- Email: [email protected]
Analysis of the Interleukin-1 Receptor Antagonist Gene Variable Number Tandem Repeats in Ischemic Stroke
Analysis of the Interleukin-1 Receptor Antagonist Gene Variable Number Tandem Repeats in Ischemic Stroke Leema Reddy Peddareddygari, MD,* Souvik Sen, MD,† Ankit Pahwa, PhD,‡ Mark A. Levenstien, PhD,x and Raji P. Grewal, MDk
Background: There is an increasing interest in the role of inflammatory mechanisms contributing to the development of stroke. Recent studies have reported an association between allele 2 of a variable number tandem repeat of the interleukin-1 receptor antagonist (IL1RN) gene in Caucasian patients with ischemic stroke. The purpose of this investigation is to independently confirm these results in our study population. Methods: We recruited and genotyped 516 Caucasian patients with ischemic stroke and 380 matched controls. Tests of association were performed to estimate odds ratio (OR) for the IL1RN gene variable number tandem repeat polymorphism with case– control status. Genotype frequencies of IL1RN gene were compared by case–control and symptom status using c2 contingency tables and logistic regression models. Results: No significant association was observed between any of the IL1RN gene genotypes and ischemic stroke. The unadjusted association model a, and the fully saturated model e, adjusted for age, gender, and stroke risk factors demonstrated no significant increase in risk associated with the IL1RN gene 2/2 genotype (a: OR, 1.11; 95% confidence interval [CI], .67-1.89; P 5 .615; and e: OR, .95; 95% CI, .461.94; P 5 .574). Analyses of genotypic and allelic frequencies of each Trial of Org 10172 in Acute Stroke Treatment subtype with control and pairwise comparison between stroke subtypes did not show any significant differences in their distributions, and all P values were greater than the significance level of .05. Conclusion: Our results do not confirm an association between the gene and ischemic stroke in Caucasian patients. Key Words: Ischemic stroke—interleukin-1 receptor antagonist gene—variable number tandem repeat polymorphism. Ó 2014 by National Stroke Association
Introduction Stroke is the third leading cause of mortality and the most common cause of disability in the United States.
From the *The Neuro-genetics Institute, Sharon Hill, Pennsylvania; †Department of Neurology, University of South Carolina School of Medicine, Columbia, South Carolina; ‡Superior Research Associates, Sharon Hill, Pennsylvania; xDivision of Genetics, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania; and kNeuroscience Institute at Saint Francis Medical Center, Seton Hall University, Trenton, New Jersey. Received September 19, 2013; accepted December 29, 2013. Address correspondence to Raji P. Grewal, MD, Neuroscience Institute, Saint Francis Medical Center, 601 Hamilton Ave, Trenton, NJ. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.12.045
The commonly accepted risk factors of stroke include hypertension, diabetes, and cigarette smoking. However, these 3 major risk factors account for only about 69% of the population-attributable risk for stroke.1 There has been increasing interest in the role of genes as risk factors for stroke. Although a number of single-gene disorders are known to cause cerebrovascular disease, collectively, these are uncommon. Genetic risk for stroke may result from the additive influence of minor risk alleles in multiple genes. Consequently, in the majority of cases, ischemic stroke can be considered a polygenic disorder. There are a number of approaches that have been undertaken to identify these genes ranging from genome-wide association to case–control studies. In a case–control study, a candidate gene is selected for investigation based on biological plausibility, and gene frequencies of affected cases are compared with unaffected controls.
Journal of Stroke and Cerebrovascular Diseases, Vol. -, No. - (---), 2014: pp 1-5
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L.R. PEDDAREDDYGARI ET AL.
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A number of potential candidate genes contributing to the pathogenesis of stroke have been studied and among them are those encoding proteins involved in inflammatory mechanisms.2 Interleukin-1 (IL-1) is one of the key modulators of the inflammatory response, and its activity is regulated by its receptor antagonist, interleukin-1 receptor antagonist, an anti-inflammatory cytokine.3 An 86-bp variable number tandem repeat polymorphism (VNTR) has been reported in the intron 2 of the interleukin-1 receptor antagonist (IL1RN) gene, the number of repeats varies between 2 and 6. Allele 1 (IL1RN*1) with 4 repeats is more common than allele 2 (IL1RN*2) with 2 repeats. The remaining alleles, 3 (IL1RN*3) with 5 repeats, 4 (IL1RN*4) with 3 repeats, and 5 (IL1RN*5) with 6 repeats occur in less than 1% of the population.4 Polymorphisms in the IL1RN gene have been shown to cause variations in the modulation of IL-1 expression and function, thereby affecting the inflammatory response.5 The IL1RN*2 polymorphism has been implicated as a genetic risk factor for carotid atherosclerosis6 and coronary artery disease.7 In a follow-up publication, Worrall et al8 extended their original study of IL1RN*2 in carotid atherosclerosis to investigate a potential etiologic role in ischemic stroke. They reported that the IL1RN*2 polymorphism confers a greater risk for ischemic stroke among Caucasians (odds ratio [OR] 5 2.80; 95% confidence interval [CI], 1.29-6.11; P 5 .009). To independently confirm and extend the results of this report, we studied the IL1RN gene in ischemic stroke in our population of Caucasian stroke patients and controls.
Methods For this study, patients were recruited and samples were obtained from sites located in New Jersey and North Carolina: The Middlesex County Ischemic Stroke Study based at John F. Kennedy Hospital/New Jersey Neuroscience Institute, Edison, NJ (447 patients), and the secondary site at the Department of Neurology, University of North Carolina, Chapel Hill, NC (69 patients). At both institutions, patients diagnosed with a clinical suspicion of stroke were admitted to a stroke unit and enrolled in studies of the genetics of ischemic stroke. All procedures, including the generation of the databases, recruitment of the stroke patients, and controls were done according to protocols and methods approved by the local institutional review boards, at the New Jersey Neuroscience Institute JFK Hospital, Edison, NJ, and the University of North Carolina, Chapel Hill, NC. Informed consent was obtained from the patients or their proxies during hospitalization or in the clinic when evaluated in neurologic follow-up. The control cohort of stroke-free patients/ volunteers were recruited from the offices of local primary care physicians in Middlesex County, NJ, and from a general neurology clinic based at the New Jersey Neuroscience Institute, Edison, NJ (381 controls).
The clinical data collected include the patient’s gender, ethnicity, the presence or absence of the common risk factors (diabetes mellitus, smoking, hypertension, atrial fibrillation, and cholesterol levels), and the vascular distribution of the stroke. All patients were subjected to cranial magnetic resonance imaging (MRI), magnetic resonance angiogram, diffusion-weighted MRI examination, carotid duplex ultrasound, electrocardiography, and an echocardiogram. If the MRI examination was contraindicated, cranial computed tomography scans were performed. Ischemic strokes were classified into etiologic subtypes using the Trial of Org 10172 in Acute Stroke Treatment (TOAST).9
IL-1 Genotyping DNA was extracted from blood samples (Gentra Puregene Blood Kit; Qiagen, Valencia, CA), and genotyping of 86-bp VNTR polymorphism in intron 2 of IL1RN gene was performed as previously described.10 Briefly, each reaction was carried out in a final volume of 50 mL consisting of 13 Taq buffer (50 mM KCl, 10 mM Tris–HCl, 1.5 mM MgCl2, and .1% Triton X-100), 2.5 units Taq DNA polymerase (Promega, Madison, WI), 0.4 mM dNTP (Applied Biosystems, Life Technologies, Grand Island, NY), and .5 mM of each primer (F: 50 TCCTGGTCTGCAGGTAA-30 and R: 50 -CTCAGCAACACTCCTAT-30 ). The cycling parameters were 35 cycles of 94 C for 45 seconds, 50 C for 45 seconds, and 70 C for 45 seconds. Ten microliters of the polymerase chain reaction product was electrophoresed on a 2% agarose gel and run in TBE (Tris-borate-EDTA) buffer at 96 V for 1 hour to resolve the products, stained with SYBR Green I (Molecular Probes, Life Technologies, Grand Island, NY), for visual analysis of product size and allele counting. The genotypes obtained with this method were confirmed by direct DNA sequencing.
Statistical Analysis All statistical analyses were performed using IBM SPSS Statistics, version 21.0, IBM Corporation (Armonk, NY). The control group was analyzed for deviations from Hardy–Weinberg Equilibrium, and none were found. For the analyses of the study groups, categorical data were reported as frequencies and percentages, and continuous data were reported as means with standard deviations. A 2-sample test for binomial proportions between cases and controls was reported using a c2 test of independence. The Fisher exact test was used where appropriate. A 2-sample t test for independent samples was used to compare age among groups. Association analyses were performed to estimate OR for the IL1RN VNTR polymorphism with case–control status. Genotype frequencies of IL1RN were compared by case–control and symptom status using contingency
INTERLEUKIN-1 RECEPTOR ANTAGONIST GENE POLYMORPHISM IN ISCHEMIC STROKE
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Table 1. Demographic, stroke risk factor, and subtype characteristics of the study population
Age, mean 6 SD, y Female, n (%) Risk factors, n (%) Hypertension Diabetes mellitus Hypercholesterolemia Carotid stenosis . 70% Atrial fibrillation CAD/MI Cardiomyopathy PVD Ever smoker Ever drinker TOAST classification Large-vessel atherosclerosis Cardioembolism Small-vessel occlusion Others Undetermined etiology
Cases (n 5 516)
Controls (n 5 380)
71.6 6 13.12 272 (52.7)
68.7 6 12.95 227 (59.7)
417 (80.8) 173 (33,5) 266 (51.6) 143 (27.7) 138 (26.7) 211 (40.9) 107 (20.7) 125 (24.2) 194 (37.6) 137 (26.6)
220 (57.9) 81 (21.3) 122 (32.1) 5 (1.3) 42 (11.1) 62 (16.3) 23 (6.1) 23 (6.1) 108 (28.4) 85 (22.4)
104 (20.2) 149 (28.9) 79 (15.3) 14 (2.7) 170 (32.9)
— — — — —
P value .001 .041 ,.001 ,.001 ,.001 ,.001 ,.001 ,.001 ,.001 ,.001 .02 .425
Abbreviations: CAD, coronary artery disease; MI, myocardial infarction; PVD, peripheral vascular disease; TOAST, Trial of Org 10172 in Acute Stroke Treatment.
tables and logistic regression models. Individuals with rare alleles (3, 4, and 5) were excluded from the analyses. For the case–control data, generalized model was used and a series of linear regression equations were computed that included relevant covariates (age, gender, atrial fibrillation, carotid stenosis, cardiomyopathy, hypercholesterolemia, hypertension, and peripheral vascular disease). All modeling was performed in a hierarchical manner, with a baseline model that included only the single-nucleotide polymorphism (SNP) as the predictor. Additional models were tested with age and gender; further models were then tested by adding an individual stroke risk factor variable as a covariate. A final, fully saturated model that included stroke risk factors was also used. Probability values were computed using the 2 degrees of freedom generalized test of association.
Dominant and recessive genetic models were tested for estimation of best fit for risk. Results were examined only when the generalized test reached statistical significance. Differences in the distribution of IL1RN genotypes between each TOAST subtype of stroke with control, and pairwise comparison between stroke subtypes was tested using c2 likelihood ratio test. Each pair is also tested for differences in the distribution of alleles using Fisher exact test.
Results Analysis of the IL1RN 87-bp VNTR polymorphisms was performed by comparing 516 stroke cases and 380 controls. For this analysis, only Caucasian patients and controls were studied. Demographic and risk factor
Table 2. Genotypes distribution in controls, ischemic stroke and TOAST subtypes of stroke cases TOAST classification of stroke cases
Control All stroke Large-vessel IL1RN (n 5 380), cases (n 5 516), atherosclerosis Cardioembolic genotype n (%) n (%) (n 5 104), n (%) (n 5 149), n (%) 1/1 1/2 2/2 Rare*
181 (47.6) 158 (41.6) 26 (6.8) 15 (3.9)
250 (48.4) 195 (37.8) 40 (7.8) 31 (6.0)
48 (46.2) 46 (44.2) 5 (4.8) 5 (4.8)
Abbreviation: TOAST, Trial of Org 10172 in Acute Stroke Treatment. *Rare genotypes contain alleles 3, 4, or 5.
68 (45.6) 56 (37.6) 15 (10.1) 10 (6.7)
Small-vessel Occlusion (n 5 79), n (%)
Others (n 5 14) n (%)
Undetermined (n 5 170), n (%)
39 (49.4) 32 (40.5) 5 (6.3) 3 (3.8)
3 (21.4) 11 (78.6) — —
92 (54.1) 50 (29.4) 15 (8.8) 13 (7.6)
L.R. PEDDAREDDYGARI ET AL.
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Table 3. Logistic regression models: all cases and controls, rare alleles removed P value
IL1RN Model*
Genotypes
Count
OR (95% CI)
General association
Dominant
Recessive
a
2/2 1/2 1/1 2/2 1/2 1/1 2/2 1/2 1/1 2/2 1/2 1/1 2/2 1/2 1/1
66 353 431 66 353 431 66 353 431 66 353 431 66 353 431
1.11 (.67-1.89) .89 (.67-1.19) 1.00 (1-1) 1.06 (.62-1.81) .91 (.69-1.22) 1.00 (1-1) 1.11 (.65-1.87) .89 (.67-1.18) 1.00 (1-1) 1.05 (.61-1.79) .91 (.68-1.22) 1.00 (1-1) .95 (.46-1.94) .82 (.56-1.19) 1.00 (1-1)
.615
.572
.543
.772
.631
.711
.606
.553
.550
.774
.614
.736
.574
.328
.921
b
c
d
e
Abbreviations: CI, confidence interval; OR, odds ratio. *Logistic regression model: a, unadjusted; b, age; c, gender; d, age and gender; e, atrial fibrillation, carotid stenosis, cardiomyopathy, hypercholestrolemia, hypertension, and peripheral vascular disease.
characteristics by stroke cases and control status are described in Table 1. The mean age of stroke (case) and control subjects was 71.6 6 13.12 and 69.7 6 12.95 years, respectively. The proportion of women in the case and control groups was 52.7% and 59.7%, respectively. Cases were significantly more likely to have a history of commonly accepted risk factors including hypertension (P , .001), diabetes mellitus (P , .001), hypercholesterolemia (P , .001), carotid stenosis (P , .001), atrial fibrillation (P , .001), coronary artery disease/myocardial infarction (P , .001), cardiomyopathy (P , .001), peripheral vascular disease (P ,.001), and to ever been smokers (P 5 .02), which are the commonly accepted risk factors for stroke. The allelic and genotypic frequencies (Table 2) were consistent with Hardy–Weinberg expectations (data not shown). As shown in Table 2, the IL1RN 1/1 genotype was present in 250 (48.4%) of 516 cases and 181
(47.6%) of 380 controls. The IL1RN 1/2 genotype occurred in 195 (37.8%) of 516 cases and 158 (41.6%) of 380 controls. The frequency of the IL1RN 2/2 genotype was 40 (7.8%) of 516 cases and 26 (6.8%) of 380 controls. The frequency of IL1RN genotypes containing at least one of the rare alleles (3, 4, or 5) was 31 (6%) of 516 cases and 15 (3.9%) of 380 controls. Table 2 also shows allelic and genotypic frequencies of TOAST subtypes of stroke cases. In Table 3, the unadjusted association model a, and all the adjusted models including fully saturated model e adjusted for age, gender, and stroke risk factors (atrial fibrillation, carotid stenosis, cardiomyopathy, hypercholesterolemia, hypertension, and peripheral vascular disease) demonstrated no significant increase in risk associated with the IL1RN 2/2 genotype (Model a: OR, 1.11; 95% CI, .67-1.89; P 5 .615 and Model e: OR, .95; 95% CI, .46-1.94; P 5 .574).
Table 4. P values for genotype association test for comparisons of control group to stroke subtypes and pairwise comparison between the stroke subtype (all cases and controls included, rare alleles removed) Group
Atherothrombosis (n 5 99)
Genotype association: c2 likelihood ratio test Control (n 5 365) .694 Atherothrombosis (n 5 99) Cardioembolic (n 5 139) Allelic association: Fisher exact test Control (n 5 365) .930 Atherothrombosis (n 5 99) Cardioembolic (n 5 139)
Cardioembolic (n 5 139)
Small-vessel occlusion (n 5 76)
.407 .232
.958 .808 .581
.536 .544
.844 .905 .509
INTERLEUKIN-1 RECEPTOR ANTAGONIST GENE POLYMORPHISM IN ISCHEMIC STROKE
Analyses of genotypic and allelic frequencies of each TOAST subtype with control and pairwise comparison between stroke subtype did not show any significant differences in their distributions, and all P values were greater than the significance level of .05 (Table 4).
Discussion IL1RN*2 has been reported to be a genetic risk factor for ischemic stroke in white patients in a previous study involving cases from 3 American ischemic stroke cohorts.8 This study demonstrated an association of IL1RN*2 genotype with ischemic stroke risk in white but not in nonwhite participants. More recently, a case–control study involving only white Swedish patients with ischemic stroke and matched controls was performed investigating the IL-1 gene cluster. The sample size was large consisting of 844 ischemic stroke cases and 668 controls and a replication sample of 1793 cases and 3145 controls. They found no association of 2 single-nucleotide polymorphisms rs 454078 and rs 419598 that are in linkage disequilibrium with the 86-bp VNTR and any of the major stroke subtypes.11 Although they did not specifically study the IL1RN 86-bp VNTR and ischemic stroke, this suggests that there is a lack of correlation between the 86-bp VNTR and ischemic stroke. We studied this 86-bp VNTR polymorphism in white participants and did not observe any association with ischemic stroke. Our inability to confirm the results of Worrall et al could be related to several factors. These include population stratification that can lead to false-positive results. It is possible that the Caucasian populations of the 2 studies are genetically disparate thereby accounting for the differences in the results. In addition, it should be noted that our sample size of 516 cases is larger than that studied by Worrall et al of 375. It is possible that with the increased sample size there is an increased power and, therefore, increases the validity of our study. Currently, there is an international effort investigating the genetics of ischemic stroke.12,13 In the near future, with these large collaborative studies, hopefully, the
5
issue of whether the IL1RN gene is associated with ischemic stroke will be resolved.
References 1. Hassan A, Markus HS. Genetics and ischaemic stroke. Brain 2000;123(Pt 9):1784-1812. 2. DeGraba TJ. Immunogenetic susceptibility of atherosclerotic stroke: implications on current and future treatment of vascular inflammation. Stroke 2004;35(11 Suppl 1):2712-2719. 3. Dinarello CA. Biologic basis for interleukin-1 in disease. Blood 1996;87:2095-2147. 4. Tarlow JK, Blakemore AI, Lennard A, et al. Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of an 86-bp tandem repeat. Hum Genet 1993;91:403-404. 5. Witkin SS, Gerber S, Ledger WJ. Influence of interleukin-1 receptor antagonist gene polymorphism on disease. Clin Infect Dis 2002;34:204-209. 6. Worrall BB, Azhar S, Nyquist PA, et al. Interleukin-1 receptor antagonist gene polymorphisms in carotid atherosclerosis. Stroke 2003;34:790-793. 7. Francis SE, Camp NJ, Dewberry RM, et al. Interleukin-1 receptor antagonist gene polymorphism and coronary artery disease. Circulation 1999;99:861-866. 8. Worrall BB, Brott TG, Brown RD Jr, et al. IL1RN VNTR polymorphism in ischemic stroke: analysis in 3 populations. Stroke 2007;38:1189-1196. 9. Adams HP Jr, Bendixen BH, Kappelle LJ, et al. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial.TOAST.Trial of Org 10172 in Acute Stroke Treatment. Stroke 1993; 24:35-41. 10. Mansfield JC, Holden H, Tarlow JK, et al. Novel genetic association between ulcerative colitis and the antiinflammatory cytokine interleukin-1 receptor antagonist. Gastroenterology 1994;106:637-642. 11. Olsson S, Holmegaard L, Jood K, et al. Genetic variation within the interleukin-1 gene cluster and ischemic stroke. Stroke 2012;43:2278-2282. 12. The NINDS Stroke Genetics Network (SiGN) Study Investigators. Stroke Subtypes in Hispanic Patients in the NINDS Stroke Genetics Network (SiGN): a comparison of two classification systems. International Stroke Conference in February 2012. Abstract# 3884. 13. Markus HS. Stroke genetics: prospects for personalized medicine. BMC Med 2012;10:113.
Background: There is an increasing interest in the role of inflammatory mechanisms contributing to the development of stroke. Recent studies have reported an association between allele 2 of a variable number tandem repeat of the interleukin-1 receptor antagonist (IL1RN) gene in Caucasian patients with ischemic stroke. The purpose of this investigation is to independently confirm these results in our study population. Methods: We recruited and genotyped 516 Caucasian patients with ischemic stroke and 380 matched controls. Tests of association were performed to estimate odds ratio (OR) for the IL1RN gene variable number tandem repeat polymorphism with case– control status. Genotype frequencies of IL1RN gene were compared by case–control and symptom status using c2 contingency tables and logistic regression models. Results: No significant association was observed between any of the IL1RN gene genotypes and ischemic stroke. The unadjusted association model a, and the fully saturated model e, adjusted for age, gender, and stroke risk factors demonstrated no significant increase in risk associated with the IL1RN gene 2/2 genotype (a: OR, 1.11; 95% confidence interval [CI], .67-1.89; P 5 .615; and e: OR, .95; 95% CI, .461.94; P 5 .574). Analyses of genotypic and allelic frequencies of each Trial of Org 10172 in Acute Stroke Treatment subtype with control and pairwise comparison between stroke subtypes did not show any significant differences in their distributions, and all P values were greater than the significance level of .05. Conclusion: Our results do not confirm an association between the gene and ischemic stroke in Caucasian patients. Key Words: Ischemic stroke—interleukin-1 receptor antagonist gene—variable number tandem repeat polymorphism. Ó 2014 by National Stroke Association
Introduction Stroke is the third leading cause of mortality and the most common cause of disability in the United States.
From the *The Neuro-genetics Institute, Sharon Hill, Pennsylvania; †Department of Neurology, University of South Carolina School of Medicine, Columbia, South Carolina; ‡Superior Research Associates, Sharon Hill, Pennsylvania; xDivision of Genetics, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania; and kNeuroscience Institute at Saint Francis Medical Center, Seton Hall University, Trenton, New Jersey. Received September 19, 2013; accepted December 29, 2013. Address correspondence to Raji P. Grewal, MD, Neuroscience Institute, Saint Francis Medical Center, 601 Hamilton Ave, Trenton, NJ. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.12.045
The commonly accepted risk factors of stroke include hypertension, diabetes, and cigarette smoking. However, these 3 major risk factors account for only about 69% of the population-attributable risk for stroke.1 There has been increasing interest in the role of genes as risk factors for stroke. Although a number of single-gene disorders are known to cause cerebrovascular disease, collectively, these are uncommon. Genetic risk for stroke may result from the additive influence of minor risk alleles in multiple genes. Consequently, in the majority of cases, ischemic stroke can be considered a polygenic disorder. There are a number of approaches that have been undertaken to identify these genes ranging from genome-wide association to case–control studies. In a case–control study, a candidate gene is selected for investigation based on biological plausibility, and gene frequencies of affected cases are compared with unaffected controls.
Journal of Stroke and Cerebrovascular Diseases, Vol. -, No. - (---), 2014: pp 1-5
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L.R. PEDDAREDDYGARI ET AL.
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A number of potential candidate genes contributing to the pathogenesis of stroke have been studied and among them are those encoding proteins involved in inflammatory mechanisms.2 Interleukin-1 (IL-1) is one of the key modulators of the inflammatory response, and its activity is regulated by its receptor antagonist, interleukin-1 receptor antagonist, an anti-inflammatory cytokine.3 An 86-bp variable number tandem repeat polymorphism (VNTR) has been reported in the intron 2 of the interleukin-1 receptor antagonist (IL1RN) gene, the number of repeats varies between 2 and 6. Allele 1 (IL1RN*1) with 4 repeats is more common than allele 2 (IL1RN*2) with 2 repeats. The remaining alleles, 3 (IL1RN*3) with 5 repeats, 4 (IL1RN*4) with 3 repeats, and 5 (IL1RN*5) with 6 repeats occur in less than 1% of the population.4 Polymorphisms in the IL1RN gene have been shown to cause variations in the modulation of IL-1 expression and function, thereby affecting the inflammatory response.5 The IL1RN*2 polymorphism has been implicated as a genetic risk factor for carotid atherosclerosis6 and coronary artery disease.7 In a follow-up publication, Worrall et al8 extended their original study of IL1RN*2 in carotid atherosclerosis to investigate a potential etiologic role in ischemic stroke. They reported that the IL1RN*2 polymorphism confers a greater risk for ischemic stroke among Caucasians (odds ratio [OR] 5 2.80; 95% confidence interval [CI], 1.29-6.11; P 5 .009). To independently confirm and extend the results of this report, we studied the IL1RN gene in ischemic stroke in our population of Caucasian stroke patients and controls.
Methods For this study, patients were recruited and samples were obtained from sites located in New Jersey and North Carolina: The Middlesex County Ischemic Stroke Study based at John F. Kennedy Hospital/New Jersey Neuroscience Institute, Edison, NJ (447 patients), and the secondary site at the Department of Neurology, University of North Carolina, Chapel Hill, NC (69 patients). At both institutions, patients diagnosed with a clinical suspicion of stroke were admitted to a stroke unit and enrolled in studies of the genetics of ischemic stroke. All procedures, including the generation of the databases, recruitment of the stroke patients, and controls were done according to protocols and methods approved by the local institutional review boards, at the New Jersey Neuroscience Institute JFK Hospital, Edison, NJ, and the University of North Carolina, Chapel Hill, NC. Informed consent was obtained from the patients or their proxies during hospitalization or in the clinic when evaluated in neurologic follow-up. The control cohort of stroke-free patients/ volunteers were recruited from the offices of local primary care physicians in Middlesex County, NJ, and from a general neurology clinic based at the New Jersey Neuroscience Institute, Edison, NJ (381 controls).
The clinical data collected include the patient’s gender, ethnicity, the presence or absence of the common risk factors (diabetes mellitus, smoking, hypertension, atrial fibrillation, and cholesterol levels), and the vascular distribution of the stroke. All patients were subjected to cranial magnetic resonance imaging (MRI), magnetic resonance angiogram, diffusion-weighted MRI examination, carotid duplex ultrasound, electrocardiography, and an echocardiogram. If the MRI examination was contraindicated, cranial computed tomography scans were performed. Ischemic strokes were classified into etiologic subtypes using the Trial of Org 10172 in Acute Stroke Treatment (TOAST).9
IL-1 Genotyping DNA was extracted from blood samples (Gentra Puregene Blood Kit; Qiagen, Valencia, CA), and genotyping of 86-bp VNTR polymorphism in intron 2 of IL1RN gene was performed as previously described.10 Briefly, each reaction was carried out in a final volume of 50 mL consisting of 13 Taq buffer (50 mM KCl, 10 mM Tris–HCl, 1.5 mM MgCl2, and .1% Triton X-100), 2.5 units Taq DNA polymerase (Promega, Madison, WI), 0.4 mM dNTP (Applied Biosystems, Life Technologies, Grand Island, NY), and .5 mM of each primer (F: 50 TCCTGGTCTGCAGGTAA-30 and R: 50 -CTCAGCAACACTCCTAT-30 ). The cycling parameters were 35 cycles of 94 C for 45 seconds, 50 C for 45 seconds, and 70 C for 45 seconds. Ten microliters of the polymerase chain reaction product was electrophoresed on a 2% agarose gel and run in TBE (Tris-borate-EDTA) buffer at 96 V for 1 hour to resolve the products, stained with SYBR Green I (Molecular Probes, Life Technologies, Grand Island, NY), for visual analysis of product size and allele counting. The genotypes obtained with this method were confirmed by direct DNA sequencing.
Statistical Analysis All statistical analyses were performed using IBM SPSS Statistics, version 21.0, IBM Corporation (Armonk, NY). The control group was analyzed for deviations from Hardy–Weinberg Equilibrium, and none were found. For the analyses of the study groups, categorical data were reported as frequencies and percentages, and continuous data were reported as means with standard deviations. A 2-sample test for binomial proportions between cases and controls was reported using a c2 test of independence. The Fisher exact test was used where appropriate. A 2-sample t test for independent samples was used to compare age among groups. Association analyses were performed to estimate OR for the IL1RN VNTR polymorphism with case–control status. Genotype frequencies of IL1RN were compared by case–control and symptom status using contingency
INTERLEUKIN-1 RECEPTOR ANTAGONIST GENE POLYMORPHISM IN ISCHEMIC STROKE
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Table 1. Demographic, stroke risk factor, and subtype characteristics of the study population
Age, mean 6 SD, y Female, n (%) Risk factors, n (%) Hypertension Diabetes mellitus Hypercholesterolemia Carotid stenosis . 70% Atrial fibrillation CAD/MI Cardiomyopathy PVD Ever smoker Ever drinker TOAST classification Large-vessel atherosclerosis Cardioembolism Small-vessel occlusion Others Undetermined etiology
Cases (n 5 516)
Controls (n 5 380)
71.6 6 13.12 272 (52.7)
68.7 6 12.95 227 (59.7)
417 (80.8) 173 (33,5) 266 (51.6) 143 (27.7) 138 (26.7) 211 (40.9) 107 (20.7) 125 (24.2) 194 (37.6) 137 (26.6)
220 (57.9) 81 (21.3) 122 (32.1) 5 (1.3) 42 (11.1) 62 (16.3) 23 (6.1) 23 (6.1) 108 (28.4) 85 (22.4)
104 (20.2) 149 (28.9) 79 (15.3) 14 (2.7) 170 (32.9)
— — — — —
P value .001 .041 ,.001 ,.001 ,.001 ,.001 ,.001 ,.001 ,.001 ,.001 .02 .425
Abbreviations: CAD, coronary artery disease; MI, myocardial infarction; PVD, peripheral vascular disease; TOAST, Trial of Org 10172 in Acute Stroke Treatment.
tables and logistic regression models. Individuals with rare alleles (3, 4, and 5) were excluded from the analyses. For the case–control data, generalized model was used and a series of linear regression equations were computed that included relevant covariates (age, gender, atrial fibrillation, carotid stenosis, cardiomyopathy, hypercholesterolemia, hypertension, and peripheral vascular disease). All modeling was performed in a hierarchical manner, with a baseline model that included only the single-nucleotide polymorphism (SNP) as the predictor. Additional models were tested with age and gender; further models were then tested by adding an individual stroke risk factor variable as a covariate. A final, fully saturated model that included stroke risk factors was also used. Probability values were computed using the 2 degrees of freedom generalized test of association.
Dominant and recessive genetic models were tested for estimation of best fit for risk. Results were examined only when the generalized test reached statistical significance. Differences in the distribution of IL1RN genotypes between each TOAST subtype of stroke with control, and pairwise comparison between stroke subtypes was tested using c2 likelihood ratio test. Each pair is also tested for differences in the distribution of alleles using Fisher exact test.
Results Analysis of the IL1RN 87-bp VNTR polymorphisms was performed by comparing 516 stroke cases and 380 controls. For this analysis, only Caucasian patients and controls were studied. Demographic and risk factor
Table 2. Genotypes distribution in controls, ischemic stroke and TOAST subtypes of stroke cases TOAST classification of stroke cases
Control All stroke Large-vessel IL1RN (n 5 380), cases (n 5 516), atherosclerosis Cardioembolic genotype n (%) n (%) (n 5 104), n (%) (n 5 149), n (%) 1/1 1/2 2/2 Rare*
181 (47.6) 158 (41.6) 26 (6.8) 15 (3.9)
250 (48.4) 195 (37.8) 40 (7.8) 31 (6.0)
48 (46.2) 46 (44.2) 5 (4.8) 5 (4.8)
Abbreviation: TOAST, Trial of Org 10172 in Acute Stroke Treatment. *Rare genotypes contain alleles 3, 4, or 5.
68 (45.6) 56 (37.6) 15 (10.1) 10 (6.7)
Small-vessel Occlusion (n 5 79), n (%)
Others (n 5 14) n (%)
Undetermined (n 5 170), n (%)
39 (49.4) 32 (40.5) 5 (6.3) 3 (3.8)
3 (21.4) 11 (78.6) — —
92 (54.1) 50 (29.4) 15 (8.8) 13 (7.6)
L.R. PEDDAREDDYGARI ET AL.
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Table 3. Logistic regression models: all cases and controls, rare alleles removed P value
IL1RN Model*
Genotypes
Count
OR (95% CI)
General association
Dominant
Recessive
a
2/2 1/2 1/1 2/2 1/2 1/1 2/2 1/2 1/1 2/2 1/2 1/1 2/2 1/2 1/1
66 353 431 66 353 431 66 353 431 66 353 431 66 353 431
1.11 (.67-1.89) .89 (.67-1.19) 1.00 (1-1) 1.06 (.62-1.81) .91 (.69-1.22) 1.00 (1-1) 1.11 (.65-1.87) .89 (.67-1.18) 1.00 (1-1) 1.05 (.61-1.79) .91 (.68-1.22) 1.00 (1-1) .95 (.46-1.94) .82 (.56-1.19) 1.00 (1-1)
.615
.572
.543
.772
.631
.711
.606
.553
.550
.774
.614
.736
.574
.328
.921
b
c
d
e
Abbreviations: CI, confidence interval; OR, odds ratio. *Logistic regression model: a, unadjusted; b, age; c, gender; d, age and gender; e, atrial fibrillation, carotid stenosis, cardiomyopathy, hypercholestrolemia, hypertension, and peripheral vascular disease.
characteristics by stroke cases and control status are described in Table 1. The mean age of stroke (case) and control subjects was 71.6 6 13.12 and 69.7 6 12.95 years, respectively. The proportion of women in the case and control groups was 52.7% and 59.7%, respectively. Cases were significantly more likely to have a history of commonly accepted risk factors including hypertension (P , .001), diabetes mellitus (P , .001), hypercholesterolemia (P , .001), carotid stenosis (P , .001), atrial fibrillation (P , .001), coronary artery disease/myocardial infarction (P , .001), cardiomyopathy (P , .001), peripheral vascular disease (P ,.001), and to ever been smokers (P 5 .02), which are the commonly accepted risk factors for stroke. The allelic and genotypic frequencies (Table 2) were consistent with Hardy–Weinberg expectations (data not shown). As shown in Table 2, the IL1RN 1/1 genotype was present in 250 (48.4%) of 516 cases and 181
(47.6%) of 380 controls. The IL1RN 1/2 genotype occurred in 195 (37.8%) of 516 cases and 158 (41.6%) of 380 controls. The frequency of the IL1RN 2/2 genotype was 40 (7.8%) of 516 cases and 26 (6.8%) of 380 controls. The frequency of IL1RN genotypes containing at least one of the rare alleles (3, 4, or 5) was 31 (6%) of 516 cases and 15 (3.9%) of 380 controls. Table 2 also shows allelic and genotypic frequencies of TOAST subtypes of stroke cases. In Table 3, the unadjusted association model a, and all the adjusted models including fully saturated model e adjusted for age, gender, and stroke risk factors (atrial fibrillation, carotid stenosis, cardiomyopathy, hypercholesterolemia, hypertension, and peripheral vascular disease) demonstrated no significant increase in risk associated with the IL1RN 2/2 genotype (Model a: OR, 1.11; 95% CI, .67-1.89; P 5 .615 and Model e: OR, .95; 95% CI, .46-1.94; P 5 .574).
Table 4. P values for genotype association test for comparisons of control group to stroke subtypes and pairwise comparison between the stroke subtype (all cases and controls included, rare alleles removed) Group
Atherothrombosis (n 5 99)
Genotype association: c2 likelihood ratio test Control (n 5 365) .694 Atherothrombosis (n 5 99) Cardioembolic (n 5 139) Allelic association: Fisher exact test Control (n 5 365) .930 Atherothrombosis (n 5 99) Cardioembolic (n 5 139)
Cardioembolic (n 5 139)
Small-vessel occlusion (n 5 76)
.407 .232
.958 .808 .581
.536 .544
.844 .905 .509
INTERLEUKIN-1 RECEPTOR ANTAGONIST GENE POLYMORPHISM IN ISCHEMIC STROKE
Analyses of genotypic and allelic frequencies of each TOAST subtype with control and pairwise comparison between stroke subtype did not show any significant differences in their distributions, and all P values were greater than the significance level of .05 (Table 4).
Discussion IL1RN*2 has been reported to be a genetic risk factor for ischemic stroke in white patients in a previous study involving cases from 3 American ischemic stroke cohorts.8 This study demonstrated an association of IL1RN*2 genotype with ischemic stroke risk in white but not in nonwhite participants. More recently, a case–control study involving only white Swedish patients with ischemic stroke and matched controls was performed investigating the IL-1 gene cluster. The sample size was large consisting of 844 ischemic stroke cases and 668 controls and a replication sample of 1793 cases and 3145 controls. They found no association of 2 single-nucleotide polymorphisms rs 454078 and rs 419598 that are in linkage disequilibrium with the 86-bp VNTR and any of the major stroke subtypes.11 Although they did not specifically study the IL1RN 86-bp VNTR and ischemic stroke, this suggests that there is a lack of correlation between the 86-bp VNTR and ischemic stroke. We studied this 86-bp VNTR polymorphism in white participants and did not observe any association with ischemic stroke. Our inability to confirm the results of Worrall et al could be related to several factors. These include population stratification that can lead to false-positive results. It is possible that the Caucasian populations of the 2 studies are genetically disparate thereby accounting for the differences in the results. In addition, it should be noted that our sample size of 516 cases is larger than that studied by Worrall et al of 375. It is possible that with the increased sample size there is an increased power and, therefore, increases the validity of our study. Currently, there is an international effort investigating the genetics of ischemic stroke.12,13 In the near future, with these large collaborative studies, hopefully, the
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issue of whether the IL1RN gene is associated with ischemic stroke will be resolved.
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