Association between serotonin 2A receptor (HTR2A) genetic variations and risk of hypertension in a community-based cohort study

Background Hypertension is one of the risk factors for obesity-related cardiovascular diseases. We investigated whether genetic variations in serotonin 2A receptor (HTR2A) were associated with hypertension. Methods We carried out a cross-sectional study in cohorts A (Ansan-Ansung cohort, N = 6039) and B (Wonju-Pyengchang cohort, N = 7524). Several genetic variants in HTR2A including rs7330636, rs9590999, rs2183057, and rs4942595 were selected and genotyped. Results In hypertensive participants in cohort A, the baseline systolic blood pressure and body mass index were 141.80 ± 17.20 mg/dL and 24.48 ± 4.75 kg/m2, respectively, which were higher than in those without hypertension (p < 0.001). rs4942595TC genotype was associated with hypertension in cohort A (OR = 0.739), after adjusting for variables. Subjects with rs4942578AA genotype had a decreased risk of hypertension after adjusting for clinical factor (OR = 0.735) in cohort B, and an elevated risk of hypertension in cohort A (OR = 1.562). The logistic regression analysis showed that participants with rs4941573TC genotype were 1.327 times more likely to have a higher blood pressure than those with TT genotype (95% CI 1.101–1.599) in cohort B. Whereas, the OR for developing hypertension in subjects with rs17069883CC genotype compared to those with AA genotype was 1.447 (95% CI 1.018–2.056; p for trend = 0.040) in cohort A. Conclusions HTR2A genetic variations were associated with hypertension risk in our study.


Background
Hypertension is considered not only a risk factor contributing to cardiovascular disease, but also a health issue in the community [1,2]. The known risk factors for hypertension are age, low physical fitness, tobacco usage, unhealthy eating, and high salt consumption [1]. Hypertension is a complex disease affected by genes, environmental factors, and their interactions [3,4].
Twin and family studies have shown that generally 20-60% of blood pressure variations could be ascribed to genetics [3,5]. Until now, many candidate genes have been shown to be involved in the regulation of blood pressure and susceptibility of hypertension [3,6,7]. Moreover, several genome-wide association studies demonstrated multiple susceptibility loci, associated with hypertension and blood pressure [8,9].
Among the physiological factors for regulating blood pressure, a neurotransmitter and hormone serotonin (5hydroxytryptamine, 5-HT) were considered. Its precise function in normal or altered blood pressure is unclear, likely because of the complex events of blood 5-HT in vitro [10,11]. The association of serotonin and development of hypertension was investigated by the blood platelets, which served as a primary storage location, and were easily accessible source of serotonin [10]. Blood platelet serotonin 2A receptor (HTR2A, also known as 5-HTR2A), regulates platelet activation and serotonin release; whereas 5-HTR2A located in vascular smooth muscle, endothelial cells, and cardiomyocytes regulate blood pressure and heart rate [12]. HTR2A encodes the 5HT2a receptor, which is expressed in the peripheral vasculature, and is involved in hypertension due to its vasoconstrictive effect [3,13].
Recent studies suggest that genetic variants in the HTR2A gene may be associated with hypertension [14], and abdominal obesity [15,16]. However, no associations were described between HTR2A genetic variations and hypertension in smaller case control studies in Chinese or Japanese subjects [17,18]. We investigated whether genetic variations of HTR2A were associated with the risk of hypertension in two different community-based cohorts.

Study population
This study was performed with individuals from a community-based cohort (the Ansan-Ansung and Wonju-Pyengchang cohort) within the Korean Genome Epidemiology Study (KoGES). The Ansan-Ansung cohort is a longitudinal survey supported by the Korean government (Korean National Research Institute of Health, Korean Centers for Disease Control and Prevention, and the Ministry of Health and Welfare), to examine the genetic and environmental factors of chronic disease in Koreans [19][20][21]. Korean adults was recruited from the following two sites: Ansung and Ansan, which is a rural district with roughly 190,000 and 693,000 residents, respectively [22]. A total of 10,038 adults (5018 from Ansung and 5020 from Ansan) had a baseline health examination at the Ajou University Medical Center and the Korea University Ansan Hospital from June 2001 to January 2003. Follow-up surveys were performed biennially [22]. Additionally, all adults residing in rural areas of Wonju and Pyengchang in South Korea, where demographic changes are uncommon, participated in the study [23][24][25]. The study protocol was approved by the Institutional Review Board of Wonju Christian Hospital and Ajou University Medical Center, and the Korea University Ansan Hospital. All participants provided written informed consent. We excluded the participants with incomplete data and without the result of sequencing in HTR2A gene ( Figs. 1 and 2). In total, 6039 participants (cohort A) and 7524 (cohort B) were eligible for this study. Hypertension was defined as a systolic blood pressure (SBP) ≥ 140 mmHg, and/or diastolic blood pressure (DBP) ≥ 90 mmHg, and/or current treatment with antihypertensive medications. All the participants were examined after fasting.

Anthropometric and laboratory measurements
Anthropometric indices including the body mass index (BMI), smoking status, regular physical exercise and several other parameters were investigated. The waist circumference was obtained using a tape measure (SECA-200, SECA, Hamburg, Germany). Systolic (SBP) and diastolic blood pressure (DBP) were measured twice by a standardized mercury sphygmomanometer (Baumanometer, Copiague, New York). Blood sample was extracted from participants after fasting. Fasting glucose was estimated by a glucose oxidase-based assay. The serum concentrations of low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides (TGs) were examined using the enzymatic calorimetric method (Advia 1650, Siemens, Tarrytown, New York). Alcohol and smoking habits were identified by self-questionnaires [26].

Sequencing and genotype
We analyzed several genetic variants in HTR2A gene. PCR primers were designed to amplify HTR2A fragments. PCR products were sequenced by a BigDye Terminator Cycle Sequencing Kit (version 3.1, ABI, Foster City, CA, USA), and an ABI 3730 × 1 automated sequencer (Applied Biosystems, Foster City, CA, USA). SNPs confirmed in the HTR2A gene were genotyped. Genomic DNA was drawn from 5 mL of peripheral venous blood using an isolation kit (QuickGene SP Kit DNA whole blood, Fujifilm, Tokyo, Japan). Genotyping was performed using the TaqMan fluorogenic 5′ nuclease assay (ABI) [27].
We evaluated the association between genetic variants in HTR2A and risk of hypertension in each cohort, however, some of them did not show the association of genetic variants of HTR2A and risk of hypertension. In cohort A and B, several single nucleotide polymorphisms (SNPs) which shown significant and non-significant associations of risk of hypertension were reported (e.g. rs7330636, rs9590999, rs2183057) in this study.

Statistical analysis
Group differences for categorical variables were analyzed using chi-square test, and the continuous variables were estimated by Student's t-test. The association between genotype and the risk of hypertension was assessed using odds ratios (ORs) and 95% confidence intervals (CIs), from chi-square tests and logistic regression analyses. The results are shown as mean ± standard error range. The p-values for each genotype were calculated by ANOVA test. A p-value with three decimal places was considered as statistically significant difference between no hypertension and hypertensive individuals. All analyses were determined using SPSS software, version 23.0 (SPSS Inc., Chicago, IL, USA). We adjusted for age, gender, smoking status, alcohol consumption and regular exercise in Model 1. Model 2 was adjusted for Model 1 and systolic blood pressure, total cholesterol and baseline body mass index, additionally.

Results
Characteristics of the study population according to the genotype of HTR2A Characteristics of the population are shown in Table 1. Total 1418 subjects, who developed hypertension in cohort A, were aged 61.59 ± 6.12 years, and 46.8% (n = 664) of the total population were men. In the participants who developed hypertension in cohort B, the baseline SBP and fasting glucose levels were 140.06 ± 16.21 mmHg and 91.18 ± 30.71 mg/dL, respectively, which were higher than in those without hypertension (p < 0.001) ( Table 1).
The genotype frequency of the studied loci is shown in Table 2. Genotype frequencies for all SNPs were in accordance with the Hardy-Weinberg equilibrium. Distribution of HTR2A rs2183057 genotype frequencies differed significantly between patients with or without hypertension. We observed a statistically significant increase in rs17069883CC genotype frequency in the group of patients with hypertension (23.3 vs. 19.3% in no hypertension group, p = 0.041), and a decrease in the  Table 2).
The analysis of the data showed that carriers of rs7330636GA genotype had a lower risk of hypertension (OR = 0.644, 95% CI 0.437-0.949). The rs9590999CT genotype was associated with a decreased risk of hypertension (OR = 0.738 95% CI 0.551-0.989) in cohort A.
Subjects with rs4942578AA genotype had a decreased risk of hypertension after adjusting for clinical factor such as age and gender (OR = 0.735, p = 0.010) in cohort B, while, those with rs4942578AA genotype had an elevated risk of hypertension in cohort A (OR = 1.562, p = 0.020). The logistic regression analysis showed that participants with rs6313GA genotype were 1.365 times more likely to have a higher blood pressure than those with GG genotype (OR = 1.365; 95% CI 1.135-1.643; p = 0.003) in cohort B. Meanwhile, the odds ratio for developing hypertension in subjects with rs4941573TC genotype compared to those with TT genotype was 1.327 (95% CI 1.101-1.599; p for trend = 0.003) in cohort B (Table 3).

Discussion
Genetic variations in the serotonin 2A receptor gene were found to be associated with hypertension, in a communitybased cohort study. Heterozygosity of T102C (rs6313) was associated with the blood pressure of 140/95 mmHg or higher, after adjustment for demographic covariates, such as age, gender and BMI. We identified an association between rs4942578 and rs6313, located in intron, and an elevated risk of hypertension. Even though the mechanism of these connection exists uncertain, there is sufficient evidence available ascertaining that serotonin and serotonergic neurotransmission play a crucial role in cardiovascular regulation. Serotonin is a vasoactive monoamine, having amphibolic features demonstrated by its capability to induce either vasoconstriction and blood pressure enhancement [16,28]. The human brain serotonergic inductivity in the hypothalamic-pituitary axis has been founded to increase with a decrease in resting blood pressure, and vice versa [16]. The blood pressure increasing effects of serotonin seem to be regulated by 5-HT 2A receptor, while 5-HT 1A receptor activation leads to sympathoinhibition and vasodilation [16]. Therefore, the HTR2A polymorphisms might play a role in the blood pressure regulation by serotonin, most likely by central mechanisms [14,16] and recent studies suggested that polymorphic variations in HTR2A gene may be associated with hypertension [14].
Association between HTR2A SNPs and hypertension has been demonstrated in different ethnic groups [3,18,29,30]. SNPs in several other genes have also been shown to be associated with hypertension such as A1161 polymorphism in angiotensin II type I receptor gene (AGTR1) and CYP11B2 gene polymorphism [14,[31][32][33]. The ß2 adrenergic receptor has been involved in the cause of hypertension owing to its role in vasodilatation, and a significant association was verified between the genetic polymorphism of ß2 adrenergic receptor and hypertension [14]. A meta-analysis has shown that RGS2 1891-1892del TC polymorphism and CYP4A11 T8590C polymorphism were associated with hypertension risk [3]. Also, the T58C promoter polymorphism of the bradykinin ß2 receptor gene was associated with hypertension in a Japanese population [14,34]. Halder I et al. suggested that elevated blood pressure was associated with both -1438GG and 2416TT genotypes in HTR2A gene [16]. Similarly, we identified significant associations between genetic variants in HTR2A and risk of hypertension.
There are few strengths and limitations of our study. HTR2A gene polymorphisms were evaluated in two larger community-based cohorts compared to earlier studies, thus, the sample size was large enough to reach adequate statistical power. Moreover, association of HTR2A rs4942578 and rs4941573 polymorphisms with hypertension had not been investigated before. However, our findings may not be applicable to other populations,  especially different ethnicities or with younger age. Additionally, we did not find sufficient studies to corroborate the association between HTR2A gene polymorphisms and the risk of hypertension found in this communitybased cohort study. In cohort A, basic characteristics of populations included in the analyses and those excluded from the analyses were significantly different including age, gender, SBP, DBP, BMI, glucose, smoker and exercise and this might lead to selection bias.

Conclusions
We identified significant associations between several HTR2A SNPs and the risk of hypertension. Further studies are required to elucidate the in vivo effects and identify functional loci in order to explain a mechanistic basis for observed associations. Confirmatory studies are also warranted using larger sample size and definitive genetic methodologies, such as family-based designs or populations with genetic stratification, along with application of genomic control procedures.
Additional file 1: Table S1. Basic characteristics of participants included and excluded. Table S2. Non-significant association of genetic polymorphisms of HTR2A gene and risk of hypertension. Table S3. Nonsignificant association of genetic polymorphisms of HTR2A gene and risk of hypertension. Table S4. Distribution of HTR2A genetic variations according to development of hypertension. Table S5. Association of genetic polymorphisms of HTR2A gene and risk of hypertension. Table S6. Association of genetic polymorphisms of HTR2A gene and risk of hypertension.