Angiotensin-converting enzyme insertion/deletion gene polymorphism and risk of intracranial aneurysm in a Chinese population

Abstract

Objective

The relationship between angiotensin-converting enzyme (ACE) insertion/deletion (I/D) gene polymorphisms and intracranial aneurysm (IA) has been studied in Caucasian and Japanese populations. The present study aimed to investigate this association in a Chinese population.

Methods

Patients with confirmed IA and age- and sex-matched control subjects without evidence of IA were enrolled. ACE I/D gene polymorphisms were analysed using polymerase chain reaction–restriction fragment length polymorphism.

Results

A total of 220 patients with IA and 220 matched controls were enrolled. In the IA group, 64, 106 and 50 patients were of the II, ID and DD genotypes, respectively, compared with 44, 99 and 77 subjects in the control group. The ACE DD genotype and D allele frequencies were significantly lower in the IA group compared with the control group. There were no statistically significant differences in the site, shape, size and Fisher Grade of aneurysms between genotypes in patients with IA.

Conclusion

The ACE DD genotype may be a protective factor for IA in a Chinese population.

Keywords

Intracranial aneurysm angiotensin-converting enzyme gene polymorphism

Introduction

Intracranial aneurysm (IA) occurs in ∼2% of the population worldwide, meaning that over 20 million people in China will experience an IA.¹ It is ∼1.6-times more common in women than in men, and the majority of ruptured IAs is observed in people between the ages of 40 and 60 years.² Rupture of an IA accounts for >90% of subarachnoid haemorrhage (SAH) cases.³ Despite diagnostic and therapeutic advances, ∼50% of SAH cases are fatal and cause considerable disability in ∼30% of affected individuals.^4–6 IA is a complex disease with both environmental and genetic components,^7–9 the latter of which has prompted many studies on the genetic determinants of IA in the last 10 years.^7–9

The renin–angiotensin–aldosterone system (RAAS) is essential to cardiovascular haemodynamics and plays an important role in the development of hypertension and cerebrovascular disease.¹⁰ Angiotensin-converting enzyme (ACE) is an important circulating enzyme in the RAAS that catalyses the conversion of angiotensin I to angiotensin I, and the degradation of bradykinin.¹⁰ Plasma and tissue concentrations of ACE are determined by the ACE gene (chromosome 17q23).¹⁰ The ACE gene has a functional insertion/deletion (I/D) polymorphism of a 287 base pair (bp) Alu sequence within intron 16.¹¹ ACE DD carriers have higher local levels of ACE than individuals with the ID and II genotypes.^11,12 These polymorphisms have, therefore, been extensively studied in several adult cardiovascular diseases including ischaemic stroke,¹³ coronary heart disease¹⁴ and intracerebral haemorrhage.^15–20

Until now, the relationship between ACE I/D polymorphisms and IA has only been studied in Caucasian populations and in a single study from Japan.^20–24 The aim of the present hospital-based, case–control study was to investigate the association between ACE I/D polymorphisms and the risk of IA in a Chinese population.

Patients and methods

Study population

Peripheral blood specimens, demographic information and medical and family histories were obtained from sequentially ascertained, unrelated patients admitted to the West China Hospital of Sichuan University, Chengdu, China between July 2010 and June 2012. Aneurysms in cases were confirmed and classified by magnetic resonance imaging (MRI) or computed tomography (CT)²⁵ and cerebral angiography. Age- and sex-matched control subjects met the following criteria: confirmation of the absence of IA by digital subtraction angiography, MR angiography, or three-dimensional CT angiography; no medical history of any vascular disease, including IA or SAH; no family history of IA or SAH in first-degree relatives; were from the same geographic region as the cases. Healthy control subjects were recruited from the Department of Neurosurgery, West China Hospital of Sichuan University where they were attending a clinic for routine examination.

The Ethical Committee of the West China Hospital of Sichuan University approved the study protocols, and all participants gave written informed consent according to the Declaration of Helsinki.

DNA extraction and genotyping

Leucocyte DNA was extracted from 10 ml of venous blood, anticoagulated with 1.6 mg/ml ethylenediaminetetra-acetic acid using a commercially available kit (Wizard® Genomic DNA Purification Kit, Promega, Madison, WI, USA) and stored at 4℃ until analysis. ACE I/D gene polymorphisms were analysed using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). PCR was performed in a final volume of 100 µl containing 1 µg genomic DNA, 100 pmol of each primer, 3 mmol/l magnesium chloride, 50 mmol/l potassium chloride, 10 mmol/l Tris-HCl (pH 8.4) and 0.5 U of Taq polymerase (Perkin Elmer, Foster City, CA, USA). Based on the GenBank reference sequence, the following PCR primers were used: forward-5′-CTGGAGACCACTCCCATCCTTTCT-3′ and reverse-5′-GATGT GGCCATCACATTCGTCAGAT-3′. DNA was denatured at 94℃ for 5 min, followed by 30 cycles of denaturation at 94℃ for 1 min, annealing at 67℃ for 1 min and extension at 72℃ for 2 min, with a final extension step of 5 min at 72,℃ using an Applied Biosystems® GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). PCR products (490-bp insertion and 190-bp deletion) were separated by 2% agarose gel electrophoresis, stained with ethidium bromide and viewed with ultraviolet light. β-actin was used as an internal control gene. To confirm the genotyping results, > 10% of PCR-amplified DNA samples were randomly selected by computer and sent for DNA sequencing.

Statistical analyses

All statistical analyses were carried out using SPSS® statistical software, version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Differences between continuous variables and between categorical variables were assessed by Student's t-test and Pearson’s Χ²-test, respectively. Pearson’s Χ²-test was also used to examine any differences in allelic and genotypic frequencies between different groups, and odds ratios (OR) and 95% confidence intervals (CI) were calculated. Two-sided P-values were used, with P < 0.05 considered statistically significant. Genotype distribution and allele frequencies were compared to the Hardy–Weinberg equilibrium model using Pearson’s Χ²-test.

Results

The study population consisted of 220 patients with IA and 220 matched control subjects. Demographic characteristics of patients with IA and controls are summarized in Table 1. There was no statistically significant difference between groups with respect to age, sex, or prevalence of risk factors including hypertension, smoking and alcohol consumption. Clinical characteristics of aneurysms including location, site, shapes, sizes and Fisher Grade²⁵ are shown in Table 1.

Table 1.

Demographic and clinical characteristics of Chinese patients with intracranial aneurysm (IA), and age-and sex-matched control subjects.

Characteristic	Patients with IA n = 220	Controls n = 220
Sex
Male	95 (43.2)	103 (46.8)
Female	125 (56.8)	117 (53.2)
Age, years	47.4 ± 11.3	45.6 ± 10.7
Hypertension
Yes	82 (37.3)	75 (34.1)
No	138 (62.7)	145 (65.9)
Smoking
Ever	56 (25.5)	54 (24.5)
Never	164 (74.5)	166 (75.5)
Alcohol consumption
Ever	47 (21.4)	45 (20.5)
Never	173 (78.6)	175 (79.5)
Family history of IA
Yes	5 (2.3)
No	215 (97.7)
Site of aneurysm
ACA	98 (44.6)
ICA	76 (34.5)
MCA	46 (20.9)
Shape of aneurysm
Saccular	178 (80.9)
Fusiform	33 (15.0)
Dissecting	9 (4.1)
Size of aneurysm
Small, <15 mm	155 (70.5)
Larger, 15 – 25 mm	56 (25.4)
Giant, >25 mm	9 (4.1)
Fisher Grade^a
1	24 (10.9)
2	101 (45.9)
3	65 (29.6)
4	30 (13.6)

Data presented as n (%) of patients or mean ± SD.

Fisher Grade classifies the appearance of subarachnoid haemorrhage (SAH) on computed tomography scan: 1, no haemorrhage evident; 2, SAH < 1-mm thick; 3, SAH > 1-mm thick; 4, SAH of any thickness with intraventricular haemorrhage or parenchymal extension.²⁵

There were no statistically significant differences (P ≥ 0.05) between study groups with respect to any demographic variable (determined by Student's t test for continuous variables and Pearson’s Χ²-test for categorical variables).

ACA, anterior cerebral artery; ICA, internal carotid artery; MCA, middle cerebral artery.

The ACE genotypes of patients with IA and matched controls are presented in Table 2. All individual single nucleotide polymorphisms (SNPs) were within the Hardy–Weinberg equilibrium model in both study groups. The ACE DD genotype (P = 0.003) and D allele frequencies (P = 0.002) were significantly less frequently found in patients with IA compared with controls (Table 2). When stratified by the site, shape, size and Fisher Grade, no statistically significant differences between genotypes were observed in patients with IA (Table 3).

Table 2.

Genotype frequencies of angiotensin-converting enzyme gene insertion/deletion (I/D) polymorphisms among Chinese patients with intracranial aneurysm (IA), and age- and sex-matched control subjects.

Genotype	Patients with IA n = 220	Controls n = 220	OR (95% CI)	Statistical significance^a
II	64 (29.1)	44 (20.0)	1.00 (Ref)
ID	106 (48.2)	99 (45.0)	0.74 (0.46, 1.18)	NS
DD	50 (22.7)	77 (35.0)	0.45 (0.26, 0.75)	P = 0.003
I allele frequency	234 (53.2)	187 (42.5)	1.00 (Ref)
D allele frequency	206 (46.8)	253 (57.5)	0.65 (0.50, 0.85)	P = 0.002

Data presented as n (%) of patients.

Pearson’s Χ²-test.

OR, odds ratio; CI, confidence interval; NS, not statistically significant (P ≥ 0.05).

Table 3.

Stratification analysis of angiotensin-converting enzyme gene insertion/deletion (I/D) genotype frequency in Chinese patients with intracranial aneurysm.

Variable	II, n = 64		ID, n = 106		DD, n = 50
Variable	OR (95% CI)	OR (95% CI)	OR (95% CI)
Site of aneurysm	1 (Ref)	1 (Ref)	1 (Ref)
ACA, n = 98	31 (31.6)	1.09 (0.67, 1.78)	43 (43.9)	0.91 (0.59, 1.40)	24 (24.5)	1.08 (0.63, 1.85)
ICA, n = 76	25 (32.9)	1.13 (0.66, 1.92)	35 (46.1)	0.96 (0.60, 1.52)	16 (21.0)	0.93 (0.50, 1.72)
MCA, n = 46	8 (17.4)	0.60 (0.27, 1.33)	28 (60.9)	1.26 (0.75, 2.13)	10 (21.7)	0.96 (0.45, 2.02)
Shape of aneurysm	1 (Ref)	1 (Ref)	1 (Ref)
Saccular, n = 178	51 (28.6)	0.98 (0.65, 1.50)	87 (48.9)	1.01 (0.72, 1.43)	40 (22.5)	0.99 (0.62, 1.57)
Fusiform, n = 33	10 (30.3)	1.04 (0.49, 2.23)	15 (45.5)	0.94 (0.49, 1.81)	8 (24.2)	1.07 (0.47, 2.45)
Dissecting, n = 9	3 (33.3)	1.15 (0.30, 4.36)	4 (44.4)	0.92 (0.28, 3.06)	2 (22.2)	0.98 (0.21, 4.67)
Size of aneurysm	1 (Ref)	1 (Ref)	1 (Ref)
Small, <15 mm, n = 155	46 (29.7)	1.02 (0.66, 1.57)	74 (47.7)	0.99 (0.69, 1.42)	35 (22.6)	0.99 (0.62, 1.60)
Larger, 15–25 mm, n = 56	16 (28.6)	0.98 (0.53, 1.83)	27 (48.2)	1.00 (0.60, 1.67)	13 (23.2)	1.02 (0.52, 2.01)
Giant, >25 mm, n = 9	2 (22.2)	0.76 (0.16, 3.63)	5 (55.6)	1.15 (0.38, 3.53)	2 (22.2)	0.98 (0.21, 4.67)
Fisher Grade^a	1 (Ref)	1 (Ref)	1 (Ref)
1, n = 24	7 (29.2)	1.00 (0.41, 2.43)	11 (45.8)	0.95 (0.45, 2.01)	6 (25.0)	1.10 (0.43, 2.83)
2, n = 101	28(27.7)	0.95 (0.58, 1.58)	51 (50.5)	1.05 (0.70, 1.58)	22 (21.8)	0.96 (0.55, 1.67)
3, n = 65	20 (30.8)	1.06 (0.60, 1.88)	30 (46.2)	0.96 (0.59, 1.57)	15 (23.1)	1.02 (0.54, 1.93)
4, n = 30	9 (30.0)	1.03 (0.47, 2.28)	14 (46.7)	0.97 (0.49, 1.90)	7 (23.3)	1.03 (0.43, 2.47)

Data presented as n (%) of patients.

There were no statistically significant differences (P ≥ 0.05) between genotypes with respect to any clinical variable as determined by Pearson’s Χ²-test.

OR, odds ratio; CI, confidence interval; ACA, anterior cerebral artery; ICA, internal carotid artery; MCA, middle cerebral artery.

Discussion

The relationship between ACE I/D polymorphisms and frequency of IA has previously been studied in Caucasian populations.^20–23 Takenaka et al.²⁴ were the first to investigate the association between ACE I/D polymorphisms and IA, and found that the frequency of the DD genotype in patients with IA was significantly lower compared with control subjects. These results suggested that genetic heterogeneity of the ACE gene may be correlated with the aetiology of IA. Other studies, conducted in UK and Polish patient populations, have demonstrated that the ACE I allele is associated with an increased risk of ruptured IA and that the II genotype is a risk factor for aneurysmal SAH, respectively.^20,23 These reports are consistent with the results from the present study. Contrary to these findings, however, a US study in a Caucasian population failed to find any evidence of an association between the allelic or genotypic distribution of the ACE I/D polymorphism and IA.²²

In the last decade, many studies have been conducted to investigate the genetic factors associated with IA. A genetic meta-analysis of eight genes and 13 polymorphisms in ∼20000 individuals showed that there was a likely genetic basis for sporadic IA.²¹ The evidence base was, however, small compared with other complex disorders.²¹ The interleukin (IL)-6-572GG genotype has been shown to be associated with a heightened risk of IA in a Chinese population,²⁶ and this finding is consistent with other published reports in Chinese Han and Cantonese populations.^27,28 A region between introns 7 and 15 of the cyclin-dependent kinase inhibitor 2B antisense RNA-1 (CDKN2BAS) gene, carrying the rs1333040-T allele, has also been associated with an increased risk of IA in Japanese patients.⁵ Furthermore, there is evidence of an association between the development of aneurysms and a polymorphism at a genetic variant of endoglin, a glycoprotein expressed on the surface of human vascular endothelial cells, in Japanese patients with IA.²⁹ In another Chinese case–control study, it was suggested that the IL-12A and IL-12B genes are both associated with a greater risk of developing an IA.³⁰ An increased risk of IA in a Korean population has been associated with the rs2621215 SNP in intron 46 of the collagen, type I, alpha-2 (COL1A2) gene.³¹ The rs42524 polymorphism of COL1A2 could also be a genetic risk factor for sporadic IA among individuals of Chinese Han ethnicity.³² Another study in a Chinese population suggested that susceptibility to IA may be affected by the miR-34b/c rs4938723CC and TP53 Arg72-Pro polymorphisms.³³ Polymorphisms of homocysteine metabolism are also possible risk factors for the development of IA.³⁴ The endothelial nitric oxide synthase T-786C SNP may be a genetic marker that differentiates between small and large ruptured IA,³⁵ although other studies failed to confirm this finding.^36,37 Finally, polymorphisms in the matrix metalloproteinase-9 and the kallikrein gene cluster have been linked to the pathogenesis of IA.^38–40

Molecular mechanisms underlying the association between ACE I/D polymorphism and the risk of IA remain unclear. ACE DD carriers have higher local levels of ACE than those with the ID and II genotypes.^11,12 Several studies in different populations have found that ACE I/D polymorphisms are strongly associated with hypertension, which is a known risk factor for IA.^41–44ACE I/D polymorphisms have also been extensively studied in several cardiovascular adult diseases, such as thoracic aortic dissection,⁴⁵ ischaemic stroke,¹³ coronary heart disease,^14,46 left ventricular hypertrophy⁴⁷ and intracerebral haemorrhage.^15–20

In conclusion, data from the present study suggest that the ACE DD genotype may be a protective factor against IA in a Chinese population. A larger, multicentre study, which is statistically powered to detect differences in the risk of IA between different ACE genotypes, is required in order to confirm the present results.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This work was supported by the National Natural Science Foundation of China General Program (No.30872673 to C.Y.) and Youth Project (No. 30801185 to Y.L.).

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