© 1997 American Heart Association, Inc.
Articles |
The Apolipoprotein E and ß-Fibrinogen G/A-455 Gene Polymorphisms Are Associated With Ischemic Stroke Involving Large-Vessel Disease
From the Department of Neurology (C.S., C.K.), Institute of Clinical Chemistry (D.S.), Institute of Physiology (S.M., R.R.), and Institute of Biochemistry (R.W.), Ernst-Moritz-Arndt-University, Greifswald, and the Technology Transfer Unit Biotechnology (J.S., R.W.), Greifswald, Germany.
Correspondence to Christof Kessler, Department of Neurology, Ernst-Moritz-Arndt-University, Ellernholzstrasse 1/2, 17487 Greifswald, Germany. E-mail kessler{at}neurologie.uni-greifswald.de
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Abstract |
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Abstract The relationship between the apolipoprotein E (apoE) and ß-fibrinogen G/A-455 polymorphisms and cerebrovascular disease (CVD) was examined in the present study. We compared 227 patients with the subtypes of CVD (large-vessel disease, lacunar stroke, cardiac embolism, or undetermined pathomechanisms) with 225 control subjects. The occurrence of apoE isoforms (E2, E3, and E4) and the ß-fibrinogen G/A-455 genotype was determined in these individuals. No differences in apoE polymorphisms or allele frequencies between the CVD patients and control subjects were found. However, analysis of apoE genotypes as a function of stroke subtype revealed that the apoE4 allele was significantly more common in those patients with macroangiopathy-associated CVD. The only CVD risk factor that distinguished patients with the E4 allele from those with other apoE genotypes was elevated cholesterol. No association between the ß-fibrinogen G/A-455 polymorphism and CVD was found. However, homozygosity for the A allele was more common in patients with CVD resulting from large-vessel disease. These data demonstrate that the apoE4 allele and the AA genotype of the ß-fibrinogen G/A-455 polymorphism occur significantly more frequently in patients with CVD resulting from stenosis of large, brain-supplying vessels. Such genetic analyses may further our understanding of the etiology of cerebrovascular disease.
Key Words: stroke • genetics • apoE • ß-fibrinogen • polymorphisms
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Introduction |
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Stroke and ischemic heart disease are the major causes of death and disability in industrialized countries.1,2 Classic vascular risk factors, such as arterial hypertension, diabetes mellitus, and cigarette smoking,3,4 are closely associated with the incidence of CVD, whereas the role of hypercholesterolemia is still undetermined.5 CVD is a complex condition influenced not only by conventional vascular risk factors but by genetic factors as well.6,7 Molecular biology has advanced our understanding of diseases at the level of DNA. With regard to cardiovascular and stroke research, genes can directly affect the occurrence of vascular risk factors and the development of atherosclerosis. In the past, several potential gene polymorphisms have been investigated in patients with myocardial infarctions, whereas such information with regard to stroke is rare.
Several studies have dealt with apolipoprotein (apo) E polymorphism and atherosclerosis. ApoE affects hepatic binding, uptake, and catabolism of several classes of lipoproteins and is associated with serum lipoprotein levels.8–10 The gene for apoE is located on chromosome 19; three common polymorphisms designated as E2, E3, and E4 code for the major apoE protein isoforms in the plasma. There is evidence that the apoE2 allele is associated with low LDL levels, whereas the apoE4 allele is associated with high LDL levels. Clinical and postmortem studies have demonstrated a close relationship between the apoE4 allele and the occurrence of myocardial infarction and coronary atherosclerosis.11–13 In patients with cerebrovascular disease, only a limited number of studies have been performed, and these have produced mainly contradictory results.14–16 In one cross-sectional study, the E2 allele was found to be associated with stroke.14 A second, similarly designed, investigation reported a higher frequency of the E4 allele in stroke patients.15 In contrast, E4 was shown to be unrelated to the incidence of stroke in elderly subjects.16
Another important risk factor for cardiovascular disease and stroke is hyperfibrinogenemia.17–21 Fibrinogen influences platelet aggregation as well as blood viscosity. Fibrinogen and its degradation product, fibrin, accumulate in the atherosclerotic plaque at a rate proportional to the level of plasma fibrinogen.22 There are 10 known ß-fibrinogen polymorphisms, and recently the ß-fibrinogen G/A-455 (ß Hae III) polymorphism was reported to influence plasma fibrinogen concentrations.23–25 As demonstrated clinically, this polymorphism is associated with coronary atherosclerotic disease,26,27 as well as myocardial infarction.28,29 Up to now, no study has been done to explore the association between ß-fibrinogen G/A-455 polymorphism and the occurrence of CVD.
In the present case-control study, we determined the distribution of both the apoE and the ß-fibrinogen G/A-455 polymorphisms in a cohort of patients with CVD.
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Methods |
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Patients
Blood samples were collected from a consecutive series of 227 CVD patients admitted to the stroke unit of the Department of Neurology. We included only patients with neurologic symptoms resulting from focal cerebral ischemia and excluded patients with intracerebral hemorrhage. Patients with stroke resulting from vasculitis or migraine were also excluded. All CVD patients underwent ultrasound sonography (both extracranial and transcranial), CT or MRI, and ECG. Transesophageal echocardiography and digital subtraction angiography were performed optionally. On the basis of clinical symptoms, CT/MRI findings, and cardiac diagnostic criteria, the patients were assigned to the following CVD categories: (1) large-vessel disease—stroke due to significant (>70% stenosis) of the extracranial symptomatic artery; (2) lacunar stroke—due to small deep infarctions, the location of the lacunae seen in the CT/MRI matched the clinical symptoms; (3) cardiac embolism—source positively diagnosed by EKG or transesophageal echocardiography; or (4) other—eg, arterial dissection or unknown pathomechanism despite intensive investigation. In all patients we also measured blood pressure and obtained, via routine laboratory examination, plasma levels of lipids, fibrinogen, fasting glucose, hematocrit, and red and white cell counts. Fibrinogen determinations were made using the standard technique after von Clauss.30 Diluted citrate plasma was titrated with high concentrations of thrombin. The onset of clotting is proportional to fibrinogen concentrations.
In the CVD group, 42 (18.5%) of the 227 patients had transient focal symptoms (transient ischemic attack or persisting reversible ischemic neurologic deficit) and 185 (81.5%) had a completed stroke. CVD causes were large vessel atherosclerosis in 70 (31%) patients, lacunar stroke in 34 (15%) patients, cardiac embolic sources in 53 (23%) patients, and in 69 (30%) patients, the cause remained undetermined.
Control Subjects
The CVD patients were compared with a control group of 225
inpatients showing no history of CVD or coronary heart disease.
Persons with other conditions in which vascular pathologic conditions,
might play a role, eg, migraine or arteriovenous malformations, were
excluded from the control group. Another exclusion criterion was a
definite or probable diagnosis of Alzheimer's disease, which
is known to be associated with the E4
allele.31 After initial screening of
potential control subjects, a pool of consenting control subjects was
created. At the end of the study, a matched control subject was
selected from this pool for each CVD patient. CVD patients and control
subjects were matched for sex and age. All participants were whites,
who provided informed consent as a precondition for participation in
the study.
Of the 452 participants in the present study, 216 were men (108
both in the CVD sample and the control group) and 236 were women (119
CVD patients and 117 control subjects). The mean age of the CVD group
was 62.3±14.2 years and of the control group was 62.6±14.0 years.
Characteristics of the two groups are presented in Table 1
.
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DNA Preparation and Genotyping
DNA was prepared by a modification of the method of Miller et
al32 from whole blood. After erythrocyte lysis,
lymphocytes were recovered and lysed, and the DNA was liberated by
proteinase K digestion. Protein was subsequently removed by
precipitation with NaCl, and the DNA was recovered by ethanol
precipitation.
For typing of the common apoE isoforms (E2, E3, and E4), amplification of apoE sequences and restriction enzyme isotyping were carried out by a modification of the method of Hixson and Vernier33 Amplified apoE sequences containing nucleotide substitutions that result in arginine-cysteine interchanges in different alleles at positions 112 and 158 yielded different c-fos I digestion fragments. Electrophoresis of the digested samples was performed on a 15% polyacrylamide gel with detection of the restriction fragments by silver staining. For ß-fibrinogen G/A-455 polymorphism genotyping, the 5'-flanking region and the first exon of the ß-fibrinogen gene were enzymatically amplified using PCR and oligonucleotides, as described previously.34 The amplified fragment contains a common restriction site for Hae III, while the presence of guanine nucleotide at position-455 creates an additional Hae III restriction site. Digestion of PCR products with the Hae III isoschizomer BsuRI (MBI Fermentas) was performed according to the manufacturer's instructions. The DNA fragments were separated by electrophoresis through a 2% agarose gel containing 0.5 µg/mL of ethidium bromide and visualized under UV light. Digestion of the PCR products gives bands of 343 and 383 bp in GG-455 homozygotes, 343 and 958 bp in AA-455 homozygotes, and all four bands in the heterozygotes. The G-455 and A-455 alleles were previously designated H1 and H2, respectively.
Statistics
Data analyses were performed with the Statistical
Package for the Social Sciences (SPSS PC+, version 4.0) software. Data
are expressed as mean±SD. Nonparametric procedures and
2 test were performed where appropriate. For all tests, the
significance level was established at a value of
P
.05.
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Results |
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Participant Characteristics
As depicted in Table 1
, hypertension and diabetes mellitus were
significantly more frequent in the CVD group than in the control
subjects (2=43.4, P.001 and 2=5.0,
P.024, respectively). Smoking status (both current and
ex-smokers) was also more frequent in CVD patients than in control
subjects (both categories together: 2=10.5, P.002).
Other possible risk factors such as high cholesterol
(defined as known high levels of plasma cholesterol,
current medication with lipid-lowering drugs, and/or plasma
cholesterol at admission 
6.5 mmol/L) or a
positive family history for cardiovascular and
cerebrovascular events in first-degree relatives were not significantly
more common in the CVD group than in control subjects.
ApoE Genotypes
As shown in Table 2, there was no
significant difference in the apoE polymorphism genotypes
or allele frequencies between the CVD patients and the control
subjects. Individuals with the E4 allele were not more frequently
represented in the entire CVD sample than in the control
group. However, analysis by subtype of CVD showed that the E4
allele was more frequent in CVD patients with large-vessel
atherosclerosis (2=4.8, P.029). This
was not found for any other CVD subtype.
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Participants carrying the E4 allele (n=113; 25.0%) did not show
significantly increased frequencies of a positive family history of
cardiovascular or cerebrovascular events, hypertension,
diabetes mellitus, or smoking (Table 3.).
A high cholesterol level was significantly more common in
E4 carriers (2=42.0, P.001). The level of plasma
cholesterol was significantly higher in individuals with
the E4 allele, compared with those without this allele
(6.92±1.48 versus 6.07±1.51 mmol/L; z=-5.2,
P.001).
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ß-Fibrinogen Genotype
The ß-fibrinogen G/A 455 polymorphism was unable to be
genotyped in 15 participants (3.3%) because blood samples were
not available. Subsequent analysis is, therefore, based on the
remaining 437 individuals.
We found no association between the ß-fibrinogen G/A-455
polymorphism and CVD, as depicted in Table 4. When the sample was stratified
according to AA and GA/GG genotypes, however, homozygosity for
the A allele was more common in patients with CVD resulting from
large-vessel disease (2=4.0, P.045). No such
relationship was found for any other subtype of CVD.
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None of the conventional risk factors was significantly associated with
the A allele (cf. Table 3). Fibrinogen levels tended to be higher
in participants carrying the A allele, but did not reach
statistical significance. Fibrinogen levels were significantly higher
in CVD patients than in control subjects (3.57 versus 3.20 g/L;
z=-2.6, P<.01). Fibrinogen was also not
significantly higher in smokers (3.62 g/L) than in
nonsmokers (3.32 g/L). The highest concentration of fibrinogen
was observed in smokers carrying the A allele (3.76 g/L).
Within this group, CVD patients with large- vessel
atherosclerosis had the highest level of fibrinogen
(3.99 g/L).
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Discussion |
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We studied the association between two genetic polymorphisms, the apoE polymorphism and the ß-fibrinogen gene 455-G/A polymorphism and CVD. Our results suggest that these genetic markers distinguish a subgroup of CVD patients, specifically those with macroangiopathy. The apoE polymorphism is a major determinant of interindividual variation in the initiation and progression of atherosclerosis.12,35,36 This is especially true for the occurrence and severity of coronary atherosclerotic disease.16,36 However, its association with stroke has only been investigated in a small number of studies, which have yielded mixed results.14–16 It is likely that these conflicting results are due to methodologic differences, eg, no differentiation between thromboembolic or hemorrhagic stroke.16 Moreover, these studies failed to subdivide stroke patients according to their pathogenesis. Such a differentiation is important because there are different pathogenic mechanisms involved in cerebral ischemia. For example, stroke resulting from large-vessel disease usually is the consequence of arterioarterial embolism, whereas cardiac embolism is often due to atrial fibrillation unrelated to atherosclerosis. Lacunar stroke and cerebral microangiopathy, on the other hand, can also result in cerebral ischemic symptoms.37
Our results do not indicate an association between CVD, in general, and the apoE polymorphism, because the genotype distribution did not distinguish CVD patients and control subjects. As in other studies,14 the E4/E4 genotype was less frequent in CVD patients, resulting in an increase in other genotypes, with the exception of the E4/E2 genotype, compared with control subjects. It has been previously suggested that the E3/E2 genotype is associated with cerebral ischemia, because it was found in 10.1% of CVD patients and in only 1.4% of control subjects.14 In contrast, our study found the E3/E2 genotype in 13.7% of CVD patients and in 10.7% of control subjects. Comparing our data with recently published studies on genotype frequencies of the apoE polymorphism,31,36 which reported prevalences of 8.8% and 9%, respectively, suggests that our data may be more representative of the white population. Allele frequencies in the control groups of our study differ from those of an earlier study,14 although genotype distribution in the CVD patients is nearly identical.
We found that the E4 allele is more common in CVD patients with large-vessel atherosclerosis than in those with other types of CVD. This finding supports a previous study reporting that E4 carriers have a significantly increased segment-specific extracranial carotid artery intima-media thickening, as measured by B-mode ultrasound, thus indicating more atherosclerosis.38 In contrast, a study of participants free of clinically manifest CVD suggested that the E3/2 and not the E4 genotype is associated with preclinical carotid atherosclerosis.39 In addition, a neuropathologic postmortem examination failed to confirm an association between the E4 allele and cerebral atherosclerosis in patients with Alzheimer's disease.11 Perhaps, methodologic differences can, again, best account for these apparently discrepant findings.
As in other studies,9,10,12,13,37,40 we observed that individuals carrying the E4 allele had a significantly higher level of total plasma cholesterol. No such relationship was found for the level of triglycerides, in accordance with other investigations.41,42 We know from epidemiologic studies that high cholesterol levels are closely related to myocardial infarction, whereas the role played by hypercholesterolemia in CVD is still under discussion.5 It may have no direct influence on stroke, but in the Multiple Risk Factors Intervention Study,43 ischemic stroke correlated positively with cholesterol levels in 450 000 men, and cerebral hemorrhage showed an inverse correlation. This example illustrates the importance of regarding "stroke" not as an unique entity but as the sequelae of multiple diseases. Despite these uncertainties, we know that atherosclerosis of the large, brain-supplying vessels and intracerebral arteries remains the most common cause of cerebral ischemia. Moreover, we have evidence that the apoE locus modulates the susceptibility to atherosclerosis in a complex multifactorial interaction.33,35 Our results provide support for the concept that the apoE polymorphism, and consequently the related lipids and lipoproteins, play an important role in cerebral atherogenesis.
Elevated fibrinogen levels are a well-known and important cardiovascular and cerebrovascular risk factor.17–21 It is reasonable, then, to expect genetic factors which account for variability in plasma fibrinogen concentrations, such as the ß-fibrinogen G/A-455 polymorphism,24–28 to be associated with vascular disease. Our study is the first to investigate this relationship. We did not find an association between CVD, in general, and the ß-fibrinogen polymorphism. However, analyses by subtypes of CVD yielded a statistically significant association between this polymorphism and large-vessel atherosclerosis. This particular association might possibly be mediated by the complex process of atherothrombosis: fibrinogen is fundamentally involved in platelet aggregation and blood rheology, and thus plays a major role in thrombogenesis.17–19 Moreover, there is also evidence indicating that fibrinogen promotes atherosclerosis.17,23
We found higher concentrations of fibrinogen in our CVD patients than in control subjects, supporting earlier reports.18,20 Fibrinogen was also higher in smokers and participants carrying the A allele, again confirming previous work.34 The highest levels of fibrinogen were found in CVD patients with large-vessel atherosclerosis who both smoked and carried the A allele. This finding indicates that there may be a complex interaction between smoking and the G/A-455 polymorphism, which results in elevated fibrinogen concentrations and eventually the promotion of atherosclerosis.
In conclusion, our data demonstrate that the apoE4 allele of the apoE polymorphism and the AA genotype of the ß-fibrinogen G/A-455 polymorphism are more common in the large-vessel atherosclerosis subtype of CVD. Elucidation of the functional relationship between gene polymorphisms, the related proteins, and their potential contribution to the pathogenesis of atherosclerotic CVD will facilitate the development of rational, cost-effective prevention strategies.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported, in part, by the Community Medicine Program of the Medical Faculty, Ernst-Moritz-Arndt-University Greifswald.
Received May 13, 1997; accepted August 27, 1997.
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 J. P. Casas, A. D. Hingorani, L. E. Bautista, and P. Sharma Meta-analysis of Genetic Studies in Ischemic Stroke: Thirty-two Genes Involving Approximately 18 000 Cases and 58 000 Controls Arch Neurol, November 1, 2004; 61(11): 1652 - 1661. [Abstract] [Full Text] [PDF]
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P. Jerrard-Dunne, G. Cloud, A. Hassan, and H. S. Markus Evaluating the Genetic Component of Ischemic Stroke Subtypes: A Family History Study Stroke, June 1, 2003; 34(6): 1364 - 1369. [Abstract] [Full Text] [PDF]
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M. Martiskainen, T. Pohjasvaara, J. Mikkelsson, R. Mantyla, T. Kunnas, P. Laippala, E. Ilveskoski, M. Kaste, P.J. Karhunen, and T. Erkinjuntti Fibrinogen Gene Promoter -455 A Allele as a Risk Factor for Lacunar Stroke Stroke, April 1, 2003; 34(4): 886 - 891. [Abstract] [Full Text] [PDF]
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 J. B. Braunstein, D. W. Kershner, P. Bray, G. Gerstenblith, S. P. Schulman, W. S. Post, and R. S. Blumenthal Interaction of Hemostatic Genetics With Hormone Therapy : New Insights To Explain Arterial Thrombosis in Postmenopausal Women Chest, March 1, 2002; 121(3): 906 - 920. [Abstract] [Full Text] [PDF]
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 L. Iacoviello, M. Vischetti, F. Zito, and M. Benedetta Donati Genes Encoding Fibrinogen and Cardiovascular Risk Hypertension, November 1, 2001; 38(5): 1199 - 1203. [Abstract] [Full Text] [PDF]
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F. Harrington, B. K. Saxby, I. G. McKeith, K. Wesnes, and G. A. Ford Cognitive Performance in Hypertensive and Normotensive Older Subjects Hypertension, December 1, 2000; 36(6): 1079 - 1082. [Abstract] [Full Text] [PDF]
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 T. J. Tegos, E. Kalodiki, S.-S. Daskalopoulou, and A. N. Nicolaides Stroke: Epidemiology, Clinical Picture, and Risk Factors: Part I of III Angiology, October 1, 2000; 51(10): 793 - 808. [Abstract] [PDF]
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M. O. McCarron, K. W. Muir, J. A. R. Nicoll, J. Stewart, Y. Currie, K. Brown, and I. Bone Prospective Study of Apolipoprotein E Genotype and Functional Outcome Following Ischemic Stroke Arch Neurol, October 1, 2000; 57(10): 1480 - 1484. [Abstract] [Full Text] [PDF]
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 A. Hassan and H. S. Markus Genetics and ischaemic stroke Brain, September 1, 2000; 123(9): 1784 - 1812. [Abstract] [Full Text] [PDF]
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Y. Kokubo, A. H. Chowdhury, C. Date, T. Yokoyama, H. Sobue, and H. Tanaka Age-Dependent Association of Apolipoprotein E Genotypes With Stroke Subtypes in a Japanese Rural Population Stroke, June 1, 2000; 31(6): 1299 - 1306. [Abstract] [Full Text] [PDF]
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M. O. McCarron, D. Delong, and M. J. Alberts APOE genotype as a risk factor for ischemic cerebrovascular disease: A meta-analysis Neurology, October 1, 1999; 53(6): 1308 - 1308. [Abstract] [Full Text]
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M. O. McCarron, K. W. Muir, C. J. Weir, A. G. Dyker, I. Bone, J.A.R. Nicoll, and K.R. Lees The Apolipoprotein E {epsilon}4 Allele and Outcome in Cerebrovascular Disease Stroke, September 1, 1998; 29(9): 1882 - 1887. [Abstract] [Full Text] [PDF]
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