EVALUATION OF THE ASSOCIATION BETWEEN AT 1 R 1166 C POLYMORPHISM AND THE INCIDENCE OF CAD AND CAC SCORE IN THE IRANIAN POPULATION

Most of the physiological effects of Ag II are mediated by the angiotensin II type 1 receptor. Polymorphisms of the AT1R gene can affect the function of this receptor and subsequent atherogenic activity. In this study we investigated the correlation between AT1R A1166C polymorphism and coronary artery calcification (CAC), a marker of the coronary artery burden. Fifty CAD patients and fifty healthy individuals fulfilled the inclusion and exclusion criteria entered this study. CAC was determined in the left main coronary artery (LMCA), left coronary artery (LCA), right coronary artery (RCA) and CX by CT-angiography and a blood sample was taken at this time. DNA extracted from whole blood leukocytes was analyzed by the polymerase chain reaction – restriction fragment-length polymorphism (PCR-RFLP) assay. There were no significant differences in genotype and allele frequencies between the CAD and control groups. The mean calcium score was compared in genotypes and alleles and no significant difference was seen. In addition, the frequency of genotypes and alleles was not significantly different in the calcium score groups (low<100, medium= 100-400, high >400). An analysis was performed separately in males and females and no significant correlation was found. According to our results, no association was found between AT1R1166C polymorphism and the incidence of CAD and CAC score in our study population.


INTRODUCTION
Coronary artery disease (CAD) continues to be the main cause of death and a major cause of morbidity and decline in quality of life.Atherosclerosis, the main cause of coronary artery disease (CAD), is influenced by a complex interaction among various environmental and genetic factors (Peyser, 1997).Familial incidence of CAD has been known for almost 100 years, and shows the genetic basis of many recognized CAD risk factors (Lusis, 2000).
Even after managing CAD risk factors, there is a great risk of CAD associated with a family history of the disease.This suggests that numerous genetic factors underlying disease susceptibility are yet to be identified (Devereux and Alderman, 1993).One of the markers of coronary artery atherosclerosis is coronary artery calcification (CAC).Coronary calcium is a degenerative process with active rule in atherosclerotic plaque development (Rumberger et al. 1999).The degree of calcification in atherosclerotic plaques in coronary arteries, after management of the well-known CAD risk factors, is an indicator of plaque development and the calcium score of coronary arteries is related to atherosclerosis improvement and thus to CAD severity (Guerci et al., 1998;Bielak et al., 2000;Keelan et al., 2001).The renin-angiotensin system (RAS), which regulates blood pressure, has an important role in the pathogenesis of the different stages of atherosclerosis and accelerates the disease process (MacGregor et al., 1981;Cusi, 1997).Angiotensin II, its receptors and the angiotensin-converting enzyme (ACE), are the main components of the renin-angiotensin system.Angiotensin II is an effective regulator of cardiovascular homeostasis and acts through two different G-protein-coupled receptors.Most of the known effects of angiotensin II are mediated through the angiotensin II type 1 receptor (AT1R), which mediates the cardiovascular procedures of angiotensin II and it has been studied the most extensively (Takayanagi et al., 1992;Herzig et al., 1997).It is expressed in vascular smooth muscle cells and also in the myocardium, thus a possible relationship between the AT1R gene and myocardial infarction has been investigated (Berk and Corson, 1997).There are controversial reports regarding the role of the AT1 receptor gene A/C polymorphism as a risk factor for MI (Tiret et al., 1994;Ulgen et al., 2006).Also, ACE gene polymorphism may be associated with severe aortic valve calcification (AVC) and the risk of coronary artery disease, and development of the stenosis of coronary artery.The AT1 receptor CC genotype is also associated with coronary disease, hypertension, and ischemic heart disease in Caucasian populations (Gardemann et al., 1998;Jones et al., 2003;Pullareddy et al., 2009).The AT1R gene exhibits polymorphism, and an A/C transverse at the 3′-untranslated region has been related to essential hypertension (Bonnardeaux et al., 1994).The most widely studied polymorphism in the AT1R gene is the A1166C variant.
In different studies the correlation between AT1R gene polymorphism and CAD and atherosclerosis, which are linked to CAC, has been investigated, however, the probable relationship with coronary artery calcification has not been studied yet.We have examined this relationship in the present study.

Patients
This study was carried out in the cardiovascular and pharmaceutical research center of the Mashhad University of Medical Science in Iran from November 2008 to September 2009.50 CAD patients and 50 healthy volunteers entered the study.All of the patients fulfilled the following inclusion criteria: people aged 30-70 years (average age 43 years, range 26-50).The exclusion criteria were: patients using ACE inhibitors and ARBs, and patients with calcium and phosphor metabolism disorders, primary and secondary hyperparathyroidism, chronic renal failure, bone disorders and malignancies.

Blood sampling and procedure
CT angiography was performed for all patients and CAC was determined in the left main coronary artery (LMCA), left coronary artery (LCA), right coronary artery, (RCA) and CX.A peripheral venous blood sample was collected from each patient for biochemical measurement and the extraction of genomic DNA.Biochemical tests, including plasma lipid profiles, the presence of previous disease and administered drugs, FBS, ESR, PTH, hsCRP, Ca, phosphorus and Cr were determined.DNA was extracted from whole blood leukocytes by a salting-out method and samples were analyzed by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) assay.

Genetic analysis
Peripheral leukocytes, taken after centrifugation of 2 mL blood, were used to isolate genomic DNA using the proper nonenzymatic extraction kit (BioGene Ltd, UK).DNA concentrations and purification percentages were determined by gel electrophoresis and spectrophotometry.
The determination of the A/C-polymorphism at the 3′-untranslated region of the AT1R gene was based on the triple-primer polymerase chain reaction (PCR) method that used a primer including a mismatch, introducing a site for the Ded I restriction enzyme.A 540 bp fragment was amplified under the following conditions: 200 ng/ µL target DNA in a final volume of 20 µL containing 15pM (1μL) of each primer, 1.5 mM MgCl2 (1.2µL), 0.2 mM (0.4 μL) of each dNTP, and 1-2 U/mlTaq polymerase, 1X PCR buffer 10X (2 µL) and distilled water to 20 μL.The amplification profile included an initial denaturation at 95°C for 5 min and 30 cycles of denaturation at 95°C for 60 seconds, annealing at 55°C for 60 seconds, and extension at 72°C for 60 seconds with a final extension time of 5 min.The primers were as follows: 5′-GCACCATGTGAGGTTG-3′ (named AT1RM) and 5′-CGACTACTGCTTAGCATA-3′ (named AT1RH).The PCR yields of the expected size (540bp) were evaluated on 0.8% agarose gels.To illustrate the polymorphism, PCR products were processed overnight (24 h) with the restriction endonuclease Dde I at 37ºC, which cuts the product into two pieces, 430 bp and 110 bp long.An additional Dde I detection site was created in the C-type variant at nucleotide 1166.Thus, the homozygote CC created two bands (430 and 110 bp long), the homozygote AA produced one band (540 bp long), and the heterozygote AC produced all three bands (540,110 and 430 bp long).Digested products were detected on 3% agarose gel and by staining with ethidium bromide.

Statistical analysis
Statistical analysis was performed with SPSS 11.5 software.Differences in allele frequencies and genotype distribution between cases and controls and in the different groups according to their calcium scores were analyzed by chi-squared statistics.Comparison of the mean calcium scores in different genotypes and alleles in the CAD patients and control was performed by one-way ANOVA and Tukey post test.The results were recorded as mean ± SD, a p-value of 0.05 or less was considered as significant.

Population study
The main characteristics of the study population include demographic characteristics, biochemical parameters, CAC and population AT1R phenotype and genotype frequencies and are shown in Table 1.

CAC comparison between CAD patients and control
The study population (control and patients) was divided into 3 groups according to the calcium score (group 1 = <100; group 2 = 100-400, grades 2-4; group 3 = >400).The calcium score was significantly higher in the CAD patients than in the control (p<0.001) (Fig. 1).

Comparison of genotype and allele frequencies between patients and control
In the study population there was no difference in genotype and allele frequencies between the patients and controls with different calcium scores (p>0.05) (Fig. 2).

Comparison of genotype and allele frequencies in the study population
There was no significant difference in these frequencies in the whole study population, patients and controls with different calcium scores (p>0.05)(Figs. 3,4,5).This was investigated in men and women separately, and no correlation was found (data not shown).

DISCUSSION
In this study, the correlation between A1166 and C AT1R gene polymorphism with coronary artery calcification score and coronary artery diseases (CAD) in patients and healthy individuals was investigated.Genotype frequencies AA, AC and CC were similar to other studies (70%, 24% and 6%, respectively) and the CC genotype frequency was the lowest.There were no significant difference in genotype and allele frequencies in the patients and control group or in the different groups with different calcium scores.In other words, we did not find any significant correla-tion between the distribution of these genotypes and alleles and CAC and the incidence of CAD.
It is evident that both environmental risk factors and genetic factors could be the causes of CAD.Also, cardiovascular risk factors such as diabetes, dislipidemia, hypertension and obesity could have genetic   and environmental causes.One of genetic causes is common polymorphisms at several genes in reninangiotensin system (angiotensinogen, angiotensin converting enzyme and angiotensin receptor genes).For example, in several families the AT1R has been related to essential hypertension or diabetes, and its polymorphisms have been linked to an increased risk of hypertension and diabetes (Bonnardeaux et al., 1994).
It is well established that the renin-angiotensin system (mainly Angiotensin II) has a key role in the development of atherosclerosis processing.The pro-inflammatory effect of the renin-angiotensin system and following inflammatory mediators' synthesis causes an interaction between leucocytes and endothelial cells, which is an important stage in the pathogenesis of atherosclerosis (Hirata et al., 2011), and Angiotensin II is able to directly affect the endothelium.These are important factors in plaque instability and natural fibrinolytic pathway destruction.Recently, an essential role has been established for the rennin-angiotensin system in CAC promotion.Angiotensin II activates the expression of genes related to calcification such as parathyroid hormone receptors and the hepatorenal-osteocyte alkaline phosphatase gene in heart smooth muscles (Armstrong et al., 2011).Angiotensin II acts through its receptors, mainly AT1R, which changes in its expression and function and leads to changes in the RAS and atherogenic activation.Due to these mechanisms, a correlation between atherosclerosis and CAC with AT1R polymorphism is reasonable.
Several related studies in different populations have reported controversial results.Kretowski showed that in diabetic patients there was no significant correlation between AT1R and ACE gene polymorphism and CAC.In addition, it has been shown that there is no correlation between AT1R and ACE and AGT gene polymorphism with clinical signs in patients with or without cardiac arrest history (Jeunemaitre et al., 1997), which is in agreement with Joseph (Joseph et al., 1998) who demonstrated no significant differences in AT1R genotypes in patients and healthy Indians.Evidence suggests that a genetic constituent affects the frequency and the degree of arterial calcification in humans (O'Donnell et al., 2002;Peyser et al., 2002).Many studies have found an association between insertion/deletion polymorphism of the ACE gene in atherosclerosis and coronary artery calcification (Pfohl et al., 1998;Agerholm-Larsen et al., 2000;Doherty et al., 2004).The study of RAS gene interaction in CAD patients showed that ACE and AT1R gene interaction could be effective in both CAD incidence and development (Ye et al., 2003).Results from a study on Turkish patients suggest that ACE gene polymorphism may be associated with severe aortic valve calcification (Ertas et al., 2007).
Different alleles in other sites that are inherited simultaneously, and also heterogeneity and epistasis (a gene's effects hidden by another gene) could be responsible for this controversy (Carlborg and Haley, 2004).A1166C gene polymorphism occurs in the 3'-untranslated region, without alteration in receptor amino acid sequences.Functional modifications could be a result of changes in mRNA stability, processing, receptor activation, binding and in expression regulation.Thus, this polymorphism could be affected by other genes that control these pathways.According to an investigation into reninangiotensin system polymorphisms (ACE, AGT and AT1R), in separated analyses none of them had correlation with CAD or cardiac arrest in people with or without risk factors (Tsai et al. 2006).CAD has a multifactor genetic and environmental basis and factors such as race, sex, physiologic disturbances like salt and other SNPs imbalances, could alter the relation between genotype and phenotype existence.
To our knowledge, there has been no other similar study to the presented work, and this is the first time that the correlation between AT1R gene polymorphism and CAD has been investigated.According to our results, there is no correlation between AT1R gene polymorphism and CAD in Iranian patients.

Fig. 3 .
Fig. 3. Frequency of different genotypes and alleles in whole study group with different calcium scores.

Fig. 4 .
Fig. 4. Frequency of different genotypes and alleles in CAD group (patients) with different calcium scores.

Fig. 5 .
Fig. 5. Frequency of different genotypes and alleles in control group with different calcium scores.

Table 1 .
Study population characteristics