Original Articles Korean Circulation J 2000;30 6 : 심근경색의위험인자로서 Thrombomodulin 유전자변이의의의 박현영 1 김영미 1 권혁문 2 지선하 3 조승연 2 정남식 2 심원흠 2 장양수 2 Genetic

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1 Original Articles Korean Circulation J 2000;306: 심근경색의위험인자로서 Thrombomodulin 유전자변이의의의 박현영 1 김영미 1 권혁문 2 지선하 3 조승연 2 정남식 2 심원흠 2 장양수 2 Genetic Variants of Thromobomodulin Gene as Risk Factors for Myocardial Infarction Hyun Young Park 1, Young Mi Kim 1, Hyuck Moon Kwon 2, Sun Ha Jee 3, Seung Yeon Cho 2, Nam Sik Chung 2, Won Heum Shim 2 and Yang Soo Jang 2 1 Yonsei Cardiovascular Research Institute, 2 Department of Internal Medicine, 3 Department of Epidemiology and Disease Control, Yonsei University, Seoul, Korea ABSTRACT Thrombomodulin TM is thrombin receptor present on the luminal surface of endothelial cells. Because the thrombin-tm complex acts as an anticoagulant, the functional variants or deficiency of TM may lead to increment of thrombotic tendency. In this study, we screened the genetic variants of the TM gene in patients with myocardial infarction MI and analyzed the genotype to elucidate the effects of genetic variations of TM gene on the development of the MI. We screened a promoter region and coding sequence of the TM gene using single strand conformation polymorphism-heteroduplex analysis and identified three common genetic variantsthose were TM G-33A, TM Ala455Val, and TM C1922T. The genotype frequencies were investigated in the patients with MI n234 and control subjects n291 by the method of allele-specific oligomer hybridization. The frequencies of mutant genotypes TM 33A, TM 455Val, and TM 1922T were higher in patient group compared to the control subjects in males while there were no significant differences in females. In the multiple logistic regression analysis, TM 455Val and TM 1922T alleles were independent risk factors for MI OR95% CI p0.014 and , p0.048, respectively in males. However, the genetic variations were not independent risk factors for MI in females. There were significant linkage disequilibriums among three genetic variants. These linkage disequilibriums explain the similar effects of three genetic variants on the development of MI. To investigate the effect of the TM G-33A mutation on TM promoter activity, the two TM promoter constructs ptm-355 and ptm-125, bearing TM 33G or TM 33A containing of firefly luciferase gene were transfected into HepG2, BAE, and CHO cells. The promoter activities were higher in the promoter constructs with TM 33G compared to the constructs with TM 33A in ptm-355. These results suggest the possibility of the positive predisposing effect of TM 33A allele on MI in males. The functional study for TM Ala455Val and TM C1922T should be followed to elucidate the genotype effects of these mutations on the development of MI. In this study, we identified three genetic variants of TM gene and showed the significant associations between genetic variants and MI in males. These results proposed that TM gene is an attractive candidate for genetic risk factor for MI in Koreans. Korean Circulation J 2000;306: KEY WORDSThrombomodulin Gene Polymorphism Myocardial infarction. 702

2 서론 대상및방법 대상 703

3 방법 Table 1. Characteristics of study population Male Female MI 191 Control 174 MI 43 Control 117 Age years * * Smoking % Diabetes % 21.5* * 2.6 Hypertension % 40.8* * 25.6 BMI Kgm * * TC mgdl TG mgdl HDL-C mgdl MIpatients with myocardial infarction, Controlcontrol subjects, BMIbody mass index, TCtotal cholesterol, TGtriglyceride, HDL-Chigh density lipoprotein-cholesterol The number in parenthesis represents the number of subjects. *p0.05 MI vs. Control by t-test or 2 -test. 704 Korean Circulation J 2000;306:

4 Table 2. Oligonucleotide primers in the PCR amplification of the TM gene Primer name Nucleotide no* Oligonucleotide sequence TM-1S GAT GAA AGA GGG CTG CAC GC-3 TM-1AS CAT GGG ATC ACC TCG CCG-3 TM-2S CGC CAG GGC AGG GTT TAC TCA-3 TM-2AS GTG CCC GGC CCT CCC TCC C-3 TM-3S CCT TTT CCC GAA CGT CC-3 TM-3AS GCC TCT CCT GTC CGT CC-3 TM-4S CGC GCA CGG CAA GAA GTG T-3 TM-4AS AGG ACC AGG ACC CCA AGC A-3 TM-5S TGG GTA ACA TGC TTG GGG TCC T-3 TM-5AS GCC GTC GCC GTT CAG TAG CAA G-3 TM-6S GTG GCT GCC GAT GTC ATT T-3 TM-6AS CCC CAT TGA GGT CGA GCC G-3 TM-7S TTA CGG GAG ACA ACA ACA CCA GC-3 TM-7AS AGG TGA TCG AGA CGG CGG CAG-3 TM-8S CAG CAG TGC GAA GTG AAG G-3 TM-8AS AGT GCC CCT GGA CCG CTC-3 TM-9S TAA TGT GCA CCG CGC CGC CCG-3 TM-9AS CGG CTG GTC GGG GTT GGG AA-3 TM-10S CAG ACG GGC GCT CCT GCA CC-3 TM-10AS GGT CCA CGG GCT CCA CAC ACT-3 TM-11S GCC ACT GCT ACC CTA ACT ACG AC-3 TM-11AS TGC GGC TCG TGG GGA ATG-3 TM-12S CTA GCT ACC TCT GCG TCT GCG-3 TM-12AS CGC AGA TGC ACT CGA AGG T-3 TM-13S TCT GCT CCG GGG TGT GC-3 TM-13AS AGC CCG AAT GCA CGA GC-3 TM-14S GGC TCC ACC TTG ACT CCT CCG-3 TM-14AS GCC GCG CAC TTG TAC TCC AT-3 TM-15S CTG GTG GTG GCG CTT TTG GC-3 TM-15AS CAA AGC TGG GGG TGA GGA GGC A-3 *Nucleotide number is destinated from the transcriptional start point. TMthrombomodulin, Ssense oligonucleotide, ASantisense oligonucleotide 705

5 Table 3. Conditions for PCR Amplification of the TM gene Primers Sizes of PCR Annealing product temperature Addendum base pair TM-1STM-1AS TM-2STM2AS TM-3STM-3AS TM-4STM-4AS TM-5STM-5AS M Betaine TM-6STM-6AS M Betaine TM-7STM-7AS TM-8STM8-AS M Betaine TM-9STM-8AS M Betaine TM-10STM-10AS TM-11STM-11AS TM-12STM-12AS TM-13STM-13AS TM-14STM-14AS TM-15STM-15AS The thermal profile included 10 minutes of denaturation at 95, 35 cycles containing of denaturation for 1 minute at 94, annealing for 1 min at indicated temperature and extension for 1 minute at 72, followed by 5 minutes of final extension at 72. Table 4. A list of the allele-specific oligonucleotides, and hybridization and washing conditions for genotyping Position Alelle-specific oligonucleotides Hybridization Washing condition TM G-33A G5-TAAGTGCCCGGCCCTC-3 45 G2SSC0.2% SDS 60 A5-GAGGGCCAGGCACTTA-3 A2SSC0.2% SDS 60 TM Ala455Val Ala5-GCCCTTGCCCGCCACA-3 45 Ala2SSC0.2% SDS 60 Val5-TGTGGCGGACAAGGGC-3 Val2SSC0.2% SDS 60 TM C1922T C5-CCTGGCTCCGTCC-3 35 C2SSC0.2% SDS 45 T5-CCTGGCTTCGTCC-3 T2SSC0.2% SDS 43 TMthrombomodulin. Underlines indicate polymorphic nucleotides. Fig. 1. Identification of the genotype by the allele-specific oligomer ASO hybridization of G- 33A. The hot spot means the presence of the complement sequence to labeled ASO. The genotype was determined by the presence of the hot spots on the hybridized membrane. 706 Korean Circulation J 2000;306:

6 결과 TM 유전자변이 707

7 Fig. 2. Mutation detection by SSCPheteroduplexA and sequencing data B of the mutations on the TM gene. The asterisk indicates the mutation site. TM 유전자변이의유전형분포 708 TM 유전자변이의심근경색발생에서의상대위험도 Korean Circulation J 2000;306:

8 TM 유전자변이와심근경색증의위험요인과의관계 TM 유전자변이의심근경색발생위험인자로써의의의 Table 5. Genotype and allele frequencies in patients with MI and in control subjects Male Genotype frequency Allele frequency TM G-33A GG AGAA G A MI * Control TM Ala455Val A/A AVVV A V MI * * Control TM C1922T CC CTTT C T MI * Control Female TM G-33A GG AGAA G A MI Control TM Ala455Val A/A AVVV A V MI Control TM C1922T CC CTTT C T MI Control *p0.05 MI vs. Control by 2 -test. Table 6. The distributions of non-parametric risk factors for MI according to TM genotype Male GG TM G-33A TM Ala455Val TM C1922T AGAA 2 AA AVVV 2 CC CTTT Smoking % Diabetes % Hypertension % Female Smoking % Diabetes % Hypertension %

9 Table 7. The distributions of parametric risk factors for MI according to TM genotype Male TM G-33A TM Ala455Val TM C1922T t t GG AGAA AA AVVV CC CTTT Age years BMI Kgm * TC mgdl TG mgdl HDL-C mgdl * Female Age years BMI Kgm TC mgdl TG mgdl HDL-C mgdl * BMIbody mass index, TCtotal cholesterol, TGtriglyceride, HDL-Chigh density lipoprotein-cholesterol *p0.05 t 710 Table 8. Multiple logistic regression analysis of TM -33A and risk factors for MI Male Odds ratio Wald p value 95% CI Age to BMI to Hypertension to TM 33A to Female Age to TM 33A to BMIbody mass index, CIconfidence interval 각대립유전자간의연관관계 Korean Circulation J 2000;306:

10 유전자발현조절부위에서의유전자변이가유전자발현에미치는영향 Table 9. Multiple logistic regression analysis of TM 455Val and risk factors for MI Male Odds ratio Wald p value 95% CI Age to TM 455Val to Female Age to TM 455Val to CIconfidence interval Table 10. Multiple logistic regression analysis of TM 1922T and risk factors for MI Male Odds ratio Wald p value 95% CI Age to Smoking to TM 1922T to Female Age to TM 1922T to CIconfidence interval 고찰 Fig. 3. Luciferase activities of two TM promoter constructs, ptm-355 and ptm-125, in HepG2, BAE, and CHO cells. The firefly luciferase activities were corrected by the beta-galactosidase activities to control the differences of the transfection efficacy. 711

11 712 Korean Circulation J 2000;306:

12 요약 713

13 중심단어 감사문 714 REFERENCES 1) Ferguson RJ, Petitclerc R, Choquette G, Chaniotis L, Gauthier P, Huot R, et al. Effect of physical training on treadmill exercise capacity, collateral circulation and progression of coronary disease. Am J Cardiol ) Marcus ML. The coronary circulation. In Marcus ML, ed. The coronary circulation in health and disease. New York, Mc Graw-Hill Book Co p ) Davies MJ, Thomas AC. Plaque fissuring-the cause of acute myocardial infarction, sudden ischemic death, and crescendo angina. Br Heart J ) Falk E. Coronary thrombosis Pathogenesis and clinical manifestation. Am J Cardiol B-38B. 5) Falk E, Shah PK, Fuster V. Coronary plaque disruption. Circulation ) Lane DA, Mannucci PM, Bauer KA, Bertina RM, Bochkov NP, Boulyjenkov V, et al. Inherited thrombophilia Part 1. Thromb Haemost ) Lane DA, Mannuci PM, Bauer KA, Bertina RM, Bochkov NP, Boulyjenkov V, et al. Inherited thrombophilia Part 2. Thromb Haemost ) Engesser L, Brommer EJ, Kluft C, Briet E. Elevated plasminogen activator inhibitor PAI, a cause of thrombophilia? A study in 203 patients with familial or sporadic venous thrombophilia. Thromb Haemost ) Ernst E, Resch KL. Fibrinogen as a cardiovascular risk factor A meta-analysis and review of the literature. Ann Intern Med 1993; ) Jorgensen M, Bonnevie-Nielsen V. Increased concentration of fast-acting plasminogen activator inhibitor in plasma associated with familial venous thrombosis. Br J Haematol ) Berg K, Kierulf P. DNA polymorphisms at fibrinogen loci and plasma fibrinogen concentration. Clin Genet ) Pankow JS, Folsom AR, Province MA, Rao DC, Williams RR, Eckfeldt J, et al. Segregation analysis of plasminogen activator inhibitor-1 and fibrinogen levels in the NHLBI family heart study. Arterioscler Thromb Vasc Biol ) Esmon CT. The regulation of natural anticoagulant pathways. Science ) Esmon CT. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem ) Esmon CT, Esmon NL, Harris KW. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalysed fibrin formation and factor V activation. J Biol Chem ) Esmon NL, Caroll RC, Esmon CT. Thrombomodulin blocks the ability of thrombin to activate platelets. J Biol Chem ) Jackman RW, Beeler DL, Fritze L, Soff G, Rosenberg RD. Human thrombomodulin gene is intron deleted Nucleic acid sequences of the cdna and gene predict protein structure and suggest sites of regulatory control. Proc Natl Acad Sci USA ) Norlund L, Zoller B, Ohlin AK. A novel thrombomodulin gene mutation in a patient suffering from sagittal sinus thrombosis. Thromb Haemost ) Ohlin AK, Norlund L, Marlar RA. Thrombomodulin gene variations and thromboembolic disease. Thromb Haemost ) Ohlin AK, Marlar RA. The first mutation identified in the thrombomodulin gene in a 45-year-old man presenting with thromboembolic disease. Blood ) Ohlin AK, Marlar RA. Thrombomodulin gene defects in families with thromboembolic diseasea report on four families. Thromb Haemost ) Faioni EM, Merati G, Peyvandi F, Bettini PM, Mnnuccu PM. The G1456 to T mutation in the thrombomodulin gene is not frequent in patients with venous thrombosisletter. Blood ) Van der Velden PA, Krommenhoek-Van Es T, Allaart CF, Bertina RM, Reitsma PH. A frequent thrombomodulin amino acid dimorphism is not associated with thrombophilia. Thromb Haemost ) Norlund L, Holm J, Zller B, Ohlin AK. A common thrombomodulin amino acid dimorphism is associated with myocardial infarction. Thromb Haemost ) Ireland H, Kunz G, Kyriakoulis K, Stubbs PJ, Lane DA. Thrombomodulin gene mutations associated with myocardial infarction. Circulation ) Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically amplified -globin and HLA-DQ DNA with allele-specific oligonucleotide probes. Nature ) Hedrick PW. Gametic disequilibrium measuresproceed with caution. Genetics ) Thompson EA, Deeb S, Walker D, Motulsky AG. The detection of linkage disequilibrium between closely linked markers RFLPs at the AI-CIII apolipoprotein genes. Am J Hum Genet ) Xie X, Otto J. Testing linkage disequilibrium between a Korean Circulation J 2000;306:

14 disease gene and marker loci. Am J Hum Genet abstract. 30) Flem LL, Picard V, Emmerich J, Gandrille S, Fiessinger J, Aiach M, et al. Mutations in promoter region of thrombomodulin and venous thromboembolic disease. Arterioscler Thromb Vasc Biol ) Carter AM, Catto AJ, Bamford JM, Grant PJ. Genderspecific associations of the fibrinogenb448 polymorphism, fibrinogen levels, and acute cerebravascular disease. Arterioscle Thromb Vasc Biol ) Yu K, Morioka H, Fritze LM, Beeler DL, Jackman RW, Rosenberg RD. Transcriptional regulation of the thrombomodulin gene. J Biol Chem 1992;267: ) Tazawa R, Hirosawa S, Suzuki K, Hirokawa K, Aoki N. Functional characterization of the 5 -regulatory region of the human thrombomodulin gene. J Biochem

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