Effect of RAS Polymorphism on Essential Blood Pressure Essay
Effect of RAS Polymorphism on Essential Blood Pressure Essay
Pharmacogenetic Association of RAS Polymorphism on Essential Blood Pressure in Relation to Enalapril/Lisinopril among Malay male newly diagnosed hypertensives
Abstract
Objective: It has been suggested that genetic backgrounds, which have an association with essential hypertension, may also determine the responsiveness to ACE inhibitor.Effect of RAS Polymorphism on Essential Blood Pressure Essay. We determined the association of angiotensin-converting enzyme (I/D, G2350A), angiotensinogen (M235T, T174M and A-6G) and renin (Bg/I and Mbo/I) gene polymorphisms with essential hypertension and the relationship between genetic variant of interest and high blood pressure response to ACE inhibitor (enalapril, lisinopril) in patients with essential hypertension subjects from Seremban, Malaysia.
Methods: A newly hypertensive Malay male population (n=142) was recruited for a mono-trophy[ah1] pharmacogenetic study. Hypertensive patients were treated with ACEI drugs, particularly enalapril or lisinopril alone. We differentiated between those who controlled their HT with those who did not. Each group’s characteristics were compared to determine the risk of non-controlled HT associated with RAS polymorphisms by adjusting for different variables.
Results: Statistically significant associations of I, G, T and M alleles were observed with essential hypertension in I/D, G2350A, M235T, and T175M. The decrease in systolic blood pressure and diastolic blood pressure after 24 weeks of treatment of the patients carrying II, GG, and TT genotypes was greater than the groups carrying DD, AA, MM and MM genotypes. In contrast, no significant difference was shown between renin gene polymorphisms (Bg/I and MboI).
Conclusions: Although this study shows a possible association of polymorphisms of RAAS genes[ah2] with the risk of non-controlled HT in ACEI-treated patients and indicates the importance of all components in this system in regulating HT, it needs to be replicated in other data sources.
Keywords: Essential hypertension; renin-angiotensin-aldosterone system; single-nucleotide polymorphism; ACE inhibitors; pharmacogenetic
INTRODUCTION
Essential hypertension (EH) is an increasingly important medical and public health issue [1].Effect of RAS Polymorphism on Essential Blood Pressure Essay. In Malaysia, the National Health and Morbidity Survey (NHMS) 2013 has shown that the prevalence of hypertension in adults ≥18 years increased from 33.2% in 2006 to 35.7% in 2013 [2]. Furthermore, the prevalence increased from 42.6% to 43.5% for those >30 years old. Unfortunately, 60.6% of total hypertensives were “undiagnosed” [3]. These poor rates of high blood pressure (BP) control are not explained by the lack of treatment, as one study estimated approximately 30% of treated hypertensive patients take one antihypertensive drug, 40% take two antihypertensive drugs and 30% take three or more antihypertensive drugs [4]. These data suggest that the present trial and error approach for high blood pressure management is suboptimal, and alternative approaches for identifying the optimal antihypertensive regimen in a specific patient are needed. Using genetic make-up of an individual along with the association between single-nucleotide polymorphisms (SNPs) and angiotensin-converting enzyme (ACE) inhibitors for hypertensive response offers a new preventive approach to lower adverse drug interaction risk.
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A renin-angiotensin system (RAS) is an important component of blood pressure regulation, and it has been suspected to be involved in hypertension [5]. Moreover, the major active peptide of the RAS is angiotensin II. Produced from the precursor molecule, angiotensinogen (AGT), via an enzyme cascade involving ACE enzyme, angiotensin II exerts numerous effects on the homeostatic regulation of blood pressure, the vast majority of which are mediated via the angiotensin II type 1 receptor (AT1R) [6].
The presence of polymorphisms in the ACE, AGT and renin (REN) genes of the RAS has been associated with adverse EH changes in several studies [7,8,9].Effect of RAS Polymorphism on Essential Blood Pressure Essay. For example, the insertion/deletion (I/D) polymorphisms of the ACE gene have been associated with increased blood pressure, urinary albumin excretion (UAE) and target-organ damage in hypertensive patients [10]. Moreover, ACE G2350A gene polymorphisms in exon 17 were reported as a remarkable genetic variant mostly associated through hypertension with an average increase of 3.2 mmHg in SBP by having the G allele [11]. Effect of RAS Polymorphism on Essential Blood Pressure Essay.
It has been reported that the presence of A-6G polymorphism of AGT gene among Chinese hypertensives increased body weight gain in hypertensive patients [12]. The Mb/I and Bg/I polymorphisms of the REN gene have been associated with hypertension, left ventricular hypertrophy, aortic stiffness and exaggerated vasoconstriction; additionally, the Bg/I polymorphism in the same gene appears to confer protection against the development of microalbuminuria in patients with hypertension [13].
The purpose of the present study was to evaluate the impact of four RAS gene polymorphisms on the antihypertensive response in newly detected hypertensives receiving two ACEIs (enalapril and lisinopril). The polymorphisms investigated were A-6G, the A for G substitution of the AGT gene 6 nucleotides upstream from the start site; the ACE I/D polymorphism corresponding to an insertion or deletion of a 287bp alu repeat; and two polymorphisms of the REN gene, Bg/I and MboI and both in the coding area of intron 9. There are compelling reasons hypothesizing that variations in genes of the system may be predictive of variations in BP response. Therefore, genetic variation of the RAS has been investigated in relation to antihypertensive response to ACE inhibitors in various population and dosage (Table 1) as the most common lowering BP agent in Malaysia [14]; however, previously publications have had somewhat conflicting results elsewhere [15-19].
We hypothesized those genetic polymorphisms in RAS genes, including ACE I/D, G2350A, AGT M235T, T174M, A-6G along with REN MboI and Bg/I were associated with the incidence of EHT. Therefore, the aim of this pharmacogenetic study was to investigate the association between seven RAS gene polymorphisms of interest among 142 newly diagnosed Malay male hypertensives that never took BP medications. They were treated once daily for 24 weeks with 20 mg of enalapril or lisinopril. Effect of RAS Polymorphism on Essential Blood Pressure Essay.
MATERIAL AND METHODS
Patient Populations
Malay male patients >18 years of age with three-generation Malay family who were newly diagnosed with essential mild-to-moderate hypertension were collected from clinics for non-communicable disease Seremban, Malaysia. The information includes age (25-60 years old), onset (25-60 years old), systolic BP > 140 mmHg and a diastolic BP > 90 mmHg on 2 consecutive visits for those untreated, absence of secondary forms of hypertension. Subjects with a history of diabetes mellitus, renal failure and major infectious disease were excluded. Effect of RAS Polymorphism on Essential Blood Pressure Essay. They had no metabolic or endocrine disorder, as well as any acute illness. They were not on any antihypertensive treatment and were drug-naive patients. Written informed consent was obtained from each patient before being included in the clinical trial, and patient’s identity was kept strictly confidential. A specific consent form was requested for genetic testing permission.
BLOOD PRESSURE REDUCTION PATTERN
Lifestyle Modification
For patients, lifestyle modification for a period of three months was advised. The patients were seen three times during this period to assess the efficacy of the non-pharmacological management including weight loss, regular exercise, and ingestion of a high-fiber, low-fat, and low-salt diet.
Follow-up Mon-trophy[ah3] Management
Dispensed ACEIs (lisinopril or enalapril) on the same date for individuals have recorded.
Each patient received lisinopril or enalapril (20 mg, once daily) for 24 weeks on a regular basis.Effect of RAS Polymorphism on Essential Blood Pressure Essay. Patients’ BP was measured using the same device and protocol; follow-up visits were made 12 times (once per two weeks). Of the 152 patients, 10 lost to the follow-up along monitoring due to relocation, travelling and/or change of medication. Eventually 142 hypertensives (92.6%) completed the study. This subsample was divided into two groups; individuals whose HT was not controlled as the non-responded (n=35), and individuals whose HT was under control as the responded (n=107). Figure 1 presents how responded and non-responded HT groups are categorized.
Fig. 1. The flowchart of sample collection.
Genotyping procedures
A blood sample was taken in two separate tubes; one was used for colorimetric analysis of total cholesterol (TC), high density lipid profile (HDL), low density lipid profile (LDL), triacylglycerol (TG) and fasting glucose (FBG) using Diays Commercial Kits (Diagnostic System, GmBH, D66559 Holzheim, Germany), and the other test tube contained venous blood samples collected on EDTA was subjected to DNA extraction, which was obtained from individuals in the morning after a minimum of 8 hours fasting at the time of randomization. Eventually, the samples were stored at -20°C for further molecular and biochemical analysis.
DNAs were extracted from 5 mL blood samples as explained elsewhere [20]. ACE I/D polymorphism was genotyped using DNA amplification with oligonucleotides as described elsewhere [21]. Effect of RAS Polymorphism on Essential Blood Pressure Essay. For ACE G2350A, DNA amplification followed the approach by Zhu et al. [22]. Reactions were conducted using DNA amplification in a final volume of 25 mL containing 20 pmol of each primer, 0.4 mmol/L of each deoxynucleotide triphosphate (dNTP), 2 mmol/L of MgCl2, 1XTaq buffer and one unit of NEB Taq DNA polymerase (New England Biolabs, Beverly, MA, USA). The PCR cycling conditions were carried out in an iCycler machine (BioRad Laboratories, Hercules, CA, USA). Were chosen[ah4] for genotyping using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) approach, and the details are presented in Table 2. Eventually, DNA fragments were stained in ethidium bromide and visualized by Alpha Imager (Alpha Innotech, San Leandro, CA, USA) under ultraviolet (UV) light. Effect of RAS Polymorphism on Essential Blood Pressure Essay.
Statistical Analysis
SPSS 20 statistical package (SPSS, Chicago, USA) was used for analysis. Allele frequencies were calculated from the genotypes of all subjects. Hardy-Weinberg equilibrium (HWE) was assessed by χ2 analysis. Continuous data are presented as mean ± SD. Differences between groups were tested by an χ2 test for qualitative parameters and by one-way analysis of variance (ANOVA). All tests were two-tailed and the values of p<0.05 were considered to indicate statistical significance. Calculation of mean arterial pressure (MAP) was performed by using the formula given below: Equation: MAP = [(2xdiastolic)+systolic] /3 Where diastole counts twice as much as systole because 2/3 of the cardiac cycle is spent in diastole. The usual range of MAP is 70-110 and an MAP of about 60 is necessary to perfuse coronary arteries, brain, and kidneys [][ah5]. To detect false-positive results due to multiple testing, we applied the Bonferroni correction test for 94 independent genotype loci.
Results
Population Characteristics
Table 3 shows the anthropometric characteristics and the biochemical factors of the controlled hypertensives (HT) and non-controlled hypertensives via lisinopril or enalapril patient groups. This study found significant differences for the levels of total cholesterol and LDL-cholesterol, systolic blood pressure and diastolic blood pressure, in which were all higher in the non-controlled HT group. Effect of RAS Polymorphism on Essential Blood Pressure Essay. The systolic blood pressure levels for the non-controlled HT group (159.03 ± 19.46 mmHg) were slightly higher than the limit set by WHO for HT (140 mmHg) [][ah6], while the diastolic blood pressure levels (89.17 ± 10.11 mmHg) were near the limit established for the general population (90 mmHg).
Distribution of the Analyzed SNPs and the Single-SNP Association Analysis
In the association analysis of the genotypic distribution of the SNPs, through the comparison between controlled HT and non-controlled HT, conclusive results were obtained for ten risk SNPs (Table 4). For rs4646994, which belongs to the ACE I/D gene polymorphism, an adjusted Odds Ratio (OR) of 2.4460 (Cl 1.7334-3.4318, p = 0.0003) was obtained for I/I. Likewise, for rs4343 of G2350A gene polymorphism, we obtained an OR of 1.1416 (Cl 0.8098-1.6092, p= 0.0001) for genotypes G/G–G/A. Regarding the SNPs belonging to the AGT gene, we obtained an OR of 0.3506 (Cl 02496- 0.4925 p = 0.001), 0.3506 (Cl 02496- 0.4925, p = 0.001) and 2.9458 (Cl 2.0716 – 4.1888, p = 0.003) for rs11571099, rs4762, and when the -/- and m/m genotypes were expressed for the first SNP and for the second as well, respectively. Effect of RAS Polymorphism on Essential Blood Pressure Essay.
Comparison of Reductions of Blood pressure in Patients Receiving Enalapril and Lisinopril
Two ACEIs were applied for the hypertensives population equally. We have found that there were no significant differences in BP reduction within 24 weeks between two groups in response to lisinopril and enalapril (Table 5). Effect of RAS Polymorphism on Essential Blood Pressure Essay.
Discussion
The concept of “pharmacogenomics” promises to offer the ultimate in personalized medicine, and RAS is one of the most plausible candidates for the application of this approach in the area of hypertension. ACE, especially I/D and G2350A genetic variants[ah7]. Rigat et al. [21] reported that serum ACE concentration differs according to the I/D allele numbers, and this phenomenon has been reconfirmed in many studies [22]. Accordingly, the I/D variant is one of the most plausible candidates for pharmacogenomics RAS blockade mediation. Indeed, early studies showed significant differences of blood pressure reduction among the variants. A greater reduction by enalapril in II genotype compared with DD genotype was reported in 23 normotensive men [23], and a greater reduction by irbesartan in D allele compared with I allele was reported in 43 hypertensive patients [24]. However, as shown in Table 1, recent relatively well-powered studies have almost consistently shown no difference in blood pressure reduction in ACE genotypes (mainly the I/D, G2340A) variant by ACE inhibitors [25– 30]. Thus, this study concludes the ACE I/D and G2350A gene variants are associated in response to lisinopril /enalapril among Malay male hypertensives.
Another of the most evaluated genes in pharmacogenomics studies for the RAS is AGT, especially the M235T, T174M and A-6G genetic variants. Evidence of genetic linkage between the AGT gene and high BP, as well as association of AGT with the disorder, has been observed, and significant differences in plasma concentrations of angiotensinogen were found among hypertensive subjects with different AGT genotypes [31]. Effect of RAS Polymorphism on Essential Blood Pressure Essay.Thus, the AGT variant is one of the most plausible candidates for pharmacogenomic RAS blockade intervention. Although one previous study of 125 cases reported that M235T was associated with lowered blood pressure in response to ACE inhibitors [32], the recent relatively well-powered studies in Table 1 almost consistently observed no difference in blood pressure reduction with M235T and A-6G by ACE inhibitors [27, 31]. Accordingly, it seems that this study can conclude that M235T, T174M and A-6G, gene variant of AGT are associated with lisinopril and enalapril in response to hypertensives in this study.
Molecular genetic studies of humans have led to mixed reports; however studies, on rat models mainly demonstrated that genetically determined variations in the REN gene affect blood pressure. Indeed, linkage and sib-pair linkage analyses, as well as some association studies have failed to identify involvement of the REN gene in EHT [20-24].
In this study, we have found no association between Mob/I and Bg/I of REN in response to cause ACEIs among Malay male hypertensives. As the MboI polymorphism is located in an intron, it is probably not the causative mutation of the effect uncovered here. However, our results together with those of Okura et al. [10] suggest that genetic variations in linkage disequilibrium with this site (either in the REN gene itself or in a nearby gene in linkage disequilibrium with it) may not be directly implicated in an individual’s genetic susceptibility to blood pressure regulation. Effect of RAS Polymorphism on Essential Blood Pressure Essay.It is, therefore, necessary that studies which advance this particular aspect be conducted so that the efficacy of treatments that targets the genetic component of hypertension may be enhanced, resulting in more effective blood pressure control and in a lower incidence of hypertension-related morbidity and mortality. In addition to improved efficacy, screening for the genetic basis of hypertension may reduce the toxicity profile of antihypertensive drugs and improve the overall outcome and the cost-benefit ratio [32].
Limitations of this study
The fact that this study was carried out in a Malay male population is not necessarily a limitation, because according to a publication based on national studies [][ah8], the HT control index in Malay is similar to that of other Asian countries. Likewise, as the subsample of the selected population meets very specific criteria (hypertensive patients taking an ACEI treatment), the sample size of this study decreased to 142 individuals despite the fact that treatment with ACEIs is the most commonly prescribed treatment for this study population (75.3%), combined with ARA II, which is similar to the proportions found in other studies (Llisterri Caro et al., 2004). Nonetheless, coherent and significant results have been obtained in the association analysis.Effect of RAS Polymorphism on Essential Blood Pressure Essay.
Furthermore, we have determined the degree of HT control in terms of the clinical data obtained from patients’ blood pressure values and by gathering information on the type of ACEI treatment each patient takes. However, this study has not assessed the compliance with pharmacotherapy or whether the prescribed dose was adequate. However, it is worth noting that as the pathology under study is chronic and important as a risk factor in the population’s morbidity and mortality, doctors conduct a strict follow-up of progress by individually controlling the drugs prescribed and the most effective dose for each patient. Therefore, it is hard to conceive despite this tight medical control, and given the highly established pharmacotherapeutic intervention for HT that the non-control of HT is due to not establishing a sufficient dose of ACEI drugs. Even more, doctors are obliged to instill the importance of treatment compliance, particularly among prone populations such as the elderly. In light of this, we understand that the extent of therapeutic compliance is high given that the mean age in this study is 71.3 ± 12.25 years.
Epidemiological studies have demonstrated the influence of environmental factors on HBP in the population of Lithuanian children, although the role of genetic factors in hypertension has not yet been studied. The aim of this study was to assess the distribution of AGTR1, AGT, and ACE genotypes in the Lithuanian child population and to determine whether these genotypes have an impact on HBP in childhood.
Methods
This cross-sectional study enrolled 709 participants aged 12–15 years. The subjects were genotyped for AGT (M235 T, rs699), AGTR1 (A1166C, rs5186), and ACE (rs4340) gene polymorphisms using real-time and conventional polymerase chain reactions. Blood pressure and anthropometric parameters were measured.
Results
The prevalence of HBP was 38.6% and was more frequently detected in boys than in girls (47.9% vs. 29.5%; p < 0.001). No significant differences in the frequencies of the AGT or AGTR1 genotypes or alleles between boys and girls were observed, except for ACE genotypes. The mean SBP value was higher in HBP subjects with ACE ID genotype compared to those with ACE II homozygotes (p = 0.04). Effect of RAS Polymorphism on Essential Blood Pressure Essay. No significant differences in BP between different AGT and AGTR1 genotype groups were found. Boys who carried the ACE ID + DD genotypes had higher odds of having HBP than carriers of the ACE II genotype did (controlling for the body mass index (BMI): ORMH = 1.83; 95% CI, 1.11–3.02, p = 0.024; and controlling for waist circumference (WC): ORMH = 1.76; 95% CI, 1.07–2.92, p = 0.035). These associations were not significant among girls. The same trend was observed in the multivariate analysis – after adjustment for BMI and WC, only boys with ACE ID genotype and ACE ID + DD genotypes had statistically significantly increased odds of HBP (aOR = 2.05; 95% CI, 1.19–3.53 (p = 0.01) and aOR = 1.82; 95% CI, 1.09–3.04 (p = 0.022), respectively).
Conclusions
The evaluated polymorphisms of the AGT and AGTR1 genes did not contribute to the presence of HBP in the present study and may be seen as predisposing factors, while ACE ID genotypes were associated with significantly increased odds for the development of HBP in the Lithuanian child and adolescent population – especially in boys.
Background
Recent epidemiological studies have shown that the prevalence of high blood pressure or hypertension has significantly increased among children and adolescents worldwide [1–3]. Hypertension is one of the major cardiovascular risk factors and is associated with cardiovascular mortality, and cardiovascular diseases are the main cause of death in Lithuania [4, 5]. In Lithuania, the prevalence of increased BP or hypertension in children and adolescents is 21% [6] and 35% [3], respectively.Effect of RAS Polymorphism on Essential Blood Pressure Essay. In the adult population, approximately 60% of men and 45% of women aged 25–64 years have elevated BP [7]. High blood pressure in childhood commonly leads to hypertension in adulthood [8–10], which is the leading cause of premature death worldwide [11]. Hypertension in children can lead to left ventricular hypertrophy and pathologic vascular changes [12, 13].
Studies support the fact that hypertension is a complex disease resulting from the interactions of genes and environmental factors [14]. Epidemiological studies suggest that genetic factors account for approximately 30% of the variation in blood pressure [15–17]. Studies have demonstrated that hypertension is twice as common in subjects who have one or two hypertensive parents [18, 19]. Twin studies have also shown that genes could explain approximately 50% of blood pressure variation [20].
The renin-angiotensin-aldosterone system (RAAS) is a major regulator of arterial pressure and as an important endocrine and paracrine system plays a key role in the pathogenesis of essential hypertension [21]. Investigations have shown that the genetic variations of the RAAS-encoding genes have strong connections with the genetic basis of essential hypertension and antihypertensive treatment [22]. An increased risk of hypertension has been associated with angiotensinogen (AGT), angiotensin-converting enzyme (ACE), and angiotensin II receptor 1 (AGTR1). Angiotensinogen is a protein secreted by the liver and found in the α2- globulin fraction of plasma. The enzyme renin cleaves AGT forming angiotensin I, and ACE or kinase II as dipeptidyl carboxypeptidase encoded by the ACE gene produces the active hormone angiotensin II that promotes vasoconstriction. The M235 T (methionine substituted by threonine) polymorphism in the AGT gene was associated with hypertension in Caucasians and with a 10–20% increase in plasma angiotensinogen levels in individuals homozygous for the T allele [23]. ACE plays an important role in blood pressure regulation and electrolyte balance by hydrolyzing angiotensin I into angiotensin II. ACE is not only a membrane-bound enzyme on the surface of the vascular endothelial cells, but it also circulates in blood plasma [24]. The ACE insertion/deletion (I/D) polymorphism is associated with the concentration of the circulating enzyme. Individuals homozygous for the D allele have higher tissue and plasma ACE concentrations than heterozygotes and II homozygotes do [25]. Angiotensin 2 receptor type 1 encoded by AGTR1 gene mediates the major cardiovascular effects of angiotensin II, leading to such effects as vasoconstriction, increased arterial blood pressure, and myocardial contractility [26]. Studies have shown that A/C transversion at position 1166 in the 3′ untranslated region of the AGTR1 gene could be associated with hypertension [27–29]. Effect of RAS Polymorphism on Essential Blood Pressure Essay.
Although relationships between overweight and obesity with prehypertension and hypertension have been established [3], the association between polymorphisms in RAS genes and HBP among Lithuanian schoolchildren based on obesity-related traits has not been studied yet.
The aim of the present cross-sectional study was to investigate if polymorphisms of the RAS genes AGT, AGTR1, and ACE might be associated with an increased risk for higher blood pressure and hypertension in the Lithuanian children population.
Methods
Study design and population
This cross-sectional study included a randomly selected sample of 709 subjects aged 12–15 years, who had participated in the baseline survey of “Prevalence and risk factors of high blood pressure in 12–15-year-old Lithuanian children and adolescents (Study 1, 2010-2012)” in Kaunas city and Kaunas district, located in Kaunas County, Lithuania [3]. The school children who had congenital heart defects, kidney diseases, or endocrine diseases were excluded from the study.
The sample size was estimated using the formula n = z2pq/d2, where: n – sample size, z = 1.96 at the 95% confidence level, p = 0.35 (the prevalence of HBP was 35% among Lithuanian adolescents aged 12–15 years [3]), q = 1–p and d = 0.05 (the degree of precision). The minimum sample size was calculated to be 350 participants.
Measurements
Blood pressure and anthropometric measurements (weight, height, and waist circumference (WC)) performed in this study have been presented in our previous publication [3].
Blood pressure measurement
BP measurements were performed with an automatic BP monitor using a cuff of an appropriate size.Effect of RAS Polymorphism on Essential Blood Pressure Essay. The average of three BP measurements was calculated and used in the analysis, according to “The 4th Report on the Diagnosis, Evaluation, and Treatment of High Blood Pressure in Children and Adolescents” (National High Blood Pressure Education Program (NHBPEP) Working Group on High Blood Pressure in Children and Adolescents) [30]. High BP was defined as systolic blood pressure (SBP) and diastolic blood pressure (DBP) ≥90th percentile.
Anthropometric measurement
All subjects underwent anthropometric measurements. According to the cut-off points of the BMI for children and adolescents proposed by the International Obesity Task Force (IOTF) [31], the subjects were grouped into three categories: normal-weight, overweight, and obese. Using the cut-off values of the percentiles of the WC according to the criteria of the Third National Health and Nutrition Examination Survey (NHANES III) [32], the participants were classified into three groups on the basis of their WC: below the 75th percentile, 75th < 90th percentile, and ≥90th percentile. High waist value was defined as WC ≥75th percentile.
Genetic analysis
For DNA extraction, saliva samples were collected into tubes from each individual. Genomic DNA was extracted using a commercial DNA isolation kit – “Genomic DNA Purification Kit” (“Thermo Fisher Scientific”, Lithuania) according to the manufacturer’s instructions. Aliquots of purified DNA were stored at −20 °C until use in genetic analysis.
Subjects were genotyped for AGT (M235 T, rs699) and AGTR1 (A1166C, rs5186) gene polymorphisms with TaqMan allelic discrimination Assay-By-Design genotyping kits: C_1985481_20 and C_3187716_10, respectively (Applied Biosystems, Foster City, CA, USA).
To analyze ACE (ID, rs4340) polymorphism, genomic DNA was amplified by PCR reaction with primers 5′-AGGAGAGGAGAGAGACTCAAGCACG-3′ and 5′-GGCAGC CTGGTTGATGAGTTCC-3′ [33]. PCR was performed in a 25-μL volume containing approximately 50 ng of the genomic template, 0.4 μmol/L of each primers, 200 μmol/L of each deoxynucleotide triphosphate (dNTP) (Termofisher, Lithuania), 1 unit of Maxima Hot Start Taq polymerase (Termofisher, Lithuania), 2 mmol/L of MgCl2(Termofisher, Lithuania), and 1X Maxima Hot Start PCR Buffer (Termofisher, Lithuania). Cycling conditions were preceded by a denaturing step at 95 °C for 10 min, followed by 32 cycles of denaturation at 94 °C for 30 s, annealing at 68.8 °C for 30 s, and synthesis at 72 °C for 1 min and then at 59 °C for 1 min. The reaction was ended by incubation at 72 °C for 10 min. PCR products were separated by electrophoresis (100 V for 1.5 h; 1.5% agarose gel) and visualized by ethidium bromide-stained agarose gel under ultraviolet light using a video documentation system, the BioDocAnalyse 2.0 (Biometra, Göttingen, Germany). The I allele of ACE manifested as a 700 bp band, and the D allele was seen as a 400 bp band of DNA. Each ACE DD genotype was confirmed through a second PCR with primers specific for the insertion sequence as previously described [33]. Effect of RAS Polymorphism on Essential Blood Pressure Essay.
DNA sequencing for different AGT, AGTR1, and ACE gene genotypes was used for quality control.
Statistical analysis
Continuous variables were expressed as mean ± standard deviation (SD). The normality of the distribution of continuous variables was assessed by the Kolmogorov-Smirnov test. Comparisons between the groups were performed by applying the chi-square (χ2) test and Student’s t-test. The χ2 test was used for the assessment of the Hardy-Weinberg equilibrium (HWE) for the distribution of genotypes. Logistic regression analyses were used to test for the associations of AGT, AGTR1, and ACE polymorphisms with HBP under inheritance models to calculate the odds ratios (ORs, 95% CI) for AGT, AGTR1,and ACE polymorphism. ORs were adjusted for BMI and WC. The Mantel-Haenszel technique was used to estimate the associations of AGT, AGTR1, and ACEpolymorphisms with HBP while controlling for BMI and WC separately. The data were analyzed separately for boys and girls.
Results
The characteristics of the study subjects according to BP levels are presented in Table 1. Among 709 study subjects aged 12–15 years (mean ± SD age: 13.27 ± 1.14 years), 49.8% (n = 353) were boys and 50.2% (n = 356) were girls. There was no significant difference in the mean age between these groups.
Table 1
Characteristics of the study population according to the BP level
| Characteristics | HBP (n = 274) | NBP (n = 435) | P value |
|---|---|---|---|
| Age, years, mean (SD) | 13.30 (1.13) | 13.26 (1.15) | 0.647 |
| SBP, mm Hg, mean (SD) | 137.03 (10.89) | 110.94 (7.35) | <0.001 |
| DBP, mm Hg, mean (SD) | 70.17 (8.20) | 62.25 (6.59) | <0.001 |
| BMI, kg/m2, mean (SD) | 21.31 (3.54) | 18.68 (2.81) | <0.001 |
| WC, cm, mean (SD) | 72.27 (9.05) | 64.58 (6.89) | <0.001 |
| Weight, kg, mean (SD) | 58.71 (12.41) | 49.26 (10.77) | <0.001 |
| Height, cm, mean (SD) | 165.48 (8.82) | 161.67 (9.65) | <0.001 |
| Sex, n (%) | |||
| Boys | 169 (47.9) | 184 (52.1) | <0.001 |
| Girls | 105 (29.5) | 251 (70.5) | |
| Age, years, n (%) | |||
| 12–13 | 156 (36.9) | 267 (63.1) | 0.240 |
| 14–15 | 118 (41.3) | 168 (58.7) | |
| BMI categories, n (%) | |||
| Normal weight | 195 (33.0) | 396 (67.0) | <0.001 |
| Overweight | 58 (62.4) | 35 (37.6) | |
| Obesity | 21 (84.0) | 4 (16.0) | |
| WC percentile categories, n (%) | |||
| < 75th | 204 (33.2) | 411 (66.8) | <0.001 |
| 75th–<90th | 48 (69.6) | 21 (30.4) | |
| ≥ 90th | 21 (87.5) | 3 (12.5) | |
The means were compared using the t test
Categorical variables were compared using the chi-square (χ2) test
Abbreviations: NBP normal blood pressure, HBP high blood pressure, SBP systolic blood pressure, DBP diastolic blood pressure, BMI body mass index, WC waist circumference, SDstandard deviation
The overall prevalence of HBP was 38.6% in the entire study sample. HBP was more frequently detected in boys than in girls (47.9% vs. 29.5%; p < 0.001). Boys aged 14 to 15 years were more likely to have HBP than boys aged 12 to 13 years (56.8% vs. 41.5%; p = 0.005). Higher mean values of SBP and WC were detected in boys with HBP (SBP: 139.47 mmHg; WC: 73.21 cm) than in girls with HBP (SBP: 133.11 mmHg; WC: 70.77 cm), p < 0.001 and p = 0.024, respectively (data not shown). Boys with HBP had significantly higher SBP. Effect of RAS Polymorphism on Essential Blood Pressure Essay.The participants with HBP had significantly higher mean values of BMI, WC, weight, and height, compared to normotensive participants. Overweight, obesity, and high WC (≥75th percentile) were much more common in subjects with HBP than in the normal blood pressure (NBP) group for both sexes separately.
Allele and genotype frequencies in HBP and NBP groups were in the Hardy-Weinberg equilibrium (p > 0.05) (Table 2). No significant differences in the frequency of the AGTor AGTR1 genotypes or alleles were observed between normotensive subjects and those with HBP according to sex, except for ACE genotypes in boys (p = 0.021).
Table 2
Distribution of the AGT, AGTR1, and ACE genotypes and the frequency of alleles in the study population
| Characteristics | Boys | Girls | ||
|---|---|---|---|---|
| HBP | NBP | HBP | NBP | |
| AGT, M235 T genotypes, n (%) | ||||
| MM | 45 (27.3) | 49 (27.1) | 28 (27.2) | 65 (26.7) |
| MT | 82 (49.7) | 77 (42.5) | 57 (55.3) | 125 (51.2) |
| TT | 38 (23.0) | 55 (30.4) | 18 (17.5) | 54 (22.1) |
| χ2 = 2.70, p = 0.259 | χ2 = 0.99, p = 0.607 | |||
| AGT, M235 T alleles, frequency | ||||
| M | 0.52 | 0.48 | 0.55 | 0.52 |
| T | 0.48 | 0.52 | 0.45 | 0.48 |
| χ2 = 0.99, p = 0.321 | χ2 = 0.39, p = 0.531 | |||
| AGTR1, A1166C genotypes, n (%) | ||||
| AA | 87 (52.4) | 85 (47.2) | 59 (56.2) | 123 (50.2) |
| AC | 67 (40.4) | 77 (42.8) | 39 (37.1) | 107 (43.7) |
| CC | 12 (7.2) | 18 (10.0) | 7 (6.7) | 15 (6.1) |
| χ2 = 1.35, p = 0.508 | χ2 = 1.29, p = 0.524 | |||
| AGTR1, A1166C alleles, frequency | ||||
| A | 0.73 | 0.69 | 0.75 | 0.72 |
| C | 0.27 | 0.31 | 0.25 | 0.28 |
| χ2 = 1.32, p = 0.251 | χ2 = 0.55, p = 0.450 | |||
| ACE, ID genotypes, n (%) | ||||
| II | 34 (20.6) | 58 (31.6) | 25 (24.0) | 65 (26.4) |
| ID | 94 (57.0) | 79 (42.9) | 55 (52.9) | 126 (51.2) |
| DD | 37 (22.4) | 47 (25.5) | 24 (23.1) | 55 (22.4) |
| χ2 = 7.74, p = 0.021 | χ2 = 0.22, p = 0.897 | |||
| ACE, ID alleles, frequency | ||||
| I | 0.49 | 0.53 | 0.50 | 0.52 |
| D | 0.51 | 0.47 | 0.50 | 0.48 |
| χ2 = 1.06, p = 0.304 | χ2 = 0.14, p = 0.707 | |||
Comparisons between groups were performed by applying the χ2 test
Abbreviations: NBP normal blood pressure, HBP high blood pressure, AGT angiotensinogen, AGTR1 angiotensin II receptor 1, ACE angiotensin-converting enzyme
Although obesity-related traits such as BMI and WC did not differ according to AGT, AGTR1, or ACE genotypes between HBP and NBP subjects, overweight combined with obesity were much more common in boys with the ACE ID + DD genotype compared to ACE II carriers with high SBP (p = 0.04) (data not shown).
The mean SBP and DBP levels of the participants according to genotypes are shown in Table 3. The mean values of SBP were significantly higher in boys with HBP and the ACE ID and AGTR1 AC genotypes compared to the ACE II and AGTR1 AA carriers. No association was observed in the group of girls. Effect of RAS Polymorphism on Essential Blood Pressure Essay.
