Original Articles Korean Circulation J 2000;306:702-715 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% CI1.7991.1252.878 p0.014 and 5.6241.01931.025, 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;306:702-715 KEY WORDSThrombomodulin Gene Polymorphism Myocardial infarction. 702
서론 대상및방법 대상 703
방법 Table 1. Characteristics of study population Male Female MI 191 Control 174 MI 43 Control 117 Age years 57.010.9* 47.311.2 65.6 8.7* 48.212.1 Smoking % 63.4 62.1 9.3 2.6 Diabetes % 21.5* 5.7 32.6* 2.6 Hypertension % 40.8* 28.7 48.8* 25.6 BMI Kgm 2 24.4 2.7* 23.7 2.7 24.6 4.2* 23.2 3.3 TC mgdl 193.438.8 189.336.5 196.848.7 192.738.4 TG mgdl 149.276.9 143.081.0 121.663.8 131.783.9 HDL-C mgdl 32.311.1 40.910.9 41.112.7 43.510.5 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;306:702-715
Table 2. Oligonucleotide primers in the PCR amplification of the TM gene Primer name Nucleotide no* Oligonucleotide sequence TM-1S 306287 5-GAT GAA AGA GGG CTG CAC GC-3 TM-1AS 146163 5-CAT GGG ATC ACC TCG CCG-3 TM-2S 187167 5-CGC CAG GGC AGG GTT TAC TCA-3 TM-2AS 2846 5-GTG CCC GGC CCT CCC TCC C-3 TM-3S 6448 5-CCT TTT CCC GAA CGT CC-3 TM-3AS 5672 5-GCC TCT CCT GTC CGT CC-3 TM-4S 2947 5-CGC GCA CGG CAA GAA GTG T-3 TM-4AS 188170 5-AGG ACC AGG ACC CCA AGC A-3 TM-5S 161182 5-TGG GTA ACA TGC TTG GGG TCC T-3 TM-5AS 405384 5-GCC GTC GCC GTT CAG TAG CAA G-3 TM-6S 364382 5-GTG GCT GCC GAT GTC ATT T-3 TM-6AS 556538 5-CCC CAT TGA GGT CGA GCC G-3 TM-7S 500522 5-TTA CGG GAG ACA ACA ACA CCA GC-3 TM-7AS 745725 5-AGG TGA TCG AGA CGG CGG CAG-3 TM-8S 634652 5-CAG CAG TGC GAA GTG AAG G-3 TM-8AS 871854 5-AGT GCC CCT GGA CCG CTC-3 TM-9S 833854 5-TAA TGT GCA CCG CGC CGC CCG-3 TM-9AS 10771058 5-CGG CTG GTC GGG GTT GGG AA-3 TM-10S 9921011 5-CAG ACG GGC GCT CCT GCA CC-3 TM-10AS 12701249 5-GGT CCA CGG GCT CCA CAC ACT-3 TM-11S 12141236 5-GCC ACT GCT ACC CTA ACT ACG AC-3 TM-11AS 13731356 5-TGC GGC TCG TGG GGA ATG-3 TM-12S 13191339 5-CTA GCT ACC TCT GCG TCT GCG-3 TM-12AS 15671549 5-CGC AGA TGC ACT CGA AGG T-3 TM-13S 15171533 5-TCT GCT CCG GGG TGT GC-3 TM-13AS 17171701 5-AGC CCG AAT GCA CGA GC-3 TM-14S 16721692 5-GGC TCC ACC TTG ACT CCT CCG-3 TM-14AS 18351816 5-GCC GCG CAC TTG TAC TCC AT-3 TM-15S 17501769 5-CTG GTG GTG GCG CTT TTG GC-3 TM-15AS 19571936 5-CAA AGC TGG GGG TGA GGA GGC A-3 *Nucleotide number is destinated from the transcriptional start point. TMthrombomodulin, Ssense oligonucleotide, ASantisense oligonucleotide 705
Table 3. Conditions for PCR Amplification of the TM gene Primers Sizes of PCR Annealing product temperature Addendum base pair TM-1STM-1AS 161 65 TM-2STM2AS 160 65 TM-3STM-3AS 136 60 TM-4STM-4AS 160 65 TM-5STM-5AS 245 55 1M Betaine TM-6STM-6AS 193 55 1M Betaine TM-7STM-7AS 246 65 TM-8STM8-AS 238 55 1M Betaine TM-9STM-8AS 245 55 1M Betaine TM-10STM-10AS 279 65 TM-11STM-11AS 160 65 TM-12STM-12AS 249 65 TM-13STM-13AS 201 60 TM-14STM-14AS 164 65 TM-15STM-15AS 208 65 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;306:702-715
결과 TM 유전자변이 707
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;306:702-715
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 191 0.775 0.225* 0.882 0.118 Control 174 0.868 0.132 0.923 0.077 TM Ala455Val A/A AVVV A V MI 191 0.560 0.440* 0.754 0.246* Control 174 0.678 0.322 0.822 0.178 TM C1922T CC CTTT C T MI 191 0.953 0.047* 0.977 0.023 Control 174 0.989 0.011 0.994 0.006 Female TM G-33A GG AGAA G A MI 43 0.767 0.233 0.872 0.128 Control 117 0.821 0.179 0.902 0.098 TM Ala455Val A/A AVVV A V MI 43 0.628 0.372 0.767 0.233 Control 117 0.564 0.436 0.761 0.239 TM C1922T CC CTTT C T MI 43 0.884 0.116 0.942 0.058 Control 117 0.966 0.034 0.983 0.017 *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 % 63.6 59.1 0.46 60.9 65.7 0.86 63.6 36.4 3.38 Diabetes % 13.0 18.2 1.19 13.3 15.0 0.20 14.1 9.1 0.23 Hypertension % 32.8 45.5 3.82 33.3 37.9 0.78 34.8 45.5 0.54 Female Smoking % 4.6 3.2 0.12 4.3 4.5 0.00 4.64 0 0.44 Diabetes % 11.6 6.5 0.71 11.8 9.0 0.33 10.6 11.1 0.00 Hypertension % 31.0 35.5 0.23 33.3 29.9 0.22 31.8 33.3 0.01 2 709
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 51.912.0 54.012.6 1.30 51.912.2 52.812.0 0.69 52.212.6 55.615.1 0.93 BMI Kgm 2 23.9 2.7 24.7 2.4 2.21* 23.9 2.8 24.1 2.5 0.59 24.0 2.7 24.4 2.3 0.41 TC mgdl 190.337.4 196.439.2 1.17 191.838.3 190.836.8 0.25 190.837.8 211.527.6 1.79 TG mgdl 145.579.2 149.378.0 0.34 145.982.6 146.873.0 0.10 144.877.9 191.598.3 1.94 HDL-C mgdl 40.911.1 36.710.1 2.78* 40.510.6 39.511.7 0.84 40.211.1 36.0 8.9 1.24 Female Age years 52.813.5 53.314.8 0.18 54.113.4 51.214.0 1.32 52.613.8 58.010.8 1.15 BMI Kgm 2 23.4 3.4 24.3 4.2 1.25 23.4 3.2 23.7 3.9 0.59 23.4 3.5 25.5 4.4 1.62 TC mgdl 191.240.3 205.044.1 1.66 193.240.9 194.642.0 0.20 193.642.0 197.621.8 0.27 TG mgdl 130.481.2 123.770.0 0.41 133.484.3 123.071.2 0.81 129.680.0 119.862.6 0.34 HDL-C mgdl 42.010.6 46.512.9 2.03* 41.810.1 44.312.4 1.37 42.811.0 44.520.0 0.24 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 1.550 44.217 0.0001 1.362 to 1.764 BMI 1.119 5.763 0.016 1.021 to 1.226 Hypertension 1.014 0.003 0.956 0.608 to 1.693 TM 33A 1.784 3.276 0.070 0.953 to 3.341 Female Age 2.371 32.079 0.0001 1.759 to 3.197 TM 33A 1.589 0.671 0.4130 0.525 to 4.811 BMIbody mass index, CIconfidence interval 각대립유전자간의연관관계 Korean Circulation J 2000;306:702-715
유전자발현조절부위에서의유전자변이가유전자발현에미치는영향 Table 9. Multiple logistic regression analysis of TM 455Val and risk factors for MI Male Odds ratio Wald p value 95% CI Age 1.570 53.437 0.0001 1.391 to 1.771 TM 455Val 1.799 6.005 0.0143 1.125 to 2.878 Female Age 2.368 31.892 0.0001 1.756 to 3.193 TM 455Val 0.957 0.010 0.9920 0.393 to 2.330 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 1.703 91.195 0.0001 1.527 to 1.900 Smoking 4.468 38.041 0.0001 2.777 to 7.190 TM 1922T 5.624 3.929 0.0475 1.019 to 31.025 Female Age 2.375 31.242 0.0001 1.754 to 3.216 TM 1922T 3.449 1.886 0.1700 0.589 to 20.188 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
712 Korean Circulation J 2000;306:702-715
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