M vs. T), MM vs. TT, TM vs. TT, recessive model (MM vs. TT+TM) and dominant model (MM+TM vs. TT), respectively. The significance of pooled ORs was determined by a Z-test and a p-value < 0.05 was considered as statistically significant. We assessed the within-and between-study variation or heterogeneity by Cochran's Q-statistic testing (Deeks et al., 2001) and calculating the I 2 index (I 2 = 100%(Q-df)/Q) (Higgins et al., 2003). A significant Q-statistic (p < 0.10) or I 2 > 50% indicated heterogeneity across studies, so the pooled OR estimate was calculated using the random-effects (DerSimonian and Laird, 1986). Otherwise, the fixed-effect (Mantel and Haenszel, 1959) was used. Publication bias was evaluated by using the Egger's linear regression test (Egger et al., 1997), which measures funnel plot asymmetry on the natural logarithm scale of the effect size. Analyses were performed using the STATA software package v.11.0 (Stata Corporation, College Station, TX, USA).

Characteristics of the eligible studies
We retrieved 1037 records that were potentially relevant to the search terms (PubMed: 312; MEDLINE: 136; Springer: 198; Elsevier Science Direct: 105; Cochrane Library: 21; Google Scholar: 265). The study selection process is summarized in Figure 1. After duplicates were removed, this number went down to 129 potentially relevant studies. By screening the abstracts, 89 of these articles were excluded, these including 31 review articles, 23 articles irrelevantto the T174M gene, and 35 articles irrelevantto essential hypertension. The remaining 40 studies were then examined in detail by full text review, this leading to the exclusion of 31 articles, including 17 publications that not about the T174M gene and 14 for not showing available data.
The final result were nine studies (Iso et al., 2000;Sato et al., 2000;Vasku et al., 2002;Wang et al., 2002;Nair et al., 2003;Tsai et al., 2003;Nejatizadeh et al.,2008;Jiang et al., 2009;Yuan et al., 2009). These involved 2188 essential hyper cases and 2459 controls, and by meeting the inclusion criteria they were included in this meta-analysis. All of them were case-control studies. The characteristics of these selected studies are summarized in Tables 1 and 2. The included studies were published between 2000 and 2009. The sample sizes ranged from 203 to 919, and source of control groups were normotensives. Eight studies were conducted in Asian populations and one in a European. Furthermore, the observed genotype counts for T174M polymorphisms in each study were all consistent with Hardy-Weinberg disequilibrium (p > 0.05).

Meta-analysis of the association between T174M polymorphism and risk for essential hypertension
Crude ORs with 95% CIs were used to assess the association between T174M polymorphism and risk of essential hypertension. Significant heterogeneities were observed across the studies for M-allele comparison (M vs. T, p < 0.01 and I 2 = 88.2%), MM vs. TT comparison (p < 0.01 and I 2 = 73.0%), TM vs. TT comparison (p < 0.01 and I 2 = 82.9%), recessive model (MM vs. TT+TM, p < 0.01 and I 2 = 65.4%) and dominant model (MM+TM vs. TT, p < 0.01 and I 2 = 87.1%) (Table 3). Therefore, the random effect model was applied to estimate the overall ORs.
In the present study, no significant difference was observed in the risk of essential hypertension between the genotypes M vs. We also performed a subgroup analysis stratified by geographic location (ethnicity) and sample size. As shown in Table 3, this refined further analysis still did not indicated an association between T174M polymorphism and essential hypertension risk.

Publication bias
The Egger's test was performed to assess the publication bias of the published studies. For all comparisons, the p value of the Egger's test was higher than 0.05 (Table 4), this indicating that there was no evident publication bias in this meta-analysis.

Discussion
The association between the T174M polymorphism and risk for essential hypertension was not clear due to inconsistent data generated by a range of independent studies (Nair et al., 2003;Tsai et al., 2003;Jiang et al., 2009;Nejatizadeh et al., , 2008). Therefore, we performed a meta-analysis of published studies to clarify the inconsistency and to establish a comprehensive picture of this gene-disease association. All studies included in this analysis were conducted in Asian or European populations with high prevalence of essential hypertension. In our metaanalysis, a total of nine studies were included, these comprising 2188 cases and 2459 controls. The conclusion is that no significant association was detected between the T174M polymorphism and essential hypertension risk.
The findings of the present study were different from the results of a previous meta-analysis conducted by Pereira et al. (2008), in which a codominant model of T174M polymorphism demonstrated a significant increase in the risk of essential hypertension in Asians (10 studies were included and four of them were Chinese) and in mixed/other populations (including African, Indo-European/East Asian, Russian Arab and Turkish) but not in a population of European descendants. Upon comparing these two metaanalyses, we found that two studies (Morise et al., 1995;Yi-Yang et al., 2006) conducted among Asian populations (one in Japan and one in Chain) were included in the analysis of Pereira et al. (2008) but not in ours. These two studies indicated a significantly increased risk of essential hypertension among populations carrying the 174M allele. This may be one reason for the different outcomes of these two meta-analyses. Furthermore, it should be noted that in the study of Pereira et al. (2008) a potential publication bias was present.
The AGT gene T174M polymorphism was first reported by Jeunemaitre et al. (1992) to be related to the prevalence of essential hypertension. Since then several studies on this relationship, enrolling various ethnic groups, have been published. Some studies confirmed the association between T174M variant and hypertension (Gardemann et al., 1999;Procopciuc et al., 2005), but others did not (Nair et al., 2003;Renner et al., 2005). Moreover, some studies T174M polymorphism with essential hypertension 477 reached different conclusions, even when they were performed among the same ethnic populations (Qi et al., 2007;Gu et al., 2011). There are several potential explanations for the discrepancies among these studies. The first possible cause may be genetic differences in the population samples and phenotypic differences in the hypertensive populations analyzed (Marco et al., 2005). Second, the effect of the T174M variant on plasma AGT levels may vary among different ethnic groups so that the association between T174M polymorphism and risk of essential hypertension may not be detected in every ethnic group, just as shown in the study of Pereira et al. (2008). Most studies included in current meta-analysis were conducted in Asian populations and only one in a European population, this meaning that the final result may not be generalizable. Therefore, more studies need to be performed in various ethnic groups and subgroup analysis stratified by ethnicity should be conducted in future research. Second, the reliability of the findings of a study may be questionable if it lacks an appropriate control group. In some studies, the age and percentage of subjects with alcohol consumption or smoking were significantly different between the hypertensive and control groups (Hingorani et al., 1996;Kiema et al., 1996). A third possible cause of discrepancy may be a multiple interaction of polymorphisms or genes. These gene-to-gene interactions make the association of hypertension with any single candidate gene more complex (Wang et al., 2002). In addition, the numbers of cases were different in various studies, this leading to variation in precision. Finally, hypertension is an acknowledged multifactorial disease. Genetic factors may interact with several other factors, such as salt intake, body mass index, and smoking in the development of hypertension.
Certain limitations of this study need to be mentioned. First of all, heterogeneity across the studies was observed. One possible reason for the presence of heterogeneity is the wide variation in populations included (77.7% in Asian and 11.1% in European). Second, the effect of genetic and environmental interactions was not considered. In addition, all the included articles were case-control studies and the number of studies was small (nine). Therefore, more and high-quality case-control studies are still needed to test and verify the results of this metaanalysis. Finally, our analysis was limited to eight studies on Asian and one on European populations, which is certainly not representative of all ethnicities. The association between the T174M polymorphism and risk of essential hypertension still needs to be clarified.
Despite these limitations, this meta-analysis suggests that the T174M polymorphism of the angiotensinogen gene is not associated with susceptibility of essential hypertension, and the allele M may not increase the risk of essential hypertension in Asian or European populations. Considering the different genetic and phenotypic features among different ethnic groups, larger and well-designed studies based on different populations are needed to confirm our results.