Abstract
The aim of this study was to assess the associations between single nucleotide polymorphisms (SNPs) of the apelin and APJ (apelin receptor) genes and the risk of hypertension in people living in the south-east coastal area of China. A cross-sectional study involving 1031 participants was performed. Genotypes of the apelin (rs3115757, rs56204867 and rs3761581) and APJ (rs7119375 and rs9943582) genes were determined by the TaqMan® MGB probe method. For male patients, the frequencies of mutant alleles in the three apelin gene SNPs were significantly different between the hypertension and control groups (all p < 0.05), while no significant difference was obtained for frequencies of mutant alleles in the two APJ gene SNPs (p > 0.05). For females, the frequencies of mutant alleles in all five SNPs were not significantly different between the hypertension and control groups (all p > 0.05). After adjusting for several factors, the risk of developing hypertension increased significantly in patients, regardless of gender, carrying rs3115757-C, rs56204867-C or rs3761581-A allele (all p < 0.05). The optimal gene–gene interaction model for both males and females with regard to hypertension was apelin rs3761581–apelin rs3115757–APJ rs7119375. In conclusion, gene polymorphisms of the apelin–APJ system are associated with susceptibility to hypertension.
Introduction
Recent studies have found that apelin,[Citation1] a new bioactive peptide, and its receptor APJ [Citation2] are involved in blood pressure regulation. Intravenous injection of apelin effectively reduced the blood pressure in spontaneous hypertensive rats.[Citation3,Citation4] Human studies also found that plasma apelin level is associated with hypertension.[Citation5] Other studies have demonstrated that apelin can reduce blood pressure by relaxing vascular smooth muscles, also antagonizing hypertension and cardiovascular fibrosis induced by angiotensin II.[Citation6] The Ang II inhibition-mediated beneficial effects are likely attributable, at least in part, to the restoration of apelin/APJ expression in diet-induced obesity-related hypertension.[Citation7] These findings suggest that this peptide could be used for the treatment of hypertension.[Citation8,Citation9] In our previous cross-sectional study undertaken in the south-east coastal area of China, we found a negative association between plasma apelin level and hypertension.[Citation10]
Primary hypertension is a complex polygenic disease caused by the combined effects of genetic and environmental factors. Genetic factors have been estimated to account for about 30–50% of the pathogenesis of primary hypertension.[Citation11] The heredity of hypertension is reflected not only by the prevalence, but also by its severity and complications.[Citation12] Accumulating evidence has shown that single nucleotide polymorphisms (SNPs) of the apelin–APJ system’s genes are associated with hypertension. In a hypertension pedigree-based study performed by Li et al.,[Citation13] it was found that the rs56204867 and rs3761581 polymorphisms of the apelin gene and rs7119375 polymorphism of the APJ gene are associated with hypertension: the A-T apelin haplotype was shown to be involved in the development of hypertension, while the C allele in rs9943582 of the APJ gene conferred susceptibility to primary hypertension, abnormal body mass index (BMI) and early-onset hypertension. Another study, performed by Liao et al.,[Citation14] showed that the apelin rs3115757 gene polymorphism is closely associated with waist circumference and BMI in Chinese women; the percentage of women with higher waist circumference or BMI was significantly higher in individuals carrying the CC genotype than in those with the GG or GC genotype (1.07- and 1.29-fold increase, respectively). Zhao et al. [Citation15,Citation16] performed a GenSalt study to assess the effects of apelin gene polymorphisms, low sodium and potassium supplementation on blood pressure; they found that the rs2235306 polymorphism in women and rs4646174 polymorphism in men are associated with potassium supplementation in improving the susceptibility to blood pressure responses, while rs2282623 and rs746886 polymorphisms were shown to be associated with a low-sodium diet in improving the susceptibility to blood pressure responses.
In the present study, we determined the genotypes of three apelin gene SNPs, namely rs3115757, rs56204867 and rs3761581, as well as two APJ gene SNPs, namely rs7119375 and rs9943582, to assess the associations between the apelin–APJ system’s gene polymorphisms and hypertension in people living in the south-east coastal area of China.
Methods
Study population
Inhabitants of four villages in Haidao, Xiapu County, and three villages of Tailu, Lianjiang County, in Fujian Province, China, were included in this cross-sectional study, between July and November 2011. In total, 3344 subjects, comprising 1076 individuals with hypertension and 2268 non-hypertensive patients, were enrolled. Subjects with diabetes, secondary hypertension, severe liver or renal dysfunction, coronary heart disease, heart failure or stroke were excluded. Finally, 556 subjects with hypertension and 475 without hypertension were randomly selected and included. All of the included subjects signed informed consent forms.
Blood examination
Subjects were asked to fast for at least 10 h, and 5 ml and venous blood was collected from the forearm; serum was obtained by centrifugation and stored at –20 °C. Total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and fasting blood glucose (FBG) levels were measured on an automatic biochemical analyser (DxC800; Beckman Coulter Inc., Brea, CA).
Genotyping
A DNA extraction kit (DN01; Ed Biological Technology Co., Beijing, China) was used to extract DNA from blood samples A quantitative polymerase chain reaction (qPCR) was performed with TaqMan® MGB probes, which were provided by Applied Biosystems (Foster City, CA) for all five sites; the corresponding primers were purchased from Applied Biosystems (Foster City, CA) as well. A 20 μl reaction system was used for qPCR and included 10 μl of TaqMan® SNP genotyping PCR mix (2 × Master-Mix), 1 μl of mixture of probes and primers (20 ×), 4 μl of DNA template (10 ng/μl) and 5 μl of sterile water. The reaction was carried out at 95 °C for 10 min, 92 °C for 15 s and 60 °C for 1 min, for a total of 40 cycles; both pre- and post-plate readings were performed at 60 °C for 1 min. Both PCR and genotyping were performed using an ABI 7300 real-time PCR instrument (Applied Biosystems, Foster City, CA); the PCR program was designed with software provided by Applied Biosystems and the genotypes were determined automatically by reading the fluorescence signals.
Definitions
Hypertension was defined as a systolic blood pressure of ≥ 140 mmHg and/or diastolic blood pressure of ≥ 90 mmHg in the absence of antihypertensive drug therapy. Subjects with a history of hypertension and who were currently using antihypertensive drugs were also defined as hypertensive.[Citation17]
Diabetes was defined as a glycosylated haemoglobin (HbA1c) of ≥ 6.5%. Patients with a history of diabetes or who were currently using antidiabetic drugs were also considered diabetic.[Citation18]
Statistical analysis
The SPSS software (IBM Corp., Armonk, NY) was used for statistical analysis. The apelin gene is located on the X chromosome,[Citation19] and the results were displayed by gender. Means and standard deviations were used to describe quantitative data, while rates were employed for qualitative data. The independent t test was used to compare the quantitative data between different groups, while the chi-squared test was used for rate comparisons. A recessive model was employed by combining the mutant homozygotes and heterozygotes as mutant alleles and comparing to wild type; age, BMI and FBG were adjusted, and binary logistic regression was performed to assess the associations between the five SNPs of the apelin–APJ system’s genes and the risk of hypertension. Two-sided p values < 0.05 were considered statistically significant.
Hardy–Weinberg equilibria of the five apelin–APJ system gene SNPs were evaluated by HaploView v4.2 software (http://www.broadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview).
The latest version of multifactor dimensionality reduction (MDR) 2.0 software [Citation20] was used to evaluate the gene–gene interactions among the apelin–APJ system’s genes.
Results
Clinical characteristics of the subjects
The 1031 included subjects comprised 398 males and 633 females, aged 53.9 ± 11.6 years (range 30–84 years); of the subjects, 556 and 475 were hypertensive and controls, respectively. Age, systolic blood pressure, diastolic blood pressure, waist circumference, BMI, total cholesterol, LDL-C and FBG were significantly higher in the hypertension group compared with control individuals, while HDL-C was significantly lower in the hypertension group than in the control group (all p < 0.05), irrespective of subjects’ gender. However, smoking status was not significantly different between these two groups (p > 0.05) ().
Table 1. Clinical characteristics of the included subjects.
Genotype and allele distribution
The apelin gene is located on the X chromosome; thus, the Hardy–Weinberg equilibrium for apelin was only tested in female subjects, while the equilibrium for the APJ gene was assessed in both males and females. The distribution of all genotypes in the three apelin gene SNPs (rs3115757, rs56204867 and rs3761581) and two APJ gene SNPs (rs7119375 and rs9943582) was in accordance with the Hardy–Weinberg equilibrium (all p > 0.05), suggesting that the sample was from a large, randomly distributed, equilibrated population, and thus constituted a representative sample.
For male subjects, the frequencies of mutant alleles of the three apelin gene SNPs were significantly different between the hypertension and control groups (rs3115757-C: 53.8% vs 15.1%; rs56204867-C: 48.4% vs 27.7%; and rs3761581-A: 72.8% vs 53.3%; all p < 0.05), while the frequencies of mutant alleles of the two APJ gene SNPs were not significantly different between hypertensive and control individuals (rs7119375-A: 29.2% vs 31.1%; rs9943582-T: 33.2% vs 34.4%; both p> 0.05). On the other hand, for female subjects, the frequencies of mutant alleles of all five SNPs were similar between the hypertension and control groups (all p > 0.05) ().
Table 2. Genotype and allele distribution of the apelin–APJ system’s genes in the hypertension and control groups.
Associations between the five SNPs of the apelin–APJ system’s genes and the risk of hypertension
After adjusting for several factors, including age, BMI and FBG, the logistic regression data showed that, irrespective of gender, individuals carrying the rs3115757-C allele were at significantly higher risk of developing hypertension than those with the wild-type G allele [odds ratio (OR) = 6.341, 95% confidence interval (CI) 3.739–10.753, p < 0.001 for males; OR = 2.069, 95% CI 1.025–4.177, p = 0.042 for females]; individuals carrying the rs56204867-C allele had a significantly higher risk of developing hypertension than subjects with the wild-type T allele (OR = 2.410, 95% CI 1.490–3.891, p < 0.001 for males; OR = 2.052, 95% CI 1.213–3.470, p = 0.007 for females); patients harbouring the rs3761581-A allele were at significantly higher risk of developing hypertension than those with the wild-type C allele (OR = 1.949, 95% CI 1.205–3.154, p = 0.007 for males; OR = 2.000, 95% CI 1.327–3.013, p = 0.001 for females). For the APJ gene, no significant difference in the risk of developing hypertension was found between individuals (males or females) with the rs7119375-A allele and those carrying the wild-type G allele (OR = 1.441, 95% CI 0.740–2.805, p = 0.283 for males; OR = 1.076, 95% CI 0.617–1.878, p = 0.795 for females), or between patients (males or females) carrying the rs9943582-T allele and those with the wild-type C allele (OR = 1.355, 95% CI 0.703–2.612, p = 0.365 for males; OR = 0.916, 95% CI 0.535–1.570, p = 0.750 for females) ().
Table 3. Associations between the five SNPs of the apelin–APJ system and the risk of hypertension.
Gene–gene interactions among the apelin–APJ system’s genes
The optimal MDR model with regard to hypertension for males was the combination of apelin rs3761581, apelin rs3115757 and APJ rs7119375 genotypes. The error of the training sample for this model was low (1 – 0.733 = 0.267); a consistency of cross-validation of 10/10 was obtained and the sign test showed that the results were statistically significant (χ2 = 8.611, p = 0.003). Similarly, the optimal MDR model with regard to hypertension for females was also the combination of apelin rs3761581, apelin rs3115757 and APJ rs7119375 genotypes. The error of the training sample for this model was also low (1 – 0.743 = 0.257), a consistency of cross-validation of 10/10 was obtained and the sign test showed that the results were statistically significant (χ2 = 13.207, p = 0.001) ().
Table 4. Multifactor dimensionality reduction analysis of hypertension.
Discussion
The human apelin gene consists of three exons and two introns, and is located at q25-26.1 of the X chromosome. The length of the human apelin mRNA is 2673 bp.[Citation16] The APJ gene is located on chromosome 11, and the amino acid sequence of the encoded protein shares 54% homology with the transmembrane domain of AT1R. Although an overlapping zone exists with regard to the anatomical distribution in the cardiovascular system of the products encoded by these two genes, the APJ gene products do not combine with AngII. In 2009, Li et al. [Citation13] first analysed the associations between the apelin–APJ system and hypertension in a study based on hypertension pedigrees. Since then, multiple studies have been performed to investigate the associations between apelin–APJ gene SNPs and the risk of cardiovascular diseases.[Citation15,Citation16] In the present study, we performed a cross-sectional study to investigate the associations between the five apelin–APJ gene SNPs and the risk of hypertension, and found that the three apelin gene SNPs (rs3115757, rs56204867 and rs3761581) and one APJ gene SNP (rs7119375) could be among the multiple candidate SNPs for hypertension; no difference was found for these four SNPs between males and females, as shown above. However, no statistically significant difference was obtained for the other APJ gene SNP (rs9943582) in our study. After adjusting for several factors, including age, BMI and FBG, males or females carrying the rs3115757-C, rs56204867-C or rs3761581-A allele had significantly higher risk of developing hypertension than individuals with wild-type alleles (the risk of developing hypertension increased by 5.341-, 1.410- and 0.949-fold for males, and 1.069-, 1.052- and 1.000-fold for females, respectively), suggesting that apelin polymorphisms could be associated with the genetic mechanisms of hypertension in the people in this area, in accordance with previous findings.[Citation13] We speculated that mutation at rs3115757, rs56204867 or rs3761581 of the apelin gene could affect or induce the generation of new binding regions for transcriptional regulators, which could influence apelin gene transcription, and the expression of the encoded protein. In addition, the alterations of protein expression could, in turn, change the phenotype, thus further delaying the pathogenesis of hypertension.
Minor gene effects play major roles in the genetic mechanisms of primary hypertension. As a polygenetic disease, the heredity of hypertension does not always obey the Mendelian inheritance pattern; other genetic sites and multiple environmental risk factors also affect the inheritance and even cause complex high-level interactions.[Citation21] In other words, the independent effects of the SNP of some minor genes could be covered by the effects of adjacent SNPs.[Citation22] In the present study, analysis of single sites showed that the polymorphisms of the ligand apelin gene are associated with the risk of hypertension, while the receptor APJ gene was not significantly associated with hypertension. Only after combination with the receptor can apelin exert its endothelium-dependent vascular dilatation and blood pressure reduction effects; however, whether the association differences between apelin and APJ gene SNPs and hypertension in the studied population are related to the interactions between these two genes remains unclear.
MDR, a non-parametric, model-free method, was first described by Ritchie et al. [Citation23] in 2001 to investigate gene–gene and gene–environment interactions. When using this method to investigate the interactions among different factors at different levels, the main effects are not considered. Therefore, when the potential main effects are not statistically significant, the high-level interactions can still be found; however, the main effects cannot be identified when they show statistical significance; meanwhile, logistic regression can effectively identify the main effects. In the present study, the latest MDR software was used for analysis, and we found that for both males and females, a three-site model, namely apelin rs3761581–apelin rs3115757–APJ rs7119375, was the best MDR model; logistic regression found no main effect of the APJ rs7119375 polymorphism. These two methods demonstrated from different aspects that the effects of the apelin–APJ system on blood pressure require both apelin and APJ in the costal Chinese population, which could also partially explain why the effects of apelin gene polymorphisms were relatively pronounced, while those of APJ gene polymorphisms were not statistically significant.
Several limitations of the present study should be discussed. First, this was a cross-sectional study, and subjects in the hypertension and control groups were not matched by gender and age. Secondly, only five SNPs of the apelin–APJ system were investigated, and no further studies involving more SNPs of this system or other classic systems (such as the renin–angiotensin–aldosterone system) were conducted.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding information
This study was supported by the Fujian Major Program of Basic Science Project Foundation entitled “Epidemiologic survey of hypertension and prehypertension intervention research in HaiDao County of Fujian Province” [2010Y1103] and the Fujian Medical Innovation Project entitled “The association of plasma apelin levels and apelin polymorphism with hypertension and hypertensive vascular injury” [2012-CX-1].
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