| Since coronary artery spasm (CAS) was described by Prinzmetal in 1959, its crucial role in the pathogenesis of coronary heart disease, especially in the acute coronary syndrome has been widely recognized. CAS always occurs in the basis of atherosclerosis, but it is not positively correlated to the degree of coronary artery stenosis. Usually, CAS was provoked in the vascular segments with mild or moderate stenosis. Epidemiological studies in population of CAS have revealed that smoking and dyslipidemia are important risk factors of CAS. Smoking is an independent risk factor of CAS, which can increase the risk of CAS by 4.2 times and dyslipidemia can increase the risk of CAS by 2.3 times, while gender, age, hypertension and the result of ECG treadmill test were not significantly correlated with CAS. However, the other two major risk factors of atherosclerosis, diabetes and hypertension, do not seem to increase the risk of CAS significantly. Therefore, smoking cessation and lipid lowering therapy are effective not only in prevention of arteriosclerosis, but also in prevention of CAS. In addition, the patients with coronary artery myocardial bridge were highly potential to be provoked CAS during provocation tests. However, those patients with myocardial bridge may have significant systolic stenosis and probably present the characteristics of typical exertional angina pectoris. The evidences about the relationship between CAS and diabetes, insulin resistance and hypertension are not entirely consistent, and need to be further defined.The exact mechanism of CAS has not been totally clarified. But it is accepted that CAS may be related to the following pathophysiologic mechanisms:1. Vascular endothelial cells confuse in both structure and function. It may decrease nitric oxide, increase endothelin in plasma and then cause dysfunction of vasomotor. The related molecular biological mechanisms include oxidative stress, endothelin-1 and nitric oxide synthase gene polymorphism. With the hazard of dyslipidemia or smoking, the process of injuring vascular endothelial cell will be triggered by oxidative stress.2. Contraction activity of vascular smooth muscle cells increases. Vascular smooth muscle cells were excessively contractive with agonist, which may be related to gene mutation of myosin light chain phosphorylation-related enzymes or to inflammatory cytokines such as interleukin-6.3. Function of autonomic nervous system is unbalanced, which lead to reduced activity of sympathetic nervous system and increased activity of vagal nervous system. Without corresponding match of sympathetic activity, CAS occurs with high vagal activity. Severe case may present a typical variant angina.Among all of these, the disorder of vascular endothelial cells in structure and function, which causes the imbalance between vasoconstrictors and vasodilators by increasing endothelin-1 and decreasing NO, is considered as one of the most important mechanisms of CAS. Many studies have focused on the increased levels of ET-1 in plasma. Our previous study has shown that the basic level of ET-1 in plasma is much higher in the CAS group than in the healthy controls (108.71±8.26 vs. 93.02±8.13, t=2.219, P<0.05). EDN1 gene is encoding for ET-1. Human EDN1 gene links closely with D6S89 and locates at chromosome 6 p23-p24, which is the end of chromosome 6 and near to HLA,B and DRB. EDN1 gene includes 5 exons and 4 introns. Its full-length is 5.5 kb. Mature peptide sequence of ET-1 is encoding in exon 2. ET-1 gene has been found to have six polymorphisms. There are T/G transversion in 1370 point in the transcription starting site, insertions or deletions of A in +138 point in exon 1, G/A converter in +1932 point in intron 1, T/C conversion in +3539 point in intron 2, G/T transversion in +5665 point in exon 5 and T/C conversion in 8000 point in intron 4. ET-1 level in plasma is mainly decided by regulator of gene expression. Some single nucleotide polymorphisms have been identified. Gene polymorphisms in intron and exon have been found to increase the risk of cardiovascular diseases, such as chronic heart failure, hypertension and ventricular arrhythmia. Lee's research found that there are some gene polymorphisms of ET-1 in Korean patients with CAS. This report indicates that the change of ET-1 level in plasma may be associated with gene polymorphisms. Foreign studies have shown that the level of ET-1 in plasma are closely related with gene polymorphisms including +138 delA in exon-1, G8002A in intron -4 and G5665T in exon -5. However, there is no any study of ET-1 gene polymorphism in Chinese CAS population. Therefore, we select these three polymorphism sites to evaluate ET-1 gene polymorphisms.Base on the above findings,100 CAS patients and 120 healthy controls are enrolled in this study to evaluate the relevance between ET-1 gene polymorphism and CAS.Objective1. To explore the frequencies of ET-1 gene polymorphisms in Chinese population. 2. To investigate the relationship between the ET-1 gene polymorphylisms and CAS.Subjects and methods1. SubjectsAll subjects were chosen from hospitalized patients or outpatients in cardiovascular department and the healthy people for medical check-up in our hospital from December 2008 to March 2010. One hundred CAS patients who meet one of the following two diagnostic criteria of CAS are enrolled:the positive acetylcholine provocation test and/or non-invasive diagnostic criterion. The positive criteria of acetylcholine provocation test includes three conditions as below:firstly, clinical manifestations of chest pain or chest tightness at rest; secondly, no significant coronary stenosis by angiography; thirdly, CAS induced by intracoronary injection of acetylcholine that could result in more than 90% coronary stenosis, with similar symptoms of chest pain or chest tightness as usual, which would disappear after the spasm is relieved automatically or by intracoronary nitroglycerol. The criterion of non-invasive diagnostic method is described as below:the patients are diagnosed as CAS if the following three conditions are met at same time:①the clinical manifestation of resting chest pain or chest tightness,②negative treadmill exercise electrocardiogram or ischemic electrocardiographic changes during recovery period after exercise,③reverse redistribution on myocardial perfusion scintigraphy. One hundred and twenty health controls were selected from healthy people for medical check-up in our hospital. The criteria of healthy control group are described as:the subjects with the following three conditions at the same time:①without the clinical manifestations of chest pain and chest tightness,②excluding coronary heart disease by routine examinations, treadmill electrocardiographic exercise test and radionuclide perfusion myocardial imaging,③with matched age and sex with the subjects in the CAS group. All subjects are natural and unrelated Han people, who don't have serious systemic diseases such as bronchial asthma, systemic infection and gastrointestinal tract diseases.2. MethodsOne milliliter of blood was taken from cubital venous in all subjects at optional time. The venous blood samples were anticoagulated with EDTA-Na2 to extract leukocyte genomic DNA according to the instruction of Human blood DNA extraction kit. Polymerase chain reaction (PCR) was performed on the ABi9700 system by Shanghai Invitrogen Biotechnology Co. Ltd. ET-1 gene type was defined by direct sequencing on the 3730XL DNA sequencing instrument. General demographic and clinical characteristics such as age, sex and so forth were compared between the two groups by independent-sample T test andχ2 test. Frequency of gene type and allele frequency in the two groups were compared byχ2 test. A P<0.05 was considered to be statistically significant difference.Results1. Three gene types of ET-1 gene were detected at following three polymorphisms sites in both of CAS group and control group:4A/4A wild type,4A/3A heterozygous mutation and 3A/3A homozygous mutation in exon -1 at +138 site; G/G wild type,G/A heterozygous mutation and A/A homozygous mutation in intron -4 at 8002 site; G/G wild type,G/T heterozygous mutation and T/T homozygous mutation in exon -5 at 5665 site.2. The frequencies of 4A/4A,4A/3A,3A/3A gene type of ET-1 gene are 2.5%, 20.8%,76.7% in in the CAS group and 8%,30%,62% in the control group respectively. The frequencies of 4A,3A allele are 12.9% and 87.1% in the CAS group and 23% and 77% in the control group, respectively (Wald=9.133, P=0.003;χ2= 7.683, P=0.006, respectively). The frequencies of G/G,G/A,A/A gene type are 63.3%,33.4%,3.3% in the CAS group and 47%,41%,12% in the control group, respectively (Wald=0.333, P=0.564). And the G, A allele frequencies are 80% and 20% in the CAS group and 67.5%and 32.5% in the control group(χ2= 8.931, P= 0.003). The frequencies of G/G,G/T,T/T gene type are 65%,29.2% and 5.8% in in the CAS group and 43%,46% and 11% in the control group respectively. The G,T allele frequencies are 79.6% and 20.4% in the CAS group and 66% and 34% in the control group (Wald=6.916, P=0.009;χ2=10.311, P=0.001, respectively).3. The frequency of 3A/G8002/G5665 haplotype in the CAS group is lower than that in the healthy control group (0.47 vs.0.65,χ2=14.406, P< 0.001, odds ratio =0.475).Conclusion1. BMI and smoking are important risk factors of CAS (Wald=24.161, P<0.001,95%CI 2.657-9.751; Wald=22.998, P<0.001,95%CI 3.427-18.805 respectively), but age, gender, lipid level in plasma, drinking diabetes and hypertension do not seem to increase the risk of CAS significantly.2. In Chinese population, the exon -1 at +138 site of ET-1 gene includes three gene types:4 A/4 A wild type,4G/3 A heterozygous mutation and 3A/3A homozygous mutation. The intron -4 at 8002 site of ET-1 gene includes three gene types:G/G wild type, G/A heterozygous mutation and A/A homozygous mutation. The exon -5 at 5665 site includes three gene types:G/G wild type, G/T heterozygous mutation and T/T homozygous mutation.3. The distributing frequencies of ET-1 allelet at exon -1 of +138 site (rs 10478694), intron -4 G8002A (rs2071942), and exon -5 G5665T (rs5370) are significant different between the CAS group and the healthy control group. The distributing frequencies of ET-1 genotype at exon-1 of +138 site (rs10478694) and exon -5 G5665T (rs5370) are also significant different between the two groups. However, there is no remarkable difference in the distributing frequency of ET-1 genotype at intron -4 G8002A (rs2071942) between the two groups. These results suggest that CAS might be related to ET-1 gene polymorphisms.4. The haplotype analysis indicated that 3A/G8002/G5665 haplotype might be a protective genotype to against CAS. |
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