Association of Angiotensin II Type 1 Receptor (A1166C) Gene Polymorphism and Its Increased Expression in Essential Hypertension: A Case-Control Study

<div><p>Objectives</p><p>Hypertension is one of the major cardiovascular diseases. It affects nearly 1.56 billion people worldwide. The present study is about a particular genetic polymorphism (A1166C), gene expression and protein expression of the angiotensin II type I receptor (AT1R) (SNP ID: rs5186) and its association with essential hypertension in a Northern Indian population.</p><p>Methods</p><p>We analyzed the A1166C polymorphism and expression of AT1R gene in 250 patients with essential hypertension and 250 normal healthy controls.</p><p>Results</p><p>A significant association was found in the AT1R genotypes (AC+CC) with essential hypertension (χ² = 22.48, p = 0.0001). Individuals with CC genotypes were at 2.4 times higher odds (p = 0.0001) to develop essential hypertension than individuals with AC and AA genotypes. The statistically significant intergenotypic variation in the systolic blood pressure was found higher in the patients with CC (169.4±36.3 mmHg) as compared to that of AA (143.5±28.1 mmHg) and AC (153.9±30.5 mmHg) genotypes (p = 0.0001). We found a significant difference in the average delta-CT value (p = 0.0001) wherein an upregulated gene expression (approximately 16 fold) was observed in case of patients as compared to controls. Furthermore, higher expression of AT1R gene was observed in patients with CC genotype than with AC and AA genotypes. A significant difference (p = 0.0001) in the protein expression of angiotensin II Type 1 receptor was also observed in the plasma of patients (1.49±0.27) as compared to controls (0.80±0.24).</p><p>Conclusion</p><p>Our findings suggest that C allele of A1166C polymorphism in the angiotensin II type 1 receptor gene is associated with essential hypertension and its upregulation could play an important role in essential hypertension.</p></div

Fold change expression and Quantitative PCR results of Angiotensin II type 1 receptor gene.

<p>CT, Threshold cycle.</p><p>*Data are means ± SD, *p = 0.0001. 18S rRNA gene expression of the same samples was used for calculations.</p

Intergenotypic (A1166C variant of the Angiotensin II type 1 receptor gene) variations in gene expression (delta-CT values) in study subjects.

<p>Patients and controls were compared with respect to genotypes with t-test of significance test at one degree of freedom adjusted for age and sex. p<0.05 is considered to be significant.</p><p>*analysis of variance (ANOVA) using Bonferroni’s method for multiple comparisons between genotype classes.</p

Semi-quantitative analysis of Angiotensin II type 1 receptor gene expression.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101502#pone-0101502-g002" target="_blank">Figure 2A</a> represents the semi-quantitative analysis of AT1R gene. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101502#pone-0101502-g002" target="_blank">Figure 2B</a> illustrates the relative AT1R gene expression in terms of integrated densitometric value (IDV) of patients (2.66±0.62) and normal healthy controls (2.15±0.28). Results were expressed as average densitometric ratio (AT1R to 18S rRNA gene) in patients and controls group ± SD. p<0.0001.</p

Genotype and Allele Frequencies of A1166C variant of the Angiotensin II type 1 receptor gene in the study subjects.

<p>Patients groups were compared with controls with chi-square (χ²) test at one degree of freedom with Odds ratio adjusted for age and sex in both genotypes and alleles. p<0.05 is considered to be significant.</p

Baseline characteristics of the study subjects.

<p>BMI, Body mass index; HDL, High density lipoprotein; LDL, Low density lipoprotein.</p><p>Patients group were compared with controls with t-test of significance or by chi-square test;</p><p>*p<0.05 is considered to be significant.</p

Western blot analysis of Angiotensin II type 1 receptor protein expression.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101502#pone-0101502-g005" target="_blank">Figure 5A</a> represents the western blots of AT1R and β-actin protein in controls and patients respectively. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101502#pone-0101502-g005" target="_blank">Figure 5B</a> shows the relative AT1R protein expression in terms of integrated densitometric value (IDV) of patients (1.49±0.27) and normal healthy controls (0.80±0.24). Results were expressed as average densitometric ratio (AT1R to β-actin protein) in patients and controls group ± SD. p = 0.0001.</p

Intergenotypic (A1166C variant of the Angiotensin II type 1 receptor gene) variations in systolic and diastolic blood pressure in patients with essential hypertension.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101502#pone-0101502-g001" target="_blank">Figure 1A</a> represents the scatter plot between Intergenotypic (A1166C variant of the Angiotensin II type 1 receptor gene) variations and systolic blood pressure. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101502#pone-0101502-g001" target="_blank">Figure 1B</a> shows the scatter plot between Intergenotypic (A1166C variant of the Angiotensin II type 1 receptor gene) variations and diastolic blood pressure. Results were expressed as mean ± SD.</p

delta-CT values for study subjects.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101502#pone-0101502-g003" target="_blank">Figure 3</a> represents the scatter plot of delta-CT values for patients and controls group. The higher delta-CT value represents the lower expression of gene at mRNA level.</p