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T polymorphism of the intercellular adhesion molecule-1 gene (exon 6, codon 469). A risk factor for coronary heart disease and myocardial infarction
International Journal of Cardiology 84 (2002) 171–177 www.elsevier.com / locate / ijcard
C / T polymorphism of the intercellular adhesion molecule-1 gene (exon 6, codon 469). A risk factor for coronary heart disease and myocardial infarction a,c ,
a
b
b,c
a
Hong Jiang *, Rolf Michael Klein , Dieter Niederacher , Ming Du , Roger Marx , Mark Horlitz a , Guido Boerrigter a , Harald Lapp a , Thomas Scheffold a , Ingo Krakau a , a ¨ Hartmut Gulker a Heart Center Wuppertal, University of Witten-Herdecke, Arrenbergerstr. 20, 42117 Wuppertal, Germany Molecular Genetics Laboratory of the Gynecology Division, Henrich-Heine University, Duesseldorf, Germany c Union Hospital, Tongji Medical College, HuaZhong University of Science and Technology, HuaZhong, PR China b
Received 7 August 2001; received in revised form 8 March 2002; accepted 10 March 2002
Abstract Background: The intercellular adhesion molecule-1 (ICAM-1) mediates the interaction of activated endothelial cells with leukocytes and plays a fundamental role in the pathogenesis of coronary atherosclerosis. ICAM-1 single-base C / T polymorphism, which determines an amino acid substitution in the ICAM-1 protein in exon 6 codon 469, has been described. Our purpose was to determine whether this C / T polymorphism influences the risk of coronary heart disease (CHD) and myocardial infarction (MI) in humans. Methods and results: We enrolled 349 patients with angiographically documented CHD, including a sub-group of 179 patients with acute or chronic MI. The control group consisted of 213 patients with normal left ventricular function and no documented evidence of CHD. All patients and controls were Germans genotyped by polymerase chain reaction and allele-specific oligonucleotide techniques for the ICAM-1 polymorphism. In the patients with CHD and MI the frequencies of the T genotype (TT1TC) were significantly higher than the CC genotype compared to the control subjects (P,0.001). With the additional use of multivariable logistic regression analysis for CHD (TT1TC versus CC; P50.011, odds ratio 2.21, 95% CI 1.20–4.07), we found a significant association between CHD and MI and the TT and TC genotype of the ICAM-1 gene polymorphism. Conclusions: These results suggest that the TT and TC genotype of the ICAM-1 gene polymorphism in codon 469 is a genetic factor that may determine an individual’s susceptibility for CHD and MI. 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: ICAM-1; Genes; Coronary heart disease; Myocardial infarction
1. Introduction Inflammation plays an important role throughout all stages of coronary heart disease (CHD). The intercellular adhesion molecule-1 (ICAM-1, CD54) is *Corresponding author. Heart Center Wuppertal, University of WittenHerdecke, Arrenbergerstr. 20, 42117 Wuppertal, Germany. Tel.: 149202-896-5702; fax: 149-202-896-5713. E-mail address: [email protected] (H. Jiang).
a cell surface glycoprotein that mediates adhesion of circulating leukocytes to the activated endothelium, which plays a role in inflammation processes and is one of the earliest events in the pathogenesis of atherosclerosis. ICAM-1 is expressed widely on nonhematopoietic and hematopoietic cells, but at a low level on normal endothelium. Its expression can be rapidly upregulated several fold in atherosclerotic lesions by inflammatory mediators [1,2]. Enzymatic
0167-5273 / 02 / $ – see front matter 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 02 )00138-9
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H. Jiang et al. / International Journal of Cardiology 84 (2002) 171 – 177
cleavage of the extracellular portion of ICAM-1 or alternative splicing of mRNA encoding for ICAM-1 can yield circulating forms which can be measured in blood samples of the coronary and the peripheral circulation [3]. The finding of increased soluble forms of ICAM-1 in patients with various coronary artery disease processes, including atherosclerosis, thrombosis, reperfusion injury and restenosis after coronary angioplasty, directly reflects levels of soluble forms of ICAM-1 within the coronary circulation at the site of endothelial inflammation [4–7]. Several studies have reported an increase in the expression of local membrane bound ICAM-1 on endothelial plaque in CHD, myocardial infarction (MI) and in patients undergoing coronary angioplasty [1,8–10]. Detection of the expression of ICAM-1 on smooth muscle cells in the atherosclerotic vascular wall has also been reported [9], whereas ICAM-1 expression could not be detected on smooth muscle cells in the normal adult aorta. In these studies, upregulated expression of ICAM-1 was thought to be a potential mechanism of the progression of atherosclerosis. Correlations between several polymorphisms in the ICAM-1 gene and rheumatoid arthritis and inflammatory diseases have been reported [11–14], including a single base C to T transition polymorphism which results in an amino acid substitution glutamic acid (E) to lysine (K) in the ICAM protein in exon 6 codon 469, which has been found to be related to Behcet’s disease [14]. However, their role in mediating the risk of CHD and MI is still unknown. Therefore, the purpose of our study was to determine whether the C / T polymorphism of the ICAM-1 gene plays a role in CHD and MI in humans.
2. Methods
2.1. Study population The study population consisted of three groups, a control group, a CHD group and an MI group (subgroup of CHD) (Table 1). The CHD group consisted of 349 patients (239 males, 110 females; median age 62.2611.3) with angiographically documented CHD. The inclusion criteria were stenosis of more than 50% of at least one major coronary vessel.
Table 1 Characteristics of the study population Variable
Control group (n5213)
CHD group (n5349)
MI group (n5179)
Age (years) Gender (female / male) BMI (kg / m 2 ) Habitual smoking (%) Hypertension (%) Diabetes mellitus (%) Hypercholesterolemia (%)
60.6611.9 63 / 150 27.065.0 27 73 17 59
62.2611.3 110 / 239 27.263.9 54* 70 24 81*
60.9611.6 41 / 138 27.264.2 59* 66 21 81*
The age and BMI values are presented as mean6S.D. BMI, body mass index; CHD, coronary heart disease; MI, myocardial infarction. *P,0.001 compared with control group.
The MI group consisted of 179 patients with acute or chronic MI from the CHD group. The diagnosis of MI was based on typical electrocardiographic changes and increases in serum enzyme activities, including those of creatinine kinase (CK), CKMB, and lactate dehydrogenase (LDH). The control group comprised 213 patients (150 males, 63 females; median age 60.6611.9) with normal left ventricular function and no documented evidence of CHD. There were no significant differences in age and gender between CHD, MI and controls. The volunteers had no history of heart disease or systemic disease and were found to be normal by physical examination, electrocardiogram, and echocardiogram. The blood samples were obtained in the Heart Center Wuppert from November 1999 through November 2001. All patients and control subjects were Germans and had given written informed consent.
2.2. Isolation of DNA EDTA blood samples (5 ml) were obtained from peripheral venous blood of all subjects after cardiac catheterization. All blood sample were stored at 4 8C until DNA extraction was performed with a QIA amp DNA blood midi kit (Qiagen, Germany).
2.3. Determination of biochemical parameters Total, low-density lipoprotein (LDL) and highdensity lipoprotein (HDL) cholesterol were measured in blood samples using the Boehringer Enzymatic Colorimetric method with Boehringer CHOD PAP
H. Jiang et al. / International Journal of Cardiology 84 (2002) 171 – 177
enzymatic colorimetric reagents [15] (Boehringer Mannheim, Mannheim, Germany).
2.4. Polymerase chain reaction and genotyping The polymerase chain reaction (PCR) was used to amplify a 331 bp fragment of exon 6. Analyses were performed using 25 ng template DNA in a final reaction volume of 25 ml, containing 13PCR reaction buffer, 0.2 mM of each deoxynucleotide, and 2.0 U of TaqDNA polymerase (Pharmacia) with 15 pmol of each upstream and downstream primer (S1 and S2), as previously published [16]. Thermal cycling was carried out with an initial 95 8C denaturation step for 10 min, followed by 36 cycles of denaturation at 95 8C 30 s, annealing at 60 8C 30 s, extension at 72 8C 30 s and a final extension of 7 min at 72 8C. To determine the ICAM-1 alleles, we performed specially conditioned nested PCR with the use of allele-specific primers (S-t, S-c) with the allele-differentiating base located at the 39 position. The sequences of the primers are shown in Table 2. The nested PCR conditions were: amount of template 1 ml of a 1:50 to 1:100 dilution of PCR products. Thermal cycling was carried out with a 95 8C denaturation step for 10 min, followed by 15 cycles of denaturation at 95 8C 30 s, annealing at 56 8C 5 s, extension at 72 8C 30 s and a final extension of 7 min at 72 8C. The genotype was determined according to the presence of the specific PCR products of expected length (237 bp) after 2% agarose gel electrophoresis followed by ethidium bromide staining and ultraviolet visualization.
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the unpaired Student’s t-test. Frequencies were compared using the Chi-squared test and Fisher’s exact test. The ICAM genotype was calculated according to a dominant (TT1TC vs. CC) or additive (TC vs. CC, and TT vs. CC) genetic effect of the T allele. Associations between gene alleles and CHD or MI risk were assessed as odds ratio (OR) and 95% confidence intervals (CI), which are interpreted as the relative risk of disease for ‘exposed’ compared to ‘unexposed’ persons. Alleles at each polymorphism were said to be in Hardy–Weinberg equilibrium if the observed homozygote frequencies did not differ significantly (P.0.05) from those expected when analyzed by the x 2 test. We performed multivariable logistic regression analysis for the effect of the ICAM-1 polymorphism and other coronary risk factors for CHD, where CHD was a dependent variable and independent variables included ICAM-1 genotype [0 for CC, 1 for TC and TT combined (dominant effect of the T allele)], smoking status (05 nonsmoker, 15smoker), and hypercholesterolemia (05for absence, 15for presence). P#0.05 was taken as the level of significance.
3. Results
3.1. Comparison of CHD, MI and control group for coronary risk factors As expected, smokers and patients with hypercholesterolemia were more frequent in the CHD and MI groups compared with controls (Table 1).
2.5. Statistical analysis
3.2. Coronary risk factors by ICAM-1 genotype
Statistical analyses were performed with the SPSS (version10.0) package. Data were expressed as mean6S.D. Continuous variables were compared by
The different characteristics of CHD and MI patients were analyzed with respect to ICAM-1 genotypes. No statistically significant association
Table 2 Oligonucleotide primers for ICAM-1 genotyping Gene
ICAM-1 Exon 6 codon 469
S1 S2 S-t S-c
Primer sequence
Product size (bp)
59 CCC CGA CTG GAC GAG AGG 39 59 GGG GCT GTG GGG AGG ATA 39 59 CAC ATT CAC GGT CAC CTT 39 59 CAC ATT CAC GGT CAC CTC 39
331 237
ICAM-1, intercellular adhesion molecule-1; S1 and S2, upstream and downstream primer for PCR; S-t and S-c, allele-specific primer for nested PCR.
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H. Jiang et al. / International Journal of Cardiology 84 (2002) 171 – 177
Table 3 Characteristics of CHD patients by ICAM-1 genotype Variable
CC (n562)
CT1TT (n5287)
P
Age (years) Gender (female / male) BMI (kg / m 2 ) Habitual smoking (%) Hypertension (%) Diabetes mellitus (%) Hypercholesterolemia (%)
62.7610.1 19 / 43 26.863.7 50 77 21 88
62.1611.6 91 / 196 27.463.9 55 70 24 81
0.72 0.87 0.46 0.56 0.29 0.66 0.24
Table 5 Allele and genotype frequencies of E / K 469 ICAM-1 polymorphism in patients and controls Variable Genotypes n (%) TT TC CC
The age and BMI values are presented as mean6S.D. BMI, body mass index; CHD, coronary heart disease; ICAM-1, intercellular adhesion molecule-1.
Dominant effects TT1TC CC P OR (95% CI)
between the genotypes and coronary risk factors was found (Tables 3 and 4).
Additive effects P (TC and CC) OR (95% CI) P (TT and CC) OR (95% CI)
3.3. Distribution of allele and genotype frequencies The distribution of the C / T genotype in exon 6 of the ICAM-1 gene is shown in Table 5. We found that the genotype frequencies were 18% (CC), 42% (TC) and 40% (TT) in the patients with CHD, and 21% (CC), 44% (TC) and 35% (TT) in the patients with MI, thus differing from those in the control subjects, which were 41% (CC), 31% (TC) and 28% (TT). Analysis of the genotype with respect to dominant and additive effects of the T allele showed that individuals with the T allele (TT and TC) had a significantly increased risk of CHD and MI.
3.4. Multiple variable logistic regression analysis ( Table 6) This analysis showed that independent risk factors for CHD were cigarette smoking (OR53.32, 95% Table 4 Characteristics of MI patients by ICAM-1 genotype Variable
CC (n538)
CT1TT (n5141)
P
Age (years) Gender (female / male) BMI (kg / m 2 ) Habitual smoking (%) Hypertension (%) Diabetes mellitus (%) Hypercholesterolemia (%)
61.2610.4 8 / 30 26.363.4 53 71 21 85
60.8612.0 33 / 108 27.564.2 62 65 20 81
0.84 0.76 0.22 0.35 0.55 0.89 0.54
The values are presented as mean6S.D. BMI, body mass index; MI, myocardial infarction; ICAM-1, intercellular adhesion molecule-1.
Allele frequency (%) T C P
Controls (n5213)
CHD (n5349)
MI (n5179)
60 (28) 66 (31) 87 (41)
139 (40) 148 (42) 62 (18)
63 (35) 78 (44) 38 (21)
0.59 0.41
0.82 0.18 ,0.001* 3.20 (2.17–4.71)
0.79 0.21 ,0.001* 2.56 (1.63–4.02)
,0.001* 3.15 (2.03–4.87) ,0.001* 3.25 (2.08–5.07)
,0.001* 2.71 (1.64–4.47) 0.001* 2.40 (1.43–4.04)
0.61 0.39 ,0.001*
0.57 0.43 ,0.001*
0.44 0.56
*Compared with controls. The presence of the T allele of the ICAM-1 gene was significantly associated with CHD and MI. CHD, coronary heart disease; MI, myocardial infarction; ICAM-1, intercellular adhesion molecule-1; OR, odds ratio; CI, confidence intervals.
CI51.84–5.97), hypercholesterolemia (OR53.41, 95% CI51.93–6.02), and the TT and TC genotype of the ICAM-1 gene (OR52.21, 95% CI51.20–4.07).
4. Discussion To our knowledge, the present study is the first to examine the K469E polymorphism of the ICAM-1 gene in patients with CHD and MI. In this study, we have shown that the K469E polymorphism of the human ICAM-1 gene may be Table 6 Results of multiple logistic regression analysis: final significant variables in equation Variable
b Coefficient
S.E.
P
OR
95% CI
Smoker Hypercholesterolemia ICAM-1
1.199
0.315
0.000
3.32
1.84–5.97
1.226 0.793
0.290 0.311
0.000 0.011
3.41 2.21
1.93–6.02 1.20–4.07
ICAM-1, intercellular adhesion molecule-1; OR, odds ratio; CI, confidence intervals; S.E., standard error.
H. Jiang et al. / International Journal of Cardiology 84 (2002) 171 – 177
involved in the pathogenesis of coronary atherosclerosis. The genotype distributions in our control group are in accordance with Hardy–Weinberg expectations. Although the frequencies of several classical risk factors for CHD and MI, including smoking and hypercholesterolemia, were higher compared with controls (Table 1), no statistically significant association between the genotypes and coronary risk factors was found (Tables 3 and 4). With the additional consideration of our multiple variable logistic regression analysis this indicates that the TT and TC genotype of the ICAM-1 K469E polymorphism is an independent risk factor for CHD and MI. In addition to classical risk factors, genetic predisposition may thus play an important role in the pathogenesis of CHD and MI. Atherosclerosis is regarded as a chronic inflammatory process [17]. One of the earliest events in this process is the expression of the endothelial cell adhesion molecule, which plays a fundamental role in the pathogenesis of coronary atherosclerosis and inflammatory disease [10]. The ICAM-1 (CD54) gene is one of these adhesion molecules, a cell surface glycoprotein of 95 kD belonging to the immunoglobulin gene superfamily. Increased expression of membrane bound ICAM-1 has been found in all periods of atherogenesis. Membrane ICAM-1 expression was found in all blood vessels with fatty streaks and fibrotic plaques in subjects aged from 25 to 61 years [9]. ICAM-1 can be expressed on fibroblasts, macrophages, circulating leukocytes, endothelial cells, smooth muscle cells in fatty streaks and fibrotic plaques, and enhanced expression of ICAM-1 has also been shown on macrophages, endothelial cells, and smooth muscle cells in human atherosclerotic plaques [8]. Soluble forms of ICAM-1 (sICAM-1) have also been detected. Two prospective cohort studies noted that baseline levels of sICAM-1 are elevated many years before a first MI occurs [18,19]. ICAM-1 in the coronary sinus and aortic root was higher in patients with unstable angina than in those with stable exertional angina and in controls [7]. Further, sICAM-1 is increased in the serum of CHD patients, MI patients [4,5] and after angioplasty [6]. Recently, several polymorphisms of the ICAM-1 gene have been reported, and K469E polymorphism has been found to be related to inflammatory pro-
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cesses of multiple sclerosis in the Polish population [16], and Behcet’s disease in a Jordanian Palestinian population [14]. Our study provides no information about the mechanisms by which the K469E polymorphism of the ICAM-1 gene predisposes patients to CHD and MI. However, a recent study showed that mice with ICAM-1 deficiency had normal endothelial function [vasorelaxation in response to acetylcholine (Ach)] after ischemia-reperfusion, whereas wild-type mice had impaired vasorelaxation in response to Ach [20], indicating that ICAM-1 gene function may be related to impaired endothelium-dependent vasodilatation. This dysfunction of the endothelium plays a key role in all stages of atherosclerosis. ICAM-1 is produced as a consequence of inflammation. The mechanisms of the proinflammatory effects of ICAM-1 on endothelial cells are not completely clear. It may be that ICAM-1 binds and interacts with leukocyte integrin receptors such as LFA-1 (a L b 2 , CD11a / CD18) and Mac-1 (a M b 2 , CD11b / CD18) [21]. This provides an adaptive alternative in the adhesion process between circulating cells and the endothelium, leading to the attachment of leukocytes to endothelial cells and the transendothelial migration of leukocytes into the intima and thus to the accumulation of leukocytes in the vascular wall [22,23]. In addition to its above role in cell-to-cell adhesion by integrin / ICAM-1 interaction, ICAM-1 also serves as a receptor for soluble fibrinogen. It has been reported that fibrinogen mediates leukocyte adhesion to the endothelium through an ICAM-1-dependent pathway [24]. ICAM-1 may therefore play a major role in the pathogenesis of the dysfunction of the endothelium and CHD. The K469E polymorphism represents a change of amino acid, which occurs in Ig domain 5 of ICAM-1. An immunodominant epitope was found in domain 5 [25], while Joling et al. [26] reported that domain 5 was involved in adhesion of follicular dendritic cells and B-lymphocytes. It is thus possible to hypothesize that domain 5 may be important in maintaining normal protein structure, affecting the adhesion of circulating leukocytes to the activated endothelium. An increased expression of the other cell adhesion molecules, such as endothelial leukocyte adhesion molecule-1 E-selectin (ESEL), has also been observed in patients with unstable angina pectoris [27]. The nucleotide polymorphism in E-selectin was
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analysed by Wenzel et al. [28]. They suggested that the S128R variant is associated with a higher risk for CHD. They further discovered that this polymorphism influenced the E-selectin function in vitro and may be considered as one of the risk factors involved in the pathogenesis of atherosclerosis [29]. It is known that the ICAM-1 gene locates very tightly linked to the LDL receptor on chromosome 19 region p13.3–p13.2, and the selectin clusters have been clustered in tandem within a 220 kb region of chromosome 1q23 [30]. Selectins are suited for mediating the rapid cycle of attachment, detachment and reattachment underlying leukocyte rolling, support rolling of neutrophils, monocytes, eosinophils, and some lymphocytes. ICAM-1 can mediate adhesion of circulating leukocytes to the activated endothelium. It plays roles not only in the firm attachment but also in transendothelial migration of leukocytes. E-selectin is only expressed transiently on the endothelium after activation. ICAM-1 is expressed widely on the surface of non-hematopoietic and hematopoietic cells, including fibroblasts, macrophages, activated monocytes, lymphocytes, follicular dendrite cells, and epithelial cells, but at very low levels on normal endothelium, and could not be detected on smooth muscle cells in the normal adult aorta. ICAM-1 expression can be rapidly upregulated several fold in atherosclerotic lesions by inflammatory mediators. The concentration of ICAM and ESEL together contributes to CHD through their inflammatory effects on the vascular endothelium. This should be a specific finding for coronary heart disease. In conclusion, our results suggest that ICAM-1 gene K469E polymorphism is a genetic factor that may determine an individual’s susceptibility for CHD and MI.
Acknowledgements We thank statistician Dr. Q. Yang of the Coordination Center for Clinical Trials of the HHU Duesseldorf for help with the statistical analysis.
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[26] Joling P, Boom S, Johnson J et al. Domain 5 of the intercellular adhesion molecule-1 (ICAM-1) is involved in adhesion of B-cells and follicular dendritic cells. Adv Exp Med Biol 1994;355:131–5. [27] Atalar E, Aytemir K, Haznedaroglu I et al. Increased plasma levels of soluble selections in patients with unstable angina. Int J Cardiol 2001;78:69–73. [28] Wenzel K, Blackburn A, Ernst M et al. Relationship of polymorphisms in the renin–angiotensin system and in E-selectin of patients with early severe coronary heart disease. J Mol Med 1997;75(1):57–61. [29] Wenzel K, Stahn R, Speer A et al. Functional characterization of atherosclerosis-associated Ser128Arg and Leu554Phe E-selectin mutations. Biol Chem 1999;380(6):661–7. [30] Vora DK, Rosenbloom CL, Beaudet AL, Cottingham RW. Polymorphisms and linkage analysis for ICAM-1 and the selectin gene cluster. Genomics 1994;21(3):473–7.
C / T polymorphism of the intercellular adhesion molecule-1 gene (exon 6, codon 469). A risk factor for coronary heart disease and myocardial infarction a,c ,
a
b
b,c
a
Hong Jiang *, Rolf Michael Klein , Dieter Niederacher , Ming Du , Roger Marx , Mark Horlitz a , Guido Boerrigter a , Harald Lapp a , Thomas Scheffold a , Ingo Krakau a , a ¨ Hartmut Gulker a Heart Center Wuppertal, University of Witten-Herdecke, Arrenbergerstr. 20, 42117 Wuppertal, Germany Molecular Genetics Laboratory of the Gynecology Division, Henrich-Heine University, Duesseldorf, Germany c Union Hospital, Tongji Medical College, HuaZhong University of Science and Technology, HuaZhong, PR China b
Received 7 August 2001; received in revised form 8 March 2002; accepted 10 March 2002
Abstract Background: The intercellular adhesion molecule-1 (ICAM-1) mediates the interaction of activated endothelial cells with leukocytes and plays a fundamental role in the pathogenesis of coronary atherosclerosis. ICAM-1 single-base C / T polymorphism, which determines an amino acid substitution in the ICAM-1 protein in exon 6 codon 469, has been described. Our purpose was to determine whether this C / T polymorphism influences the risk of coronary heart disease (CHD) and myocardial infarction (MI) in humans. Methods and results: We enrolled 349 patients with angiographically documented CHD, including a sub-group of 179 patients with acute or chronic MI. The control group consisted of 213 patients with normal left ventricular function and no documented evidence of CHD. All patients and controls were Germans genotyped by polymerase chain reaction and allele-specific oligonucleotide techniques for the ICAM-1 polymorphism. In the patients with CHD and MI the frequencies of the T genotype (TT1TC) were significantly higher than the CC genotype compared to the control subjects (P,0.001). With the additional use of multivariable logistic regression analysis for CHD (TT1TC versus CC; P50.011, odds ratio 2.21, 95% CI 1.20–4.07), we found a significant association between CHD and MI and the TT and TC genotype of the ICAM-1 gene polymorphism. Conclusions: These results suggest that the TT and TC genotype of the ICAM-1 gene polymorphism in codon 469 is a genetic factor that may determine an individual’s susceptibility for CHD and MI. 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: ICAM-1; Genes; Coronary heart disease; Myocardial infarction
1. Introduction Inflammation plays an important role throughout all stages of coronary heart disease (CHD). The intercellular adhesion molecule-1 (ICAM-1, CD54) is *Corresponding author. Heart Center Wuppertal, University of WittenHerdecke, Arrenbergerstr. 20, 42117 Wuppertal, Germany. Tel.: 149202-896-5702; fax: 149-202-896-5713. E-mail address: [email protected] (H. Jiang).
a cell surface glycoprotein that mediates adhesion of circulating leukocytes to the activated endothelium, which plays a role in inflammation processes and is one of the earliest events in the pathogenesis of atherosclerosis. ICAM-1 is expressed widely on nonhematopoietic and hematopoietic cells, but at a low level on normal endothelium. Its expression can be rapidly upregulated several fold in atherosclerotic lesions by inflammatory mediators [1,2]. Enzymatic
0167-5273 / 02 / $ – see front matter 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 02 )00138-9
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cleavage of the extracellular portion of ICAM-1 or alternative splicing of mRNA encoding for ICAM-1 can yield circulating forms which can be measured in blood samples of the coronary and the peripheral circulation [3]. The finding of increased soluble forms of ICAM-1 in patients with various coronary artery disease processes, including atherosclerosis, thrombosis, reperfusion injury and restenosis after coronary angioplasty, directly reflects levels of soluble forms of ICAM-1 within the coronary circulation at the site of endothelial inflammation [4–7]. Several studies have reported an increase in the expression of local membrane bound ICAM-1 on endothelial plaque in CHD, myocardial infarction (MI) and in patients undergoing coronary angioplasty [1,8–10]. Detection of the expression of ICAM-1 on smooth muscle cells in the atherosclerotic vascular wall has also been reported [9], whereas ICAM-1 expression could not be detected on smooth muscle cells in the normal adult aorta. In these studies, upregulated expression of ICAM-1 was thought to be a potential mechanism of the progression of atherosclerosis. Correlations between several polymorphisms in the ICAM-1 gene and rheumatoid arthritis and inflammatory diseases have been reported [11–14], including a single base C to T transition polymorphism which results in an amino acid substitution glutamic acid (E) to lysine (K) in the ICAM protein in exon 6 codon 469, which has been found to be related to Behcet’s disease [14]. However, their role in mediating the risk of CHD and MI is still unknown. Therefore, the purpose of our study was to determine whether the C / T polymorphism of the ICAM-1 gene plays a role in CHD and MI in humans.
2. Methods
2.1. Study population The study population consisted of three groups, a control group, a CHD group and an MI group (subgroup of CHD) (Table 1). The CHD group consisted of 349 patients (239 males, 110 females; median age 62.2611.3) with angiographically documented CHD. The inclusion criteria were stenosis of more than 50% of at least one major coronary vessel.
Table 1 Characteristics of the study population Variable
Control group (n5213)
CHD group (n5349)
MI group (n5179)
Age (years) Gender (female / male) BMI (kg / m 2 ) Habitual smoking (%) Hypertension (%) Diabetes mellitus (%) Hypercholesterolemia (%)
60.6611.9 63 / 150 27.065.0 27 73 17 59
62.2611.3 110 / 239 27.263.9 54* 70 24 81*
60.9611.6 41 / 138 27.264.2 59* 66 21 81*
The age and BMI values are presented as mean6S.D. BMI, body mass index; CHD, coronary heart disease; MI, myocardial infarction. *P,0.001 compared with control group.
The MI group consisted of 179 patients with acute or chronic MI from the CHD group. The diagnosis of MI was based on typical electrocardiographic changes and increases in serum enzyme activities, including those of creatinine kinase (CK), CKMB, and lactate dehydrogenase (LDH). The control group comprised 213 patients (150 males, 63 females; median age 60.6611.9) with normal left ventricular function and no documented evidence of CHD. There were no significant differences in age and gender between CHD, MI and controls. The volunteers had no history of heart disease or systemic disease and were found to be normal by physical examination, electrocardiogram, and echocardiogram. The blood samples were obtained in the Heart Center Wuppert from November 1999 through November 2001. All patients and control subjects were Germans and had given written informed consent.
2.2. Isolation of DNA EDTA blood samples (5 ml) were obtained from peripheral venous blood of all subjects after cardiac catheterization. All blood sample were stored at 4 8C until DNA extraction was performed with a QIA amp DNA blood midi kit (Qiagen, Germany).
2.3. Determination of biochemical parameters Total, low-density lipoprotein (LDL) and highdensity lipoprotein (HDL) cholesterol were measured in blood samples using the Boehringer Enzymatic Colorimetric method with Boehringer CHOD PAP
H. Jiang et al. / International Journal of Cardiology 84 (2002) 171 – 177
enzymatic colorimetric reagents [15] (Boehringer Mannheim, Mannheim, Germany).
2.4. Polymerase chain reaction and genotyping The polymerase chain reaction (PCR) was used to amplify a 331 bp fragment of exon 6. Analyses were performed using 25 ng template DNA in a final reaction volume of 25 ml, containing 13PCR reaction buffer, 0.2 mM of each deoxynucleotide, and 2.0 U of TaqDNA polymerase (Pharmacia) with 15 pmol of each upstream and downstream primer (S1 and S2), as previously published [16]. Thermal cycling was carried out with an initial 95 8C denaturation step for 10 min, followed by 36 cycles of denaturation at 95 8C 30 s, annealing at 60 8C 30 s, extension at 72 8C 30 s and a final extension of 7 min at 72 8C. To determine the ICAM-1 alleles, we performed specially conditioned nested PCR with the use of allele-specific primers (S-t, S-c) with the allele-differentiating base located at the 39 position. The sequences of the primers are shown in Table 2. The nested PCR conditions were: amount of template 1 ml of a 1:50 to 1:100 dilution of PCR products. Thermal cycling was carried out with a 95 8C denaturation step for 10 min, followed by 15 cycles of denaturation at 95 8C 30 s, annealing at 56 8C 5 s, extension at 72 8C 30 s and a final extension of 7 min at 72 8C. The genotype was determined according to the presence of the specific PCR products of expected length (237 bp) after 2% agarose gel electrophoresis followed by ethidium bromide staining and ultraviolet visualization.
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the unpaired Student’s t-test. Frequencies were compared using the Chi-squared test and Fisher’s exact test. The ICAM genotype was calculated according to a dominant (TT1TC vs. CC) or additive (TC vs. CC, and TT vs. CC) genetic effect of the T allele. Associations between gene alleles and CHD or MI risk were assessed as odds ratio (OR) and 95% confidence intervals (CI), which are interpreted as the relative risk of disease for ‘exposed’ compared to ‘unexposed’ persons. Alleles at each polymorphism were said to be in Hardy–Weinberg equilibrium if the observed homozygote frequencies did not differ significantly (P.0.05) from those expected when analyzed by the x 2 test. We performed multivariable logistic regression analysis for the effect of the ICAM-1 polymorphism and other coronary risk factors for CHD, where CHD was a dependent variable and independent variables included ICAM-1 genotype [0 for CC, 1 for TC and TT combined (dominant effect of the T allele)], smoking status (05 nonsmoker, 15smoker), and hypercholesterolemia (05for absence, 15for presence). P#0.05 was taken as the level of significance.
3. Results
3.1. Comparison of CHD, MI and control group for coronary risk factors As expected, smokers and patients with hypercholesterolemia were more frequent in the CHD and MI groups compared with controls (Table 1).
2.5. Statistical analysis
3.2. Coronary risk factors by ICAM-1 genotype
Statistical analyses were performed with the SPSS (version10.0) package. Data were expressed as mean6S.D. Continuous variables were compared by
The different characteristics of CHD and MI patients were analyzed with respect to ICAM-1 genotypes. No statistically significant association
Table 2 Oligonucleotide primers for ICAM-1 genotyping Gene
ICAM-1 Exon 6 codon 469
S1 S2 S-t S-c
Primer sequence
Product size (bp)
59 CCC CGA CTG GAC GAG AGG 39 59 GGG GCT GTG GGG AGG ATA 39 59 CAC ATT CAC GGT CAC CTT 39 59 CAC ATT CAC GGT CAC CTC 39
331 237
ICAM-1, intercellular adhesion molecule-1; S1 and S2, upstream and downstream primer for PCR; S-t and S-c, allele-specific primer for nested PCR.
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Table 3 Characteristics of CHD patients by ICAM-1 genotype Variable
CC (n562)
CT1TT (n5287)
P
Age (years) Gender (female / male) BMI (kg / m 2 ) Habitual smoking (%) Hypertension (%) Diabetes mellitus (%) Hypercholesterolemia (%)
62.7610.1 19 / 43 26.863.7 50 77 21 88
62.1611.6 91 / 196 27.463.9 55 70 24 81
0.72 0.87 0.46 0.56 0.29 0.66 0.24
Table 5 Allele and genotype frequencies of E / K 469 ICAM-1 polymorphism in patients and controls Variable Genotypes n (%) TT TC CC
The age and BMI values are presented as mean6S.D. BMI, body mass index; CHD, coronary heart disease; ICAM-1, intercellular adhesion molecule-1.
Dominant effects TT1TC CC P OR (95% CI)
between the genotypes and coronary risk factors was found (Tables 3 and 4).
Additive effects P (TC and CC) OR (95% CI) P (TT and CC) OR (95% CI)
3.3. Distribution of allele and genotype frequencies The distribution of the C / T genotype in exon 6 of the ICAM-1 gene is shown in Table 5. We found that the genotype frequencies were 18% (CC), 42% (TC) and 40% (TT) in the patients with CHD, and 21% (CC), 44% (TC) and 35% (TT) in the patients with MI, thus differing from those in the control subjects, which were 41% (CC), 31% (TC) and 28% (TT). Analysis of the genotype with respect to dominant and additive effects of the T allele showed that individuals with the T allele (TT and TC) had a significantly increased risk of CHD and MI.
3.4. Multiple variable logistic regression analysis ( Table 6) This analysis showed that independent risk factors for CHD were cigarette smoking (OR53.32, 95% Table 4 Characteristics of MI patients by ICAM-1 genotype Variable
CC (n538)
CT1TT (n5141)
P
Age (years) Gender (female / male) BMI (kg / m 2 ) Habitual smoking (%) Hypertension (%) Diabetes mellitus (%) Hypercholesterolemia (%)
61.2610.4 8 / 30 26.363.4 53 71 21 85
60.8612.0 33 / 108 27.564.2 62 65 20 81
0.84 0.76 0.22 0.35 0.55 0.89 0.54
The values are presented as mean6S.D. BMI, body mass index; MI, myocardial infarction; ICAM-1, intercellular adhesion molecule-1.
Allele frequency (%) T C P
Controls (n5213)
CHD (n5349)
MI (n5179)
60 (28) 66 (31) 87 (41)
139 (40) 148 (42) 62 (18)
63 (35) 78 (44) 38 (21)
0.59 0.41
0.82 0.18 ,0.001* 3.20 (2.17–4.71)
0.79 0.21 ,0.001* 2.56 (1.63–4.02)
,0.001* 3.15 (2.03–4.87) ,0.001* 3.25 (2.08–5.07)
,0.001* 2.71 (1.64–4.47) 0.001* 2.40 (1.43–4.04)
0.61 0.39 ,0.001*
0.57 0.43 ,0.001*
0.44 0.56
*Compared with controls. The presence of the T allele of the ICAM-1 gene was significantly associated with CHD and MI. CHD, coronary heart disease; MI, myocardial infarction; ICAM-1, intercellular adhesion molecule-1; OR, odds ratio; CI, confidence intervals.
CI51.84–5.97), hypercholesterolemia (OR53.41, 95% CI51.93–6.02), and the TT and TC genotype of the ICAM-1 gene (OR52.21, 95% CI51.20–4.07).
4. Discussion To our knowledge, the present study is the first to examine the K469E polymorphism of the ICAM-1 gene in patients with CHD and MI. In this study, we have shown that the K469E polymorphism of the human ICAM-1 gene may be Table 6 Results of multiple logistic regression analysis: final significant variables in equation Variable
b Coefficient
S.E.
P
OR
95% CI
Smoker Hypercholesterolemia ICAM-1
1.199
0.315
0.000
3.32
1.84–5.97
1.226 0.793
0.290 0.311
0.000 0.011
3.41 2.21
1.93–6.02 1.20–4.07
ICAM-1, intercellular adhesion molecule-1; OR, odds ratio; CI, confidence intervals; S.E., standard error.
H. Jiang et al. / International Journal of Cardiology 84 (2002) 171 – 177
involved in the pathogenesis of coronary atherosclerosis. The genotype distributions in our control group are in accordance with Hardy–Weinberg expectations. Although the frequencies of several classical risk factors for CHD and MI, including smoking and hypercholesterolemia, were higher compared with controls (Table 1), no statistically significant association between the genotypes and coronary risk factors was found (Tables 3 and 4). With the additional consideration of our multiple variable logistic regression analysis this indicates that the TT and TC genotype of the ICAM-1 K469E polymorphism is an independent risk factor for CHD and MI. In addition to classical risk factors, genetic predisposition may thus play an important role in the pathogenesis of CHD and MI. Atherosclerosis is regarded as a chronic inflammatory process [17]. One of the earliest events in this process is the expression of the endothelial cell adhesion molecule, which plays a fundamental role in the pathogenesis of coronary atherosclerosis and inflammatory disease [10]. The ICAM-1 (CD54) gene is one of these adhesion molecules, a cell surface glycoprotein of 95 kD belonging to the immunoglobulin gene superfamily. Increased expression of membrane bound ICAM-1 has been found in all periods of atherogenesis. Membrane ICAM-1 expression was found in all blood vessels with fatty streaks and fibrotic plaques in subjects aged from 25 to 61 years [9]. ICAM-1 can be expressed on fibroblasts, macrophages, circulating leukocytes, endothelial cells, smooth muscle cells in fatty streaks and fibrotic plaques, and enhanced expression of ICAM-1 has also been shown on macrophages, endothelial cells, and smooth muscle cells in human atherosclerotic plaques [8]. Soluble forms of ICAM-1 (sICAM-1) have also been detected. Two prospective cohort studies noted that baseline levels of sICAM-1 are elevated many years before a first MI occurs [18,19]. ICAM-1 in the coronary sinus and aortic root was higher in patients with unstable angina than in those with stable exertional angina and in controls [7]. Further, sICAM-1 is increased in the serum of CHD patients, MI patients [4,5] and after angioplasty [6]. Recently, several polymorphisms of the ICAM-1 gene have been reported, and K469E polymorphism has been found to be related to inflammatory pro-
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cesses of multiple sclerosis in the Polish population [16], and Behcet’s disease in a Jordanian Palestinian population [14]. Our study provides no information about the mechanisms by which the K469E polymorphism of the ICAM-1 gene predisposes patients to CHD and MI. However, a recent study showed that mice with ICAM-1 deficiency had normal endothelial function [vasorelaxation in response to acetylcholine (Ach)] after ischemia-reperfusion, whereas wild-type mice had impaired vasorelaxation in response to Ach [20], indicating that ICAM-1 gene function may be related to impaired endothelium-dependent vasodilatation. This dysfunction of the endothelium plays a key role in all stages of atherosclerosis. ICAM-1 is produced as a consequence of inflammation. The mechanisms of the proinflammatory effects of ICAM-1 on endothelial cells are not completely clear. It may be that ICAM-1 binds and interacts with leukocyte integrin receptors such as LFA-1 (a L b 2 , CD11a / CD18) and Mac-1 (a M b 2 , CD11b / CD18) [21]. This provides an adaptive alternative in the adhesion process between circulating cells and the endothelium, leading to the attachment of leukocytes to endothelial cells and the transendothelial migration of leukocytes into the intima and thus to the accumulation of leukocytes in the vascular wall [22,23]. In addition to its above role in cell-to-cell adhesion by integrin / ICAM-1 interaction, ICAM-1 also serves as a receptor for soluble fibrinogen. It has been reported that fibrinogen mediates leukocyte adhesion to the endothelium through an ICAM-1-dependent pathway [24]. ICAM-1 may therefore play a major role in the pathogenesis of the dysfunction of the endothelium and CHD. The K469E polymorphism represents a change of amino acid, which occurs in Ig domain 5 of ICAM-1. An immunodominant epitope was found in domain 5 [25], while Joling et al. [26] reported that domain 5 was involved in adhesion of follicular dendritic cells and B-lymphocytes. It is thus possible to hypothesize that domain 5 may be important in maintaining normal protein structure, affecting the adhesion of circulating leukocytes to the activated endothelium. An increased expression of the other cell adhesion molecules, such as endothelial leukocyte adhesion molecule-1 E-selectin (ESEL), has also been observed in patients with unstable angina pectoris [27]. The nucleotide polymorphism in E-selectin was
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analysed by Wenzel et al. [28]. They suggested that the S128R variant is associated with a higher risk for CHD. They further discovered that this polymorphism influenced the E-selectin function in vitro and may be considered as one of the risk factors involved in the pathogenesis of atherosclerosis [29]. It is known that the ICAM-1 gene locates very tightly linked to the LDL receptor on chromosome 19 region p13.3–p13.2, and the selectin clusters have been clustered in tandem within a 220 kb region of chromosome 1q23 [30]. Selectins are suited for mediating the rapid cycle of attachment, detachment and reattachment underlying leukocyte rolling, support rolling of neutrophils, monocytes, eosinophils, and some lymphocytes. ICAM-1 can mediate adhesion of circulating leukocytes to the activated endothelium. It plays roles not only in the firm attachment but also in transendothelial migration of leukocytes. E-selectin is only expressed transiently on the endothelium after activation. ICAM-1 is expressed widely on the surface of non-hematopoietic and hematopoietic cells, including fibroblasts, macrophages, activated monocytes, lymphocytes, follicular dendrite cells, and epithelial cells, but at very low levels on normal endothelium, and could not be detected on smooth muscle cells in the normal adult aorta. ICAM-1 expression can be rapidly upregulated several fold in atherosclerotic lesions by inflammatory mediators. The concentration of ICAM and ESEL together contributes to CHD through their inflammatory effects on the vascular endothelium. This should be a specific finding for coronary heart disease. In conclusion, our results suggest that ICAM-1 gene K469E polymorphism is a genetic factor that may determine an individual’s susceptibility for CHD and MI.
Acknowledgements We thank statistician Dr. Q. Yang of the Coordination Center for Clinical Trials of the HHU Duesseldorf for help with the statistical analysis.
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