Herein we describe the protocol of an ongoing prospective, multicenter study, the Ischemic Stroke Genetics Study (ISGS). This study uses a candidate gene approach in which rates of variant polymorphisms of the candidate genes are compared between patients with ischemic stroke and stroke-free control subjects.
Selection of Candidate Genes for ISGS
The candidate genes for this study include the gene encoding β-fibrinogen and the genes encoding platelet glycoprotein (GP) receptors Ia/IIa, Ib/IX/V, and IIb/IIIa. In selecting candidate genes for an association study, effects of polymorphisms on structure, function, or expression of a gene product should be considered. Failure to consider the underlying pathophysiologic mechanism when searching for polymorphisms associated with stroke might result in mistaking association for causation [2
]. We studied genes related to thrombosis because the importance of thrombosis in acute ischemic stroke has been established conclusively in numerous clinical trials of treatment and prevention [3
]. We focused on genetic variations in the fibrinogen gene cluster because of the efficacy of fibrinolytic agents in the acute treatment of ischemic stroke. In addition, we studied genes encoding for platelet receptors because of the efficacy of platelet anti-aggregant therapy in preventing first-time and recurrent ischemic strokes.
To restrict the choice of polymorphisms worthy of further study, we constructed an evidence table from reports appearing in MEDLINE-indexed English language journals describing cross-sectional or longitudinal studies of at least one thrombosis gene polymorphism in at least 100 patients with stroke. Results were classified as positive or negative according to whether a significant association (P < 0.05) was found between stroke (or carotid atherosclerosis) and a polymorphism. Because it is biologically plausible that a prothrombotic polymorphism may exert a differential effect across different ages, sexes, and ethnic groups, we classified studies as having positive results even if they had only one positive subgroup. We considered a polymorphism worthy of further study if it was not already a clearly established stroke risk factor and if at least one association study was positive.
Regarding a possible relationship to stroke risk, most studies of hemostasis genes have been inconclusive at best and unconvincing at worst. On the basis of the evidence, we concluded that the polymorphisms of factor VII R353Q, factor XIII Val34Leu, plasminogen activator inhibitor-1 4G/5G, and prothrombin G20210A were not worthy of further investigation because large studies had consistently yielded negative results (Table ). For similar reasons, we decided not to study factor V R506Q (G1691A; i.e., the factor V Leiden mutation), despite its apparent association with cerebral vein thrombosis [9
]. Although unknown point mutations in the coding regions of these genes may relate to stroke and relevant variations in gene expression elements may exist, we decided to focus on more immediately high-yield candidate genes.
Association Studies of Thrombosis Genes in Ischemic and Carotid Atherosclerosis
The results of three large European studies listed in Table led us to conclude that the β-fibrinogen gene might be a promising candidate. Fibrinogen is a 340,000-Da GP consisting of three polypeptide chains: α, β, and γ. The genes that encode these polypeptides reside on chromosome 4q in a cluster. In a study of the β-fibrinogen G455A polymorphism, Kessler et al. [10
] did not find an overall association between genotype and stroke, but heterozygosity for the A allele was associated with large-vessel ischemic stroke (P
= 0.045). Schmidt et al. [11
] observed an association between carotid atherosclerosis and the C148T polymorphism in a population-based cross-sectional study of persons with normal neurologic status. Carotid atherosclerosis was seen in 53.6% of persons with the C/C genotype, 54.1% of those with the C/T genotype, and 88% of those with the T/T genotype (P
= 0.003). Abnormal results on carotid ultrasonography were significantly more common in the T/T genotype group (OR, 6.29; 95% CI, 1.91 to 20.71). Data from the study by Carter et al. [12
] on the G448A polymorphism of the β-fibrinogen gene suggested that mechanisms linking fibrinogen and the development of cerebrovascular disease may be different in men and women.
Several studies listed in Table suggested that polymorphisms of genes controlling the three platelet glycoprotein receptors Ia/IIa, Ib/IX/V, and IIb/IIIa, which play a role in adhesion, might also be promising candidate risk factors for stroke. GP Ia/IIa (integrin α2
) is involved in collagen-induced platelet aggregation. It does not bind collagen monomers, but it does bind collagen fibrils and immobilized collagen. Binding of GPIa/IIa to collagen induces a conformational change in receptor structure that enhances affinity. Thus, one platelet GP of interest is GPIa. Carlsson et al. [13
] compared the GPIa (α2
) C807T genotype distribution in patients with ischemic stroke or transient ischemic attacks with that in hospitalized patients without cerebrovascular disease and in healthy blood donors. An association between the polymorphism and stroke was not seen overall. However, there was an overrepresentation of the C807T polymorphism in patients with stroke age 50 years or younger (n
= 45) versus age-matched controls (OR, 3.02; 95% CI, 1.20 to 7.61). No such overrepresentation was detected in older patients.
The second platelet GP of interest is GPIbα, a transmembranous platelet GP (molecular weight, 143,000) that forms noncovalent complexes with GPIbβ, GPIX, and GPV to form the GPIb/IX/V receptor, which is involved in shear stress-induced platelet activation by binding to von Willebrand factor (vWF). This receptor may be particularly relevant in large-vessel atherosclerotic ischemic stroke because high shear stresses like those seen in atherosclerotic arteries increase ligand-receptor affinity. The receptor may also have a role in so-called aspirin failure, in which patients suffer stroke despite taking daily aspirin prophylaxis. Cyclooxygenase inhibition by aspirin has little effect on initial aggregation in response to shear forces. One GPIbα polymorphism is referred to as "VNTR" because it consists of a variable number of tandem repeats of 39 base pairs, each repeat leading to a 13-amino acid addition that pushes a vWF-binding domain further away from the platelet membrane surface. Another is human platelet antigen-2 (HPA-2), a mutation that codes for either a thr (HPA-2a) or met (HPA-2b) at position 145. The HPA-2 site resides next to the vWF and high-affinity thrombin binding sites.
In a case-control study of these polymorphisms, Gonzalez-Conejero et al. [14
] found that cerebrovascular disease was associated with both the C/B genotype of the VNTR polymorphism (OR, 2.83; 95% CI, 1.16 to 7.07; P
= 0.0114) and the β allele of the HPA-2 polymorphism. Of the 104 patients with cerebrovascular disease, 22.11% carried at least one β allele compared with 10.58% of controls (OR, 2.40; 95% CI, 1.04 to 5.63; P
= 0.0244). Neither polymorphism showed significant differences related to age, sex, or type of cerebrovascular disease. Both polymorphisms also correlated with coronary artery disease, but neither correlated with deep vein thrombosis. This is the converse of what Ridker et al. [15
] and others found for factor V Leiden. Taken together, the studies suggest that polymorphisms predisposing to arterial thrombosis may differ from polymorphisms predisposing to deep vein thrombosis. This hypothesis supports the rationale for a hemostasis candidate-gene association study such as ISGS, which investigates ischemic stroke specifically and does not regard all acute thrombotic events, whether arterial or venous, as a single clinical entity.
The third candidate platelet GP gene controls GPIIb/IIIa (integrin αIIb
), a transmembranous heterodimer with several ligands, including fibrinogen, fibrin, fibronectin, and vWF. Many receptors are involved in platelet adhesion and many agonists stimulate platelet aggregation, but platelet aggregation requires GPIIb/IIIa. When platelets aggregate, GPIIb/IIIa binds to fibrinogen and vWF. Binding to vWF gains importance under conditions of high shear stress. Carter et al. [16
] found no overall association between the P1A2 polymorphism of the GPIIb/IIIa gene and cerebral infarction confirmed by computed tomography (CT). However, a subgroup analysis showed significant genotype distribution differences in nonsmokers. The risk of stroke was greater in nonsmokers heterozygous for the P1A2 allele than in those homozygous for P1A2 (OR, 2.37; 95% CI, 1.19 to 4.74; P
= 0.01). Information on young stroke patients was limited (n
= 37), but in a logistic regression model that included P1A genotype status, smoking, hypertension, and diabetes, the OR for stroke in those possessing the A2 allele was 1.68 (95% CI, 1.00 to 2.82; P
= 0.05). This study highlights the need for further studies of the interaction between genes and environmental factors, in this case smoking, in attempts to elucidate inherited stroke risk.