Introduction

Essential hypertension is a complex syndrome determined by both genetic and environmental factors. The response of patients to antihypertensive treatment is diverse (Materson 2004). Past effort to identify responders to therapy include phenotypic (age, race) (Preston et al. 1998) and biochemical (rennin profile, insulin sensitivity) (Laragh et al. 1988; Lind et al. 1995) methods, but none of these have found extensive clinical utility. The advent of the Human Genome Project (Venter et al. 2001) has generated rejuvenated interest in the pursuit of pharmacologic therapy targeted to individuals genetically identified as most likely to benefit from treatment. The rennin–angiotensin–aldosterone system (RAAS) plays a central role in the modulation of blood pressure (BP). Genetic variation in the genes encoding products of the RAAS has been studied extensively as candidate genes for essential hypertension (EH), such as ACE I/D gene (Patnaik et al. 2014), ATIR gene (Liu et al. 2015), CYP11B2 gene (Alvarez-Madrazo et al. 2013), AGT gene (Kim et al. 2015) and so forth. It is reasonable to hypothesize that the RAAS gene polymorphism may be predictive of variation in BP response. Variant in genes of the RAAS has been investigated to influence the therapeutic responsiveness to antihypertensive drugs. However, the results of other studies are controversial (Taverne et al. 2010; Li et al. 2012; Do et al. 2014; Gupta et al. 2015).

Angiotensin receptor blockers (ARBs) are a kind of widely used antihypertensive drugs. ARBs act directly on the angiotensin II type 1 receptor to block the vasoconstrictive response to angiotensin II and inhibit angiotensin II-stimulated release of aldosterone. The aim of this study was to investigate the association between the CYP11B2 –344C/T and ACE I/D polymorphism and the BP response to angiotensin II type 1 receptor blocker telmisartan in Chinese hypertensive patients.

Methods

Study subjects

In this study both male and female Chinese Han patients who met the following criteria were included: aged 18–80 years; history of essential hypertension; diastolic blood pressure (DBP) greater than or equal to 95 mmHg and lower than or equal to 109 mmHg; systolic blood pressure (SBP) lower than 180 mmHg. The exclusion criteria were as follows: secondary hypertension, congestive heart failure, cerebrovascular accident, myocardial infarction within the past three months; a documented history of unstable angina pectoris within the past three months; any clinically important abnormal laboratory findings, such as alanine aminotransferase (ALT) or creatinine level twice the upper limit of normal; pregnant or lactating in women. This study was approved by the appropriate Ethics Committees. All of the participating patients gave informed consent before any study procedures.

Study design

BP was measured by a well-trained doctor or nurse using a mercury sphygmomanometer after the patient had rested for at least 10 min in a seated position and was determined as the mean of three measurements taken 2 min apart. In this study, a total of 164 patients were recruited with essential hypertension. All antihypertensive agents were withdrawn before the start of a two-week placebo period. At the end of the placebo period, 11 patients were excluded and a total of 153 qualified patients were given telmisartan, 80 mg orally, once daily for eight weeks. Five patients were withdrawn from the study because of failing to follow up. Thus, 148 patients completed the eight-week trial, and their data were used for the present study. Blood was collected from all patients for genotype analysis.

Table 1 Changes of patient characteristics and BP response to the treatment.
Full size table
Table 2 Genotype and allele frequencies of the CYP11B2 –344C/T and ACE I/D gene polymorphisms in the study population.
Full size table

Definition of study variables

Baseline information on the following variables was included in the analysis: gender, age, body mass index (BMI \(\hbox {kg/m}^{2}\)), BP, heart rate (HR), laboratory variables included the serum levels of ALT, creatinine, glucose, uric acid, total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL) and low-density lipoprotein (LDL), sodium, potassium and angiotensin II.

Detection of CYP11B2 gene polymorphisms

Genomic DNA was isolated from peripheral leucocytes separated from the blood. The CYP11B2 –344C/T polymorphism was determined through PCR/RFLP. PCR reaction volume of \(20\,\mu \hbox {L}\) contained \(10\times \hbox {PCR}\) buffer, \(1.5\,\hbox {mM}\,\hbox {MgCl}_{2}\), 0.1 mM of each dNTP, \(0.5\,\mu \hbox {M}\) of each primer, 1 unit of Taq enzyme (Takara) and 100 ng of genomic DNA. The sequence of the sense oligonucleotide primer was 5-ATGTTGACCACCAGGAGGAGAC-3, and that of the antisense primer was 5-CCAGGGCTGAGAGGAGTAAAATG-3. The PCR cycling conditions consisted of initial denaturing step at \(95^{\circ }\hbox {C}\) for 5 min, followed by 35 cycles of \(95^{\circ }\hbox {C}\) for 30 s, \(62^{\circ }\hbox {C}\) for 30 s and \(72^{\circ }\hbox {C}\) for 30 s, then a final elongation step at \(72^{\circ }\hbox {C}\) for 7 min. The PCR products were then digested overnight with 5 units of HaeIII (New England Biochemicals), as described previously (Kupari et al. 1998), before separation on a 3% agarose gel and visualization with ethidium bromide. The amplification was cut into 273-bp as the –344 T allele and of 214 bp and 59 bp as the –344C allele after HaeIII digestion.

Detection of ACE I / D gene polymorphisms

The genotype of the ACE gene was determined according to the method of Tiret et al. (1992). A 287-bp insertion or deletion polymorphism in intron 16 of the ACE gene was identified by the PCR method. The PCR reaction volume of \(20\,\mu \hbox {L}\) contains \(10\times \hbox {PCR}\) buffer, \(1.5\,\hbox {mM}\,\hbox {MgCl}_{2}\), 0.1 mM of each dNTP, \(0.5\,\mu \hbox {M}\) of each primer, 1 unit of Taq enzyme (Takara) and 100 ng of genomic DNA. The sequence of the sense oligonucleotide primer was 5-CTGGAGACCACTCCATCCTTTCT-3 and that of the antisense primer was 5-GATGTGGCCATCACATTCGTCAGAT-3. The PCR cycling conditions consisted of initial denaturing step at \(95^{\circ }\hbox {C}\) for 5 min, followed by 35 cycles of \(95^{\circ }\hbox {C}\) for 30 s, \(60^{\circ }\hbox {C}\) for 30 s and \(72^{\circ }\hbox {C}\) for 30 s, then a final elongation step at \(72^{\circ }\hbox {C}\) for 7 min. The PCR products were separated on a 2% agarose gel and visualization with ethidium bromide. The ACE gene polymorphism was classified into three genotypes: the 190-bp deletion homozygous (DD) genotype, the 490-bp insertion homozygous (II) genotype, and the 490-bp insertion, 190-bp deletion heterozygous (ID) genotype.

Statistical analysis

Data were reported as means ± standard deviation (SD). The \(\chi ^{2}\) test was used to evaluate categorical variables and test for Hardy–Weinberg equilibrium (HWE) of polymorphisms. Differences in biochemical parameters and BP were assessed using a paired t-test. Linear regression modelling was used to determine the correlation of BP response with genotypes, following normalization with pretreatment BP, age, gender, BMI, blood glucose, TC, TG, HDL and LDL. The SPSS 18 software was applied for statistical analysis. A two-tailed P value less than 0.05 was considered to be significant.

Results

Data from a total of 148 patients was analysed in this study. All patients had mild-to-moderate hypertension. The basic characteristics of patients are presented in table 1. After eight-week treatment with telmisartan, both SBP and DBP were remarkably decreased \((P < 0.001)\). The serum angiotensin II level was obviously increased at the end of the 8-week treatment \((P< 0.001)\). No significant differences were observed for the other parameters.

CYP11B2 –344C/T and ACE I / D gene polymorphism and antihypertensive response to telmisartan

Genotype and allele of CYP11B2 –344C/T and ACE I/D gene, distributions of the study population are summarized in table 2. Genotype and allele frequencies of each polymorphism in the study population were in Hardy–Weinberg equilibrium \((P > 0.05)\). The changes in BP response to antihypertensive treatment in relation to genotypes of CYP11B2 –344C/T and ACE I/D gene polymorphisms are providedin table 3. Seven subjects carrying homozygous CYP11B2 –344C allele were found, thus CC and CT genotypes were used in combination for analyses. We did not find any association of ACE I/D gene polymorphisms with SBP or DBP response to telmisartan. Surprisingly, linear regression analysis indicated that CYP11B2 –344C/T was associated with DBP response to telmisartan, after correction with covariates, including pretreatment DBP, age, gender, BMI, blood glucose, TC, TG, HDL and LDL \((P= 0.005)\). CYP11B2 –344C/T accounted for 18.1% of inter-individual variation in the DBP response to telmisartan. The patients carrying the CYP11B2 –344C allele (CC+CT) showed a greater reduction in DBP than those carrying the TT genotype (figure 1).

Table 3 SNPs of CYP11B2 –344C/T and ACE I/D gene in linear regression and BP response to antihypertensive drugs.
Full size table

Discussion

Aldosterone is one of the main effectors of the RAAS. Aldosterone acts on the distal nephron to regulate sodium resorption, potassium excretion and intravascular volume. CYP11B2 is the key rate-limiting enzyme in the final steps of aldosterone biosynthesis. A few polymorphisms in CYP11B2 have recently been identified (White and Slutsker 1995; Fardella et al. 1996). Among them, –344C/T is a single-nucleotide polymorphism (SNP) located at a putative binding site for the transcription factor steroidogenic factor -1 (SF-1) (Fardella et al. 1996), 344 nucleotides from the transcription start site, where the residue could be a cytosine (–344C) or thymine (–344 T). This polymorphism has been associated with hypertension (Cheng and Xu 2009; Alvarez-Madrazo et al. 2013), coronary heart disease (Boduła et al. 2007) and has shown to influence aldosterone levels (Brand et al. 1998).

Fig. 1
figure 1

DBP response to telmisartan based on CYP11B2 –344C/T genotypes after adjustment for pretreatment DBP, age, gender, BMI, glucose, TG, TC, HDL and LDL.

Full size image

Several studies evaluated the impact of –344C/T polymorphism of CYP11B2 on the response to antihypertensive drugs. One study on the association between the CYP11B2–344C/T gene polymorphism and antihypertensive response to hydrochlorothiazide (HCTZ) found that the BP response in patients with CC genotype was less obvious than that in others for male patients (Li et al. 2012). Another study indicated that the patients with TC and TT genotypes had significant reduction in BP after ACE inhibitor treatment (Yu et al. 2006). But the studies on the antihypertensive response to telmisartan were few. In our study, the reduction in DBP after eight weeks of telmisartan treatment was significantly greater in patients carrying the C allele (CC+CT) compared with that carrying the TT genotype \((P\,{=}\,\,0.043)\). We did not find an association between the –344C/T polymorphism and the reduction in SBP after treatment. ARBs, which inhibit the combination of angiotensin II and angiotensin II type 1 receptor, led to marked peripheral vasodilation and BP-lowering effects. Angiotensin II is a stimulus regulator of CYP11B2, which is the key rate-limiting enzyme in the final steps of aldosterone biosynthesis. Aldosterone controls sodium balance and intravascular volume and helps to regulate BP. It could be proposed that the C allele is associated with a higher expression of the CYP11B2 gene and increased CYP11B2 activity, which leads to higher aldosterone secretion and individuals with a variant in this gene could respond differently to ARBs.

The ACE I/D polymorphism is one of the more extensively studied genes in hypertension. This polymorphism consists of the insertion or deletion of some portion of a 287-bp sequence of nonsense DNA in intron 16 of the ACE gene. Some studies had investigated the relationship between the ACE I/D polymorphism and BP response to antihypertensive drugs. However, the results have been conflicting. A meta-analysis demonstrated that there was a significant association between ACE I/D polymorphism and BP responses to HCTZ (Choi et al. 2013). But the GENRES study reported that ACE I/D polymorphism does not markedly predict BP response to antihypertensive agents involved amlodipine, bisoprolol, HCTZ and losartan in white hypertensive men (Suonsyrjä et al. 2009). In this study, there were no significant differences in the reduction of SBP and DBP after treatment. However, there was also a tendency for a higher SBP reduction in patients carrying the DD genotype compared with those carrying the II or ID genotype after treatment.

In summary, the CYP11B2 –344C/T polymorphism but not ACE I/D polymorphism was shown to influence the response to telmisartan in Chinese patients. Thus, specific genotypes might predict the response to specific antihypertensive treatment. This study has limitations because of the relatively small size. Thus our study should be viewed as hypothesis-generating and should be followed by larger prospective studies to confirm the results.