The angiotensin converting enzyme insertion/deletion polymorphism and intracranial aneurysm: A meta-analysis of case-control studies
Correspondence Address: Source of Support: The Youth Project No. 30801185 (to Y.L.) and the General Program No.30872673 (to C.Y.) from the National Natural Science Foundation of China, Conflict of Interest: None DOI: 10.4103/0028-3886.115071
Source of Support: The Youth Project No. 30801185 (to Y.L.) and the General Program No.30872673 (to C.Y.) from the National Natural Science Foundation of China, Conflict of Interest: None
Context: Previous studies investigating the association between angiotensin converting enzyme (ACE) insertion (I)/deletion (D) polymorphism and intracranial aneurysm (IA) have provided inconsistent results and no large systematic review or meta-analyses have been conducted regarding this issue. Aim: To confirm whether the ACE I/D polymorphism correlates with risk of IA. Settings and Design: We conducted a meta-analysis to increase the statistical power by using all the available published data. Materials and Methods: Two investigators independently searched the PubMed, Medline, Embase, China National Knowledge Infrastructure and Chinese Biomedicine Databases for studies published before December 2012. For included studies, we performed meta-analyses using the Cochrane RevMan software. Statistical Analysis: Summary odds ratios (ORs) and 95% confidence intervals (CIs) for ACE I/D polymorphisms and IA were calculated in a fixed-effects model or a random-effects model when appropriate. We used Cochran's Q statistic and the I 2 statistic to assess heterogeneity and funnel plot to assess potential publication bias. We also carried out stratified analyses and sensitivity analyses by ethnicity, country and source of control group, sample size and Hardy-Weinberg equilibrium (HWE) in controls. Results: Six eligible studies were reviewed and analyzed, involving 854 cases and 1280 controls. Overall, compared with D allele, there was a close relationship between I allele and IA risk (OR: 1.21, 95% CI: 1.07-1.37, P = 0.003; Pheterogeneity = 0.56; I² = 0%). ACE I+ (I/I and I/D) genotype had significantly increased risk for IA (OR: 1.27, 95% CI: 1.03-1.57, P = 0.03; Pheterogeneity = 0.14; I² = 40%). This association remained consistently strong when analyses were limited to studies, in which genotype frequencies were in HWE. No publication bias was found in the present study. Conclusions: Our meta-analysis suggests, there is a close relationship between ACE I/D polymorphism and IA risk. Since limited studies and subjects were included, it is critical that larger and especially well-designed multicentric studies, which based on interactions of ACE and different confounding factors should be performed to re-evaluate the association.
Keywords: Angiotensin converting enzyme, deletion, insertion, intracranial aneurysm, polymorphisms
Intracranial aneurysm (IA) is a common disease characterized by balloon-like dilations of cerebral arteries. IA occurs in approximately 2% of the population. ,, Although most of IAs are clinically silent, rupture of these IAs cause subarachnoid hemorrhage (SAH) and result in high incidences of morbidity and mortality. ,
Patient characteristics such as cigarette smoking, hypertension and heavy alcohol consumption have been identified as risk factors of IA. , Furthermore, familial aggregation of IA indicates genetic factors may play a role in IA formation and rupture. , Due to the poor prognosis of ruptured IA, identification of genetic variants associated with increased risk of this disease may help improve diagnosis and treatment for this disease. However, the genetic and molecular pathogenesis of IA is still poorly understood and no single disease-causing gene variant has been identified. 
Over the last two decades, a number of studies have been conducted to investigate the association between angiotensin converting enzyme (ACE) insertion (I)/deletion (D) polymorphism and IAs. ,,,, A meta-analysis of two case-control studies in the year of 2000 showed that the I allele of the ACE gene might be a risk factor for this disease.  However, the results of this meta-analysis conflicted with subsequent studies.  And until recently, no large systematic review or updated meta-analysis had been conducted to examine association between ACE I/D polymorphism and IA risk. By using all the available published data to increase the statistical power, we conducted an updated meta-analysis to confirm whether the ACE I/D polymorphism increased the risk of IA.
Literature search strategy
We searched the PubMed, Medline, Embase, China National Knowledge Infrastructure and Chinese Biomedicine databases for all articles on the association between ACE I/D polymorphism and IA (last search update 11 th December 2012) with the combination of the key terms: "ACE" or "angiotensin converting enzyme" or "insertion/deletion" or "polymorphism" or "genotype" or "allele" and 'intracranial aneurysm' or 'subarachnoid hemorrhage.' All published papers in English language and Chinese language with available full text matching the eligible criteria were retrieved. The reference lists of all relevant papers and literature reviews were checked.
Study selection and inclusion criteria
Two review authors (ZC and JM) independently screened the titles, abstracts and keywords of citations obtained from the searches of the electronic databases and excluded studies that were clearly irrelevant. We obtained the full text of the remaining studies and the same two authors independently assessed, which trials met the predefined inclusion criteria, including: (1) Case-control studies conducted to evaluate the association between ACE I/D polymorphism and the risk of IA; (2) sufficient genotype data presented to calculate the odds ratios (ORs) and 95% confidence intervals (CIs); (3) papers should clearly describe the diagnosis of IA and the sources of cases and controls. Major reasons for exclusion of studies were: (1) Duplicated studies; (2) review or editorial or comment; (3) limited sample size. Any disagreements were resolved by consensus between investigators.
The following data of included studies were extracted independently by two review authors (YL and YC): The first author's name, year of publication, country/region of participant, ethnicity, source of control group (population-or hospital-based controls), number of cases and controls, genotypes, genotyping methods, treatment of IA and evidence of Hardy-Weinberg equilibrium (HWE). Population-based case-control study was defined as control from healthy people and hospital-based case-control study was defined as control from hospitalized patients. 
We performed meta-analyses using the Cochrane RevMan software (version 5.1; Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2011) and STATA software (version 11.0; STATA Corp., College Station, TX, USA). The strength of the association between the risk of IA and the ACE I/D polymorphism were estimated using ORs, with the corresponding 95% CIs.
As there are obviously ethnic variations in ACE I/D polymorphism (D/D is the wild genotype in Caucasians and I/I is the wild genotype in Asian), we used D/D as wild-type in this meta-analysis. ,
For ACE I/D polymorphism, we calculated directly study-specific ORs for the presence versus absence of IA, comparing the I allele versus D allele. We also examined the risk of the genotypes I/I or I/D on IA compared with the wild-type D/D homozygote. The risk of (I/I + I/D) versus D/D and I/I versus (I/D + D/D) for IA was evaluated in dominant and recessive models. Where feasible, we also carried out stratified analyses by ethnicity, country, source of control group, sample size and HWE in controls. We assessed and quantified statistical heterogeneity for each pooled summary estimate using Cochran's Q statistic and the I 2 statistic, respectively. Substantial heterogeneity was considered to exist when I 2 > 50% and Chi-square test P < 0.1. Meta-analysis was performed through effect model (the Mantel-Haenszel method) if there is no evidence of statistical heterogeneity. The random-effects model was employed to pool studies when statistical heterogeneity occurred. Potential publication bias was estimated by the funnel plot. Visual inspection of funnel plot asymmetry was conducted. The Egger's weighted regression method was used to statistically assess publication bias and P < 0.05 was considered representative of statistically significant publication bias.
The combined search strategy identified 137 citations. After title, abstract and full text screen, six case-control studies satisfied all inclusion criteria for final analysis, including 2134 patients. ,,,,, The literature search and study selection procedures are shown in [Figure 1].
The characteristics of selected studies are summarized in [Table 1]. Three studies were carried out in Europe and one study in USA, consisting mostly of Caucasians. The remaining two studies were carried out in Japan and China respectively, including subjects of East Asian ethnicity. The diagnosis of IA was based on digital subtraction angiography or computerized tomography angiography. Most of studies extracted deoxyribonucleic acid (DNA) from peripheral blood, with allele specific oligonucleotide-polymerase chain reaction (PCR) and Taqman PCR used for genotyping. In accordance with previous studies, , the frequency distribution of the ACE I/D polymorphism among the controls of Caucasians is different from that in Asians (II, ID, DD: 21.7%, 51.9%, 26.4% for Caucasians and 42.5%, 39.2%, 18.4% for those of Asians descent). The genotype distributions among the controls of all studies followed HWE except for one study. 
Association between ACE I/D polymorphism and IA
In the six studies with dichotomous data on IA, pooled results with 854 cases and 1280 controls showed that compared with the D allele, the I allele of ACE gene had significant increased odds of having IA (OR: 1.21, 95% CI: 1.07-1.37, P = 0.003; heterogeneity: Pheterogeneity = 0.56; I 2 = 0%) [Figure 2]. The OR of genotypes I/I or I/D versus D/D for risk of IA was 1.27, 95% CI: 1.03-1.57, P = 0.03; heterogeneity: Pheterogeneity = 0.14; I 2 = 40%. The OR of genotypes I/I versus I/D or D/D for the risk of IA was 1.31, 95% CI: 0.90-1.91, P = 0.16; heterogeneity: Pheterogeneity = 0.005, I 2 = 70%.
The association between I allele of ACE gene and risk of IA was more statistically significant in Caucasians than in Asians (OR: 1.21, 95% CI: 1.06-1.40, P = 0.006 in Caucasians versus OR: 1.20, 95% CI: 0.89-1.62, P = 0.23 in East Asians) [Table 2].
Heterogeneity and sensitivity analyses
There was substantial heterogeneity among studies in the dominant model (Pheterogeneity = 0.14) and significant heterogeneity among studies in the recessive model (Pheterogeneity = 0.005). To explore sources of heterogeneity across studies, we assessed all of the comparison models by ethnicity (Asian/Caucasian), region (USA/Europe/East Asia), source of control group (hospital-based/population-based), study sample size (>300 subjects or <300 subjects) and any source of heterogeneities when necessary. As a result, the study by Slowik et al., conducted in USA was found to contribute to substantial heterogeneity. When excluding the studies that were not in HWE, the estimated pooled OR still did not change at all [Table 2].
Begg's Funnel plot and Egger's test were performed to evaluate publication bias of the literature on IA. [Figure 3] displays a funnel plot that examines the ACE I/D polymorphism and overall IA risk included in the meta-analysis. The shape of funnel plot did not reveal any evidence of funnel plot asymmetry. The Egger's test suggested that there was no publication bias for studies of ACE associations with IA risk in the current meta-analysis (P = 0.612).
ACE is an exopeptidase that participates in the body's renin-angiotensin system (RAS) and arterial vasoconstriction by catalyzing the conversion of angiotensin I to angiotensin II. The ACE gene is located on chromosome 17q23 and is characterized by a major polymorphism involving the presence (I) or absence (D) of a 287-bp sequence of DNA in intron 16 of the gene (National Center for Biotechnology Information ref. single nucleotide polymorphisms ID: Rs. 1799752).  As a potent vasoconstrictor, ACE plays a crucial role in vascular remodeling, endothelial function and hemodynamic regulation. For this reason, a vast number of studies have investigated the association between the I/D polymorphism of ACE gene and numerous vascular disease. 
The present meta-analysis, including six case-control studies, explored the association between I/D polymorphisms of the ACE gene and IA risk. The pooled result with 854 cases and 1280 controls demonstrated there was a close relationship between I allele of ACE gene and IA risk, OR: 1.21, 95% CI: 1.07-1.37, P = 0.003. ACE I + (I/I and I/D) genotype correlates with significantly increased risk for IA, OR: 1.27, 95% CI: 1.03-1.57, P = 0.03.
Although, the association between the I allele of ACE gene and risk of IA was more statistically significant in Caucasians in stratified analysis (OR: 1.21, 95% CI: 1.06-1.40, P = 0.006 in Caucasians vs. OR: 1.20, 95% CI: 0.89-1.62, P = 0.23 in East Asians), we did not consider this to be a difference between ethnicities as the sample size of Asians was far less than Caucasians (sample size: 382 Asians vs. 1752 Caucasians). This sizable difference in sample size, may have contributed to the difference in statistical significance.
The mechanism by which the ACE I + genotype may contribute to IAs is unclear.  The study by Ohkuma et al., on patients with ruptured and unruptured IA revealed that local RAS was significantly down-regulated in ruptured and unruptured aneurysmal walls.  Ishibashi et al., also found there was no up-regulated RAS in an animal model of IA. It suggests the pathophysiological mechanism of IA is different from aortic aneurysms, which is associated with atherosclerosis induced by an up-regulated RAS.  We hypothesize that a low expression of ACE-messenger ribonucleic acid as seen with the I allele, in the aneurysmal wall may lead to increased inflammation and dysfunction of RAS, causing dilation and weakening of the injured vessel. However as IA is a complex disease, one particular genetic factor is not likely to possess a predominant role. Further large scale genetic association studies, preferentially with the analysis of gene-gene interactions are needed.
In the present meta-analysis, we searched as many publications as we could and we believe that most of the related literature have been obtained and screened in our study.
The test for detection of publication bias indicated that bias from publications, if any, might not have a significant effect on the results of our meta-analysis. However, in this meta-analysis, three limitations should be noted: Firstly, IA is a complex disease that results from the combined effects of multiple factors, including multi-genetic and environmental factors. Some factors (such as inflammation) may strongly influence the development of IA.  Neglecting to consider these factors may affect the significance for the independent role of ACE polymorphisms in IA development. Secondly, there was substantial heterogeneity among studies in the dominant model and significant heterogeneity among studies in recessive model. Although the study by Slowik et al., conducted in USA was found to contribute to this substantial heterogeneity, the reason of the heterogeneity cannot be determined in available data. Thirdly, the number of studies and the number of subjects in the studies included in the meta-analysis were limited, especially for stratified analysis of each ethnicity and area. Therefore, our results in relation to the association of ACE I/D polymorphisms and IA risk should be treated as preliminary and additional meta-analyses with a large number of papers are necessary to validate the association.
Our meta-analysis suggests there is a close relationship between ACE I/D polymorphism and IA risk. The I allele of ACE gene correlates with increased the risk for IA. Since limited studies and subjects were included, it is critical that larger and especially well-designed multicentric studies based on different ethnic populations should be performed to re-evaluate the association. Moreover, further researches to explore the gene-gene interactions and gene-environment interactions should be carried out to provide a better, comprehensive understanding of the pathogenesis of IA.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]