Association of genetic polymorphisms of CYP 2C19 with hypertension in a Chinese Han population

Abstract

Objectives. Cytochrome P450 (CYP) 2C19 is expressed in vascular endothelium and metabolizes arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs), which play a centralrole in regulating renal tubular fluid-electrolyte transport and vascular tone. The purpose of this study is to explore the relationship between the genetic polymorphism and hypertension risk in a Chinese Han population. Methods. There were 1120 Chinese Han hypertension patients and 1249 age- and sex-matched control subjects genotyped for two tagging single nucleotide polymorphism (rs10509676 and rs11568732) of the human CYP2C19 gene by the use of polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis. Haplotypes were constructed and their frequencies compared between the hypertension patients and the controls. Results. The AA genotype of rs10509676 was found more frequently in the hypertension group than in the control group (p < 0.001). The frequency of the A-G haplotype was significantly higher in hypertensive patients than in control subjects (p < 0.001). The frequency of the T-T genotype was markedly higher in control subjects than in hypertensive patients (p < 0.001). Conclusions. The present results indicate that hypertension is associated with the AA genotype of rs10509676 in the human CYP2C19 gene. The A-G haplotype appears to be a risk factor and the T-T haplotype may be a protective factor of hypertension in Chinese Han people.

Key Words::

• Cytochrome P450
• epoxyeicosatrienoic acids
• hypertension

Introduction

Cytochrome P450 epoxygenases, such as CYP1A (1), CYP2B (2), CYP2C (3) and CYP2D (4), metabolize arachidonic acid (AA) to four regioisomeric cis-epoxyeicosatrienoic acids (EETs) including 5,6-, 8,9-, 11,12- and 14,15-EETs (5–7). CYP2C19 is one of the human cytochrome P450 enzymes, and is abundantly expressed in artery endothelial, smooth muscle cells and cardiac myocytes (8,9). It is also an important enzyme response for EETs synthesis. The potent effects of EETs on vascular tone and renal tubular fluid-electrolyte transport (10) indicate that they might play a role in blood pressure regulation. Indeed, the well documented natriuretic and vasodilatory actions of the EETs would be predicted to lower blood pressure and produce an overall antihypertensive effect (11–13). In addition, the vasodilator properties of EETs have given rise to a hypothesis that they serve as endothelium-derived hyperpolarizing factor (EDHF), which contributes to the vasodilator response to acetylcholine and radykinin. EDHF could increase the express and activity of the eNOS and enhanced to produce the nitric oxide (14). More recently, the additional vascular protective effects of EETs, including antithrombotic, antimigratory, antioxidant and antiapoptotic effects, have also been reported (15,16). However, the EETs have also been found to cause vasoconstriction in renal and extrarenal vascular beds, suggesting that they may also possess prohypertensive actions under certain conditions (17,18). Therefore, the role of P450-derived EETs in the pathogenesis of hypertension remains highly controversial.

Recently, a few studies into the polymorphisms of CYP2C19 gene revealed a positive association with cardiovascular diseases such as coronary artery disease (19) and atherosclerosis (20). However, the role of this gene variant in hypertension has not been sufficiently investigated.

In this study, we investigated potential associations between CYP2C19 single nucleotide polymorphism (SNPs) and hypertension in a group of hypertensive patients and normotensive controls assembled in Chinese Han.

Materials and methods

Subjects

Subjects diagnosed with hypertension were recruited at the Changhai Hospital Affiliated Second Military Medical University from 2007 to 2009. We enrolled 1120 hypertension patients and 1249 control subjects in the present study. All patients and controls included in this study were Chinese Han from the same geographic area in Shanghai.

Hypertensive subjects defined by antihypertensive treatment of greater than 6 months duration or a seated systolic and/or diastolic blood pressure of greater than 140 and/or 90 mmHg, respectively, on at least two separate occasions. Secondary hypertension was excluded after clinical and laboratory work up (such as renal, renovascular or endocrine disease) as described by Kitsios & Zintzaras (21). Subjects recruited through local advertisement were defined as normotensive if they had seated systolic and diastolic pressures of less than 140 and 90mmHg, respectively, and no history of or treatment for hypertension. Cases and controls were frequency matched for age and gender. Positive family history of hypertension was defined by the proband's report of hypertension in one or more first-degree relatives (parent sibling or offspring). Mean arterial pressure (MAP) was defined as follows:

MAP=[(2×diastolic pressure)+systolic pressure]/3.

Informed consent was obtained from each individual according to a protocol approved by the Ethics Committee of the Changhai Hospital Affiliated Second Military Medical University.

Biochemical analysis

The plasma concentrations of total cholesterol, triglyceride and glucose and the serum concentration of uric acid were measured by standard methods in the Central Laboratory of Changhai Hospital Affiliated Second Military Medical University.

Sample DNA extraction

Blood samples were collected with a standard venipuncture technique and EDTA-containing tubes. DNA was extracted from peripheral vein blood leukocytes using a whole blood genome extraction kit (Beijing Boiteke Corporation) according to the manufacturer's instructions. Samples were coded to protect donor identity and to allow blinding of the investigators who carried out the genotyping.

Genotyping

There are 451 SNPs for the human CYP2C19 gene listed in the National Center for Biotechnology Information SNP database (www.ncbi.nlm.nih.gov/SNP). We also screened the data for the tagging SNPs on the International HapMap Project website (www. hapmap.org). We obtained seven SNPs (rs4532967, rs3814637, rs11568732, rs4986894, rs10509676, rs12773342 and rs12768009) for Chinese Hans using minor allele frequency (MAF) ≥0.05 and linkage disequilibrium (LD) patterns with r²≥ 0.8 as a cut-off. Because high r² values were shown between rs4532967 and rs4986894, rs10509676, rs12773342 (r²=1) and rs12768009 (r² >0.8), we selected rs10509676 from these five SNPs in this study; also as higher r² value was shown between rs3814637 and rs11568732 (r²=0.8), we selected rs3814637.

Genotyping was confirmed by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) analysis. The primers of CYP2C19 were designed by the Primer Premier 5.0 software. Their synthesis was performed by Shanghai Biological Engineering Company Limited (Shanghai, China). The primer pair sequences, annealing temperatures and restriction enzymes for two SNPs were described in Table I. Restriction enzyme digestion was performed according to the manufacturer's instructions. To ensure the results to be verified, we used sequenced genomic DNAs as positive controls in our assays.

Table I. Primer sequence of each single nucleotide polymorphism (SNP).

SNPs	Polymerase chain reaction primers	Denaturation temperature	Products length	Restriction enzyme
rs10509676	Sense: 5′AATTTTTGGCTGAGGGTAA3′ Antisense: 5′CCTAAGGTCATTTTGGGGT3′	52°C	286 bp	Ase I
rs11568732^a	Sense: 5′TATATTCAGAATAACTAGG3′	48°C	455 bp	Spe I

^aThe underline indicated mismatched base.

Statistical analysis

Data analysis was performed using the computer software Statistical Package for Social Sciences-SPSS for Windows (version 13.0, SPSS Institute, Chicago, IL). To determine whether the polymorphisms were in Hardy–Weinberg equilibrium, the actual and predicted genotype counts for controls were compared using χ² analysis with two degrees of freedom. The effects of the variant allele frequency or genotype on the probability of having hypertension were assessed using χ² analysis with one and two degrees of freedom, respectively. Measurement data are shown as means± SD, and the differences between the hypertensive patients and the control subjects were assessed by independent-sample T test. Differences in enumeration data between hypertensive patients and control subjects were analyzed using the χ² test. Differences in distributions of genotypes and alleles between hypertensive patients and control subjects were analyzed using χ² test. Allele frequencies distribution was analyzed by 2×2 contingency tables using a 5% level of significance. To compute odds ratios (OR) and 95% confidence intervals (CI) for hypertension, the effects of genotype were adjusted for body mass index (BMI), and family history of hypertension using logistic regression with multivariate analysis. Based on the genotype data of the genetic variations, we performed LD analysis and haplotype-based case–control analysis, using the expectation maximization (EM) algorithm (21) and the SHEsis software (22). The pairwise LD analysis was performed using two SNP pairs. We used |D′| values of >0.5 to assign SNP locations to one haplotype block. SNPs with an r² value of <0.5 were selected as tagged.

Results

Baseline characteristics of normotensive and hypertensive subjects

Table II shows baseline characteristics of the study population as a function of hypertensive status. Data are presented as mean± SD. BMI, fasting blood glucose percentages with smoking and hypertension family history were significantly greater in hypertensive vs normotensive subjects. Therefore, we adjusted for these variables in our multivariable models.

Table II. Characteristics of study participants.

	Hypertensive group	Controls	p
Number of subjects	1120	1249
Uric acid (mmol/l)	339.4 ± 112.3	338.1 ± 97.7	0.765
Triglyceride (mmol/l)	1.71 ± 0.40	1.70 ± 0.31	0.500
Total cholesterol (mmol/l)	4.6 ± 1.7	4.5 ± 1.4	0.121
Smoking (n, %)	504 (45.0)	493 (39.5)	0.007
Fasting blood glucose (mmol/l)	5.77 ± 1.19	5.41 ± 1.01	<0.001
Hypertension family history	475 (42.4)	101 (8.1)	<0.001
Body mass index (kg/m^2)	25.7 ± 2.4	23.1 ± 2.3	<0.001

CYP2C19 gene polymorphisms and risk of hypertension

Table III shows the distribution of the genotypes and alleles of the two SNPs. The genotype distribution of each SNP did not show significant difference from the Hardy–Weinberg equilibrium values (p> 0.05). The genotype and the allele distribution of rs10509676 differed significantly between the hypertensive patients and control subjects (p<0.01 respectively). The AA genotype and A allele were more common in the hypertensive patients than in the control subjects, but the genotype and the allele distributions of rs11568732 was not different between the hypertensive patients and control subjects.

Table III. Genotypes and alleles distribution of hypertensive patients and control subjects.

SNP	Group	n		Genotype		p	Allele		p
rs10509676			AA	AT	TT		A	T
	Hypertension	1120	840 (0.75)	224 (0.20)	56 (0.05)	<0.001	0.85	0.15	<0.001
	Control	1249	686 (0.55)	499 (0.40)	64 (0.05)		0.75	0.25
rs11568732			GG	GT	TT		G	T
	Hypertension	1120	28 (0.03)	156 (0.14)	936 (0.83)	0.461	0.09	0.91	0.378
	Control	1249	23 (0.02)	165 (0.13)	1061 (0.85)		0.08	0.92

SNP, single nucleotide polymorphism.

Table IV shows patterns of linkage disequilibrium in the CYP2C19 gene, with their |D′| and r² values. All two SNPs are located in one haplotype block because all |D′| are beyond 0.5. All two SNPs were available for the performance of a haplotype-based case–control study because all of the r² values were below 0.5.

Table IV. Pairwise linkage disequilibrium for the two single nucleotide polymorphism (SNPs).

		|D'|
		Hypertensive patients		Control subjects
	Sites	rs10509676	rs11568732	rs10509676	rs11568732
r^2	rs10509676		0.743		0.694
	rs11568732	0.121		0.087

|D'| above diagonal and r² below diagonal; |D'|>0.5.

In the haplotype-based case–control analysis, haplotypes were established through the use of two SNPs (Table V). The frequency of the A-G haplotype was significantly higher for hypertensive patients than for control subjects (p< 0.001), whereas the frequency of the T-T haplotype was significantly lower for hypertensive patients than for control subjects (p<0.001).

Table V. Haplotype distribution of hypertensive patients and control subjects.

	Haplotypes frequencies
Hplotypes	Hypertensive patients (n = 1120)	Control (n = 1249)	x^2	p
A-G	0.334	0.199	15.969	<0.001
A-T	0.549	0.557	1.237	0.079
T-G^a	0.005	0.006	−	−
T-T	0.038	0.113	13.688	<0.001

Global x² is 24.129, while df = 2 (frequency <0.03 in both control and case has been dropped). Fisher's p-value is 2.41 × 10⁻⁵.

Limitations

This study has several limitations. First, the present study was limited by the relatively small sample size. This may have led to weak statistical significance and wide CIs when estimating ORs. Second, in the present study, we only investigated two tagging SNPs of CYP2C19 gene; other SNPs of cytpchrome P450 gene may be more interesting for the future studies. Third, we selected the control subjects based on the blood pressure less than 140/90 mmHg rather than 130/85 mmHg, this fact may underestimate or overestimate the association of CYP2C19 genetic polymorphisms with hypertension.

Discussion

It is generally accepted that hypertension is a complex multifactorial and polygenic disorder in which multiple environmental and genetic factors are simultaneously involved. The foundation for human studies examining putative causative genes that may be involved in hypertension is based on a candidate gene approach. This involves selecting a functionally relevant gene to study and subsequently investigating its association with the hypertension phenotype.

Previous study indicated that endothelial dysfunction is associated with increased risk of cardiovascular events (23), and is typically manifested by impairment in endothelial-dependent vasodilation (24). The vasodilatory and anti-inflammatory properties of CYP-derived EETs are important mediators of this process (25,26). Soluble epoxide hydrolase (EPHX2) rapidly hydrolyzes EETs and is integrally involved in regulation of their cellular levels and vascular effects (27).

Human P450 genes involved in metabolism of endogenous substrates such as AA indicating they may play important role in hypertension. In the current study, we used a case–control design to investigate potential associations between functionally relevant P450 2C19 polymorphisms and hypertension in a population from Chinese Han. Our major findings are that in Chinese Han, hypertensive patients had higher frequencies of AA genotype of the rs10509676 than did controls.

Morris et al. (28) found that for genes with multiple susceptibilities, analysis based on haplotypes has advantages over analysis based on individual SNPs, particularly when linkage disequilibria between SNPs are weak (29). Consequently, in the present study, we successfully established haplotypes for the CYP2C19 gene from the different combination of the two SNPs. The frequency of the A-G haplotype was significantly higher for hypertensive patients than for control subjects (p< 0.001), and the T-T haplotype was markedly higher for control than for hypertensive patients (p< 0.001).

Conclusions

The present results indicate that hypertension is associated with the AA genotype of rs10509676 in the human CYP2C19 gene. The A-G haplotype appears to be a risk factor and the T-T haplotype may be a protective factor of hypertension in Chinese Han people. However, these findings need further be replicated in a different cohort in the future.

Declaration of interest: The author report no conflicts of interest. The author alone is responsible for the content and writing of the paper.