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Table of Contents    
Year : 2012  |  Volume : 60  |  Issue : 5  |  Page : 504-509

Association of CYP11B2 gene polymorphism with ischemic stroke in the north Chinese Han population

Department of Neurology, The Affiliated Hospital of Qingdao University Medical College, P.R. China

Date of Submission05-Feb-2012
Date of Decision04-Mar-2012
Date of Acceptance13-May-2012
Date of Web Publication03-Nov-2012

Correspondence Address:
Yan Wang
16 Jiangsu Road, Qingdao, Shandong Province, Peoples Republic of China

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Source of Support: The Natural Science Foundation of Shandong Province (ZR2010HM021),, Conflict of Interest: None

DOI: 10.4103/0028-3886.103196

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 » Abstract 

Background: Genetic variations of renin-angiotensin-aldosterone system play an important role in the pathogenesis of hypertension and stroke. Aim: To investigate the -344C/T and intron 2 conversion polymorphisms of aldosterone synthase gene (CYP11B2) for an association with stroke and hypertension in the North Chinese Han population. Materials and Methods: This case-control study included 332 patients and 250 controls. Genotypes of -344C/T polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the intron 2 conversion polymorphism was genotyped using two separated PCRs. Results: There were significant differences in genotype frequencies of -344C/T polymorphism between stroke patients and controls (P = 0.002). An association was found between TT genotype and ischemic stroke [odds ratio = 1.572, 95%CI (1.095-2.258), P = 0.014]. However, there was no significant association of intron 2 polymorphism with stroke. Furthermore, when the ischemic stroke patients were classified according to Trial of Org 10172 in Acute Stroke Treatment classification, TT genotype was found to be associated with large artery atherosclerosis [odds ratio = 1.747, 95%CI (1.182-2.584), P = 0.005] and small vessel disease [odds ratio = 1.781, 95%CI (1.134-2.796), P = 0.012]. The intron 2 polymorphism failed to show relationship with any specific stroke subtype. Conclusions: Our findings suggest a significant association of CYP11B2 (-344C/T) polymorphism with stroke but intron 2 polymorphism is not associated with increased stroke susceptibility.

Keywords: Aldosterone synthase gene, ischemic stroke, polymorphism

How to cite this article:
Yan G, Wang Y. Association of CYP11B2 gene polymorphism with ischemic stroke in the north Chinese Han population. Neurol India 2012;60:504-9

How to cite this URL:
Yan G, Wang Y. Association of CYP11B2 gene polymorphism with ischemic stroke in the north Chinese Han population. Neurol India [serial online] 2012 [cited 2020 Sep 23];60:504-9. Available from:

 » Introduction Top

Ischemic stroke (IS) is a heterogenous disease caused by the combination of several environmental and genetic factors. [1] Epidemiologic studies in families and in twins have indicated a distinctive genetic component predisposing to stroke. [2] Identification of susceptibility genes to IS might enhance the prediction of disease risk. Aldosterone, one of the main effectors of the renin- angiotensin-aldosterone system (RAAS), affects sodium balance, blood volume, and blood pressure. Recently it has been related to vascular inflammation, [3] endothelial dysfunction, [4] and vascular remodeling. [5] These changes are considered to be involved in increasing risk of stroke by initiation and progression of atherosclerosis and cerebral vascular structural alterations. Elevated plasma aldosterone or mineralocorticoid receptor (MR) was found to be associated with increased risk of stroke/transient ischemic attacks (TIA). [6] Additionally, previous evidence in the rat models suggested that an individual's risk of having a stroke can be reduced by MR antagonism. [7],[8]

CYP11B2, expressed mainly in the zona glomerulosa of the adrenal cortex, catalyzes the final steps of aldosterone biosynthesis. [9] Among various candidate genes that might be involved in stroke pathogenesis, we focused on the CYP11B2 gene, which is located on chromosome 8q22 and encodes CYP11B2. [10] Several genetic variants of CYP11B2 gene have now been identified and two of them have been most studied: one in the transcriptional regulatory region (promoter) and the other in the second intron. The first polymorphism involves a C to T substitution at position -344 in the promoter region, which disrupts a putative binding site for steroidogenic factor-1 (SF-1). [11] The second is a gene conversion of CYP11B2, in which most of the intron 2 is replaced by the corresponding intron of the adjacent and homologous CYP11B1 gene. [11] To date, association studies of the -344C/T polymorphism with stroke and hypertension have yielded conflicting results. [12],[13],[14],[15] Fewer investigations have examined the association between the intron 2 conversion polymorphism and essential hypertension, and contrasting results have also been reported. [16],[17] To our knowledge, no report has so far been published examining the intron 2 conversion and its association with stroke. Hence, we sought to examine whether the above two polymorphisms in the gene were associated with stroke and hypertension in a case-control association study.

 » Materials and Methods Top


Study population included 332 ischemic stroke patients (males: females = 200:132) presenting with new or recurrent stroke treated at the Department of Neurology, Affiliated Hospital of Qingdao University Medical College (Shandong, China) between July 2009 and March 2011. Each patient was examined by a qualified stroke neurologist to confirm the diagnosis, both clinically and by computed tomography (CT) scans and magnetic resonance imaging (MRI). Ischemic stroke was classified into stroke subtypes by Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification. [18] Only strokes due to the three main categories of the TOAST classification were included. Patients with major cardiac, renal, or hepatic diseases and cancer were excluded from this study. The control group included 250 individuals matched for gender and age (males: females = 143:107). The controls were volunteers recruited simultaneously from the outpatient department. By extensive investigation none of them were found to have any history or occurrence of stroke or cardiovascular diseases. Details on demographic characteristics and risk factors (age, gender, hypertension, diabetes mellitus, hypercholesterolemia, smoking, and alcohol use) were collected by using a structured questionnaire. Arterial hypertension was defined as having been previously diagnosed as having hypertension or with a systolic blood pressure > 140 mmHg and/or diastolic blood pressure > 90 mmHg on at least two occasions. Diabetes mellitus was defined if fasting blood glucose level > 7.0 mmol/L or a previous diagnosis of the disease. Hypercholesterolemia was determined as a total fasting plasma cholesterol > 5.2 mmol/L. The study was approved by the ethical committee of the study hospital and informed consent was obtained from all participants included in the study.

DNA isolation and genotyping

DNA for CYP11B2 genotyping was extracted from white blood cells using a standard protocol. The region of DNA containing the SF-1 polymorphism was amplified by polymerase chain reaction (PCR) using conditions similar to those previously described. [10] The sequences of primer pair were 5′-CAGGAGGAGACCCCATGTGAC-3′(forward) and 5′-CCTCCACCCTGTTCAGCCC-3′(reverse). PCR products were digested with restriction endonuclease HaeIII (TaKaRa, Dalian, China), then visualized with GelRed after electrophoresis in 3% agarose gel. The -344T allele lacks an HaeIII site (GGCC) present in the -344C allele, so -344T alleles were detected as fragments of 273,138, and 126 bp, whereas -344C alleles as fragments of 202,71,138, and 126 bp.

The intron 2 conversion polymorphism was genotyped using two separate PCRs, one that amplifies the no-conversion allele (Iw) and one that amplifies the intron conversion allele (Ic), with the primers reported previously by Davies et al. [19] Both reactions used the same reverse primer: 5′-AGGAACCTCTGCACGGCC-3′. Reactions to detect the Ic also contained the forward primer 5′-CAGAAAATCCCTCCCCCCTA-3′, whereas reactions to detect the Iw instead used the forward primer 5′-TGGAGAAAAGCCCTACCCTGT-3′. The size of the amplicon in each reaction is approximately 418 bp. The CYP11B2 polymorphisms were confirmed by sequencing the PCR products. The sequencing was done by Sangon Biotech (Shanghai, China).

Statistical analysis

Statistical analysis was performed on SPSS 17.0 software. Data were expressed as mean ± SD (continuous variables), or as percentages of total (categorical variables). Pearson χ2 was used to assess intergroup significance, and Student's t test was used to determine differences in means. Hardy-Weinberg equilibrium was tested for CYP11B2 gene polymorphism. Association between genotypes and stroke was examined by odds ratio (OR) with 95% confidence interval (CI) and Chi-square analysis. Multiple logistic regression analysis with forward stepwise selection (Wald) was performed to assess the independent contribution of genotype to stroke. Statistical significance was defined as P values less than 0.05.

 » Results Top

The clinical characteristics of the stroke patients and controls are given in [Table 1]. The stroke patients had higher serum triglycerides (P = 0.001) than controls. More patients than controls had diabetes, high blood pressure, and were smokers.
Table 1: Baseline characteristics of stroke patients versus matched controls

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The genotypic frequencies of -344C/T and intron 2 conversion polymorphisms were in agreement with the Hardy-Weinberg equilibrium. The genotypic distribution of the -344C/T polymorphism and allelic frequencies for C and T alleles in patients and controls are given in [Table 2]. The genotypes determined by PCR and agarose gel electrophoresis were confirmed by sequencing [Figure 1],[Figure 2],[Figure 3],[Figure 4] and [Figure 5]. The frequency of patients with TT genotype and the T allele of CYP11B2 (-344C/T) was significantly higher than in control subjects (P = 0.001 and P = 0.002, respectively). After adjustment for the conventional risk factors with multiple logistic regression analysis, the TT genotype (OR = 1.572; 95% confidence intervals (CI) = 1.095-2.258; P = 0.014), hypertension, diabetes, were all associated with ischemic stroke [Table 3]. However, there was no significant difference in the distribution of intron 2 conversion genotypes between IS patients and control subjects.
Table 2: Distribution of CYP11B2 genotypes and allelic frequencies of the study population

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Table 3: Logistic regression analysis

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Figure 1: Showing the amplification of region bearing -344C/T polymorphism

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Figure 2: Polymerase chain reaction products (-344C/T) digested with H and E. Lanes 1 and 5 represent CC homozygotes. Lanes 2, 4, and 7 represent TT homozygotes. Lanes 3 and 6 represent CT heterozygotes. Lane 8 represents 100 bp ladder

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Figure 3: Sequenced polymerase chain reaction products of the region bearing -344C/T showing TC heterozygote, CC homozygote, and TT homozygote, respectively

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Figure 4: The amplification for CYP11B2 intron 2 conversion genotyping. Lanes 1, 3, and 5: products of polymerase chain reaction (PCR), which amplify the normal gene. Lanes 2, 4, and 6: Products of PCR, which amplify the conversion gene. Lanes 1 and 2 represent IwIc. Lanes 3 and 4 represent IwIw. Lanes 5 and 6 represent IcIc. Lane 7 represents 100 bp ladder

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Figure 5: Sequenced polymerase chain reaction products of the region bearing intron 2 conversion polymorphism. Capital characters represent part sequence of CYP11B1 gene while italic ones represent part sequence of CYP11B2 gene. Nucleotides of no-conversion allele were identical with specific regions of CYP11B2. Nucleotides of conversion allele intron 2 were identical to those of specific regions of CYP11B1 and exon 3 identical to corresponding region of CYP11B2

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To evaluate the relationship of -344C/T, intron 2 conversion polymorphisms with hypertension, we grouped 250 controls into 126 hypertensives and 124 normotensives. The distribution of genotypes and allele frequencies in the two groups are shown in [Table 4]. As for the -344C/T polymorphism, the frequency of hypertensives with the TT genotype and the T allele was significantly higher than in normotensives (P = 0.01 and P = 0.005, respectively). After adjustment for confounders, the TT genotype was associated with hypertension (OR = 1.90; 95%CI = 1.131-3.193; P = 0.015). For intron 2 conversion polymorphism, our results failed to demonstrate its association with the risk of hypertension.
Table 4: Distribution of CYP11B2 genotypes and allelic frequencies in hypertensives and nonhypertensives

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Further analyzing the association of CYP11B2 -344C/T polymorphism with stroke subtypes classified according to TOAST classification, we found significant association of TT genotype with large artery atherosclerosis (OR = 1.747; 95%CI = 1.182-2.584; P = 0.005), small vessel disease subtype patients (OR = 1.781; 95%CI: 1.134-2.796; P = 0.012). No association of TT genotype was found with cardioembolic stroke. As to intron 2 polymorphism, we did not find significant association with any specific stroke subtype [Table 5].
Table 5: CYP11B2 genotypic frequencies in stroke patients according to TOAST classification

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 » Discussion Top

The relationship between -344C/T polymorphism with hypertension has been widely studied while results remain controversial. [12],[13],[20] Few studies have investigated the association of -344C/T polymorphism with stroke. The -344C/T polymorphism has previously been described to be related to the risk of stroke. [14],[21] However, findings from Sardinians failed to find the association. [15] Recently, -344C/T has been found associated with IS in a Southern Chinese population. [22] The disparity of the SNP of CYP11B2 (-344C/T) may be attributed to different ethnic populations. It cannot be ruled out the presence of another functional site with which -344C/T is in close linkage disequilibrium. Intron 2 conversion polymorphism has been studied less frequently than the -344C/T variant but might be a functional variant, since it might lead to overexpression of the gene. [23] The literature presents conflicting reports on association of this polymorphism with hypertension. [16],[17] To our knowledge, this study appears to be the first report on the association of intron 2 conversion polymorphism of the CYP11B2 gene and stroke.

This study has characterized the genotypes of CYP11B2 (-344C/T) and (intron 2 conversion) in groups of 332 IS patients and 250 unrelated healthy controls, and found that the frequencies of TT genotype and T allele of the CYP11B2 (-344C/T) in IS patients were significantly higher than that in controls. Our data were consistent with the findings from Munshi et al. [14] and Saidi et al. [21] and similar to that of a recent study in a Southern Chinese population. [22] Further analysis by subtypes of IS revealed that this variant was associated with large artery atherosclerosis and small vessel disease. One study revealed that genetic variations in CYP11B2 gene are associated with the progression of carotid atherosclerotic plaque size. [24] This finding suggests that the CYP11B2 polymorphism might act through carotid atherosclerosis to enhance susceptibility to the large-artery subtype of IS. Mechanisms underlying the association of -344C/T polymorphism with small vessel disease stroke subtype are not clear. However, the intron 2 conversion polymorphism is not associated with stroke in the study population. Intron 2 conversion polymorphism is probably not a major risk factor for IS in our population. Further studies of this variant with different ethic groups is warranted to explore the association. In addition, we further examined the association of the two polymorphisms with hypertension. -344C/T polymorphism was found to be associated with hypertension while no association was found between intron 2 conversion polymorphism and hypertension.

There are few limitations for this study. Two polymorphisms in CYP11B2 gene studied may not present the whole gene function. Further studies are needed to investigate the association of multiple variants in the gene with stroke. Specific mechanisms by which the studied variants exert negative effects on cerebrovascular diseases remain to be explored. In summary, our data suggest that the -344C/T polymorphism of CYP11B2 is associated with IS in the Northern Han Chinese. However, we did not find any correlation between intron 2 conversion polymorphism and stroke or hypertension. Results further suggest a tight link between -344C/T genotype and two forms of ischemic stroke: large artery atherosclerosis and small vessel disease subtypes of ischemic stroke.

 » References Top

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3.Joffe HV, Adler GK. Effect of aldosterone and mineralocorticoid receptor blockade on vascular inflammation. Heart Fail Rev 2005;10:31-7.  Back to cited text no. 3
4.White PC. Mechanism of disease: Disorders of aldosterone biosynthesis and action. N Engl J Med 1994;331:250-8.  Back to cited text no. 4
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6.Vyssoulis GP, Karpanou EA, Tzamou VE, Kyvelou SM, Michaelidis AP, Gialernios TP, et al. Aldosterone levels and stroke incidence in essential hypertensive patients. Int J Cardiol 2010;144:171-2.  Back to cited text no. 6
7.Dorrance AM, Osborn HL, Grekin R, Webb RC. Spironolactone reduces cerebral infarct size and EGF-receptor mRNA in stroke-prone rats. Am J Physiol Regul Integr Comp Physiol 2001;281:944-50.  Back to cited text no. 7
8.Rigsby CS, Pollock DM, Dorrance AM. Spironolactone improves structure and increases tone in the cerebral vasculature of male spontaneously hypertensive stroke-prone rats. Microvasc Res 2007;73:198-205.  Back to cited text no. 8
9.Clyne CD, Zhang Y, Slutsker L, Mathis JM, White PC, Rainey WE. Angiotensin II and potassium regulate human CYP11B2 transcription through common cis-elements. Mol Endocrinol 1997;11:638-49.  Back to cited text no. 9
10.Kuperi M, Hautanen A, Lankinen L, Koskinen P, Virolainen J, Nikkila H, et al. Associations between human aldosterone synthase gene polymorphisms and left ventricular size, mass and function. Circulation 1998;97:569-75.  Back to cited text no. 10
11.White PC, Slutsker L. Haplotype analysis of CYP11B2. Endocr Res 1995;21:437-42.  Back to cited text no. 11
12.Brand E, Chatelain N, Mulatero P, Fery I, Curnow K, Jeunemaitre X, et al. Structural analysis and evaluation of the aldosterone synthase gene in hypertension. Hypertension 1998;32:198-204.  Back to cited text no. 12
13.Tsukada K, Ishimitsu T, Teranishi M, Saitoh M, Yoshii M, Inada H, et al. Positive association of CYP11B2 gene polymorphism with genetic predisposition to essential hypertension. J Hum Hypertens 2002;16:789-93.  Back to cited text no. 13
14.Munshi A, Sharma V, Kaul S, Rajeshwar K, Babu MS, Shafi G, et al. Association of the -344C/T aldosterone synthase (CYP11B2) gene variant with hypertension and stroke. J Neurol Sci 2010;296:34-8.  Back to cited text no. 14
15.Rubattu S, Di Angelantonio E, Stanzione R, Zanda B, Evangelista A, Pirisi A, et al. Gene polymorphisms of the rennin-angiotensin-aldosterone system and the risk of ischemic stroke: A role of the A1161C/AT1 gene variant. J Hypertens 2004;22:2129-34.  Back to cited text no. 15
16.Nejatizadeh A, Kumar R, Stobdan T, Goyal AK, Gupta M, Tyaqi S, et al. CYP11B2 gene haplotypes independently and in concurrence with aldosterone and aldosterone to rennin ratio increase the risk of hypertension. Clin Biochem 2010;43:136-41.  Back to cited text no. 16
17.Davies E, Holloway CD, Ingram MC, Inglis GC, Friel EC, Morrison C, et al. Aldosterone excretion rate and blood pressure in essential hypertension are related to polymorphic differences in the aldosterone synthase gene CYP11B2. Hypertension 1999;33:703-7.  Back to cited text no. 17
18.Adams HP Jr, Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, et al. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke 1993;24:35-41.  Back to cited text no. 18
19.Davies E, Holloway CD, Ingram MC, Friel EC, Inglis GC, Swan L, et al. An influence of variation in the aldosterone synthase gene (CYP11B2) on corticosteroid responses to ACTH in normal human subjects. Clin Endocrinol (Oxf) 2001;54:813-7.  Back to cited text no. 19
20.Patel S, Steeds R, Channer K, Samani NJ. Analysis of promoter region polymorphism in the aldosterone synthase gene (CYP11B2) as a risk factor for myocardial infarction. Am J Hypertens 2000;13:134-9.  Back to cited text no. 20
21.Saidi S, Mahjoub T, Almawi WY. Aldosterone synthase gene (CYP11B2) promoter polymorphism as a risk factor for ischaemic stroke in Tunisian Arabs. J Renin Angiotensin Aldosterone Syst 2010;11:180-6.  Back to cited text no. 21
22.Tu Y, Cui G, Xu Y, Bao X, Wang X, Wang DW. Genetic polymorphism of CYP11B2 gene and stroke in the Han Chinese population and a meta-analysis. Pharmacogenet Genomics 2011;21:115-20.  Back to cited text no. 22
23.Pascoe L, Curnow KM. Genetic recombination as a cause of inherited disorders of aldosterone and cortisol biosynthesis and a contributor to genetic variation in blood pressure. Steroids 1995;60:22-7.  Back to cited text no. 23
24.Sharma R, Katz J. Preliminary studies on human aldosterone synthase (CYP11B2) gene polymorphism, matrix metalloprotease-9, apoptosis, and carotid atherosclerosis plaque size by proton magnetic resonance imaging. J Renin Angiotensin Aldosterone Syst 2010;11:198-204.  Back to cited text no. 24


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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