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A2 polymorphism of GpIIIa gene and a risk of aneurysmal subarachnoid haemorrhage
Biochemical and Biophysical Research Communications 383 (2009) 228–230
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
A1/A2 polymorphism of GpIIIa gene and a risk of aneurysmal subarachnoid haemorrhage Mateusz G. Adamski a,*, Anna Borratynska a, Mariusz Krupa b, Dorota Wloch-Kopec a, Wojciech Turaj a, Pawel Wolkow c, Marcin Wnuk a, Andrzej Urbanik d, Marek Moskala b, Andrzej Szczudlik a, Agnieszka Slowik a a
Department of Neurology, Jagiellonian University Medical College, 3 Botaniczna St., 31-503 Krakow, Poland Department of Neurosurgery and Neurotraumatology, Jagiellonian University Medical College, Krakow, Poland Department of Pharmacology, Jagiellonian University Medical College, Krakow, Poland d Department of Radiology, Jagiellonian University Medical College, Krakow, Poland b c
a r t i c l e
i n f o
Article history: Received 9 March 2009 Available online 5 April 2009
Keywords: GpIIIa GpIIIa A1/A2 polymorphism Aneurysmal SAH
a b s t r a c t Platelet glycoproteins are involved in pathophysiology of cerebrovascular diseases. The aim of this study was to investigate the association between the GpIIIa gene A1/A2 polymorphism and a risk of aneurysmal subarachnoid haemorrhage (SAH) in a Polish population. In a case-control study we genotyped 288 Caucasian patients with aneurysmal SAH and 457 age-, gender- and race-matched controls. The GpIIIa A1/A2 polymorphism was genotyped with RFLP technique. No difference was found in the distribution of the polymorphism between the cases and controls (cases: A1A1—201 (69.8%), A1A2—83 (28.8%) and A2A2—4 (1.4%) vs. controls: A1A1—323 (70.7%); A1A2—128 (28.0%); A2A2—6 (1.3%), P > 0.05. In a multivariate analysis female gender (OR = 1.950; 95%CI: 1.308–2.907), hypertension (OR = 4.774; 95%CI: 3.048–7.478) and smoking (OR = 2.034; 95%CI: 1.366–3.030), but not GpIIIa A1/A2 polymorphism, were independent risk factors for aneurysmal SAH. The GpIIIa A1/A2 polymorphism is not a risk factor of aneurysmal SAH in a Polish population. Ó 2009 Elsevier Inc. All rights reserved.
This research was supported by State Committee for Scientific Research Grant NN402083934 and K/ZDS/000377, to Agnieszka Slowik, M.D., Ph.D. Introduction Subarachnoid hemorrhage (SAH) from ruptured intracranial aneurysm is the most common cause of all SAHs with a mortality rate as high as 45–50% [1,2]. Several modifiable risk factors for the aneurysm rupture, including hypertension [3,4], heavy alcohol consumption [3,5,6], and smoking [3–6], have been identified. There is also data that genetic factors may play a role in the development of aneurysms [4]. Glycoprotein (Gp) IIb/IIIa is a platelet receptor involved in a final step of platelet-mediated thrombus formation on the injured vessel wall [7]. It binds fibrinogen and von Willebrand factor, causing platelet aggregation [8]. The GpIIb/IIIa receptor consists of a 2-chain GpIIb subunit noncovalently associated with a single-chain GpIIIa subunit [7]. The genes encoding GpIIb and IIIa are located on
* Corresponding author. Fax: +48 12 424 86 26. E-mail address: [email protected] (M.G. Adamski). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.156
chromosome 17q21 [9]. The GpIIIa A1/A2 is a common GPIIIa gene polymorphism. GPIIIa A1 has leucine whereas GPIIIa A2 izoform harbors proline, what determines a conformational variation in amino-terminal disulfide loop, related to fibrinogen binding [10]. Single study by Iniesta et al. [11] reported a protective role of the platelet GPIIIa A2 allele in SAH in a Spanish population. We investigated the association between GpIIIa A1/A2 polymorphism and a risk of aneurysmal SAH in an independent Polish population. Patients and methods Subjects. We included 288 patients with aneurysmal SAH admitted to the Stroke Unit and the Department of Neurosurgery, Jagiellonian University, Krakow, Poland who were recruited between 2001 and 2007. This unit serves as a stroke emergency center for one district of Krakow and as a referral center for South East Poland (10–15% of patients). We also included 457 controls matched by gender, race and age (±10 years) without any history of cerebrovascular disease. They were recruited from consecutive spouses of the patients with disorders other than SAH (30%), or from the community. All SAH patients and controls were White.
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M.G. Adamski et al. / Biochemical and Biophysical Research Communications 383 (2009) 228–230
We included 71% of all aneurysmal SAH patients. Others died before blood sampling or we were unable to obtain informed consent. SAH was diagnosed by cranial computed tomography (CT) and/ or lumbar puncture. The diagnosis of single or multiple saccular aneurysms was established by digital subtraction angiography or angio-CT. This study was performed according to the Helsinki Declaration with approval of the Ethical Committee of the Jagiellonian University. Informed consent was obtained for each individual before inclusion in this study. Methods. The information regarding gender, age and presence of major vascular risk factors (hypertension, diabetes mellitus, smoking habits and obesity) was collected from aneurysmal SAH patients and controls. An individual was classified as having arterial hypertension if he/she met 1 of the following criteria: (1) the diagnosis of hypertension in the medical history; (2) antihypertensive treatment before entry into the study; or (3) systolic or diastolic blood pressure P 140 mm Hg or P 90 mm Hg, respectively, on at least 2 different occasions (the first 3 days of hospitalization were not considered for the SAH patients). Diabetes mellitus was defined by WHO criteria.[12] Smoking habits were defined as current smokers of P 1 cigarette per day, former smokers, or nonsmokers. For statistical analysis, ‘‘current” and ‘‘former” smokers were pooled together. Obesity was defined as the Body Mass Index P 30 kg/m2. The history was taken from patients or their proxies. To determine patient’s ability to be interviewed, we asked questions about their name, date, and orientation. Irrespective of the information we were able to obtain from the patients, we also questioned all the proxies and studied medical documentation to assemble the most detailed medical history. Family history of SAH was defined as the occurrence of SAH in first-degree relatives. An individual was classified as having hyperlipidemia if he/she met 1 of the following criteria: (1) the diagnosis of hyperlipidemia in the medical history; (2) lipid-lowering treatment before entry into the study; or (3) LDL cholesterol P 3.4 mmol/L or total cholesterol P 5.2 mmol/L. We were not able to collect complete data for all study participants. Absolute numbers are provided in table 1. For the control subjects, data were collected according to a detailed clinical questionnaire, including information on vascular risk factors, current medication, and physical examination at the time of drawing the blood sample for DNA analysis. Uncuffed venous blood samples for extraction of DNA were drawn from each subject within 2 days of stroke onset. Leukocyte DNA was extracted using a commercially available kit (High Pure PCR template Preparation Kit; Boehringer Mannheim). The A1/A2 genotyping for the GpIIIa gene polymorphism was established using PCR and RFLP method, modified from Weiss et al. [13]. Power analysis revealed that a study of this size (288 cases and 457 controls) has 80% power at type I error probability a = 0.05 and a probability of event (joint probability of being GpIIIa polymorphism heterozygote or rare homozygote) in the control group of 29.3% to detect odds ratio lower than 0.6 or higher than 1.58 in the case group. Statistical analysis. Differences in subjects and controls characteristics were tested with Wilcoxon test (age) or with v2 test (categorical variables). The deviation from Hardy–Weinberg equilibrium for tested polymorphisms was examined by v2 test. We also preformed logistic regression analysis in which we calculated odds ratios (OR) with the 95% confidence intervals (95%CI) for selected SAH risk factors. For this analysis we have included only patients (n = 191) and controls (n = 347) with complete data regarding measured variables. A value of P < 0.05 was considered significant. The calculations were performed by software SPSS 10 for Windows.
Table 1 Distribution of risk factors, GPIIIa genotypes in patients and controls. SAH patients (n = 288)
Controls (n = 457)
P-value
Age, years; mean (± SD) Male gender, n (%)
50.9 (±12.9) 162 (56.2)
57.8 (±17.7) 236 (51.6)
<0.001* 0.21
Hypertension, n (%) Yes No Unknown
165 (60.0) 110 (40.0) 13 (4.5)
179 (40.6) 262 (59.4) 16 (3.5)
<0.001
Hipercholesterolemia, n (%) Yes No Unknown
38 (15.0) 215 (85.0) 35 (12.1)
109 (28.7) 271 (71.3) 77 (16.8)
<0.001
Obesity, n (%) Yes No Unknown
21 (10.9) 171 (89.1) 95 (33.0)
82 (18.8) 353 (81.2) 22 (4.8)
0.012
Diabetes mellitus, n (%) Yes No Unknown
20 (7.7) 239 (92.3) 29 (10.0)
43 (9.5) 411 (90.5) 3 (0.6)
0.43
Smoking habits, n (%) Yes No Unknown
128 (50.8) 124 (49.2) 36 (12.5)
160 (35.6) 290 (64.4) 7 (1.5)
<0.0001
GPIII genotypes, n (%) A1A1, n (%) A1A2, n (%) + A2A2, n (%)
201 (69.8) 3 (1.0) + 84 (29.2)
323 (70.7) 134 (29.3)
0.8
P-values for the differences between groups assessed with v2 test or with *Wilcoxon test; SAH, subarachnoid haemorrhage.
Results Demographic data and risk factor profiles of patients and controls are presented in Table 1. Patients and controls did not differ regarding gender or presence of diabetes mellitus. Hypertension and smoking were more prevalent in SAH patients (Table 1). In contrast hypercholesterolemia and obesity were more prevalent in the controls than in the cases (Table 1). SAH patients were significantly younger than controls (Table 1). History of familial SAH was positive in 3.7% (n = 9) and unknown for 15.2% (n = 44) of SAH patients. The allele distribution for the investigated GpIIIa A1/A2 polymorphism did not deviate from Hardy–Weinberg equilibrium for the controls and stroke patients. The frequencies of the studied genotypes were similar in aneurysmal SAH patients and controls (Table 1). We did not find any difference in the distribution of the studied polymorphism neither between males and females, nor after adjusting for common SAH risk factors. Adjusting for size (>7 mm in diameter) and number (P2) of aneurysms did not show difference in the GpIIIa A1/A2 allele distribution in all aneurysmal SAH patients or in male and female patients separately (data not shown). The GpIIIa A1/A2 polymorphism was not a risk factor of aneurysmal SAH in a multivariate analysis (OR = 0.922; 95%CI: 0.783– 1.085). Whereas female gender (OR = 1.950; 95%CI: 1.308–2.907), hypertension (OR = 4.774; 95%CI: 3.048–7.478) and smoking (OR = 2.034; 95%CI: 1.366–3.030) appeared to be independent risk factors of aneurysmal SAH. Discussion We did not find any difference in the distribution of the studied polymorphism between the cases and controls. We also performed
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a separate analysis in males and females, because GpIIIa A1A2 polymorphism had a deleterious, time-dependent impact on overall health and longevity in males and the prevalence of genotypes with A2 allele decreased as a function of male population age [14]. We did not find the impact of gender on the relation between the GpIIIa A1/A2 polymorphism and a risk for aneurysmal SAH. We did not find the relation between the size or number of the aneurysms and the GpIIIa A1/A2 polymorphism. The question arises what could be responsible for the discrepancies between our results and the results of Iniesta et al. [11]. The controls from a general population and controls recruited at traumatology and ophthalmology wards included by Iniesta et al. presented with the similar distribution of genotypes with A2 allele as compared with our controls (Spanish general population controls: 32% vs. Spanish in-hospital controls: 30% vs. our controls: 30.2%, respectively, P > 0,05). Polish and Spanish controls are of the same ethnicity and race so one could expect similar distribution of the genotypes between the two populations. We think that one of the possible reasons for these discrepancies between the conclusions may be derived from difference in selection criteria for the SAH patients. The group of patients with SAH recruited by Iniesta et al. is not homogenous [11]. Only 67% of Spanish patients with SAH presented with an aneurysm (n = 69), while our group consist of 100% aneurysmal SAH patients. It means that 33% of patients with SAH included by Iniesta had non-aneurysmal SAH. The percentage of patients with non-aneurysmal SAH in the study of Iniesta et al. is much higher than that observed in other populations (10%) [1]. In spontaneous/perimesencephalic SAH, the most common etiology of non-aneurysmal SAH, the course and outcome is usually excellent, and the pathophysiology is different [1]. Spontaneous/perimesencephalic SAH might present with different GPIIIa A1/A2 polymorphism distribution. What is more, it is possible that many aneurysmal SAH Spanish patients did not want to participate in the study or died before entering it. This information, however, was not provided by Iniesta et al. [11]. Another possible explanation for inconsistent results may come from the influence of linkage disequilibrium in the GPIIIa A1/A2 region. The GPIII A2 variant might be in linkage disequilibrium with a more functionally relevant variant, not present in our population, or present among spontaneous SAH patients. Our sample size of SAH patients, relatively small in absolute numbers, is one of the largest cohorts of SAH patients participating in genetic case–control studies in Europe. In spite of negative
results, our findings could encourage replication studies in other independent populations. However, the above-mentioned points could be taken into account while designing future case–control association studies. In conclusion, this study does not provide the evidence for the relation of the GpIIIa A1/A2 polymorphism and a risk of aneurysmal SAH in a Polish population. References [1] G. Yong-Zhong, H.A. van Alphen, Pathogenesis and histopathology of saccular aneurysms: review of the literature, Neurol. Res. 12 (1990) 249–255. [2] J.W. Hop, G.J. Rinkel, A. Algra, J. van Gijn, Case-fatality rates and functional outcome after subarachnoid hemorrhage: a systematic review, Stroke 28 (1997) 660–664. [3] B.M. Kissela, L. Sauerbeck, D. Woo, J. Khoury, J. Carrozzella, A. Pancioli, E. Jauch, C.J. Moomaw, R. Shukla, J. Gebel, R. Fontaine, J. Broderick, Subarachnoid hemorrhage: a preventable disease with a heritable component, Stroke 33 (2002) 1321–1326. [4] J.P. Broderick, M.C. Viscoli, T. Brott, W.N. Kernan, L.M. Brass, E. Feldman, L.B. Morgenstern, J.L. Wilterdink, R.I. Horwitz, Hemorrhagic stroke project investigators. Major risk factors for aneurysmal subarachnoid hemorrhage in the young are modifiable, Stroke 34 (2003) 1375–1381. [5] W.T. Longstreth Jr., L.M. Nelson, T.D. Koepsell, G. van Belle, Cigarette smoking, alcohol use, and subarachnoid hemorrhage, Stroke 23 (1992) 1242–1429. [6] S. Juvela, M. Hillbom, H. Numminen, P. Koskinen, Cigarette smoking and alcohol consumption as risk factors for aneurysmal subarachnoid hemorrhage, Stroke 24 (1993) 639–646. [7] J.J. Calvete, Clues for understanding the structure and function of a prototypic human integrin: the platelet glycoprotein IIb/IIIa complex, Thromb. Haemost. 72 (1994) 1–15. [8] D.R. Phillips, I.F. Charo, L.V. Parise, L.A. Fizgerald, The platelet membrane glycoprotein IIb/IIIa complex, Blood 71 (1988) 831–843. [9] M.A. Thornton, M. Poncz, M. Korostishevsky, E. Yakobson, S. Usher, U. Seligsohn, H. Peretz, The human platelet alphaIIb gene is not closely linked to its integrin partner beta3, Blood 94 (1999) 2039–2047. [10] A.H. Goodall, N. Curzen, M. Panesar, C. Hurd, C.J. Knight, W.H. Ouwehand, K.M. Fox, Increased binding of fibrinogen to glycoprotein IIIa-proline33 (HPA-1b PlA2 Zwb) positive platelets in patients with cardiovascular disease, Eur. Heart J. 20 (1999) 742–747. [11] J.A. Iniesta, R. Gonzales-Conejero, C. Piqueras, V. Vincente, J. Corral, Platelet GpIIIa polymorphism HpA-1 (PlA) protects against subarachnoid hemorrhage, Stroke 35 (2004) 2282–2286. [12] K.G. Alberti, P.Z. Zimmet, Definition, diagnosis, and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation, Diabet. Med. 15 (1998) 539–553. [13] E.J. Weiss, P.F. Bray, M. Tayback, S.P. Schulman, T.S. Kickler, L.C. Becker, J.L. Weiss, G. Gerstenblith, P.J. Goldschmidt-Clermont, A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis, N Engl. J. Med. 334 (1996) 1090–1094. [14] M.J. Hessner, D.M. Dinauer, R. Kwiatkowski, B. Neri, T.J. Raife, Age-dependent prevalence of vascular disease-associated polymorphisms among 2689 volunteer blood donors, Clin. Chem. 47 (2001) 1879–1884.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
A1/A2 polymorphism of GpIIIa gene and a risk of aneurysmal subarachnoid haemorrhage Mateusz G. Adamski a,*, Anna Borratynska a, Mariusz Krupa b, Dorota Wloch-Kopec a, Wojciech Turaj a, Pawel Wolkow c, Marcin Wnuk a, Andrzej Urbanik d, Marek Moskala b, Andrzej Szczudlik a, Agnieszka Slowik a a
Department of Neurology, Jagiellonian University Medical College, 3 Botaniczna St., 31-503 Krakow, Poland Department of Neurosurgery and Neurotraumatology, Jagiellonian University Medical College, Krakow, Poland Department of Pharmacology, Jagiellonian University Medical College, Krakow, Poland d Department of Radiology, Jagiellonian University Medical College, Krakow, Poland b c
a r t i c l e
i n f o
Article history: Received 9 March 2009 Available online 5 April 2009
Keywords: GpIIIa GpIIIa A1/A2 polymorphism Aneurysmal SAH
a b s t r a c t Platelet glycoproteins are involved in pathophysiology of cerebrovascular diseases. The aim of this study was to investigate the association between the GpIIIa gene A1/A2 polymorphism and a risk of aneurysmal subarachnoid haemorrhage (SAH) in a Polish population. In a case-control study we genotyped 288 Caucasian patients with aneurysmal SAH and 457 age-, gender- and race-matched controls. The GpIIIa A1/A2 polymorphism was genotyped with RFLP technique. No difference was found in the distribution of the polymorphism between the cases and controls (cases: A1A1—201 (69.8%), A1A2—83 (28.8%) and A2A2—4 (1.4%) vs. controls: A1A1—323 (70.7%); A1A2—128 (28.0%); A2A2—6 (1.3%), P > 0.05. In a multivariate analysis female gender (OR = 1.950; 95%CI: 1.308–2.907), hypertension (OR = 4.774; 95%CI: 3.048–7.478) and smoking (OR = 2.034; 95%CI: 1.366–3.030), but not GpIIIa A1/A2 polymorphism, were independent risk factors for aneurysmal SAH. The GpIIIa A1/A2 polymorphism is not a risk factor of aneurysmal SAH in a Polish population. Ó 2009 Elsevier Inc. All rights reserved.
This research was supported by State Committee for Scientific Research Grant NN402083934 and K/ZDS/000377, to Agnieszka Slowik, M.D., Ph.D. Introduction Subarachnoid hemorrhage (SAH) from ruptured intracranial aneurysm is the most common cause of all SAHs with a mortality rate as high as 45–50% [1,2]. Several modifiable risk factors for the aneurysm rupture, including hypertension [3,4], heavy alcohol consumption [3,5,6], and smoking [3–6], have been identified. There is also data that genetic factors may play a role in the development of aneurysms [4]. Glycoprotein (Gp) IIb/IIIa is a platelet receptor involved in a final step of platelet-mediated thrombus formation on the injured vessel wall [7]. It binds fibrinogen and von Willebrand factor, causing platelet aggregation [8]. The GpIIb/IIIa receptor consists of a 2-chain GpIIb subunit noncovalently associated with a single-chain GpIIIa subunit [7]. The genes encoding GpIIb and IIIa are located on
* Corresponding author. Fax: +48 12 424 86 26. E-mail address: [email protected] (M.G. Adamski). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.156
chromosome 17q21 [9]. The GpIIIa A1/A2 is a common GPIIIa gene polymorphism. GPIIIa A1 has leucine whereas GPIIIa A2 izoform harbors proline, what determines a conformational variation in amino-terminal disulfide loop, related to fibrinogen binding [10]. Single study by Iniesta et al. [11] reported a protective role of the platelet GPIIIa A2 allele in SAH in a Spanish population. We investigated the association between GpIIIa A1/A2 polymorphism and a risk of aneurysmal SAH in an independent Polish population. Patients and methods Subjects. We included 288 patients with aneurysmal SAH admitted to the Stroke Unit and the Department of Neurosurgery, Jagiellonian University, Krakow, Poland who were recruited between 2001 and 2007. This unit serves as a stroke emergency center for one district of Krakow and as a referral center for South East Poland (10–15% of patients). We also included 457 controls matched by gender, race and age (±10 years) without any history of cerebrovascular disease. They were recruited from consecutive spouses of the patients with disorders other than SAH (30%), or from the community. All SAH patients and controls were White.
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M.G. Adamski et al. / Biochemical and Biophysical Research Communications 383 (2009) 228–230
We included 71% of all aneurysmal SAH patients. Others died before blood sampling or we were unable to obtain informed consent. SAH was diagnosed by cranial computed tomography (CT) and/ or lumbar puncture. The diagnosis of single or multiple saccular aneurysms was established by digital subtraction angiography or angio-CT. This study was performed according to the Helsinki Declaration with approval of the Ethical Committee of the Jagiellonian University. Informed consent was obtained for each individual before inclusion in this study. Methods. The information regarding gender, age and presence of major vascular risk factors (hypertension, diabetes mellitus, smoking habits and obesity) was collected from aneurysmal SAH patients and controls. An individual was classified as having arterial hypertension if he/she met 1 of the following criteria: (1) the diagnosis of hypertension in the medical history; (2) antihypertensive treatment before entry into the study; or (3) systolic or diastolic blood pressure P 140 mm Hg or P 90 mm Hg, respectively, on at least 2 different occasions (the first 3 days of hospitalization were not considered for the SAH patients). Diabetes mellitus was defined by WHO criteria.[12] Smoking habits were defined as current smokers of P 1 cigarette per day, former smokers, or nonsmokers. For statistical analysis, ‘‘current” and ‘‘former” smokers were pooled together. Obesity was defined as the Body Mass Index P 30 kg/m2. The history was taken from patients or their proxies. To determine patient’s ability to be interviewed, we asked questions about their name, date, and orientation. Irrespective of the information we were able to obtain from the patients, we also questioned all the proxies and studied medical documentation to assemble the most detailed medical history. Family history of SAH was defined as the occurrence of SAH in first-degree relatives. An individual was classified as having hyperlipidemia if he/she met 1 of the following criteria: (1) the diagnosis of hyperlipidemia in the medical history; (2) lipid-lowering treatment before entry into the study; or (3) LDL cholesterol P 3.4 mmol/L or total cholesterol P 5.2 mmol/L. We were not able to collect complete data for all study participants. Absolute numbers are provided in table 1. For the control subjects, data were collected according to a detailed clinical questionnaire, including information on vascular risk factors, current medication, and physical examination at the time of drawing the blood sample for DNA analysis. Uncuffed venous blood samples for extraction of DNA were drawn from each subject within 2 days of stroke onset. Leukocyte DNA was extracted using a commercially available kit (High Pure PCR template Preparation Kit; Boehringer Mannheim). The A1/A2 genotyping for the GpIIIa gene polymorphism was established using PCR and RFLP method, modified from Weiss et al. [13]. Power analysis revealed that a study of this size (288 cases and 457 controls) has 80% power at type I error probability a = 0.05 and a probability of event (joint probability of being GpIIIa polymorphism heterozygote or rare homozygote) in the control group of 29.3% to detect odds ratio lower than 0.6 or higher than 1.58 in the case group. Statistical analysis. Differences in subjects and controls characteristics were tested with Wilcoxon test (age) or with v2 test (categorical variables). The deviation from Hardy–Weinberg equilibrium for tested polymorphisms was examined by v2 test. We also preformed logistic regression analysis in which we calculated odds ratios (OR) with the 95% confidence intervals (95%CI) for selected SAH risk factors. For this analysis we have included only patients (n = 191) and controls (n = 347) with complete data regarding measured variables. A value of P < 0.05 was considered significant. The calculations were performed by software SPSS 10 for Windows.
Table 1 Distribution of risk factors, GPIIIa genotypes in patients and controls. SAH patients (n = 288)
Controls (n = 457)
P-value
Age, years; mean (± SD) Male gender, n (%)
50.9 (±12.9) 162 (56.2)
57.8 (±17.7) 236 (51.6)
<0.001* 0.21
Hypertension, n (%) Yes No Unknown
165 (60.0) 110 (40.0) 13 (4.5)
179 (40.6) 262 (59.4) 16 (3.5)
<0.001
Hipercholesterolemia, n (%) Yes No Unknown
38 (15.0) 215 (85.0) 35 (12.1)
109 (28.7) 271 (71.3) 77 (16.8)
<0.001
Obesity, n (%) Yes No Unknown
21 (10.9) 171 (89.1) 95 (33.0)
82 (18.8) 353 (81.2) 22 (4.8)
0.012
Diabetes mellitus, n (%) Yes No Unknown
20 (7.7) 239 (92.3) 29 (10.0)
43 (9.5) 411 (90.5) 3 (0.6)
0.43
Smoking habits, n (%) Yes No Unknown
128 (50.8) 124 (49.2) 36 (12.5)
160 (35.6) 290 (64.4) 7 (1.5)
<0.0001
GPIII genotypes, n (%) A1A1, n (%) A1A2, n (%) + A2A2, n (%)
201 (69.8) 3 (1.0) + 84 (29.2)
323 (70.7) 134 (29.3)
0.8
P-values for the differences between groups assessed with v2 test or with *Wilcoxon test; SAH, subarachnoid haemorrhage.
Results Demographic data and risk factor profiles of patients and controls are presented in Table 1. Patients and controls did not differ regarding gender or presence of diabetes mellitus. Hypertension and smoking were more prevalent in SAH patients (Table 1). In contrast hypercholesterolemia and obesity were more prevalent in the controls than in the cases (Table 1). SAH patients were significantly younger than controls (Table 1). History of familial SAH was positive in 3.7% (n = 9) and unknown for 15.2% (n = 44) of SAH patients. The allele distribution for the investigated GpIIIa A1/A2 polymorphism did not deviate from Hardy–Weinberg equilibrium for the controls and stroke patients. The frequencies of the studied genotypes were similar in aneurysmal SAH patients and controls (Table 1). We did not find any difference in the distribution of the studied polymorphism neither between males and females, nor after adjusting for common SAH risk factors. Adjusting for size (>7 mm in diameter) and number (P2) of aneurysms did not show difference in the GpIIIa A1/A2 allele distribution in all aneurysmal SAH patients or in male and female patients separately (data not shown). The GpIIIa A1/A2 polymorphism was not a risk factor of aneurysmal SAH in a multivariate analysis (OR = 0.922; 95%CI: 0.783– 1.085). Whereas female gender (OR = 1.950; 95%CI: 1.308–2.907), hypertension (OR = 4.774; 95%CI: 3.048–7.478) and smoking (OR = 2.034; 95%CI: 1.366–3.030) appeared to be independent risk factors of aneurysmal SAH. Discussion We did not find any difference in the distribution of the studied polymorphism between the cases and controls. We also performed
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a separate analysis in males and females, because GpIIIa A1A2 polymorphism had a deleterious, time-dependent impact on overall health and longevity in males and the prevalence of genotypes with A2 allele decreased as a function of male population age [14]. We did not find the impact of gender on the relation between the GpIIIa A1/A2 polymorphism and a risk for aneurysmal SAH. We did not find the relation between the size or number of the aneurysms and the GpIIIa A1/A2 polymorphism. The question arises what could be responsible for the discrepancies between our results and the results of Iniesta et al. [11]. The controls from a general population and controls recruited at traumatology and ophthalmology wards included by Iniesta et al. presented with the similar distribution of genotypes with A2 allele as compared with our controls (Spanish general population controls: 32% vs. Spanish in-hospital controls: 30% vs. our controls: 30.2%, respectively, P > 0,05). Polish and Spanish controls are of the same ethnicity and race so one could expect similar distribution of the genotypes between the two populations. We think that one of the possible reasons for these discrepancies between the conclusions may be derived from difference in selection criteria for the SAH patients. The group of patients with SAH recruited by Iniesta et al. is not homogenous [11]. Only 67% of Spanish patients with SAH presented with an aneurysm (n = 69), while our group consist of 100% aneurysmal SAH patients. It means that 33% of patients with SAH included by Iniesta had non-aneurysmal SAH. The percentage of patients with non-aneurysmal SAH in the study of Iniesta et al. is much higher than that observed in other populations (10%) [1]. In spontaneous/perimesencephalic SAH, the most common etiology of non-aneurysmal SAH, the course and outcome is usually excellent, and the pathophysiology is different [1]. Spontaneous/perimesencephalic SAH might present with different GPIIIa A1/A2 polymorphism distribution. What is more, it is possible that many aneurysmal SAH Spanish patients did not want to participate in the study or died before entering it. This information, however, was not provided by Iniesta et al. [11]. Another possible explanation for inconsistent results may come from the influence of linkage disequilibrium in the GPIIIa A1/A2 region. The GPIII A2 variant might be in linkage disequilibrium with a more functionally relevant variant, not present in our population, or present among spontaneous SAH patients. Our sample size of SAH patients, relatively small in absolute numbers, is one of the largest cohorts of SAH patients participating in genetic case–control studies in Europe. In spite of negative
results, our findings could encourage replication studies in other independent populations. However, the above-mentioned points could be taken into account while designing future case–control association studies. In conclusion, this study does not provide the evidence for the relation of the GpIIIa A1/A2 polymorphism and a risk of aneurysmal SAH in a Polish population. References [1] G. Yong-Zhong, H.A. van Alphen, Pathogenesis and histopathology of saccular aneurysms: review of the literature, Neurol. Res. 12 (1990) 249–255. [2] J.W. Hop, G.J. Rinkel, A. Algra, J. van Gijn, Case-fatality rates and functional outcome after subarachnoid hemorrhage: a systematic review, Stroke 28 (1997) 660–664. [3] B.M. Kissela, L. Sauerbeck, D. Woo, J. Khoury, J. Carrozzella, A. Pancioli, E. Jauch, C.J. Moomaw, R. Shukla, J. Gebel, R. Fontaine, J. Broderick, Subarachnoid hemorrhage: a preventable disease with a heritable component, Stroke 33 (2002) 1321–1326. [4] J.P. Broderick, M.C. Viscoli, T. Brott, W.N. Kernan, L.M. Brass, E. Feldman, L.B. Morgenstern, J.L. Wilterdink, R.I. Horwitz, Hemorrhagic stroke project investigators. Major risk factors for aneurysmal subarachnoid hemorrhage in the young are modifiable, Stroke 34 (2003) 1375–1381. [5] W.T. Longstreth Jr., L.M. Nelson, T.D. Koepsell, G. van Belle, Cigarette smoking, alcohol use, and subarachnoid hemorrhage, Stroke 23 (1992) 1242–1429. [6] S. Juvela, M. Hillbom, H. Numminen, P. Koskinen, Cigarette smoking and alcohol consumption as risk factors for aneurysmal subarachnoid hemorrhage, Stroke 24 (1993) 639–646. [7] J.J. Calvete, Clues for understanding the structure and function of a prototypic human integrin: the platelet glycoprotein IIb/IIIa complex, Thromb. Haemost. 72 (1994) 1–15. [8] D.R. Phillips, I.F. Charo, L.V. Parise, L.A. Fizgerald, The platelet membrane glycoprotein IIb/IIIa complex, Blood 71 (1988) 831–843. [9] M.A. Thornton, M. Poncz, M. Korostishevsky, E. Yakobson, S. Usher, U. Seligsohn, H. Peretz, The human platelet alphaIIb gene is not closely linked to its integrin partner beta3, Blood 94 (1999) 2039–2047. [10] A.H. Goodall, N. Curzen, M. Panesar, C. Hurd, C.J. Knight, W.H. Ouwehand, K.M. Fox, Increased binding of fibrinogen to glycoprotein IIIa-proline33 (HPA-1b PlA2 Zwb) positive platelets in patients with cardiovascular disease, Eur. Heart J. 20 (1999) 742–747. [11] J.A. Iniesta, R. Gonzales-Conejero, C. Piqueras, V. Vincente, J. Corral, Platelet GpIIIa polymorphism HpA-1 (PlA) protects against subarachnoid hemorrhage, Stroke 35 (2004) 2282–2286. [12] K.G. Alberti, P.Z. Zimmet, Definition, diagnosis, and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation, Diabet. Med. 15 (1998) 539–553. [13] E.J. Weiss, P.F. Bray, M. Tayback, S.P. Schulman, T.S. Kickler, L.C. Becker, J.L. Weiss, G. Gerstenblith, P.J. Goldschmidt-Clermont, A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis, N Engl. J. Med. 334 (1996) 1090–1094. [14] M.J. Hessner, D.M. Dinauer, R. Kwiatkowski, B. Neri, T.J. Raife, Age-dependent prevalence of vascular disease-associated polymorphisms among 2689 volunteer blood donors, Clin. Chem. 47 (2001) 1879–1884.