Abstract
Background and Purpose
ICAM-1 levels are increased in pathological studies of atherosclerosis. We evaluated 13,491 participants from the Atherosclerosis Risk in Communities study to determine the association of ICAM-1 G241R and K469E polymorphisms with incident CHD and ischemic stroke.
Methods
Incidences of ischemic stroke (N=517) and CHD (N=1,629) through 2003 were determined by annual telephone calls and hospital and death certificate surveillance. Risk factors were measured at the baseline exam. Cox proportional hazards models were used to estimate HRRs.
Results
The ICAM-1 G241RR genotype was associated with significantly increased risk of ischemic stroke in both whites (HRR=2.18(1.01-4.68), P=0.05) and African Americans (HRR=7.04(3.72-13.3), P<0.001).
Conclusions
The ICAM-1 241RR genotype is associated with increased risk of incident ischemic stroke in both whites and African Americans.
Keywords: Cell adhesion molecules, CHD, Stroke, ICAM-1
INTRODUCTION
Vascular inflammation, characterized by recruitment and adhesion of circulating leukocytes by cellular adhesion molecules on the endothelium, plays an important role in the pathogenesis of atherosclerosis.1,2 Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the adhesion and subsequent transendothelial migration of circulating leukocytes into the vascular endothelium and is increased in pathological studies of atherosclerosis.1 Only a handful of studies have explored associations between ICAM-1 genetic variation and cardiovascular disease, with results proving contradictory.3-5 We evaluated participants from the large (N=15,792) bi-racial Atherosclerosis Risk in Communities (ARIC) study to determine the association of the ICAM-1 G241R and K469E polymorphisms with incident coronary heart disease (CHD) and ischemic stroke.
METHODS
Participants were selected from the ARIC study, a prospective investigation of atherosclerosis and its clinical sequelae involving 15,792 individuals aged 45 to 64 years at recruitment (1987-1989). Institutional review boards approved the ARIC study and all participants provided written informed consent. A detailed description of the ARIC study design/methods and details on ascertainment/classification of CHD and stroke events have been published elsewhere.6-8 Participants were excluded from analyses (n=2,301) if they had a positive or unknown history of prevalent stroke or CHD or a history of TIA/stroke symptoms at baseline, prohibited use of their DNA, had an ethnic background other than white or African American, or had missing information for both ICAM-1 genotypes or for the endpoints/covariates included in the analyses. Incident CHD cases (N=1,629) were defined as a definite or probable myocardial infarction (MI), a silent MI detected by ECG, a definite CHD death, or a coronary revascularization. Incident ischemic stroke cases (N=517) were defined as validated definite/probable hospitalized embolic or thrombotic brain infarctions.
Seated blood pressure was measured three times with a random-zero sphygmomanometer and the last two measurements were averaged. Hypertension was defined as SBP≥140 mmHg or DBP≥90 mmHg or current use of antihypertensive medications (assessed by questionnaires and in-person interviews). Diabetes was defined by a fasting glucose level≥126 mg/dL, a non-fasting glucose level≥200 mg/dL, and/or history of or treatment for diabetes. Cigarette-smoking status compared current smokers to former/never smokers. Body mass index (BMI, kg/m2) was calculated from height and weight measurements. Plasma total cholesterol was measured by an enzymatic method.9 High-density lipoprotein (HDL) cholesterol was measured after dextran-magnesium precipitation of non-HDL lipoproteins.10 Plasma ICAM-1 levels were determined by ELISA (R&D Systems, Abingdon, UK).
Genotyping of the ICAM-1 G241R (rs1799969) and K469E (rs5498) polymorphisms was performed using the TaqMan assay (Applied Biosystems, Foster City, CA). Primers and probes are available upon request. The percentage of agreement for blind duplicate genotyping data was 98.7% for G241R and 98.2% for K469E.
All statistical analyses were conducted utilizing STATA version 9.2 (College Station, TX). Hardy-Weinberg equilibrium expectations were tested using a χ2 goodness-of-fit test. All analyses were done separately by race due to ICAM-1 allele frequency differences between whites and African Americans. Cox proportional hazards models were used to estimate the hazard rate ratios (HRRs) of incident CHD and ischemic stroke. For incident cases, follow-up time was defined as the time between the initial clinical visit and the date of the first event. For non-cases, follow-up continued until December 31, 2003, the date of death, or the date of last contact if lost to follow-up, whichever came first. For CHD analyses, covariates included age, gender, field center and traditional CHD risk factors (BMI, HDL, total cholesterol, smoking, diabetes, hypertension). For stroke analyses, covariates included age, gender, field center, smoking, diabetes and hypertension (identified by the National Institute of Neurological Disorders and Stroke, www.ninds.nih.gov). Covariates were assessed for statistical significance by the Wald χ2 statistic.
RESULTS
No significant differences in traditional cardiovascular/cerebrovascular risk factors (i.e. BMI, HDL, total cholesterol, smoking status, diabetes, hypertension) were observed in whites or African Americans for the ICAM-1 G241R or K469E polymorphisms (data not shown). Genotype frequencies for the ICAM-1 K469E polymorphism did not differ between CHD cases and non-cases or between ischemic stroke cases and non-cases (Table 1). Genotype frequencies for the ICAM-1 G241R polymorphism did not differ between CHD cases and non-cases, but ICAM-1 241GR and 241RR genotypes were observed at a higher frequency in ischemic stroke cases compared to non-cases for both racial groups (Table 1).
Table 1.
ICAM-1 Genotype Frequencies for Incident CHD Cases, Incident Ischemic Stroke Cases and Non-Cases, the ARIC Study
| ICAM-1 G241R Genotype |
Incident CHD Cases | Non-Cases | P* | Incident Ischemic Stroke Cases |
Non-Cases | P* |
|---|---|---|---|---|---|---|
| n (%) | n (%) | n (%) | n (%) | |||
| White | ||||||
| GG | 899 (76.7) | 6194 (76.8) | Ref | 120 (64.2) | 6950 (77.1) | Ref |
| GR | 248 (21.1) | 1716 (21.3) | 1.0 | 60 (32.1) | 1894 (21.0) | 0.0001 |
| RR | 26 (2.2) | 153 (1.9) | 0.5 | 7 (3.7) | 170 (1.9) | 0.02 |
| African American | ||||||
| GG | 271 (93.8) | 2600 (94.2) | Ref | 108 (85.7) | 2736 (94.7) | Ref |
| GR | 13 (4.5) | 122 (4.4) | 0.9 | 7 (5.6) | 123 (4.3) | 0.4 |
| RR | 5 (1.7) | 37 (1.4) | 0.6 | 11 (8.7) | 29 (1.0) | <0.0001 |
|
| ||||||
| ICAM-1 K469E Genotype |
Incident CHD Cases | Non-Cases | P* | Incident Ischemic Stroke Cases |
Non-Cases | P* |
|
|
|
|||||
| n (%) | n (%) | n (%) | n (%) | |||
|
| ||||||
| White | ||||||
| KK | 419 (33.4) | 2802 (32.3) | Ref | 99 (34.1) | 3111 (32.4) | Ref |
| KE | 621 (49.4) | 4372 (50.5) | 0.4 | 138 (47.6) | 4836 (50.4) | 0.4 |
| EE | 216 (17.2) | 1491 (17.2) | 0.7 | 53 (18.3) | 1646 (17.2) | 0.9 |
| African American | ||||||
| KK | 225 (65.6) | 2085 (67.3) | Ref | 159 (71.3) | 2129 (66.9) | Ref |
| KE | 110 (32.1) | 906 (29.2) | 0.3 | 60 (26.9) | 942 (29.6) | 0.3 |
| EE | 8 (2.3) | 108 (3.5) | 0.3 | 4 (1.8) | 110 (3.5) | 0.1 |
P-value comparing genotype frequencies between cases and non-cases
Genotypes containing the ICAM-1 241R allele (GR and RR) were a significant predictor of ischemic stroke in both whites (HRR 1.80(1.34-2.43), P<0.001) and African Americans (HRR 2.77(1.68-4.57), P<0.001) (Table 2). No significant findings were observed for the ICAM-1 K469E polymorphism and incident ischemic stroke. Additionally, no significant findings were observed for analyses of incident CHD for either ICAM-1 polymorphism in whites or African Americans (data not shown).
Table 2.
Hazard Rate Ratios (HRRs) Relating ICAM-1 G241R and K469E Genotypes to Incident Ischemic Stroke, the ARIC Study
| ICAM-1 G241R Genotype Status‡ |
Model 1* | Model 2† |
|---|---|---|
| HRR (95%CI), P | HRR (95%CI), P | |
| White | ||
| GR vs. GG | 1.82 (1.34-2.49), <0.001 | 1.77 (1.29-2.41), <0.001 |
| RR vs. GG | 1.98 (0.92-4.24), 0.08 | 2.18 (1.01-4.68), 0.05 |
| GR + RR vs. GG | 1.84 (1.36-2.48), <0.001 | 1.80 (1.34-2.43), <0.001 |
| African American | ||
| GR vs. GG | 1.49 (0.69-3.20), 0.3 | 1.42 (0.66-3.07), 0.4 |
| RR vs. GG | 6.57 (3.46-12.5), <0.001 | 7.04 (3.72-13.3), <0.001 |
| GR + RR vs. GG | 2.79 (1.69-4.61), <0.001 | 2.77 (1.68-4.57), <0.001 |
|
| ||
| ICAM-1 K469E Genotype Status† |
Model 1* | Model 2** |
|
| ||
| HRR (95%CI), P | HRR (95%CI), P | |
|
| ||
| White | ||
| KE vs. KK | 0.91 (0.70-1.17), 0.5 | 0.90 (0.69-1.16), 0.4 |
| EE vs. KK | 1.01 (0.73-1.42), 0.9 | 1.04 (0.75-1.45), 0.8 |
| KE + EE vs. KK | 0.93 (0.73-1.19), 0.6 | 0.93 (0.73-1.19), 0.6 |
| African American | ||
| KE vs. KK | 0.85 (0.63-1.15), 0.3 | 0.85 (0.63-1.14), 0.3 |
| EE vs. KK | 0.51 (0.19-1.38), 0.2 | 0.59 (0.22-1.58), 0.3 |
| KE + EE vs. KK | 0.82 (0.61-1.09), 0.2 | 0.82 (0.62-1.10), 0.2 |
Adjusted for age and gender
Adjusted for age, gender, center, smoking, diabetes and hypertension status
Referent genotype: GG for G241R, and KK for K469E
ICAM-1 levels were only available on a stratified random sample of the ARIC cohort (558 whites, 244 African Americans). ICAM-1 G241R genotype distributions in this stratified random sample were similar to those observed in the entire ARIC cohort [f(R)=0.11 in whites, f(R)=0.04 in African Americans]. The ICAM-1 G241R polymorphism was associated with higher ICAM-1 levels in both whites and African Americans, but this finding was not statistically significant (Table 3).
Table 3.
Plasma ICAM-1 Levels by Race and ICAM-1 G241R Genotype
| ICAM-1 G241R Genotype | N | ICAM-1 Levels | P-value |
|---|---|---|---|
| White | |||
| GG | 445 | 288.3 (4.1) | Ref |
| GR | 101 | 291.1 (18.5) | 0.9 |
| RR | 12 | 323.8 (29.5) | 0.4 |
| African American | |||
| GG | 230 | 238.6 (6.6) | Ref |
| GR | 9 | 213.5 (18.8) | 0.1 |
| RR | 5 | 287.1 (35.1) | 0.2 |
| Races Pooled | |||
| GG | 675 | 273.3 (3.5) | Ref |
| GR | 110 | 286.3 (17.5) | 1.0 |
| RR | 17 | 312.3 (23.6) | 0.2 |
Values presented as mean (standard error of the mean) and adjusted for age, gender, case status (and race in pooled analysis)
DISCUSSION
The current study evaluated two polymorphisms located within exons 4 and 6 of the ICAM-1 gene. The ICAM-1 G241R polymorphism results in a glycine to arginine change within the Ig-like domain 3, while the ICAM-1 K469E polymorphism results in a lysine to glutamine change in the Ig-like domain 5. The Ig-like domain 3 mediates binding to macrophage antigen-1 (Mac-1) and may also affect accessibility of leukocyte function associated-antigen-1 (LFA-1) binding to the Ig-like domain 1.11-13 ICAM-1 mediates adhesive interactions of circulating leukocytes to the blood vessel wall by binding to Mac-1 and LFA-1, and increased expression of ICAM-1 has been found during all phases of atherogenesis.11-13 Additionally, molecular investigation of ICAM-1 variation has shown the G241R polymorphism to be a functional variant.14 Although our study did not investigate the mechanism(s) by which the ICAM-1 G241R polymorphism increases the risk of stroke, the observed association with stroke risk is potentially due to the functional impact of this variant on the binding affinity of ICAM-1 to both Mac-1 and LFA-1, thus possibly affecting adhesion and further interactions of circulating leukocytes to the vascular wall. Based on these results, and those of previous studies, we hypothesize that the 241R allele enhances Mac-1 and LFA-1 binding to ICAM-1 while increasing the positive feedback on ICAM-1 synthesis. Further investigation of the ICAM-1 G241R polymorphism and its functional role in ischemic disease is warranted.
ACKNOWLEDGEMENTS
The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts N01-HC-55015, N01-HC-55016, N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC-55021, and N01-HC-55022. The authors thank the staff and participants of the ARIC study for their important contributions.
Footnotes
Conflicts of Interest: None
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