Abstract
Essential hypertension (EH) is a multifactorial, polygenic condition, and is one of the most important comorbidities that contributes to stroke, myocardial infarction, cardiac failure, and renal failure. The continuous increasing rate of morbidity and mortality associated with EH presents an unmet need of population-based studies to explore pathophysiology as well as newer strategies for better diagnosis, prognosis and treatment. This study aimed to determine genotype and allele frequencies of A1166C polymorphism of AT1R gene in Indian patients with EH and correlated with serum levels of Angiotensin II. A total of 200 patients with EH and 200 age- and gender-matched control individuals were included in this study from the General Medicine Department Outpatient at Narayana Medical College and Hospital, Nellore, Andhra Pradesh, India. Patients with systolic blood pressure (SBP) ≥ 140 mmHg and/or diastolic blood pressure (DBP) ≥ 90 mmHg were considered as hypertensive. The findings of this study revealed significantly increased risk of C/A heterozygote and allele C in both men and women. Moreover, both men and women patients with EH showed higher serum levels of Angiotensin II with C/A as well as AA genotypes. These findings indicate a significant association of 1166 C/A polymorphism of the AT1R gene with increased risk of hypertension in Indian population.
Keywords: Angiotensin II, Essential hypertension, 1166 C/A single nucleotide polymorphism, PCR–RFLP, Genetic association
Introduction
Essential hypertension (EH), also known as idiopathic hypertension or primary hypertension is defined by systolic blood pressure (SBP) ≥ 130 mmHg and diastolic blood pressure (DBP) ≥ 80 mmHg [1]. It is also characterized by a constant increase in the arterial pressure. The estimated prevalence of EH in urban population is 3.80–15.63% in men and 2.00–15.38% in women, whereas in rural region the projected prevalence is 1.57–6.93% in men and 2.38–8.81% in women [2]. The primary reason of gender-wise discrimination and the incidence of EH has yet to be determined. EH is usually diagnosed through the screening of an asymptomatic person and is a key research focus in the quest for a specific diagnostic marker. EH is one of most studied diseases in the previous century. It is also one of the most crucial comorbidities that contribute to stroke, myocardial infarction, cardiac failure, and renal failure. Hence, the main objective of the EH treatment strategies has been to reduce the risk of mortality and morbidity associated with cardiovascular and renal functioning [3].
A growing body of studies supports that EH is caused by a complex interplay of genetic, epigenetic, and environmental variables. Around 30–60% variation in blood pressure is estimated due to genetic causes [4, 5]. However, known genetic factors account for only 3% of blood pressure variation [6], highlighting the fact that many genetic variants remain unknown. Furthermore, these findings suggest that other variables in the genesis of EH, such as gene–gene interactions and epigenetics, may be crucial to investigate.
Since 2007, a series of genome-wide association studies (GWAS) of blood pressure and EH encompassing one million individuals has discovered around 1477 single nucleotide polymorphisms (SNPs), accounting for about 27% of the estimated heritability of blood pressure and point to a polygenic multifactorial basis for blood pressure control and EH [7, 8]. Despite the abundance of common variants discovered through GWAS, connecting these variants to a causative mechanism in the blood pressure regulation pathway has proven to be the most difficult task. Because GWAS SNPs are preferred for screening the genome based on linkage-disequilibrium patterns, majority of the signals are found in noncoding or intergenic regions.
In the present study, we have selected the renin-angiotensin system (RAS) which plays a central role in blood pressure regulation. This enzymatic cascade in RAS pathway acts as an endocrine and paracrine system resulting in the production of active peptide Angiotensin II. Angiotensin II is a potent vasoconstrictor that exerts most of its known cellular actions through the Angiotensin II type 1 receptor (AT1RA). Several genetic variations in RAS components have been described which contribute to individual heterogeneity in the RAS status and thereby, modify the relative role of RAS in cardiovascular disease [9–11]. We have selected a single base pair change from adenine to cytosine at the 1166 position in the 3' untranslated region (UTR) of the AT1R gene. Since it was first reported that this SNP had a strong connection with EH in the Caucasian population [12], A1166C has been extensively studied worldwide among different populations. However, the results are inconclusive and there remain a lot of controversies in different studies. Hence, this study was aimed to identify the genotype and allele frequencies of A1166C polymorphism of AT1R gene in Indian patients with EH and correlated with their serum levels of Angiotensin II to disclose possible association and risk of EH. Further, gender-wise analysis of genotype and allele frequency distribution and correlation with Angiotensin II levels may provide more conclusive and better outcome.
Materials and Methods
Study Participants
A total of 200 newly-diagnosed patients with EH and 200 control individuals were included in this study from the General Medicine Department Outpatient at Narayana Medical College and Hospital, Nellore, Andhra Pradesh, India. Both men and women were enrolled with age range of 25–60 years. Patients with SBP ≥ 130 mmHg and/or DBP ≥ 80 mmHg were considered as hypertensive. Patients reporting with secondary hypertension, cardiac abnormalities, diabetes mellitus, renal, liver, and any known nutritional disorders, pregnant and lactating women were excluded from this study. Patients taking vitamin D supplements and drugs which can affect hypertension, lipids or inflammatory conditions were also excluded. Healthy controls were recruited at random from the outpatient wards, who were on routine health check-ups and were defined as participants without having any family history of hypertension or diagnosis of hypertension, not suffering from any acute or chronic disease, nor taking any drugs alleged to affect blood pressure. The demographic details and clinical characteristics of study participants were obtained using a sample collection proforma.
Sample Collection
Peripheral blood samples were collected from patients as well as control individuals after obtaining Institutional Ethics Committee approval (Letter No. IEC/NMCH/Date: 16/11/2013) from Narayana Medical College, Nellore, Andhra Pradesh, India. A total of 3 mL blood samples were collected in ethylenediaminetetraacetic acid (EDTA)-coated tubes after overnight fasting for genomic DNA extraction. Further, 5 mL blood was collected in plain tubes for serum separation and allowed for clotting adequately at room temperature. After 15 min, tubes were centrifuged at 3000 rpm for 10 min and serum was collected in a fresh tube. Serum samples were stored at −80°C and used for quantitative estimation of Angiotensin II levels.
Extraction of Genomic DNA from Blood Samples
Genomic DNA from each freshly collected peripheral blood sample was extracted using a phenol chloroform-based method as described in our earlier study [13]. In brief, red blood cells (RBCs) were removed from whole blood by performing 3–4 initial steps using RBC lysis solution. Further lymphocytes were lysed for one hour at 55°C temperature. The nucleic acid was separated from the protein using phenol–chloroform density centrifugation. Finally, DNA was precipitated using absolute alcohol and impurities were washed using a 70% ethanol solution. DNA pellets were air dried at room temperature for 10–15 min and dissolved in 100 µL of Tris–EDTA (TE) buffer. Purified DNA was quantified using nanodrop reading and integrity was confirmed using agarose gel electrophoresis. All the DNA samples were stored at −20°C until genotype analysis.
Genotype Analysis of AT1R Gene A1166C Polymorphism
PCR–RFLP of AT1R gene A1166C polymorphism was performed by PCR–RFLP method using extracted peripheral blood DNA. The amplification of AT1R gene was carried out using specific oligonucleotide primers (forward: 5'-AATGCTTGTAGCCAAAGTCACCT-3’, and reverse: 5'-GGCTTTGCTTTGTCTTGTTG-3’). PCR included one step of initial denaturation at 95°C for 8 min followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min. A final extension of 7 min at 72°C was included at the end of the 35 cycles to amplify short amplicons. A PCR product at 850 bp was confirmed with agarose gel electrophoresis under ultraviolet light using ethidium bromide staining (Fig. 1A).
Fig. 1.
A Identification of amplified AT1R gene on agarose gel in different samples through ethidium bromide staining B Identification of different genotypes of A1166C polymorphism of AT1R gene through agarose gel electrophoresis after PCR–RFLP
Following to PCR, RFLP was performed using amplified gene segments in reaction using type II restriction endonuclease DdeI (R0175S, NEB) at 37°C for 15 min. A total of 0.2 µL DdeI enzyme was taken for each reaction from 10,000 Units/mL and mixed with 20 µL of PCR amplicons, and 2.5 µL of buffer. RFLP fragments were analyzed on 2% agarose gel and designated to specific genotypes based on different gene fragment sizes. RFLP fragments at 600 bp, 140 bp and 110 bp were confirmed for the homozygote C/C genotype, fragments of 600 bp and 250 bp for homozygote A/A genotype, and the heterozygote C/A was considered for the presence of all four bands (Fig. 1B).
Quantification of Angiotensin II Serum Levels
Quantification of Angiotensin II serum levels in both patients and controls was performed using fluorometric method with the help of Angiotensin II Enzyme Immunometric Assay kit (MAK377, Sigma) as per the manufacturer's instructions. Briefly; 20 µL serum sample was added in each well of 96-well plate provided along with the kit. For background control, the same volume of lysis buffer was added. Similarly, positive and negative control standards were added in other wells. The diluted ACE2 inhibitor was added to the wells containing the sample and/or ACE2 positive/negative controls. Adjusted volume to 50 µL/well in each well using ACE2 Assay Buffer. Mixed well and incubated at room temperature for 15 min. Finally, added 50 µL of ACE2 substrate mix into each well except controls. Fluorescence of each well was measured at 320/420 nm wavelength in kinetic mode for half an hour to 2 h at room temperature. Any two time points within the linear range of plot were selected and corresponding values for the fluorescence were recorded. ACE2 Activity was calculated using following formula:
where:
B = Released MCA in the Sample based on the standard curve slope (pmol).
ΔT = Reaction time (T2—T1 in minutes).
P = Sample used (in mg).
D = Sample dilution factor (D = 1 when samples are undiluted).
Correlation Analysis of Angiotensin II Serum Levels with Genotype Frequency Distribution
The serum levels of Angiotensin II in hypertension patients were correlated with the controls based on different genotypes of the AT1R gene A1166C polymorphism using one way analysis of variance (ANOVA). Further gender-wise variations were also obtained to determine significant changes between men and women.
Statistical Analysis
All the data in this study are represented as mean ± standard deviation (SD). Sample size was calculated with the help of Creative Research Systems software (version 1.0, http://hydra.usc.edu.gxe) by considering the most plausible occurrence of EH. The sample size of 200 patients and 200 controls in this study was sufficient to attain a 90% statistical power. Hardy–Weinberg equilibrium was performed in both patients and control samples. Chi-Square test was performed to determine the distribution of risk factors in different groups. Association between genotypes and EH was identified through odds ratio (OR) with 95% confidence interval (CI) analysis. Student t-test was used to calculate the statistical significance for serum levels of Angiotensin II in EH patients compared to controls. Different genotype models such as co-dominant, dominant, over dominant and recessive were used to describe the significance of different permutations of genotype frequencies among EH patients compared to controls through SNP Stats (https://www.snpstats.net/start.htm). Pearson correlation was performed to compute correlation coefficient (r) values and generate correlation matrix for every pair of data set and calculate statistical significance among different groups. Heat map was generated for both r and p values during correlation coefficient analysis to present significance of the assay. A p value of < 0.05 was set as statistically significant for all the analyses.
Results
The distribution of different genotypes and allele frequencies of AT1R gene polymorphism, and their association with serum levels of AT1R was determined in 200 patients with EH and 200 control individuals in Indian population. This study included 51.50% of men in the control group and 42.00% of men in the patient group (Table 1). Similarly, 48.50% of women were included in the control group and 58.00% of women were included in the patient group. We didn't observe significant difference in age group between patient and control groups (44.56 ± 8.60 vs. 44.59 ± 9.58, respectively; p = 0.9781). However, SBP, DBP, and serum levels of Angiotensin II were significantly higher in patients than controls at the time of enrollment (for all p < 0.0001).
Table 1.
Demographic and biochemical characteristics of patients with EH and controls
| S. No | Demographic and Biochemical characteristics | Controls (N = 200) | Patients (N = 200) | p value |
|---|---|---|---|---|
| 1 | Men, n (%) | 103 (51.50) | 84 (42.00) | |
| 2 | Women, n (%) | 97 (48.50) | 116 (58.00) | |
| 3 | Age, years (Mean ± SD) | 44.59 ± 9.58 | 44.56 ± 8.60 | 0.9781 |
| 4 | SBP (mm Hg) | 116.65 ± 4.13 | 134.53 ± 6.78 | < 0.0001 |
| 5 | DBP (mm Hg) | 76.97 ± 4.69 | 89.32 ± 3.00 | < 0.0001 |
| 6 | Angiotensin II Levels (pg/ml) | 19.21 ± 4.61 | 57.37 ± 14.17 | < 0.0001 |
Genotype and Allele Frequency Distribution of AT1R Polymorphism
Genotype distribution analysis showed a 12% reduced frequency of homozygote A/A in EH patients; while heterozygote C/A showed 15% increased risk of EH in patients compared to controls (OR, 2.90; CI, 1.65–5.09; p < 0.0001), and homozygote C/C was absent in patients (Table 2). In the dominant model, a combination of C/A and C/C showed 12% increased risk of the disease compared to controls (OR, 2.23; CI, 1.32–3.76; p = 0.0021). Similarly, C/A genotype in over-dominant model showed 15% increased risk of the disease compared to controls (OR, 3.00; CI, 1.71–5.26; p = 1e − 04). The allele frequency distribution analysis revealed 4% increased risk of C allele in patients than controls (12% vs. 8%; p = 0.035).
Table 2.
Odd risk estimation in AT1R gene polymorphism in EH patients and controls
| Models | Genotypes | Control (N = 200) |
Patients (N = 200) |
OR (95% CI) | p value |
|---|---|---|---|---|---|
| Co-dominant | A/A | 174 (87%) | 150 (75%) | 1.00 | < 0.0001 |
| C/A | 20 (10%) | 50 (25%) | 2.90 (1.65–5.09) | ||
| C/C | 6 (3%) | – | 0.00 (0.00-NA) | ||
| Dominant | A/A | 174 (87%) | 150 (75%) | 1.00 | 0.0021 |
| C/A-C/C | 26 (13%) | 50 (25%) | 2.23 (1.32–3.76) | ||
| Recessive | A/A-C/A | 194 (97%) | 200 (100%) | 1.00 | 0.0037 |
| C/C | 6 (3%) | – | 0.00 (0.00-NA) | ||
| Over-dominant | A/A-C/C | 180 (90%) | 150 (75%) | 1.00 | 1e-04 |
| C/A | 20 (10%) | 50 (25%) | 3.00 (1.71–5.26) | ||
| Allele | A | 368 (92%) | 350 (88%) | χ2 = 4.40 | 0.035 |
| C | 32 (8%) | 50 (12%) | |||
|
Hardy–Weinberg equilibrium p value |
0.0000 | 0.049 |
Significance values are highlighted in bold
Gender-Wise Distribution of AT1R Genotype and Allele Frequencies
Gender-wise frequency distribution analysis in men patients and gender-matched controls showed 21.1% increased frequency of C/A heterozygote in co-dominant (OR, 3.37; CI, 1.24–9.13; p = 0.003), and over-dominant models (OR, 3.51; CI, 1.30–9.51; p = 0.009) in patients with EH than controls (Table 3). However, none of the allele frequencies showed significant differences between patients and control individuals (p = 0.12).
Table 3.
Genotypic and allelic frequencies of AT1R gene polymorphism in men patients with EH compared to control individuals
| Model | Genotype | Control Men (N = 103) |
Patient Men (N = 84) |
OR (95% CI) | p value |
|---|---|---|---|---|---|
| Co-dominant | A/A | 93 (90.3%) | 69 (82.1%) | 1.00 | 0.003 |
| C/A | 6 (5.8%) | 15 (17.9%) |
3.37 (1.24–9.13) |
||
| C/C | 4 (3.9%) | 0 (0%) |
0.00 (0.00-NA) |
||
| Dominant | A/A | 93 (90.3%) | 69 (82.1%) | 1.00 | 0.1 |
| C/A-C/C | 10 (9.7%) | 15 (17.9%) |
2.02 (0.86–4.77) |
||
| Recessive | A/A-C/A | 99 (96.1%) | 84 (100%) | 1.00 | 0.028 |
| C/C | 4 (3.9%) | 0 (0%) |
0.00 (0.00–NA) |
||
| Over-dominant | A/A-C/C | 97 (94.2%) | 69 (82.1%) | 1.00 | 0.009 |
| C/A | 6 (5.8%) | 15 (17.9%) |
3.51 (1.30–9.51) |
||
| Allele | A | 192 (93%) | 153 (91%) | χ2 = 2.38 | 0.12 |
| C | 14 (7%) | 5 (9%) |
Significance values are highlighted in bold
Frequency distribution analysis in women patients and gender-matched controls showed 15.8% increased frequency of C/A heterozygote in co-dominant (OR, 2.50; CI, 1.25–4.99; p = 0.005), and over-dominant models (OR, 2.56; CI, 1.28–5.11; p = 0.005) in patients with EH than controls (Table 4). Further, a combination of C/A and C/C genotypes showed 13.7% increased frequency in dominant model (OR, 2.19; CI, 1.12–4.26; p = 0.018) in patients with EH than controls. However, none of the allele frequencies showed significant differences between patients and control individuals (p = 0.07).
Table 4.
Genotypic and allelic frequencies of AT1R gene polymorphism in women patients with EH compared to control individuals
| Model | Genotype | Control Women (N = 97) |
Patient Women (N = 116) |
OR (95% CI) | p value |
|---|---|---|---|---|---|
| Co-dominant | A/A | 81 (83.5%) | 81 (69.8%) | 1.00 | 0.005 |
| C/A | 14 (14.4%) | 35 (30.2%) |
2.50 (1.25–4.99) |
||
| C/C | 2 (2.1%) | 0 (0%) |
0.00 (0.00-NA) |
||
| Dominant | A/A | 81 (83.5%) | 81 (69.8%) | 1.00 | 0.018 |
| C/A-C/C | 16 (16.5%) | 35 (30.2%) |
2.19 (1.12–4.26) |
||
| Recessive | A/A-C/A | 95 (97.9%) | 116 (100%) | 1.00 | 0.075 |
| C/C | 2 (2.1%) | 0 (0%) |
0.00 (0.00-NA) |
||
| Over-dominant | A/A-C/C | 83 (85.6%) | 81 (69.8%) | 1.00 | 0.005 |
| C/A | 14 (14.4%) | 35 (30.2%) |
2.56 (1.28–5.11) |
||
| Allele | A | 176 (91%) | 197 (85%) | χ2 = 3.27 | 0.07 |
| C | 18 (9%) | 35 (15%) |
Significance values are highlighted in bold
The statistical analyses for genotypic and allelic frequencies of AT1R gene polymorphism in men and women patients with EH were not possible due to absence of C/C homozygote.
Correlation of Angiotensin II Serum Levels with Genotype Frequency Distribution of AT1R Gene A1166C Polymorphism
Correlation analysis of genotype frequencies with Angiotensin II serum levels showed significant increase for AA homozygote (mean difference, 38.84; CI, 35.53–42.16) and CA heterozygote (mean difference, 35.84; CI, 27.96–43.72) in patients compared to controls (p = 0.0001 for both, Fig. 2A). Gender-wise correlation didn't show significant differences in Angiotensin II serum levels between men and women patients (Fig. 2B) as well as controls (Fig. 2C). In men patients, A/A homozygote (mean difference, 36.96; CI, 32.15–41.77) and C/A heterozygote (mean difference, 35.85; CI, 24.92–46.78) showed significantly increased serum levels of Angiotensin II compared to gender-matched controls (p = 0.0001 for both, Fig. 2D). Similarly, women patients also showed significantly increased serum levels of Angiotensin II for A/A homozygote (mean difference, 40.39; CI, 36.08–44.69), and C/A heterozygote (mean difference, 37.48; CI, 28.05–46.91) compared to gender-matched controls (p = 0.0001 for both, Fig. 2E).
Fig. 2.
Overall and gender-wise genotype correlation analysis showing serum levels of Angiotensin II in patients with EH and controls (***p = 0.0001)
Further person correlation analysis of Angiotensin II levels between total patients and controls with different genotypes showed negative correlation for genotypes A/A (r, − 0.01), and C/A (r, − 0.03), while no correlation was reported for C/C genotype due to absence of data for C/C patients (Fig. 3A). Correlation analysis in men patients showed positive association with Angiotensin II levels of men control individuals for A/A (r, + 0.06), and negative for C/A (r, − 0.19) genotype (Fig. 3B). Similarly, correlation analysis in women patients showed positive association of Angiotensin II levels for A/A (r, + 0.01), and negative association for C/A (r, − 0.81) genotype (Fig. 3C).
Fig. 3.
Heat map showing correlation of serum Angiotensin II levels between patients and controls with different genotypes. Each heat map represents Pearson's correlation coefficient value (r) within each block of heat map of A total study participants B men participants and C women participants. The r value towards + 1 indicates a strong positive relationship, − 1 indicates a strong negative relationship, and 0 indicates no relationship
Discussion
The present study explored association of A1166C polymorphism of AT1R gene in patients with EH, and provided correlation between different genotype frequencies and serum levels of Angiotensin II among Indian population. In our study, all the patients had SBP > 130 mmHg and DBP > 80 mmHg with significantly increased values compared to controls, representing a characteristic definition of EH. Serum Angiotensin II levels were also significantly higher in patients than controls (p < 0.0001). The mean age of patients had no significant difference from controls (p = 0.9781); hence our all the analyses report age- and gender-matched outcomes.
The effects of Angiotensin II on increasing blood pressure are mediated by AT1 receptors, which are expressed in a variety of organ systems and are thought to play crucial roles in blood pressure homeostasis. The human AT1R gene is > 55 kb encompassing five exons and four introns, and has been found to be highly polymorphic. Particularly, the SNP at position 1166 in the 3′ UTR of the AT1R gene either an adenine (A) or a cytosine (C) base (A/C transversion) is crucial to regulate blood pressure homeostasis and is associated with altered gene transcription and increased serum levels of angiotensinogen. However, different population-based studies have reported inconsistent outcomes for genotypic and allelic frequency distribution of this polymorphism and don't provide a clear correlation with serum Angiotensin II levels. Additionally, genetic association studies for A1166C polymorphism of AT1R gene have not been explored enough in Indian population in gender-based manner. The contradictory findings could be a hint of a link between the A and C alleles, indicating a separate source of hypertension.
Our genotyping study for A1166C polymorphism of AT1R gene in Indian population showed association of C/A genotype for significantly increased risk of EH. Additionally, the presence of C allele showed significantly increased risk of EH (p = 0.035). Our study findings are supported by several recent reports which have demonstrated association of A1166C polymorphism of AT1R with prevalent hypertension, increased aortic stiffness, left ventricular hypertrophy, early coronary disease, exaggerated vasoconstriction, and blood pressure response [14, 15]. The silent 1166A/C SNP in the AT1R gene is also associated with the severe form of EH, and in particular in drug-resistant hypertensive patients taking two or more antihypertensive drugs [9, 10]. The meta-analysis conducted by Fajar et al. (2019) on 11,911 cases and 1340 controls from 45 articles demonstrated 1.18 and 1.15 fold increased risk of EH for allele C and genotype C/A respectively [11]. Further in sub-group analysis, they found increased risk of EH with the presence of C/A heterozygote and CC homozygote, and allele C in Asian population.
Gender-wise frequency distribution analysis in our study population showed association of C/A genotype for significantly increased risk of EH in both men and women. However, allele C didn't show significant association with men or women patients (p > 0.05). Our study findings are further supported by study of Chandra et al. (2014) in similar population and suggests that C allele of A1166C polymorphism in AT1R gene is significantly associated with EH and its upregulation could play critical role in EH pathophysiology [9]. A study by Parchwani et al. (2018) assessed the effect of A1166C polymorphic variants of this polymorphism in patients with EH [16]. They reported significantly different genotype and allele frequencies distribution in hypertensives and normotensives. In patients with EH highest allele frequency was reported for C allele (61%) which showed significantly increased risk of EH (CI, 1.1453–2.7932; p = 0.0106). They also confirmed this association by inter-genotypic variations in the mean systolic and diastolic blood pressure in patients. Finally they concluded that genetic variation in the AT1R gene influences the risk of hypertension stratification and may serve as a predictive marker to susceptibility of hypertension among affected families.
Baltatu et al. (2001) investigated how RAS in the brain regulates blood pressure and heart rate variability, and found that RAS in the brain plays an essential role in modulating the influence of angiotensin II on blood pressure diurnal variation [17]. Further, Liu et al. (2020) studied a relationship between varying amounts of renin, angiotensin II, and aldosterone and blood pressure variability markers [18]. Their findings revealed that children with high angiotensin II levels had significantly higher levels at 24 h. As a result, SBP fluctuated a lot in children with essential hypertension and with high angiotensin II levels. Hence, in addition to genotype and allele frequency of AT1R gene polymorphism, we also correlated genotype frequencies with serum levels of Angiotensin II in patients with EH.
Our findings revealed that EH patients with C/A heterozygote and A/A homozygote represent with significantly higher serum levels of Angiotensin II compared to controls. Similarly, gender-wise analysis also revealed higher serum levels of Angiotensin II in EH patients with C/A and A/A genotypes. Further correlation analysis of Angiotensin II levels between total patients and controls with different genotypes showed negative correlation for genotypes A/A (r, − 0.01), and C/A (r, − 0.03). Gender-wise correlation in both men and women patients showed positive association with Angiotensin II levels for A/A, and negative for C/A genotypes. These findings revealed that C/A heterozygote is significantly associated with both men and women patients with EH. However, future studies are required to further explore its association with other clinical parameters of EH in early and late age of onset.
Conclusion
Our study revealed that C/A heterozygote and the presence of C allele are significantly associated with EH in both men and women in Indian population. Moreover, both men and women patients with EH represent higher serum levels of Angiotensin II having C/A heterozygote as well as A/A homozygote. These findings indicate a significant association of A1166C polymorphism of the AT1R gene in Indian patients with increased risk of EH for C allele and C/A heterozygote.
Funding
None.
Data Availability
Data will be made available when desired.
Declarations
Conflict of interest
All authors declare that they have no conflict of interest.
Human and/or Animals Participants
Study was conducted after taking Institutional Ethics Committee approval (Letter No. IEC/NMCH/Date: 16/11/2013) from Narayana Medical College Nellore, Andhra Pradesh, India.
Informed Consent
Informed consent forms were collected from each study participant during sample collection.
Consent for Publication
All the authors have read the manuscript and agreed to publish in Indian Journal of Clinical Biochemistry.
Footnotes
Publisher's Note
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Contributor Information
M. Prasad, Email: drmprasad@narayanamedicalcollege.com
Aleem Ahmed Khan, Email: aleem_a_khan@rediffmail.com.
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