DOI: 10.1111/j.1365-2362.2010.02356.x
ORIGINAL ARTICLE IL-18 gene polymorphism, cardiovascular mortality and coronary artery disease Jussi A. Hernesniemi*,†,1, Kaisa Anttila*,†,1, Tuomo Nieminen†,‡, Mika Ka¨ho¨nen†,§, Nina Mononen*,†, Kjell Nikus¶, Va¨ino¨ Turjanmaa†,§, Jari Viik**, Rami Lehtinen†,§,†† and Terho Lehtima¨ki*,† *
Laboratory of Atherosclerosis Genetics, Department of Clinical Chemistry, Tampere University Hospital, Centre for Laboratory Medicine, Tampere, Finland, †Medical School, University of Tampere, Tampere, Finland, ‡Department of Pharmacological Sciences, Medical School, University of Tampere, Tampere, Finland, §Department of Clinical Physiology, Tampere University Hospital, Tampere, Finland, ¶Heart Centre, Department of Cardiology, Tampere University Hospital, Tampere, Finland, **Department of Biomedical Engineering, Tampere University of Technology, Tampere, Finland, †† Tampere Polytechnic, University of Applied Sciences, Tampere, Finland
ABSTRACT Background Interleukin 18(IL-18) is a pro-atherosclerotic cytokine. Elevated IL-18 levels and the genetic variation of the IL-18 have been previously linked with acute coronary events and cardiovascular mortality among patients with coronary artery disease (CAD). We studied the possible association between the IL-18 gene polymorphism and cardiovascular mortality during follow-up among Finnish patients who had undergone a clinical exercise stress test, in addition to the possible effect on the expression of angiography-verified CAD. Materials and methods A total of 2152 patients of the Finnish Cardiovascular Study (cohort study) were followed up for 6Æ3 years and cardiovascular mortality was recorded. Angiography was performed on 461 patients. Genotyping of five common single nucleotide polymorphisms (SNPs) of the IL-18 gene was performed using the 5¢nuclease assay for allelic discrimination with the ABI Prism 7900HT Sequence Detection System. Results Among the study population, IL-18 gene polymorphism did not associate with cardiovascular mortality. According to adjusted binary regression analysis, the male carriers of one major haplotype (the only ones carrying the t allele of the +127 C ⁄ t SNP) had a lower occurrence rate for significant CAD defined as > 50% stenosis in at least one of the main branches of the coronary arteries (OR 0Æ495, 95% CI 0Æ862–0Æ284, P = 0Æ041). No associations were observed among women. The sex-by-genotype interaction was significant (P = 0Æ033). Conclusions The IL-18 gene was not found to associate significantly with mortality. Among patients who had coronary angiography, one major haplotype of the IL-18 gene has a gender-dependent different impact on the expression of CAD. Keywords Atherosclerosis, coronary artery disease, interleukin 18, mortality, polymorphism. Eur J Clin Invest 2010; 40 (11): 994–1001
Introduction Atherosclerosis is the leading cause of death and disability in the industrialized countries [1–3]. Atherosclerosis is not only a lipid disorder but also an inflammatory process involving the recruitment of many different cell types, including endothelial and smooth muscle cells, monocyte-derived macrophages (innate immune system) and T cells (acquired immune system) [3–5]. A considerable body of data indicates that inflammation
1
Equal contributions.
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is as a major contributor to atherogenesis from early lesion to vulnerable plaque rupture and thrombosis [6,7]. Interleukin (IL)-18, a member of the IL-1 family, is a proatherogenic and pleiotrophic (affecting both the innate and the adaptive immune system) cytokine expressed mainly in Kupffer cells and activated macrophages [8–10]. Previously, it has been shown that IL-18 receptor subunits as well as IL-18 are expressed in atherosclerotic lesions [11–13]. IL-18 has been associated with the development of subclinical atherosclerosis [14,15]. The production of IL-18 seems to affect not only the
IL-18 GENE POLYMORPHISM, CARDIOVASCULAR MORTALITY AND CORONARY ARTERY DISEASE
overall amount of atherosclerosis but also the stability of atherosclerotic lesions [12,16]. IL-18 stimulates the production of interferon c (IFN-c), and much of the pro-atherogenic and atherosclerotic plaque destabilizing influence of IL-18 [12] is likely due to this IL-18-dependent production of IFN-c [17]. Circulating levels of IL-18 carry prognostic value for cardiovascular death (CVD) among patients with coronary artery disease (CAD) [7,12,18]. Similarly, IL-18 levels are increased among patients with stable and unstable angina pectoris and among those who have suffered a myocardial infarction (MI) [15,19–22]. In a haplotype study by Tiret et al. [10], the genetic variability of the IL-18 gene was shown to associate with serum IL-18 levels and cardiovascular mortality in CAD patients. Thus far, the association between angiographically verified CAD and IL-18 gene polymorphism has been studied by Liu W et al. [23], and they demonstrated that the IL-18 promoter )137G ⁄ C polymorphism influences IL-18 levels and the occurrence of angiographically verified CAD. The Finnish Cardiovascular Study (FINCAVAS) comprises patients who underwent a clinical exercise stress test for diagnostic indications at Tampere University Hospital between October 2001 and December 2008 [24]. As the link between IL-18 and cardiovascular mortality has been previously studied among patients with diagnosed CAD, we wanted to examine whether the same impact would be seen among patients with lower risk for death by cardiovascular causes and thus replicate the finding. A subset of these patients also underwent coronary angiography, and we thus also investigated the possible effect of polymorphism of the IL-18 gene on the expression of CAD as verified by coronary angiography in this highly selected subset of patients.
Material and methods Study cohort FINCAVAS is a cohort study with the purpose of constructing a risk profile to identify individuals at a high or low risk of cardiovascular diseases, events and deaths by using genetic, haemodynamic and electrocardiographic (ECG) markers [24]. The participant pool consists of patients (n = 2152) who underwent an exercise stress test at Tampere University Hospital between October 2001 and December 2004 and were willing to participate in the study. Indications for the exercise test were as follows: suspicion of coronary heart disease (CHD, n = 959), testing vulnerability to arrhythmia during exercise (n = 465), evaluation of working capacity (n = 387) and adequacy of drug therapy (n = 330), as well as obtaining an exercise profile prior to surgery (n = 284) and post MI (n = 171). Some patients had more than one indication. Coronary angiography was performed on a subsample of the patients according to routine clinical practice of the Heart Centre at the University Hospital. The coronary angiographies (n = 461) were performed before
June 2005. The study protocol was approved by the Ethical Committee of the Hospital District of Pirkanmaa, Finland, and all patients gave informed consent prior to study initiation, as stipulated in the Declaration of Helsinki. Reporting of the study conforms to STREGA [25,26].
Collection of risk factor data Data on demographics, classical cardiovascular risk factors, lifestyles, medications and medical history were gathered using a computer-based questionnaire after the patients had signed the informed consent form and before the exercise stress test was performed. Blood samples were drawn for DNA analyses after the exercise test. The definition of risk factor data has been described in more detail earlier [27].
Genotyping of the IL-18 polymorphism Within a Caucasian population, 99% of the genetic variation of the IL-18 gene is covered by six haplotypes formed by five common SNPs (rs1946519, rs360717, rs549908, rs4937100 and rs5744292), or SNPs existing within the same bin with these five common haplotypes (SNPs in nearly complete association with each other) [10]. In this study, we genotyped the same polymorphisms. Genomic DNA was extracted from peripheral blood leucocytes by using the QIAamp DNA Blood Minikit and automated biorobot M48 extraction (Qiagen, Hilden, Germany). Genotyping was performed with the Taqman SNP Genotyping Assays C__27137505_10, C_2408550_10, C___2898462_10, C_2898459_20 for the SNPs rs4937100, rs549908, rs360717 and rs1946519, as well as the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). A description of the principles of the 5¢nuclease assay method can be found in a previous publication by Livak et al. [28]. No previous report of Taqman assay was available for the SNP rs5744292. The genotyping was performed using a custom Taqman assay designed according to the The Custom TaqMan Assays instructions and manufactured by Applied Biosystem’s The Custom TaqMan Assays Service. Parallel samples were genotyped to monitor genotyping errors. No errors were detected. The genotyping was successful in 94Æ8–98Æ8% of the cases depending on genotype.
Angiography-verified CAD The indication for coronary angiography was clinical, not investigational, in all cases. In the FINCAVAS study, the same dedicated cardiologist analysed all coronary angiographies. The percentages of stenosis in the different parts of the coronary arteries were registered. Angiography was performed on 461 of the patients. Based on the results of the angiographies, 255 (55%) patients had main branch CAD (defined as over 50% stenosis by visual estimate in at least one of the major coronary arteries including the left main, left anterior descending, right
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and left circumflex coronary artery). In addition, 37 (8%) patients had a side branch stenosis of 50% or more while none of the patients had concomitant main branch disease.
Mortality data collection Death certificates were received from the Causes of Death Register, maintained by Statistics Finland, in September 2009; this source has been proven reliable [29]. The certificates included causes of death using the tenth revision of the International Classification of Diseases (ICD-10). Diagnosis numbers and certificate texts were used to classify deaths as all-cause, cardiovascular or sudden cardiac death (SCD), i.e., cardiovascular death within 24 h after onset of symptoms. The autopsy rate was 40% for all deaths and 60% for patients with SCD.
Statistical analyses The chi-square test was applied to analyse the possible associations of IL-18 SNPs and haplotypes with categorical risk factors of CAD. ANOVA was applied for continuous risk factors with normal distribution. Non-parametric tests such as the Mann– Whitney U and Kruskall–Wallis H test were applied if the continuous variable was not normally distributed. In the case of the risk factors, Bonferroni correction was applied to correct for multiple testing. Cox regression analysis – adjusted with age, gender, use of beta-blockers, percentage of age-adjusted maximal heart rate reached as well as prior diagnoses of CHD, MI and diabetes – was used to study mortality among different genotypes and haplotypes over the follow-up period. All covariates fulfilled the proportionality assumption based on correlations of survival rankings with Schoenfeld residuals. The binary regression model was used to study whether the effect of specific genotypes would be gender-dependent. If a significant interaction was observed, the possible effect of the genotype or haplotype was studied by gender. Otherwise, no stratification by gender was performed. The risk factors preliminarily considered as covariates in each regression model were age, gender, diabetes, smoking, resting systolic blood pressure and hypercholesterolaemia. In adjusted analyses, only significant covariates were accepted in the final models, as insignificant covariates were filtered out by backward elimination. Subjects with missing data were excluded from the analyses. The proportion of missing data for the following variables was BMI 0Æ3%, age 0Æ1%, smoking 0Æ2%, the use of beta blockers 0Æ3%, the use of ACE inhibitors 0Æ3%, previous MI 0Æ1%, Diabetes 6Æ1%, the use of ATII antagonists 10Æ4%, reached percentage of age expected heart rate 4Æ3% and systolic blood pressure 10Æ8%. Otherwise there were no missing data. All of the abovementioned statistical analyses were performed with the SPSS statistical software version 14Æ0 for Windows (SPSS Inc., Chicago, IL, USA), except for the haplotype reconstruction from genotype data, which was performed by PHASE software
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(Matthew Stephens lab, University of Chicago, Chicago, IL, USA) [30,31]. The THESIAS software was used to analyse the risk factor-adjusted global association between the variation of the IL-18 gene and cardiovascular mortality as well as the occurrence of main branch CAD and all CAD [32]. A P-value of < 0Æ05 was considered statistically significant.
Results General characteristics of the study population The mean age of the whole study population (n = 2152) was 57Æ2 years (SD ± 13Æ0) and there were more men than women (n = 1379 [64%] vs. n = 773 [36%]). The demographics of the study population and of patients who had undergone angiography are presented in Table 1. During the mean follow-up of 6Æ3 years (75 months), 255 patients died. In 115 cases the cause of death was cardiovascular and 51 (44%) of these patients suffered an SCD. The prevalence of hypercholesterolaemia was higher among angiography patients than the whole population (64Æ5% vs. 49Æ5%).
Polymorphism of the IL-18 gene The distributions of the IL-18 polymorphism were in accordance with the Hardy–Weinberg equilibrium in four of the five SNPs. The frequencies of different genotypes are presented in Table 2. The distribution of the +533 (T ⁄ c) polymorphism did not follow the Hardy–Weinberg equilibrium, and it was therefore excluded from further analyses and haplotype reconstruction. According to the haplotype analysis conducted using the PHASE software, the remaining four SNPs formed five haplotypes. The haplotype agtA was the only one carrying the t allele of the +127 (C ⁄ t) polymorphism, and only the CTCg haplotype carried the g allele of the +415 (A ⁄ g) polymorphism. The haplotype construction was in accordance with the haplotype constructions of the previous studies (Table 3) [10]. The studied polymorphism or haplotypes did not associate significantly with any of the CAD risk factors (age, BMI, smoking, gender, diabetes and hypercholesterolaemia) or other baseline parameters (reached heart rate percentage of the age expected value, performance in clinical exercise test, previous diagnosis of myocardial infarction, the use of beta blockers, ACE inhibitors, AT2 antagonists, beta blockers and statins) within the whole study population. Most of the haplotypes and genotypes did not associate with the diagnostic indications for the exercise test. And we did not observe significant associations between indications such as suspicion of CHD or evaluation of performance post-MI, or testing for working capacity and IL-18 haplotypes or genotypes. However, some significant associations were observed. The evaluation of drug therapy seemed to be set less often as one of the indications for the exercise test for the carriers of the CTCg haplotype (13Æ6% vs. 16Æ7%, P = 0Æ047)
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Table 1 General characteristics of the study population. Continuous values are presented as Mean ± standard deviation
Age (years)
Whole population (n = 2152)
Angiography patients (n = 461)
Men (n = 1379)
Men (n = 319)
56Æ9 ± 13Æ0
)2
Women (n = 773)
Women (n = 142)
57Æ6 ± 13Æ0
60Æ1 ± 10Æ1
60Æ4 ± 11Æ0
BMI (kg m )
27Æ6 ± 4Æ2
27Æ4 ± 4Æ9
27Æ4 ± 4Æ2
27Æ5 ± 4Æ7
Smoker, n (%)
507 (32Æ5)
129 (14Æ9)
102 (36Æ8)
22 (18Æ0)
Diabetic, n (%)
188 (12Æ0)
93 (10Æ7)
74 (13Æ9)
34 (15Æ2)
Systolic arterial pressure
133Æ1 ± 19Æ5
136Æ5 ± 21Æ8
133Æ5 ± 20Æ3
138Æ2 ± 24Æ4
Diastolic arterial pressure
84Æ2 ± 11Æ2
84Æ1 ± 11Æ1
82Æ7 ± 11Æ3
83Æ5 ± 11Æ4
Hypercholesterolaemia, n (%)
818 (52Æ4)
386 (44Æ5)
363 (68Æ2)
125 (56Æ1)
Coronary heart disease, n (%)
507 (36Æ8)
191 (24Æ7)
123 (44Æ4)
34 (27Æ9)
Prior myocardial infarction, n (%)
346 (25Æ1)
105 (13Æ6)
80 (28Æ9)
16 (13Æ0)
Use of beta blockers, n (%)
877 (63Æ8)
421 (54Æ5)
217 (78Æ3)
82 (67Æ2)
Table 2 Distribution of IL-18 genotypes within the FINCAVAS study population
Genotype II
III
P-value by v2-test
683 (32Æ1%)
1037 (48Æ7%)
410 (19Æ2%)
0Æ860
rs549908 +35(T ⁄ g)
1058 (50Æ1%)
868 (41Æ1%)
185 (8Æ8%)
0Æ969
rs360717 +127(C ⁄ t)
1151 (54Æ2%)
821 (38Æ7%)
150 (7Æ1%)
0Æ849
rs5744292 +415 (A ⁄ g)
1156 (54Æ6%)
823 (38Æ8%)
140 (6Æ6%)
0Æ570
rs4937100 +533 (T ⁄ c)*
1076 (52Æ6%)
640 (31Æ3%)
328 (16Æ0%)
0Æ000
I Polymorphism rs1946519 )656(C ⁄ a)
*Genotype distribution not in Hardy–Weinberg equilibrium. FINCAVAS, The Finnish Cardiovascular Study.
Table 3 Frequencies of haplotypes within the FINCAVAS study population according to the PHASE software N
Frequency
Standard Error
CTCA haplotype carriers
997
0Æ2749
0Æ0006
agtA haplotype carriers
985
0Æ2655
0Æ0005
CTCg haplotype carriers
974
0Æ2605
0Æ0004
aTCA haplotype carriers
659
0Æ1708
0Æ0007
CgCA haplotype carriers
112
0Æ0267
0Æ0004
FINCAVAS, the Finnish Cardiovascular Study.
and less often for the agtA haplotype (17Æ1% vs. 13Æ8%, P = 0Æ040) when compared with non-carriers. In line, this indication was more common for the carriers of the minor g allele of the +35(T ⁄ g) (rs549908) (TT homozygotes 13Æ5%, Tg heterozygotes 17Æ6% and gg homozygotes 16Æ8%, P = 0Æ040). Interestingly, the evaluation of performance prior to an invasive
operation associated with +415 (A ⁄ g) SNP (rs5744292) (criteria in 13Æ2% cases for AA homozygotes, 14Æ6% for Ag heterozygotes and 5Æ0% for the gg homozygotes, P = 0Æ009). Finally, the carriers of the ACTA haplotype had more often the evaluation of arrhythmia as a diagnostic criteria when compared with non-carriers of the haplotype (25Æ5% vs. 20Æ0%, P = 0Æ006). Additional testing between different indication categories (for example post-MI vs. working capacity) did not reveal significant associations. Interestingly, among patients selected for angiography, the CTCA haplotype carriers seemed to be more often men (73Æ3% vs. 64Æ6%, P = 0Æ065) than women and they were more likely to suffer from hypercholesterolaemia (67Æ2% vs. 57Æ1%, P = 0Æ048) when compared with the non-carriers. The study participants were defined as having hypercholesterolaemia if they were on lipid medication or had total cholesterol over 6Æ0 mM ⁄ L. Among the agtA haplotype carriers, there seemed to be fewer men (64Æ4% vs. 74Æ5%, P = 0Æ062) when compared with non-carriers.
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IL-18 polymorphism and cardiovascular mortality According to the global association analysis, cardiovascular mortality during the follow-up was not associated with IL-18 gene polymorphism, when all five of the most common haplotypes were accounted for (P = 0Æ344). Consistently, according to the Cox regression analysis, none of the studied SNPs or haplotypes associated individually with mortality during the followup. The hazard ratios corresponding to the carriership of the haplotypes were as follows (in comparison with the non-carriers): HR 1Æ122 for CTCA (P = 0Æ588), HR 0Æ806 for atgA (P = 0Æ307), HR 1Æ355 for CTCg (P = 0Æ144), HR 0Æ803 for aTCA (P = 0Æ353) and HR 0Æ928 for CCgA (P = 0Æ924).
IL-18 and angiography-verified CAD According to the adjusted global association analysis, the genetic variation was not significantly associated with the occurrence of main branch CAD when all five of the most common haplotypes were considered in the analysis (P = 0Æ772) or with CAD defined as over 50% stenosis in any part of the coronary arteries (P = 0Æ847). According to the interaction analyses executed with the binary regression method, one haplotype seemed to associate differently among men and women in the occurrence of main branch CAD (unadjusted P = 0Æ046 [R2 = 0Æ069] and adjusted P = 0Æ039 [R2 = 0Æ237]). As statins are known to alter the inflammatory milieu within atherosclerotic plaques [33], we also repeated the analysis by introducing the use of statins into the model. The adjustment enhanced the predictive value of the statistical model and the result became even more significant (P = 0Æ033 [R2 = 0Æ257]). Further adjustments with the use of beta blockers, ATII antagonists or the use of ACE inhibitors did not alter the result significantly and failed to increase the predictive value of the model. Among men, the carriers of the agtA haplotype were less likely to suffer from main branch CAD after adjusting for risk factors (OR 0Æ574, with 95% Confidence Interval (CI) 0Æ331–0Æ996, P = 0Æ048 [R2 = 0Æ175]). Further adjustment with the use of statins improved the significance of the finding and the predictive value of the model (OR=0Æ576 with 95% CI 0Æ340–0Æ978, P = 0Æ041 [R2 = 0Æ186]). Further adjustments with the use of beta blockers, ATII antagonists or the use of ACE inhibitors did not alter the result significantly and failed to increase the predictive value of the model. Among women no significant difference was observed in the adjusted occurrence of main branch CAD (OR 1Æ562, with 95% CI 0Æ669–3Æ648, P = 0Æ303). Further adjustments with the use of medications did not alter the results significantly. Other significant interactions were not found and none of the haplotypes or SNPs was found to associate significantly with main branch CAD or with CAD in any part of the coronary arteries in the whole study population.
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Discussion In this study, the variation of the IL-18 gene was not found to associate significantly with mortality for cardiovascular causes during follow-up in a patient population undergoing exercise tests. In a coronary angiography subpopulation, a major agtA haplotype (the only one carrying the t allele of the +127 C ⁄ t polymorphism) seemed to have a significantly different effect on the occurrence of main branch CAD among men when compared with women. Among men, the carriers of the agtA haplotype had a lower risk factor-adjusted occurrence rate of main branch CAD. However, the significance of these findings is highly susceptible, because they will only qualify as significant if no correction is used for multiple hypothesis testing. The occurrence of CAD measured as over 50% stenosis in any of the branches of the coronary artery tree, including the smaller vessels, was not significantly associated with any of the IL-18 genotypes or haplotypes. The variation in the IL-18 gene was associated with IL-18 serum concentrations and cardiovascular mortality among CAD patients. Furthermore, the results of animal models have shown that atherosclerotic plaque formation decreases in the default of IL-18 or because of inhibition of IL-18 signalling, and on the other hand, the compounding of exogenous IL-18 advances atherogenesis [34–38]. The production of IL-18 seems to affect not only the overall amount of atherosclerosis but also the stability of atherosclerotic lesions [12,16]. This is one possible link between IL-18 and severe cardiovascular complications. Occlusion of one of the main branches of the coronary arteries is more likely to cause severe complications than does occlusion of smaller arteries. In this study, we found that among the men undergoing coronary angiography, the carriers of the agtA haplotype (and thus also the carriers of the t allele +127 C ⁄ t polymorphism) seemed to have a lower risk for main branch CAD. We have previously shown that among men, carriers of the c allele of the )137 (G ⁄ c) polymorphism are at a lower risk for sudden cardiac death and that hypertension is a key element conveying the risk [39,40]. These two polymorphisms are inherited in the same bin of polymorphism, making it impossible to discern which one is responsible for the observed association. Previously, the )137 (G ⁄ c) polymorphism has been associated with reduced transcriptional activity of the gene and (reduced) production of IL-18 [41,42]. Interestingly, the role of circulating IL-18 levels on angiography verified CAD is still deputed. According to the study by Espinola-Klein et al. [7], circulating IL-18 levels are not associated with the extent of coronary atherosclerosis with angiographic measurements. On the other hand, according to two other studies by Hulthe et al. and Chen et al. [15,22], IL-18 concentrations are related to coronary atherosclerosis with
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angiographic measurements. Polymorphism of the IL-18 gene was not controlled for in these studies. The main limitation of this study is the small number of angiographic patients. Furthermore, as the angiography patients were selected for the procedure according to a strict diagnostic evaluation, the expression of CAD is likely to be similar despite the differences in the genetic background of the patients. Another clear limitation is that the whole study population is very heterogeneous, consisting of patients with varying indications for the exercise stress test. It is more likely that the risk factors would be unevenly distributed among different genetic groups. Interestingly, we did observe that among patients undergoing angiography, the carriers of the most common CTCA haplotype had a higher occurrence of hypercholesterolaemia when compared with non-carriers. This haplotype was previously associated with higher cardiovascular mortality [10]. Thus it would be plausible that the occurrence of hypercholesterolaemia would be lower among the carriers of this risk haplotype in patients with suspected coronary artery disease, or who have severe stable symptoms or with high-risk features for an adverse outcome. We also observed some associations between some haplotypes and genotypes and indications but these results should be interpreted with caution as no correction for multiple testing was used. Furthermore, the indications do not define clear patient categories as many of the patients had more than one indication and some indications might be considered more secondary than primary. The overall cardiovascular mortality was also not high, as many of the patients in our study population were not high-risk individuals. This reduces the power of the current study, and therefore we can not conclude that the variation of the IL-18 gene would not, indeed, associate with cardiovascular mortality. For example, in comparison with our study, cardiovascular mortality was higher in the study by Tiret et al. in which they followed up 1,299 CAD patients for a median follow-up period of 5Æ9 years, during which 142 patients died. In this study, the follow-up period was similar (6Æ3 years) but the overall number of patients higher (2152), while the number of CV deaths was lower (n = 115). Another limitation of the present study is that we did have measurements of the circulating IL-18 levels. However, it has been shown that the circulating IL-18 levels are significantly different between subjects with or without CHD and, that also invasive operations affect the circulating IL-18 values [43,44]. Therefore, controlling for inflammatory activity in this study would have been very challenging at best, as the study population of this study is very heterogeneous. Furthermore, it is not clear that circulating IL-18 levels would be responsible for conveying the risk for atherosclerosis and for its end-points. The local production of IL-18 in atherosclerotic plaques modulated by genetic factors can also cause aggravation of the disease.
In conclusion, we did not find a significant association between the variation of the IL-18 gene and cardiovascular mortality among our study population with a clinically indicated exercise (stress) test. Within a subpopulation selected to undergo coronary angiography, one major haplotype seemed to affect the expression of the disease differently among men and women, providing some protection against occlusion of the main branches of the coronary arteries in male patients. Similar tendencies were not observed among women. Acknowledgements This study has been supported by the Academy of Finland, the Emil Aalto foundation and Tampere University Hospital Medical Research fund. The authors thank the staff of the Department of Clinical Physiology at the Tampere University Hospital for collecting the exercise test data. Address Laboratory of Atherosclerosis Genetics, Department of Clinical Chemistry, Tampere University Hospital, Centre for Laboratory Medicine, Tampere, Finland (J. A. Hernesniemi, K. Anttila, N. Mononen, T. Lehtima¨ki); Medical School, University of Tampere, Tampere, Finland (J. A. Hernesniemi, K. Anttila, T. Nieminen, M. Ka¨ho¨nen, N. Mononen, V. Turjanmaa, R. Lehtinen, T. Lehtima¨ki); Department of Pharmacological Sciences, Medical School, University of Tampere, Tampere, Finland (T. Nieminen); Department of Clinical Physiology, Tampere University Hospital, Tampere, Finland (M. Ka¨ho¨nen, V. Turjanmaa, R. Lehtinen); Heart Centre, Department of Cardiology, Tampere University Hospital, Tampere, Finland (K. Nikus); Department of Biomedical Engineering, Tampere University of Technology, Tampere, Finland (J. Viik); Tampere Polytechnic, University of Applied Sciences, Tampere, Finland (R. Lehtinen). Correspondence to: Jussi Hernesniemi, Tampere University Hospital, Finn Medi 2, 3rd floor, P.O. Box 2000, FI-33521, Tampere, Finland. Tel.: +358 3 311 74049; fax: +358 3 311 74168; e-mail: jussi.hernesniemi@uta.fi Received 1 February 2010; accepted 29 June 2010 References 1 Centers for Disease Control and Prevention (CDC). State-specific mortality from sudden cardiac death–United States, 1999. MMWR Morb Mortal Wkly Rep 2002;51:123–6. 2 Rubart M, Zipes DP. Mechanisms of sudden cardiac death. J Clin Invest 2005;115:2305–15. 3 Lusis AJ. Atherosclerosis. Nature 2000;407:233–41. 4 Daugherty A, Rateri DL. T lymphocytes in atherosclerosis: the yinyang of Th1 and Th2 influence on lesion formation. Circ Res 2002;90:1039–40.
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1000 ª 2010 The Authors. European Journal of Clinical Investigation ª 2010 Stichting European Society for Clinical Investigation Journal Foundation
IL-18 GENE POLYMORPHISM, CARDIOVASCULAR MORTALITY AND CORONARY ARTERY DISEASE
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