Lack of Association between Genetic Polymorphisms Affecting Autonomic Activity and Coronary Artery Spasm

Biomed Environ Sci, 2013; 26(8): 689-692

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Letter to the Editor Lack of Association between Genetic Polymorphisms Affecting Autonomic Activity and Coronary Artery Spasm* ZHOU Xuan1,2, XIANG Ding Cheng1,#, ZENG Jing1, YI Shao Dong1, ZHANG Jin Xia1, and LI Dan Hui1 Coronary artery spasm (CAS) is one of the leading pathological causes of a wide spectrum of ischemic heart diseases, ranging from variant angina pectoris to acute myocardial infarction and even sudden cardiac death[1]. Furthermore, Pierron et al. concluded that CAS of angiographically normal or sub-normal arteries is responsible for death or myocardial infarction in 11.6% of all cases[2]. Oddly, the incidence of CAS is remarkably higher in Asians than in Caucasians[3], suggesting genetic involvement in its pathogenesis. On the other hand, the autonomic nervous system (ANS), which is responsible for maintaining internal homeostasis and influences coronary vasomotion, has been shown by several studies to play an important role in the genesis of CAS[4]. The purpose of this study is to determine whether genetic polymorphisms affecting autonomic activity contribute to the pathogenesis of CAS. Among the numerous polymorphisms related to the ANS, we chose common and functional ones that could affect coronary vasomotor tone and may be related to cardiovascular diseases. In the ANS, sympathetic activation may result in both vasoconstriction, through α2-adrenergic receptor (AR), and vasodilation, through β2-AR. α2BDel301-303, which makes the desensitization of α2-AR decreased, has been proved to be involved in diseases associated with enhanced vasoconstriction. In addition, a strong association has been reported between α2BDel301-303 and increased risk for acute myocardial infarction in Finnish middle-aged men[5]. Meanwhile, α2CDel322-325 has been proved to result in damaged negative feedback of sympathetic activity. Neumeister et al. reported that α2CDel322-325 homozygotic volunteers have increased sympathetic drive, as reflected in three-fold higher levels of norepinephrine release in response to administration [6] of an α2 AR antagonist . Moreover, Park et al. reported α2CDel322-325 allele as a significant [7] predictor of CAS in a Korean population . On the other hand, β2Gln27 allele was suggested to be

associated with relatively decreased function of β2-AR[8]. As a result, it may cause impaired vessel dilation and increased vasomotor tone and even CAS. Correspondingly, β2Gln27 allele homozygote has also been proved by Park et al. to be associated with CAS[7]. Besides, as both of the two major receptors in the ANS, the adrenergic and muscarinic acetylcholine receptors, are G protein-coupled receptors, the C825T polymorphism of the G protein β3 subunit gene (GNB3) is expected to affect autonomic activity. Actually, Michalsen et al. have proven that GNB3 825T allele carriers had enhanced coronary vasoconstriction to sympathetic activation[9]. For these reasons, we finally chose the 4 polymorphisms: α2BDel301-303, α2CDel322-325, β2Gln27Glu, and GNB3 825T. We investigated the allele frequencies and the genotype distributions of the 4 common gene polymorphisms and examined their association with the presence of CAS in a Chinese population. A total of 109 patients who visited the department of cardiology of Guangzhou General Hospital of Guangzhou Military Command between December 2006 and December 2010 and met the following clinical and angiographic criteria were enrolled in the CAS group: (1) chest pain occurring almost exclusively during rest and not precipitated by physical exertion, (2) coronary angiography showing no significant stenosis, and (3) occlusive coronary spasm (≥90%) accompanied by usual chest pain induced by acetylcholine provocative tests. And the chest pain had been alleviated after the coronary spasm was relieved spontaneously or by intracoronary injection of nitroglycerin. The normal control group consisted of 94 apparently healthy subjects who had visited the health care center of our hospital for routine checkup during the same time period and whose histories and laboratory test results, including blood chemistry, electrocardiography, and chest radiography, did not reveal any evidence of cardiac disease. This study complied with

doi: 10.3967/0895-3988.2013.08.010 1. Department of Cardiology, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, Guangdong, China; 2. Third Military Medical University, Chongqing 400037, Chongqing, China

690 requirement of the Declaration of Helsinki and the research protocol was approved by the Ethics Committee of Guangzhou General Hospital of Guangzhou Military Command. Informed consent was obtained from all participants. One milliliter of blood was taken from the median cubital vein in all subjects at optimal time. The venous blood samples were anticoagulated with EDTA-Na2 and stored at -70 °C for the next DNA extraction. Genomic DNA was extracted from whole blood using DNAzol Reagent (Invitrogen, Carlsbad, CA, USA) as recommended by the manufacturer, and then used for gene amplification by polymerase chain reaction (PCR). The sequences for primers are shown in Table 1. All of the polymorphisms were amplified using 50 ng of genomic DNA, 50 pm of each primer, and 1 U of Platinum Taq DNA polymerase (Invitrogen) in a final volume of 25 µL. After initial denaturation at 94 °C for 5 min, the samples were amplified over 35 cycles. The PCR amplification conditions were 94 °C (30 s), 55 °C (30 s), and 72 °C (1 min). After cycling, there was a final extension at 72 °C for 5 min. The amplified products corresponding to each different polymorphism were analyzed by gel electrophoresis using 1.5% agarose and sequenced using an ABI 3730XL sequencer to obtain the genotypes. In case of discrepancies the analyses were repeated. There were no missing genotypes, as all of the patients included in the study were successfully screened for the four polymorphisms. Statistical analyses were performed using SPSS 17.0 statistical software package. Quantitative data are presented as the mean±standard deviation. The Chi-square test was used to test Hardy-Weinberg equilibrium and the association between the categorical variables, while the Student’s t-test was conducted to compare the means of continuous variables. P≤0.05 was considered as statistical significance. The baseline data of to the study groups (CAS group vs. control group) are shown in Table 2. The average age, the proportion of males, the average level of LDL-C, and the prevalence of hypertension and diabetes were not significantly different between the two groups. However, BMI (23.4±2.7 kg/m2 vs. 21.5±1.7 kg/m2; P<0.001) and the proportion of smokers (57.8% vs. 23.4%; P<0.001) were significantly higher in the CAS group than in the control group. Table 3 shows the distributions of α2BDel301- 303, α2CDel322-325, β2Gln27Glu, and GNB3 C825T polymorphisms. For α2BDel301-303 genotypes in all

Biomed Environ Sci, 2013; 26(8): 689-692 Table 1. Sequences for Primers of the Four Polymorphisms Forward Primer (5’-3’)

Reverse Primer (5’-3’)

α2BDel301-303

AGAAGGAGGGTGTTT GTGGGG

ACCTATAGCACCCACG CCCCT

α2CDel322-325

CTGCATCGGCTCCTTC TTC

ATGACCACAGCCAGCA CAA

β2Gln27Glu

ACCACAGCCGCTGAAT GAG

TCAGCACAGGCCAGTG AAGT

GNB3 825T

CAATGGAGAGGCCAT CTGC

GGCTGCTTAGAGCAAC ACTGA

Table 2. Baseline Data of CAS and Control Groups CAS

Control

109

94

56.9±11.6

56.4±8.1

0.726

67 (61.5)

59 (62.8)

0.885

BMI (kg/m )

23.4±2.7

21.5±1.7

< 0.001

LDL-C (mmol/L)

2.87±0.92

2.94±0.69

0.539

Hypertension (%)

8 (7.3)

5 (5.3)

0.583

Diabetes (%)

4 (3.7)

1 (1.1)

0.376

Smokers (%)

63 (57.8)

22 (23.4)

<0.001

No. of subjects (n) Age (y) Males (%) 2

P Value

Note. Values are presented as number (%) or mean±standard deviation. BMI: Body mass index; LDL-C: low density lipoprotein-cholesterol. participants, homozygous I/I accounted for 28.1%, I/D were 53.7% and D/D were 18.2%. For α2CDel322325 genotypes, homozygous I/I accounted for 75.9%, I/D were 22.7%, and D/D were 1.5%. For β2Gln27Glu genotypes, homozygous Gln/Gln accounted for 78.8%, Gln/Glu were 20.2% and homozygous Glu/Glu were 1.0%. For GNB3 C825T genotypes, homozygous C/C accounted for 22.2%, C/T were 50.2% and homozygous T/T were 27.6%. The distributions of α2B Del301-303, α2CDel322-325, β2Gln27Glu, and GNB3 C825T genotype in the controls had no deviation from Hardy-Weinberg equilibrium, and P values were 0.403, 0.554, 0.554, and 0.805, respectively (data not shown). For α2BDel301-303, 31.2%, 53.2%, and 15.6% of the patients with CAS were found to carry I/I, I/D, and D/D genotype, respectively, while in the controls, the percentages of the genotypes were 24.5%, 54.3%, and 21.3%, respectively. For α2CDel322-325, 77.1%, 21.1%, and 1.8% of the patients carried I/I, I/D, and D/D genotype, respectively, while the percentages of

Biomed Environ Sci, 2013; 26(8): 689-692

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Table 3. Genotype and Allele Frequency Distributions of Polymorphisms according to Groups Group

No. of Subjects Represented by Genotype (%)

P Value 0.428

No. of Subjects Represented by Allele (%)

α2BDel301-303

Wild/Wild

Wild/Del

Del/Del

Wild

Del

ALL

57 (28.1)

109 (53.7)

37 (18.2)

223 (54.9)

183 (45.1)

CAS

34 (31.2)

58 (53.2)

17 (15.6)

126 (57.8)

92 (42.2)

CONTROL

23 (24.5)

51 (54.3)

20 (21.3)

97 (51.6)

91 (48.4)

α2CDel322-325

Wild/Wild

Wild/Del

Del/Del

Wild

Del

ALL

154 (75.9)

46 (22.7)

3 (1.5)

354 (87.2)

52 (12.8)

CAS

84 (77.1)

23 (21.1)

2 (1.8)

191 (87.6)

27 (12.4)

CONTROL

70 (74.5)

23 (24.5)

1 (1.1)

163 (86.7)

25 (13.3)

β2Gln27Glu

Gln/Gln

Gln/Glu

Glu/Glu

Gln

Glu

ALL

160 (78.8)

41 (20.2)

2 (1.0)

361 (88.9)

45 (11.1)

0.846

0.320

CAS

90 (82.6)

18 (16.5)

1 (0.9)

198 (90.8)

20 (9.2)

CONTROL

70 (74.5)

23 (24.5)

1 (1.1)

163 (86.7)

25 (13.3)

GNB3 C825T

C/C

C/T

T/T

C

T

ALL

45 (22.2)

102 (50.2)

56 (27.6)

192 (47.3)

214 (52.7)

CAS

25 (22.9)

54 (49.5)

30 (27.5)

104 (47.7)

114 (52.3)

CONTROL

20 (21.3)

48 (51.1)

26 (27.7)

88 (46.8)

100 (53.2)

the genotypes in the controls were 74.5%, 24.5%, and 1.1%, respectively. For β2Gln27Glu, 82.6%, 16.5%, and 0.9% of the patients had Gln/Gln, Gln/Glu, and Glu/Glu genotype, respectively, while in the controls, the percentages of the genotypes were 74.5%, 24.5%, and 1.1%, respectively. For GNB3 C825T, 22.9%, 49.5%, and 27.5% of the patients had C/C, C/T, and T/T genotype, respectively, while the percentages of the genotypes in the controls were 21.3%, 51.1%, and 27.7%, respectively. There is no significant difference in the genotype distribution of the four polymorphisms between the patients and the controls as the P values were 0.428, 0.846, 0.320, and 0.982, respectively. The minor alleles were α2BDel301-303, α2CDel322-325, β2Glu27, and GNB3 C825, and their frequencies were 48.4%, 13.3%, 13.3%, and 46.8%, respectively, in the health control subjects while the frequencies in the patients with CAS were 42.2%, 12.4%, 9.2%, and 47.7%, respectively. There is also no significant difference in the allele frequen- cies of α2BDel301-303, α2CDel322-325, β2Glu27, and GNB3 C825 between the two groups with the P value as 0.231, 0.882, 0.207, and 0.921, respectively. In this study, we investigated the potential association of four common genetic polymorphisms with CAS. Given the important role of the ANS in CAS pathophysiology, we concentrated on genes that

0.982

P Value 0.231

0.882

0.207

0.921

encode components of the system or that may be considered as otherwise affecting it on the basis of their functional role. We found no significant difference in the genotype or allele frequencies of the 4 polymorphisms assessed between patients with CAS and healthy subjects. However, Park et al. previously found that α2CDel322-325 allele carrier state and β2Gln27 allele homozygote state were associated with CAS in a Korean population[7], which conflicts with our results in this study. This discrepancy may be due to the marked difference in frequency distributions of polymorphisms among different ethnic groups. As reported in the Korean population by Park et al., for α2CDel322-325, 86.0%, 13.2%, and 0.9% of the controls carried I/I, I/D, and D/D genotype, respectively, and the α2CDel322-325 allele frequency was 7% in the controls. After the Chi-square test, we found that both the genotype and allele frequency distributions of α2CDel322-325 in the Korean controls were different from the results of our study, with the P value as 0.036 and 0.049, respectively. Meanwhile, for β2Gln27Glu in the Korean population reported by Park et al., 56.1%, 29.8%, and 14.0% of the controls had Gln/Gln, Gln/Glu, and Glu/Glu genotype, respectively, and the β2Glu27 allele frequency was 29% in the controls. We also found significant differences between the genotype and allele

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Biomed Environ Sci, 2013; 26(8): 689-692

frequency distributions of β2Gln27Glu in the Korean controls and the results of our study, as the P values were 0.009 and less than 0.001, respectively. Moreover, the frequency of the β2Glu27 allele has been reported to be higher in other ethnicities, such [10] as Caucasians (35%) . The significant interethnic differences in frequency distributions of polymorphisms might be an important reason for the discrepancy in results from different ethnic groups. There were some limitations of this study that should be noted. Firstly, as other studies on the gene polymorphisms in CAS population, the sample size was relatively small because of the difficulty in the diagnosis of CAS. However, the genotype distributions of all the polymorphisms were within Hardy-Weinberg equilibrium and therefore the possibility of extreme selection bias is low. Secondly, CAS is unlikely to be caused by a single genetic polymorphism. The risk of CAS may arise from a large number of genes and be affected by numerous non-genetic factors. As a result, other genetic differences and/or environmental influences in which gene-gene and/or gene-environment interactions have a larger impact on predisposition than the independent effects of each locus may still account for the lack of an association in our population. Further studies using approaches with “natural” genetic cohorts, such as large pedigrees and twins, may be needed to help define contributions of particular variants. In conclusion, none of the polymorphisms affecting autonomic activity considered in this study are a major risk factor of CAS, which somewhat conflicts with previous studies of other ethnic groups to a certain extent. This result suggests that there are significant differences in the genetic background of different ethnic groups. A better understanding of the genetic determinants of CAS needs several additional studies of the interactions of the genes involved in the functioning of the ANS as well as the interactions between gene polymorphisms and environmental factors. ACKNOWLEDGEMENTS We sincerely thank the subjects who participated in the study. Besides, we appreciate

Doctor GONG Zhi Hua and WEN Yan Fei in cardiac catheterization laboratory of Guangzhou General Hospital of Guangzhou Military Command for their excellent technical assistance in our study.  This research was supported by fund from Guangdong Natural Science Foundation (No. 925100 1002000002).  Correspondence should be addressed to XIANG Ding Cheng. Tel: 86-20-88653325. Fax: 86-20-88653325. E-mail: [email protected] Biographical note for the first author: ZHOU Xuan, female, born in 1984, Ph.D candidate, majoring in cardiology. Received: March 14, 2013; Accepted: June 17, 2013 REFERENCES 1. Yasue H, Nakagawa H, Itoh T, et al. Coronary artery spasm-clinical features, diagnosis, pathogenesis, and treatment. J Cardiol, 2008; 51(1), 2-17. 2. Kiss G, Corre O, Gueret G, et al. Management of cardiac arrest caused by coronary artery spasm: epinephrine/adrenaline versus nitrates. Heart Lung, 2009; 38(3), 228-32. 3. Pristipino C, Beltrame JF, Finocchiaro ML, et al. Major racial differences in coronary constrictor response between japanese and caucasians with recent myocardial infarction. Circulation, 2000; 101(10), 1102-8. 4. Lanza GA, Careri G, Crea F. Mechanisms of coronary artery spasm. Circulation. 2011, 124(16), 1774-82. 5. Snapir A, Mikkelsson J, Perola M, et al. Variation in the alpha2B-adrenoceptor gene as a risk factor for prehospital fatal myocardial infarction and sudden cardiac death. J Am Coll Cardiol, 2003; 41(2), 190-4. 6. Neumeister A, Charney DS, Belfer I, et al. Sympathoneural and adrenomedullary functional effects of alpha2C-adrenoreceptor gene polymorphism in healthy humans. Pharmacogenet Genomics, 2005; 15(3), 143-9. 7. Park JS, Zhang SY, Jo SH, et al. Common adrenergic receptor polymorphisms as novel risk factors for vasospastic angina. Am Heart J, 2006; 151(4), 864-9. 8. Bray MS, Krushkal J, Li L, et al. Positional genomic analysis identifies the beta(2)-adrenergic receptor gene as a susceptibility locus for human hypertension. Circulation, 2000; 101(25), 2877-82. 9. Meirhaeghe A, Bauters C, Helbecque N, et al. The human G-protein beta3 subunit C825T polymorphism is associated with coronary artery vasoconstriction. Eur Heart J, 2001; 22(10), 845-8. 10.Xie HG, Stein CM, Kim RB, et al. Frequency of functionally important beta-2 adrenoceptor polymorphisms varies markedly among African-American, Caucasian and Chinese individuals. Pharmacogenetics, 1999; 9(4), 511-6.