Pharmacogenetic Association of RAS Polymorphism on Essential Blood Pressure in Relation to Enalapril/Lisinopril among Malay male newly diagnosed hypertensives
Objective: It has been suggested that genetic backgrounds, which have an association with essential hypertension, may also determine the responsiveness to ACE inhibitor. 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
Essential hypertension (EH) is an increasingly important medical and public health issue . 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 . Furthermore, the prevalence increased from 42.6% to 43.5% for those >30 years old. Unfortunately, 60.6% of total hypertensives were “undiagnosed” . 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 . 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.
A renin-angiotensin system (RAS) is an important component of blood pressure regulation, and it has been suspected to be involved in hypertension . 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) .
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]. 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 . 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 .
It has been reported that the presence of A-6G polymorphism of AGT gene among Chinese hypertensives increased body weight gain in hypertensive patients . 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 .
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 ; 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.
MATERIAL AND METHODS
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. 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
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. 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.
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 . ACE I/D polymorphism was genotyped using DNA amplification with oligonucleotides as described elsewhere . For ACE G2350A, DNA amplification followed the approach by Zhu et al. . 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.
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.
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. 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.
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).
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.  reported that serum ACE concentration differs according to the I/D allele numbers, and this phenomenon has been reconfirmed in many studies . 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 , and a greater reduction by irbesartan in D allele compared with I allele was reported in 43 hypertensive patients . 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 . 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 , 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.  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. 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 .
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.
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.
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