Association of polymorphisms in the AGT gene(M235T,T174M) with ischemic stroke in the Chinese population

Abstract

Objective:

The angiotensinogen (AGT) gene polymorphisms has been shown to be involved in the development of ischemic stroke. However, the published studies have yielded inconsistent results. We performed a meta-analysis to assess the correlation.

Methods:

Published literature from PubMed, EMBASE, CNKI and Wanfang Data was searched. The association was assessed by odds ratio (OR) with 95% confidence intervals (CI). Publication bias was also calculated.

Results:

Eight studies (1636 cases and 1433 controls) on M235T polymorphism and four studies (726 cases and 495 controls) on T174M polymorphism were included in the meta-analysis. The results showed that M235T polymorphism was significantly associated with risk of ischemic stroke risk (TT vs. MM: OR=2.60, 95% CI=1.77–3.83; MT vs. MM: OR=1.37, 95% CI=1.01–1.86; TT+MT vs. MM: OR=0.43, 95% CI=0.35–0.54; MM+MT vs. TT: OR=1.74, 95% CI=1.31–2.32). There was also significant association between T174M polymorphism and ischemic stroke risk (MM vs. TT: OR=3.66, 95% CI=1.89–7.08; TT+MT vs. MM: OR=0.28, 95% CI=0.14–0.54). Further sensitivity analysis confirmed the significant association between AGT gene polymorphisms and risk of ischemic stroke. No evidence indicated publication bias.

Conclusions:

The meta-analysis indicated the significant association of AGT gene polymorphisms (M235T, T174M) with risk of ischemic stroke in the Chinese population.

Keywords

Angiotensinogen gene polymorphism ischemic stroke meta-analysis Chinese

Introduction

Stroke has become one of the leading causes of death and disability in China. Ischemic stroke is the most common form, accounting for more than 70–75% of cases.¹ Traditional risk factors for ischemic stroke include old age, hypertension, previous stroke, high cholesterol, tobacco smoking, and atrial fibrillation.^2,3 However, traditional risk can only explain a small proportion of the observed clinical events. A large proportion of the population at attributable risk for ischemic stroke has remained unexplained.⁴ Twin and familial aggregation studies suggest that genetic factors also play key roles in the pathogenesis of this disease.⁵ However, previous studies have provided limited consistent evidence of ischemic stroke genetic loci, suggesting that very a large sample size is needed to identify variants that have only small effects.

The renin–angiotensin system (RAS) and the sympathetic nervous system maintain sodium and electrolyte balance and act as intra-cellular messengers. This has been suggested to play an important role in the regulation of blood pressure and pathogenesis of ischemic stroke.⁶ In the RAS, angiotensinogen (AGT) is converted into angiotensin I by renin and subsequently to into angiotensin II, a vasoconstrictor, by angiotensin-converting enzymes. Angiotensin II, a potent pressor substrate, stimulates aldosterone biosynthesis. In the central nervous system, angiotensin II also stimulates drinking and increases vasopressin and adrenocorticotropic hormone.⁷

Although AGT may be implicated in the pathogenesis of stroke, its genetic determinants remain unknown. The AGT gene is located at lq42-43, which contains five exons. Two variations of AGT gene loci, substitutions of methionine to threonine at residue 235 (M235T) and threonine to methionine at 174 (T174M), are the most frequent polymorphisms. Serum AGT levels were shown to be increased within the heart and kidney in subjects carrying the 235T allele.⁸ Thus, it is here speculated that the AGT gene is involved in the pathogenesis of ischemic stroke by affecting blood pressure.

To date, M235T and T174M polymorphisms have been linked to susceptibility to hypertension and coronary heart disease.^9,10 In addition, several studies have found an association between AGT gene M235T and T174M polymorphisms and ischemic stroke. However, the published findings are still controversial. One previous meta-analysis indicated that the M235T polymorphism is associated with ischemic stroke risk in Asians, but not in Caucasians.¹¹ However, that meta-analysis included only a few limited papers that covered individuals of Chinese ancestry. Most of the relevant studies performed in Chinese participants were published in Chinese in local journals. Therefore, it is very important to clarify the relationship in the Chinese Han population. In the present study, we performed a meta-analysis to further clarify the association between AGT gene M235T and T174M polymorphisms and ischemic stroke in a Chinese population.

Materials and methods

Selection of published studies

PubMed, EMBASE, CNKI and Wanfang Wan Fang Data were searched for eligible articles. The following terms were used as search keywords: [(“polymorphism” or “polymorphisms” or “variant”) and (“AGT” or “angiotensinogen”) and (“ischemic stroke” or “stroke”)]. The reference lists of the retrieved reviews and articles were hand-searched. The publication languages were restricted to English and Chinese. If more than one article was published using the same study population, only the study with largest sample size was selected. When pertinent data were not included, or data that were presented were unclear, the authors were contacted directly. The literature search was updated on 30 June 2014.

Inclusion criteria and data extraction

The resulting reports were filtered using the following inclusion criteria: (i) using a case-control or cohort design; (ii) evaluation of the association between M235T and T174M polymorphisms in the AGT gene and ischemic stroke in a Chinese population; (iii) more than 50 cases; and (iv) providing sufficient data for the calculation of an odds ratio (OR) with 95% confidence interval (CI).

The following information was extracted from each study: (i) name of the first author; (ii) year of publication; (iii) region; (iv) sample size of cases and controls; (v) genotype distributions of cases and controls; and (vi) p-value for the test of Hardy–Weinberg equilibrium (HWE) in controls. All of the data are shown in Table 1. Two investigators independently extracted the data with a standard protocol and the result was reviewed by a third investigator. Discrepancies were resolved by discussion with our research team.

Table 1.

Characteristics of papers included in the meta-analysis.

Study included	Year	Region	Cases/controls	Genotypes for cases	Genotypes for controls	HWE test
				MM/MT/TT	MM/MT/TT
M235T variant
Guan et al.¹⁴	2000	Shandong	102/82	12/37/53	10/37/35	0.96
Zhang et al.¹⁵	2001	Qingdao	75/48	6/21/48	8/22/18	0.77
Liu et al.¹⁶	2004	Shanghai	90/90	3/20/67	8/24/58	0.03
Cui et al.¹⁷	2008	Quangdong	114/76	2/37/75	3/37/36	0.08
He et al¹⁸	2008	Neimenggu	77/98	3/25/49	12/52/34	0.24
Du et al.¹⁹	2010	Beijing	664/716	8/15/59	11/35/49	0.23
Sun et al.²⁰	2010	Neimenggu	180/130	7/59/114	16/69/45	0.18
Zhang et al.²¹	2010	Hainan	334/193	227/91/16	147/40/6	0.12
T174 M variant
Liu et al.¹⁶	2004	Shanghai	90/90	5/12/73	0/11/79	0.54
Yue et al.²²	2008	Hebei	82/82	1/12/69	2/20/60	0.83
Sun et al.²⁰	2010	Neimenggu	180/130	39/22/119	9/13/108	0.00
Zhang et al.²¹	2010	Hainan	374/193	4/47/283	0/41/152	0.10

Statistical methods

The association of M235T and T174M polymorphisms in the AGT gene with ischemic stroke was estimated by calculating pooled OR and 95% CI under co-dominant, dominant and recessive genetic models. The effect of heterogeneity was quantified using I², which ranges between 0% and 100% and represents the proportion of study variability attributable to heterogeneity rather than chance. I² values of 25%, 50%, and 75% were nominally defined as low, moderate, and high estimates, respectively. When I² >50%, there was considerable indicated heterogeneity across studies, and the random-effects model was used for meta-analysis. Otherwise, the fixed-effects model was used.¹² Sensitivity analysis was performed by removing the studies not in HWE. Begg’s test was used to provide evidence of publication bias, which was shown as a funnel plot (p<0.05 was considered statistically significant).¹³ Meta-analysis was performed using the STATA package version 12.0 (Stata Corporation, College Station, TX, USA Texas).

Results

Study characteristics

The search generated 35 association studies, of which eight met the selection criteria.^14–21 The flowchart of literature screening is presented in Figure 1, and the characteristics of the selected studies included in this meta-analysis are provided in Table 1. For the M235T polymorphism, eight case-control studies were available, including a total of 1636 cases and 1433 controls. The genotype distributions among the controls of all studies were consistent with HWE except for Liu et al.¹⁶ For the T174M polymorphism, four studies involved a total of 726 cases and 495 controls. Only one of the four studies deviated from HWE.²⁰ In addition, the controls in two of these eligible studies were based on common populations, while the others were based on hospital populations.^19,22 Only one eligible study was based on common populations,¹⁹ while the others were based on hospital populations. All studies used polymerase chain reaction/restriction fragment length polymorphism with various restriction enzymes for genotyping. The publication year of the included studies ranged from 2000 to 2010.

Figure 1.

Flow chart of study selection based on the inclusion and exclusion criteria.

Quantitative synthesis

The combined results of AGT gene polymorphisms and ischemic stroke risk are summarized in Table 2. The results of the overall meta-analysis indicated a significant association between AGT M235T polymorphism and ischemic stroke risk (TT vs. MM: OR=2.60, 95% CI=1.77–3.83; MT vs. MM: OR=1.37, 95% CI=1.01–1.86; TT+MT vs. MM: OR=0.43, 95% CI=0.35–0.54; MM+MT vs. TT: OR=1.74, 95% CI=1.31–2.32, Figure 2).

Table 2.

Summary ORs and 95%CI of AGT gene polymorphisms and ischemic stroke risk.

Genetic model	Sample size	Type of model	Test of heterogeneity	Test of association	Test of publication bias
	Case Control		I², P	OR 95% CI	z, P
M235T
TT vs. MM	1636, 1433	Fixed	14.6%, 0.32	2.60 1.77–3.83	0.00, 1.00
MT vs. MM		Fixed	0.00%, 0.70	1.37 1.01–1.86	0.00, 1.00
TT+MT vs. MM		Fixed	0.00%, 0.50	0.43 0.35–0.54	0.00, 1.00
MM+MT vs.TT		Fixed	13.4%, 0.33	1.74 1.31–2.32	0.00, 1.00
T174M
MM vs. TT	726, 495	Fixed	18.1%, 0.30	3.66 1.89–7.08	0.34, 0.73
MT vs. TT		Random	51.0%, 0.11	0.84 0.51–1.38	0.34, 0.73
MM+MT vs.TT		Random	83.3%, 0.00	1.10 0.51–2.36	0.34, 0.73
TT+MT vs. MM		Fixed	8.50%, 0.35	0.28 0.14–0.54	0.34, 0.73

Figure 2.

Forest plot of the association of the AGT M235T polymorphism with ischemic stroke risk.

For the T174M polymorphism, a significant association was found between T174M polymorphism and ischemic stroke risk (MM vs. TT: OR=3.66, 95% CI=1.89–7.08; MT vs. TT: OR=0.84, 95% CI=0.51–1.38; MM+MT vs. TT: OR=1.10, 95% CI=0.51–2.36; TT+MT vs. MM: OR=0.28, 95% CI=0.14–0.54)

Sensitivity analysis

A sensitivity analysis was performed by removing the studies by Liu et al. and Sun et al. due to the genotype distribution in the control groups deviating from HWE.^16,20 No material alteration was detected, suggesting that the data in this meta-analysis were relatively stable and credible.

Publication bias analysis

The funnel plot and Begg’s test were used to assess publication bias. There was no evidence of publication bias in our study (Table 2). The results suggest that the publication bias was low in the present meta-analysis.

Discussion

Ischemic stroke is a multifactorial disease and its pathogenesis is not yet fully understood. The underlying mechanisms of ischemic stroke have been shown to comprise both genetic and environmental factors, and some inherited susceptibility genes with single nucleotide polymorphisms may exert their effects in ischemic stroke development.²³ The RAS plays an important role in the regulation of blood pressure, vascular remodeling and sodium homeostasis.²⁴ AGT is one of the components of the RAS. Over the past decade, several studies have focused on the association between two polymorphisms (M235T, T174M) in the AGT gene and ischemic stroke. However, the results have been inconsistent. The most likely reason for the inconsistencies among these studies is that they are single case-control studies with small sample sizes. We conducted this meta-analysis to combine the same kind of studies, in order to increase the sample size and statistical power, and thereby produce a more authentic result.

To our knowledge, this is the first meta-analysis that comprehensively assesses the associations between AGT gene M235T polymorphism and risk of ischemic stroke in the Chinese population. In the current meta-analysis, we examined the association between AGT gene M235T polymorphisms (M235T, T174M) and the risk of ischemic stroke by critically including all published studies. The results indicated that both M235T and T174M polymorphism were associated with risk of ischemic stroke risk in the Chinese population. Our findings were consistent with the previous meta-analysis that M235T polymorphism had a significant association with ischemic stroke risk in Asians.¹¹ Further sensitivity analysis confirmed the significant association between AGT M235T and T174M polymorphisms and risk of ischemic stroke risk. There was no evidence of publication bias in this meta-analysis with respect to the association between AGT gene M235T polymorphism and susceptibility to ischemic stroke. As the number of eligible studies was small in this meta-analysis of T174M polymorphism, these results still need further confirmation.

The mechanism by which the AGT gene M235T and T174M polymorphism affects ischemic stroke risk is currently unclear. Serum AGT levels were shown to be increased within the heart and kidney in subjects carrying the 235T allele.⁸ AGT interacts with renin to produce angiotensin II, and angiotensin II triggers vascular cell apoptosis and contributes to vascular remodeling in the ischemia-reperfusion process, thus rendering the vasculature more vulnerable to adverse cerebrovascular remodeling after hypertension.²⁵ In addition, for T174M polymorphism, the serum AGT level was not changed in subjects with the T174M variant.²⁶ The M235T polymorphism was in strong linkage disequilibrium with the T174M polymorphism.²⁷ The linkage disequilibrium of M235T and T174M may synergistically increase the risk of ischemic stroke.

The present meta-analysis has some limitations. First, it was based primarily on unadjusted effect estimates and the confounding factors were not controlled for. Second, although all cases and controls of each study were well defined with similar inclusion criteria, there may have been factors that were not taken into account that may have influenced our results. Third, the effect of potential gene–gene and gene–environment interactions could not be addressed in this meta-analysis at this time. In spite of these limitations, the current meta-analysis also had some advantages. First, a comprehensive search strategy based on computer-assisted and manual searching allowed eligible studies to be included as extensively as possible. Second, no publication bias was found, which leads to a possibly robust result.

In conclusion, results indicated the significant association of two AGT polymorphisms (M235T, T174M) with ischemic stroke risk in the Chinese population. However, further studies taking into account gene–gene and gene–environment interactions should be conducted to investigate these associations.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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