대상및방법 환자군및대조군선택 혈액채취 513

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1 1999 년도산학협동연구비 Korean Circulation J 2001;315: 한국인에서 Endothelial Nitric Oxide Synthase(eNOS) 유전자변이빈도및관동맥연축과의상관관계 김정희 2 최성준 1 김보영 2 이정우 2 강정아 2 임대승 2 이민수 2 김윤철 2 성보영 2 임병관 1 성인환 2 전은석 1 Incidence of enos Gene Mutation in Korean Patients with Coronary Artery Spasm Jung-Hee Kim, MD 2, Seong-Choon Choe, MD 1, Bo-Young Kim, MD 2, Jeong-Woo Lee, MD 2, Jung-A Kang, MD 2, Dae-Seung Lim, MD 2, Min-Soo Lee, MD 2, Yoon-Cheol Kim, MD 2, Bo-Young Sung, MD 2, Byung-Kwan Lim, MS 1, In-Whan Seong, MD 2 and Eun-Seok Jeon, MD 1 1 Department of Internal Medicine, Soonchunhyang University College of Medicine, Bucheon, 2 Department of Internal Medicine, Chungnam National University College of Medicine, Taejeon, Korea ABSTRACT BackgroundCoronary artery spasm is an important mechanism in producing myocardial ischemia. But the exact mechanism of the spasm is not well known. We investigated the mutation of endothelial nitric oxide synthase enos that produce nitric oxide and relationship between enos mutation and coronary artery spasm. Materials and MethodsBlood were drawn from the patients with angiographically proven coronary artery spasm and normal controls. DNA were extracted and polymerase chain reaction and restriction analysis with Nae I were performed to find T 786 C mutation of 5 -flanking region in enos gene. Clinical and lipid parameters were reviewed. ResultsThe prevalence of coronary artery spasm in Chungnam National University Hospital was 19% 125/664. The 85 cases of coronary artery spasm and 131 cases of normal control were compared. The men in spasm group were more prevalent than the women p and rate of smoking were higher in spasm group p The T 786 C mutation of 5 -flanking region were 20% in spasm group and 3% in normal control p There were no differences in clinial profiles between mutant group and non-mutant group. But total cholesterol p0.04, triglyceride p0.006, LDL-cholesterol p0.02 in mutant group were higher than non-mutant group. ConclusionThe T 786 C mutation of 5 -flanking region in enos gene was related to coronary artery spasm and lipid profiles in Korea. Korean Circulation J 2001;305: KEY WORDSEndothelial nitric oxide synthase Nitric oxide Coronary artery spasm Mutation Lipid. 서 론 512

2 대상및방법 환자군및대조군선택 혈액채취 513

3 Genomic DNA(gDNA) 추출 enos 유전자증폭시발체 (primer) 제작 중합효소연쇄반응과중합효소연쇄반응산물에대한제한분석 (Restriction analysis) 통계적분석 결과 대상 enos gene 변이의빈도 514 Korean Circulation J 2001;313:

4 형협심증 환자에서 돌연변이가 관찰된 군과 관찰되지 않은 군의 임상적 특징에는 큰 차이가 없었고, 남녀간 고 찰 의 차이도 없었다. 그러나 돌연변이가 있었던 군에서 총 콜레스테롤치(p 0.04), 중성지방치(p 0.006), 저밀도 한 병원에서 3년간 관동맥조영술의 19%에서 관동맥 지단백 콜레스테롤치(p 0.02)가 통계적으로 유의하게 연축이 보고되었다는 것은 상당히 의미 있는 보고로 생 높게 나타났다. 고밀도 지단백 콜레스테롤치는 통계적 각이 되는데 그 이유는 관동맥 연축을 의심하지 않고는 으로 차이가 없었다(Table 3 and 4). 유발검사를 하지 못하기 때문에 실제로 우리 나라에서 Fig. 1. Schematic diagram which demonstrated endothelial nitric oxide (enos) gene and Nae I restriction site. Fig. 2. Electrophoretic photograph of polymerase chain reaction (PCR) and restriction anlaysis by Nae I restriction enzyme (Lane 1, 3, 5, 7, 9 PCR product before Nae I restriction, Lane 2, 4, 6, 8, 10 PCR product after Nae I restriction). Lane 2, 6 were restriction fragment length polymorphism (RFLP) positive and lane 4, 8, 10 were RFLP negative. Fig. 3. Sequence of the amplified polymerase chain reaction (PCR) product with Nae I restiction sitepositive sample and -negative sample. Base thymine (T) was replaced to cytosine (C) and Nae I restriction site was appeared. Thus the PCR product was cleaved. 515

5 Table 1. Difference of clinical profiles between coronary artery spasm group and spasm-negative group Spasm Spasm p Numbers Sex M/F 74/11 81/ Age yr Diabetes 9 11% 18 14% 0.64 Hypertension 31 36% 33 25% 0.11 Obesity 11 13% 31 24% 0.08 Family history 3 4% 4 3% 0.84 Smoking 53 62% 27 21% ObesityBody mass index25 Table 2. Difference of lipid profiles and RFLP between coronary artery spasm group and spasm-negative group Unitmg/dl Spasm Spasm p Numbers Triglyceride Total cholesterol HDL-C LDL-C Nae I RFLP 17 20% 4 3% HDL-Chigh density lipoprotein-cholesterol LDL-Clow density lipoprotein-cholesterol RFLPrestriction fragment length polymorphism Table 3. Differnce of clinical profiles between mutant group and mutant-negative group in variant angina mutant grouprflp, mutant-negative group RFLP RFLP RFLP p Numbers Sex M/F 15/2 59/ Age yr Diabetes 1 6% 8 12% 0.79 Hypertension 7 41% 24 35% 0.87 Obesity 3 18% 8 12% 0.81 Family history 2 12% 1 1% 0.19 Smoking 10 59% 43 63% 0.96 ObesityBody mass index25 RFLPrestriction fragment length polymorphism Table 4. Differnce of lipid profiles between mutant group and mutant-negative group in variant angina mutant grouprflp, mutant-negative group RFLP Unitmg/dl Spasm Spasm p Numbers Triglyceride Total cholesterol HDL-C LDL-C HDL-Chigh density lipoprotein-cholesterol LDL-Clow density lipoprotein-cholesterol RFLPrestriction fragment length polymorphism Table 5. Incidence of T 786 C mutation in Japan and Korea In Japan Controls Variant /homozygote 0% 2% /heterozygote 7% 28% /homozygote 93% 70% In Korea /homozygote 0% 0% /heterozygote 3% 20% /homozygote 97% 80% 516 Korean Circulation J 2001;313:

6 요약 연구목적 : 대상및방법 : 517

7 결과 : 결론 : 중심단어 REFERENCES 1) Theroux P, Waters D. Diagnosis and management of patients with unstable angina, In Hurst s The Heart, Schlant RC, Alexander RW, 8th Ed, McGraw-Hill Inc1994. p ) Han KI, Han KH, Park SW, Kim YK, Sohn DW, Lee MM, et al. The vasomotor tone in vasospastic angina. Korean Circulation J ) MacAlpin RN, Kattus AA, Alvaro AB. Angina pectoris at rest with preservation of exercise capacity. Prinzmetal s variant angina. Circulation ) Park YB, Lee YW. The study of coronary artery spasm. Korean Circulation J ) Han KR, Choi DJ, Choi YJ, Park SS, Kim YJ, Sohn DW, et al. Intracoronary ergonovine provocation test in patients with coronary artery spasm. Korean Circulation J ) Park SW, Park SJ, Kim JJ, Song JK, Seong IW, Lee JK. 518 The provocation test of coronary artery spasm with acetylcholine and ergonovine. Korean Circulation J ) Severi S, Davies G, Maseri A, Marzullo P, L Abbate A. Long-term prognosis of variant angina with medical treatment. Am J Cardiol ) Waters DD, Szlanchcic J, Miller D, Theroux P. Clinical characteristics of patients with variant angina complicated by myocardial infarction or death within 1 month. Am J Cardiol ) Oh BH, On YK, Lim HJ, Kim DW, Sohn DW, Lee MM, et al. Follow-up provocation test in patients with coronary artery spasm. Korean Circulation J ) Song JM, Park YB, Kim SH, Sohn KS, Han KR, Han KH, et al. Effect of recurrent coronary artery spasm on left ventricular contractile function. Korean Circulation J ) Choi DJ, Cho MC, Kim JS, Kim YK, Kim CJ, Sohn DW, et al. Acute myocardial infarction with coronary artery spasm on coronary angiogram. Korean Circulation J ) Seo JD, Song JK, Kim CH, Sohn DW, Oh BH, Lee MM, et al. Role of endothelium-derived relaxing factor in the pathogenesis of coronary artery spasm and its relationship with ethanol. Korean Circulation J ) Park SW, Park DG, Choi KJ, Kwon HC, Cho MC, Kim JS, et al. Clinical features of acute myocardial infarction with normal coronary arteriogram. Kor J Intern Med ) Chung NS, Lee WK, Cho SY, Shim WH, Shin KJ, Lee WH. A case of variant angina diagnosed with provocation test using ergonovine maleate. Korean Circulation J ) Scholl JM, Benacerraf A, Ducimetiere P, Chabas D, Brau J, Chapelle J, et al. Comparison of risk factors in vasospastic angina without significant fixed coronary narrowing and no vasospastic angina. Am J Cardiol ) Ozaki Y, Keane D, Serruys PW. Progression and regression of coronary stenosis in the long-term follow-up of vasospastic angina. Circulation ) Shimabukuro M, Shinzato T, Higa S, Chibana T, Yoshida H, Nagamine F, et al. Enhanced insulin response relates to acetylcholine-induced vasoconstriction in vasospastic angina. J Am Coll Cardiol ) Shinozaki K, Suzuki M, Ikebuchi M, Takaki H, Hara Y, Tsu-shima M, et al. Insulin resistance associated with compensatory hyperinsulinemia as an independent risk factor for vasospastic angina. Circulation ) Miwa K, Miyagi Y, Fujita M. Susceptibility of plasma low density lipoprotein to cupric ion-induced peroxidation in patients with variant angina. J Am Coll Cardiol ) Kaikita K, Ogawa H, Yasue H, Sakamoto T, Suefuji H, Sumida H, et al. Soluble P-selectin is released into the coronary circulation after coronary artery spasm. Circulation ) Igawa A, Miwa K, Miyagi Y, Fugita M, Inoue H. Comparison of frequency of magnesium deficiency in patients with vasospastic angina and fixed coronary artery disease. Am J Cardiol ) Park YB. Coronary artery spasm. J Kor Med Assoc 1993 Korean Circulation J 2001;313:

8 ) Nakayama M, Tasue H, Yoshimura M, Shimasaki Y, Kugiyama K, Ogawa H, et al. T 786 C mutation in the 5 - flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation ) Yoshimura M, Yasue H, Nakayama M, Shimasaki Y, Ogawa H, Kugiyama K, et al. Genetic risk factors for coronary artery spasm Significance of endothelial nitric oxide synthase gene T 786 C and missense Glu298Asp variants. J Investig Med ) Lee WH, Hwang TH, Park JE, Choi YH, Choo JA, Kim S, et al. Analysis of the endothelial nitric oxide synthase and -fibrinogen gene polymorphism in the development of acute myocardial infarction in Korean men. Korean Circulation J ) Yoon Y, Song J, Hong SH, Kim JQ. Plasma nitric oxide concentrations and nitric oxide synthase gene polymorphisms in coronary artery disease. Clin Chem ) Choi SY, Yoo KH, Park JS, Chae IH, Kim HS, Sohn DW, et al. enos gene polymorphism in patients with acute coronary syndrome or variant angina in Korean. Korean J Med ) Tentolouris C, Tousoulis D, Crake T, Katsimaglis G, Stefanadis C, Davies G, et al. Inhibition of nitric oxide synthesis in human epicardial coronary arteries and stenoses in relation to serum lipid level. Atherosclerosis

of medication with calcium channel blocker or nitrate compared with those in previous studies. Korean Circulation J 1999;293: KEY WORDSVariant

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