C polymorphism in the interleukin-6 gene is associated with the severity of acute cerebrovascular events

Thrombosis Research 110 (2003) 181 – 186

Regular Article

The ( 174) G/C polymorphism in the interleukin-6 gene is associated with the severity of acute cerebrovascular events Stefan Greisenegger a, Georg Endler b, Daniela Haering b, Martin Schillinger c, Wilfried Lang a, Wolfgang Lalouschek a, Christine Mannhalter b,* b

a Clinical Department of Clinical Neurology, University of Vienna Medical School, Waehringer Guertel 18-20, 1097 Vienna, Austria Clinical Institute of Medical and Chemical Laboratory Diagnostics, Division of Molecular Diagnostics, University of Vienna Medical School, Waehringer Guertel 18-20, 1097 Vienna, Austria c Clinical Department of Angiology, University of Vienna Medical School, Waehringer Guertel 18-20, 1097 Vienna, Austria

Received 6 February 2003; received in revised form 26 June 2003; accepted 26 June 2003

Abstract Background and purpose: Elevated plasma levels of interleukin-6 (IL-6) are associated with an increased risk and worse outcome of acute vascular events. A common G/C promoter polymorphism at nt ( 174) of the IL-6 gene has been shown to affect basal IL-6 levels. Consequently, the IL-6 genotype may be associated with risk and outcome of ischemic stroke (IS). We investigated the statistical association between this polymorphism and cerebrovascular events, as well as the clinical outcome in patients with symptoms before the age of 60. Methods: We examined 214 patients of 60 years or less with acute ischemic stroke or transient ischemic attack (TIA) and 214 age- and sexmatched healthy control subjects for the ( 174) IL-6 G/C polymorphism by mutagenic separated polymerase chain reaction (MS PCR). Clinical severity of the vascular event was evaluated by validated scales at predefined points of time. Results: In the total group of patients, the genotype and allele frequencies in the patient group (38% GG, 45% GC, 17% CC; allelic frequency: 60% G, 40% C) did not differ significantly from the control group. However, individuals homozygous for the ( 174)G variant had significantly worse scores on the NIH Stroke Scale (NIHSS) already on admission and 1 week after the event. Also, patients with severe disability 1 week and 3 months after the event (Rankin Scale (RS) 4 or 5; NIH Stroke Scale z 6) were significantly more often carriers of the GG genotype. In a multivariate analysis, the IL-6 ( 174)GG genotype was significantly associated with severe disability after 1 week (RS 4 – 5; odds ratio (OR) = 3.2, 95% CI: 1.5 – 6.6; p = 0.002; NIHSS z 6; OR = 4.2, 95% CI: 1.6 – 11.1). Conclusions: The ( 174)GG-genotype of the IL-6 gene is associated with severe stroke in young patients with acute cerebrovascular events. Further studies with larger patient groups are warranted to confirm these findings. D 2003 Elsevier Ltd. All rights reserved. Keywords: Stroke; Interleukins; Genetics

1. Introduction Interleukin-6 (IL-6) is a pleiotropic cytokine with a broad range of effects in inflammation. It induces the secretion of monocyte chemotactic protein, an important mediator of inflammatory events in atherosclerosis and regulates the expression of adhesion molecules and the

Abbreviations: IS, ischemic stroke; TIA, transient ischemic attack; NIHSS, NIH Stroke Scale; RS, Rankin Scale; MS PCR, mutagenic separated polymerase chain reaction; bp, base pair; OR, odds ratio; MI, myocardial infarction; BMI, body mass index. * Corresponding author. Tel.: +43-1-40400-2085; fax: +43-1-404002097. E-mail address: [email protected] (C. Mannhalter). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0049-3848(03)00376-1

release of cytokines such as TNF-a and IL-1h [1,2]. Elevated plasma levels of interleukin-6 (IL-6) are associated with an increased risk and worse outcome of acute vascular events [3– 6]. A frequent G/C polymorphism at nt ( 174) in the promoter region of the interleukin-6 gene has been shown to affect basal interleukin-6 levels [7– 9]. The allelic frequency varies depending on ethnic background. In an investigation of a healthy British population, a prevalence for the GG genotype of 38% and for the CC genotype of 18% was described [7]. The role of the ( 174) IL-6 G/C polymorphism in young patients with acute cerebrovascular disease has not been investigated and no data are available. We analyzed the frequency of this polymorphism and its relation to clinical outcome in patients with acute ischemic stroke (IS) or transient ische-

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Table 1 ( 174) IL-6 G/C genotype distribution in patients and controls Patients (n = 214)

Controls (n = 214)

N

pa

%

N

%

IL-6 genotypes GG 81 GC 96 CC 37

38 45 17

76 108 30

36 50 14

1.000

IL-6 alleles G 258 C 170

60 40

260 168

61 39

0.883

a

McNemar test.

minimize cross-reactions of the PCR products generated from the two different alleles during PCR amplification. Both alleles can easily be distinguished by length on polyacrylamide gels. In every sample, one or two different products are generated depending on the genotype. In each experiment, a known heterozygous individual was included as positive

Table 2 Clinical variables and vascular risk factors in patients according to the IL-6 genotype Genotype

mic attack (TIA) before or at the age of 60 and in age- and sex-matched control subjects.

2. Materials and methods 2.1. Patients and control subjects Patients were collected in a stroke registry between October 1998 and December 1999 [10]. In total, 214 consecutive patients with acute IS (n = 168) or TIA (n = 46) between 18 and 60 years (129 males, 85 females; mean age: 49.2 F 8.7 years) for whom clinical data and blood samples were available were included in the present study. Clinical stroke severity was assessed by means of validated scales in all patients on admission (NIH Stroke Scale, NIHSS), after 1 week (NIHSS, Rankin Scale (RS)) and after 3 months (RS only). All patients underwent cranial CT or MRI, laboratory investigations for vascular risk factors, duplex sonography of the carotid and vertebral arteries, and a thorough cardiac investigation. Stroke etiology was classified according to defined criteria into large vessel disease, small vessel disease, cardioembolism, or undetermined [10]. Patients with rare causes of the cerebrovascular event, e.g. arterial dissection, were not included. The control group comprised 214 participants in an official health service program from the same geographic region who were free of clinically manifest vascular disease and were matched individually to the patients for age ( F 3 years) and gender. Informed consent to participation in the study was obtained from all individuals. The study was approved by the local ethics committee. 2.2. IL-6 genotyping For the determination of the IL-6 promoter polymorphism, a mutagenic separated polymerase chain reaction (MS PCR) assay was used essentially as described previously [11,12]. Briefly, in this MS PCR, one common and two allele specific primers (all MWG Biotech, Ebersdorf Germany) that differ in length by 9 base pairs (bp) are used in one single tube. Base mismatches at defined positions in the allele specific primers

Age (mean F S.D.) Female % Hypertension % Diabetes % BMI>27% Cigarette smoking % Previous stroke/TIA % Previous MI % Cerebrovascular stenosisb % CRP mg/100 ml (median/ 25/75 perc), n = 171 Fibrinogen mg/100 ml (median/ 25/75 perc), n = 132 Leucocyte count mU/100 ml (median/ 25/75 perc), n = 202 Blood glucose mg/100 ml (median/ 25/75 perc), n = 194 Vascular territory Hemispheric Brainstem/ cerebellum Etiology % Large vessel Cardioembolic Small vessel Undetermined

GG, n = 81

GC, n = 96

CC, n = 37

Statistical significance, pa

48 F 9

50 F 8

49 F 9

0.388

40 43 12 49 62

41 56 19 51 46

38 51 8 43 51

0.956 0.223 0.232 0.721 0.106

7

10

11

0.747

4 25

13 13

3 16

0.041 0.015

0.6/0.5/1.02

0.5/0.5/1.0

0.6/0.5/1.0

0.602

332/282/400 335/271/401 316/278/384 0.691

8.5/7.1/10.5

8.7/6.5/10.6

8.1/6.8/10.8

0.806

109/96/141

107/93/135

113/94/127

0.931

80 20

79 21

78 22

0.969

26 17 14 43

18 15 25 42

16 22 27 35

0.389

BMI: body mass index; TIA: transient ischemic attack; MI: myocardial infarction; CRP: C-reactive protein; NIHSS: NIH Stroke Scale; RS: Rankin Scale. a Pearson v2-Test (categorical data) and Kruskal – Wallis H-Test (continuous data). b >70% carotid stenosis or hemodynamically relevant stenosis of the vertebral or basilar artery.

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control to ensure amplification of both alleles. A reagent control without DNA served as a negative control. 2.3. Statistical analysis The genotype distribution in patients and controls subjects was compared using the McNemar-test. Analyses within the patient group were performed using the v2-test for binary and categorical data and the Mann – Whitney U-test for continuous variables. The severity of the cerebrovascular event at 1 week was classified according to the RS as mild (RS 0 –1; no or only mild neurological symptoms), moderate (RS 2– 3; slight or moderate disability), or severe (RS 4 –5; moderate – severe or severe disability) and according to the NIHSS (NIHSS < 6 and z 6). A multinomial analysis revealed no differences between mild and moderate events with respect to the factors associated with stroke severity. Therefore, these two groups were combined for the final multiple regression analysis. The association between the IL-6 genotype and clinical severity of the cerebrovascular event was calculated unadjusted and by multivariate logistic regression analysis following adjustment for age and sex as well as all variables associated with stroke severity with a p-value < 0.2 in the univariate analysis. Since stroke severity and other clinical parameters associated with stroke severity were very similar in carriers of the C allele (CC and GC genotype), a recessive model was applied in the multivariate analysis.

3. Results 3.1. Patients vs. control subjects Table 1 shows the distribution of the IL-6 genotypes and alleles in patients and controls. Genotypes were in accordance with the Hardy –Weinberg equilibrium. Allelic frequencies and genotype prevalences did not differ significantly between patients and controls (Table 1). Patients with large vessel disease more frequently carried the GG genotype compared to matched control subjects (59% vs. 31%; p = 0.035).

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Table 4 IL-6 genotype and stroke severitya

Patients with GG genotype univariate Adjusted for age and sexb Adjusted for age, sex, etiology, MI, BMIc,d

OR

95% CI

p

3.2 3.6 3.9

1.5 – 6.6 1.7 – 7.7 1.6 – 9.3

0.002 0.001 0.002

a

Logistic regression RS 0 to 3 vs. RS 4 to 5. Hosmer – Lemeshow-Test: C = 4.91; df = 8; p = 0.767; Nagelkerkes R2:0.136. c Hosmer – Lemeshow-Test: C = 3.66; df = 8; p = 0.886; Nagelkerkes R2:0.252. d The following variables were tested univariately for an association with stroke severity: sex, age (tertiles), hypertension, diabetes, BMI>27, cigarette smoking, atrial fibrillation, previous cerebrovascular event, previous MI, PAD, etiology, IL-6 genotype. b

3.2. IL-6-genotype and clinical variables in patients Most clinical variables and vascular risk factors were equally frequent in wildtype, heterozygous and homozygous patients (Table 2). However, carriers of the GG genotype more often had a significant stenosis of cranial arteries than carriers of the GC or CC genotype (Table 2). 3.3. IL-6-genotype and stroke severity NIHSS scores on admission were significantly higher in carriers of the GG genotype than in GC and CC carriers ( p = 0.038 for the comparison of all three genotypes). The percentage of patients with severe stroke according to the NIHSS and the RS at 1 week was also significantly higher among GG carriers (Table 3; Fig. 1). When all three genotypes were compared separately, this difference did not remain significant after 3 months; however, the v2 test for linear correlation was still significant ( p = 0.043; only RS; Fig. 1). When GC and CC carriers were combined and

Table 3 Stroke severity at 1 week in patients according to the IL-6 genotype Genotype GG, n (%)

GC, n (%)

CC, n (%)

Stroke severity (NIHSS) at 1 week 0–2 44 (54) 70 (73) 3–5 14 (17) 19 (20) z6 23 (28) 7 (7)

27 (73) 6 (16) 4 (11)

Stroke severity (RS) at 1 week 0–1 43 (53) 70 (73) 2–3 16 (20) 16 (17) 4–5 22 (27) 10 (10)

23 (62) 10 (27) 4 (11)

a

Statistical significance, p

0.003

0.014a (0.020)

Pearson v2-test; v2-test for linear correlation in parentheses.

Fig. 1. Percentage of patients with a severe stroke (RS 4 and 5) in relation to the IL-6-genotype.

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Table 5 Comparison of patients with GG genotype with patients with GC or CC genotype with respect of stroke severity after 1 weeka

GG genotype univariate Adjusted for age and sexb Adjusted for age, sex, etiology, diabetes previous cerebrovascular event, NIHSS on admissionc,d

OR

95% CI

p

4.4 5.0 4.2

2.0 – 9.6 2.2 – 11.1 1.6 – 11.1

0.001 < 0.001 0.005

a

Logistic regression NIHSS 0 – 5 vs. NIHSS z 6. Hosmer – Lemeshow-Test: C = 5.00; df = 8; p = 0.758; Nagelkerkes R2:0.171. c Hosmer – Lemeshow-Test: C = 8.02; df = 8; p = 0.431; Nagelkerkes 2 R :0.496. d The following variables were tested univariately for an association with stroke severity: sex, age (tertiles), diabetes, BMI>27, previous cerebrovascular event, etiology, NIHSS on admission, IL-6 genotype. b

compared to GG carriers, the difference after 3 months was also significant ( p = 0.014). The unadjusted odds ratio (OR) for the association of the GG genotype with severe stroke was 3.2 (95% CI: 1.5– 6.6; p = 0.002; Table 4) for a RS z 4 (Table 4) and 4.4 (95% CI: 2.0 –9.6; p = 0.001) for a NIHSS z 6 after 1 week (Table 5). This association remained stable after adjustment for age and gender, as well as after adjustment for all other variables associated with stroke severity with a p-value < 0.2 in the univariate analysis including stroke severity on admission (Table 5). (Because we had no RS on admission, we did not include initial stroke severity in the multivariate analysis based on the RS. However, we could include it in the analysis based on the NIHSS, which was performed also on admission; see Table 5.)

4. Discussion In this study, homozygosity for the GG genotype of the ( 174) IL-6 G/C polymorphism was strongly associated with the severity of stroke in patients suffering an ischemic cerebrovascular event before or at the age of 60 years. Carriers of the GG genotype had a more than threefold probability to suffer a severe stroke than carriers of at least one C allele. This association remained statistically significant even after adjustment for potential confounding factors. Previous studies have demonstrated an association of the GG genotype with asymptomatic carotid artery atherosclerosis [13], the length of hospitalization after surgical coronary revascularization [14], elevated platelet counts [15], higher levels of antibodies to heat shock proteins [16] and other diseases [3 –5,17]. We describe for the first time a relation between the interleukin-6 genotype and clinical severity of acute cerebrovascular disease in young patients. It has been reported that carriers of the IL-6 GG genotype have higher levels of circulating IL-6 [6– 9], although one study observed higher IL-6 levels in CC homozygotes [18]. This controversial finding may be explained in part by

recently published in vitro observations showing that IL-6 expression is differently regulated in different cells [19]. Previously, elevated IL-6 levels have been linked to an increased risk for acute vascular events [6,8]. Also, correlation between poor outcome and high IL-6 levels has been reported in patients with unstable angina [7,8] or with acute stroke [9]. Several mechanisms may account for the results of our study: Firstly, elevated IL-6 levels could lead to more severe atherosclerotic changes in the vasculature in individuals carrying the GG genotype and consequently to a more severe manifestation of acute vascular events. Secondly, the procoagulatory [15,20] and neurotoxic effects [21] of IL-6 could contribute to the poor outcome in carriers of the GG genotype. Surprisingly, when looking at the genotype frequencies in all patients compared to controls, we found no statistically significant difference. This association of the IL-6-genotype with stroke severity but not with stroke risk itself undoubtedly deserves attention. We cannot exclude that the inclusion of patients with TIA, who accounted for 21% of all patients, influenced the overall result as also the total number of severe cases was small. However, a very recent investigation in an older population (mean age 76 years) also observed an increased risk of ischemic stroke in carriers of the IL-6-GG genotype [22]. In contrast, another study found an association of the IL-6-CC genotype with lacunar stroke in an older population [23]. Since we investigated only patients younger than 60 years, these results cannot be directly compared to our findings. Moreover, the association between IL-6 genotype and stroke severity was not investigated in these studies. Interactions between genotype and stroke etiology could influence the association between genotype and stroke severity. In our study, the GG genotype was more prevalent in the subgroup of patients with large vessel disease than in age- and sex-matched control subjects. Moreover, we observed a statistically non-significantly higher prevalence of small vessel disease in carriers of the C allele. However, we could not find a statistically significant association of the IL-6 genotype with stroke etiology. Currently, we cannot exclude the possibility, that the lack of a statistically significant interaction between IL-6-genotype and stroke etiology (which might influence stroke severity) is due to the small numbers in etiological subgroups. But the association of the IL-6 genotype and stroke severity remained essentially unchanged after adjustment for stroke etiology. Larger studies in selected groups of patients will be needed. We did not find an association between CRP levels or other inflammatory markers and IL-6 genotype, but it has to be pointed out that we did not measure high sensitivity CRP. However, a lack of correlation of CRP and IL-6 levels has also been observed by Jones et al. [18]. 4.1. Limitations of the study In our study, patients with the most severe strokes were not included because they were either admitted to intensive care

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units not participating in the study, or died before admission. The exclusion of these patients explains why we had no deaths among our patient-population. However, still a considerable proportion of our patients suffered a severe stroke. Thus we believe that our results are relevant for this population of stroke patients. We did not quantify the infarct volume by standardized neuroradiological techniques. Previously, the usefulness of infarct volume as a surrogate marker of clinical outcome has been questioned [24] and we found no association between the IL-6 genotype and the vascular territory. Also, the association between IL-6 genotype and stroke severity remained stable after adjustment for etiology and other variables. It is therefore unlikely that the measurement of the infarct volume would have changed our results. The grouping according to stroke severity was done posthoc and the number of patients with severe stroke was relatively small. Therefore, our results have to be interpreted with caution and should be confirmed in a prospective study. We did not measure IL-6 levels in our subjects. As demonstrated in several studies, IL-6 levels are influenced by a number of exogenous factors and differ significantly from tissue to tissue. The genotype does not depend on external factors and represents a more stable marker. It cannot be excluded that the relation between IL-6-genotype and stroke severity is mediated through effects of IL-6, which are not necessarily associated with an increased risk of stroke itself (e.g. neurotoxicity). Our study design does not allow to clarify this. In conclusion, we found a significant association between the ( 174) IL-6 G/C gene polymorphism and clinical outcome in patients with acute cerebrovascular events. This association may be due to higher IL-6 levels in patients carrying the GG genotype, however, this remains to be proven. According to our results, the outcome of acute IS/ TIA might be, at least to some extent, determined by the IL-6 genotype. The role of the IL-6-genotype for stroke severity has yet to be confirmed in a large, prospective study.

[4]

[5]

[6]

[7]

[8]

[9]

[10] [11]

[12]

[13]

[14]

[15]

[16]

Acknowledgements This work has been supported by the ‘‘Jubila¨umsfonds der Oesterreichischen Nationalbank’’ (project number 9558).

[17]

[18]

References [1] Biswas P, Delfanti F, Bernasconi S, Mengozzi M, Cota M, Polentarutti N, et al. Interleukin-6 induces monocyte chemotactic protein-1 in peripheral blood mononuclear cells and in the U937 cell line. Blood 1998;91:258 – 65. [2] Mantovani A. The interplay between primary and secondary cytokines. Cytokines involved in the regulation of monocyte recruitment. Drugs 1997;54:115 – 23. [3] Ridker PM, Rifai N, Stampfer MJ, Hennekens CH. Plasma concentration of interleukin-6 and the risk of future myocardial in-

[19]

[20]

[21]

185

farction among apparently healthy men. Circulation 2000;101: 1767 – 72. Biasucci LM, Vitelli A, Liuzzo G, Altamura S, Caligiuri G, Monaco C, et al. Elevated levels of interleukin-6 in unstable angina. Circulation 1996;94:874 – 7. Biasucci LM, Liuzzo G, Fantuzzi G, Caligiuri G, Rebuzzi AG, Ginnetti F, et al. Increasing levels of interleukin (IL)-1Ra and IL-6 during the first 2 days of hospitalization in unstable angina are associated with increased risk of in-hospital coronary events. Circulation 1999; 99:2079 – 84. Vila N, Castillo J, Davalos A, Chamorro A. Proinflammatory cytokines and early neurological worsening in ischemic stroke. Stroke 2000;31:2325 – 9. Fishman D, Faulds G, Jeffery R, Mohamed Ali V, Yudkin JS, Humphries S, et al. The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 1998;102:1369 – 76. Fernandez Real JM, Broch M, Vendrell J, Richart C, Ricart W. Interleukin-6 gene polymorphism and lipid abnormalities in healthy subjects. J Clin Endocrinol Metab 2000;85:1334 – 9. Hulkkonen J, Pertovaara M, Antonen J, Pasternack A, Hurme M. Elevated interleukin-6 plasma levels are regulated by the promoter region polymorphism of the IL6 gene in primary Sjogren’s syndrome and correlate with the clinical manifestations of the disease. Rheumatology 2001;40:656 – 61. Lang W, Lalouschek W. The Vienna stroke registry—objectives and methodology. Wien Klin Wochenschr 2001;113:141 – 7. Rust S, Funke H, Assmann G. Mutagenically separated PCR (MSPCR): a highly specific one step procedure for easy mutation detection. Nucleic Acids Res 1993;21:3623 – 9. Haring D, Endler G, Hsieh K, Mannhalter C. Comparison of methods for determination of interleukin 6 G( 174)C promoter polymorphism. Biotechniques 2002;33:1114 – 7. Rauramaa R, Vaisanen SB, Luong LA, Schmidt Trucksass A, Penttila, IM, Bouchard C, et al. Stromelysin-1 and interleukin-6 gene promoter polymorphisms are determinants of asymptomatic carotid artery atherosclerosis. Arterioscler Thromb Vasc Biol 2000;20:2657 – 62. Burzotta F, Iacoviello L, Di Castelnuovo A, Glieca F, Luciani N, Zamparelli R, et al. Relation of the 174 G/C polymorphism of interleukin-6 to interleukin-6 plasma levels and to length of hospitalization after surgical coronary revascularization. Am J Cardiol 2001;88:1125 – 8. Fernandez-Real J, Vendrell J, Richart C, et al. Platelet count and interleukin 6 gene polymorphism in healthy subjects. BMC Med Genet 2001;2:6. Veres A, Prohaszka Z, Kilpinen S, Singh M, Fust G, Hurme M. The promoter polymorphism of the IL-6 gene is associated with levels of antibodies to 60-kDa heat-shock proteins. Immunogenetics 2002;53: 851 – 6. Fernandez Real JM, Broch M, Vendrell J, Gutierrez C, Casamitjana R, Pugeat M, et al. Interleukin-6 gene polymorphism and insulin sensitivity. Diabetes 2000;49:517 – 20. Jones KG, Brull DJ, Brown LC, Sian M, Greenhalgh RM, Humphries SE, et al. Interleukin-6 (IL-6) and the prognosis of abdominal aortic aneurysms. Circulation 2001;103:2260 – 5. Terry CF, Loukaci V, Green FR. Cooperative influence of genetic polymorphisms on interleukin 6 transcriptional regulation. J Biol Chem 2000;275:18138 – 44. Neumann FJ, Ott I, Marx N, Luther T, Kenngott S, Gawaz M, et al. Effect of human recombinant interleukin-6 and interleukin-8 on monocyte procoagulant activity. Arterioscler Thromb Vasc Biol 1997;17:3399 – 405. Qiu Z, Sweeney DD, Netzeband JG, Gruol DL. Chronic interleukin-6 alters NMDA receptor-mediated membrane responses and enhances neurotoxicity in developing CNS neurons. J Neurosci 1998;18: 10445 – 56.

186

S. Greisenegger et al. / Thrombosis Research 110 (2003) 181–186

[22] Pola R, Flex A, Gaetani E, Flore R, Serricchio M, Pola P. Synergistic effect of 174 G/C polymorphism of the interleukin-6 gene promoter and 469 E/K polymorphism of the intercellular adhesion molecule-1 gene in Italian patients with history of ischemic stroke. Stroke 2003;34:881 – 5. [23] Revilla M, Obach V, Cervera A, Davalos A, Castillo J, Chamorro A.

A 174G/C polymorphism of the interleukin-6 gene in patients with lacunar infarction. Neurosci Lett 2002;324:29 – 32. [24] Saver JL, Johnston KC, Homer D, Wityk R, Koroshetz W, Truskowski LL, et al. Infarct volume as a surrogate or auxiliary outcome measure in ischemic stroke clinical trials. The RANTTAS investigators. Stroke 1999;30:293 – 8.