In this study, we investigated the association of G protein β3 subunit gene (GNB 3) C825T polymorphism with ischaemic stroke and its subtypes in the Chinese Han population in a large case–control study. A total of 990 ischemic stroke patients and 1124 controls were recruited from six medical centres in China. Genotype was determined by polymerase chain reaction and restriction fragment length polymorphism (RFLP) assay. Logistic regression analysis was performed to identify the independent risk factors for stroke. The frequency of 825T carriers is significantly higher in cerebral thrombosis in male subjects (OR=1.35, 95% CI, 1.01–1.82, P=0.046). After further adjustment with traditional risk factors to stroke, the association is not significant. In conclusion, the GNB3 825T allele is not an independent risk factor to ischaemic stroke in the Chinese population.
Stroke is a major disorder that leads to mortality and morbidity all over the world. However, the epidemic pattern of stroke varies between the Chinese and Western population.1 Each year, more than 1 million residents die from stroke in China.2 Among those cases, haemorrhage strokes are as many as ischaemic strokes, while the latter subtype predominates in white subjects.3 Since stroke is a complex disease involving many risk factors, environmental influence and gene background must be taken into account in order to clarify the aetiology of this disease. Heterotrimeric guanine nucleotide-binding proteins (termed G proteins) are ubiquitously expressed, essential components that mediate signal transduction from a big family of receptors with seven transmembrane segments to several distinct intracellular signalling pathways controlling many cellular processes.4 The β3 subunit of G proteins is encoded by the GNB3 gene, which is located on chromosome 12p13. In 1998, Siffert et al5 discovered a novel C825T polymorphism in exon 10 of GNB3, and observed that the T allele was linked with a splice variant, which, by deletion of 41 amino acids, leads to increased G-protein activation. This may explain the T allele's association with enhanced cardiac potassium channel activity and α2-adrenoceptor-mediated vasoconstriction. Numerous studies have investigated the role of this SNP in various disorders, and now the 825T allele is considered to be an independent risk factor for essential hypertension in white subjects, despite the fact that it remains controversial in other ethnic groups.6, 7, 8 Pathophysiological and epidemiological study established hypertension as an essential risk factor for stroke;9 indicating C825T may play a role in the pathogenesis of clinical stroke.
Materials and methods
As a multicentre study sponsored by the Ministry of Science and Technology of China (973 Project) for assessment of risk factors in stroke, the study protocol was reviewed and approved by the review board of the Ministry of Public Health, Ministry of Science and Technology of China, and admission was gained from the ethics committees at all participating hospitals, while informed consent was obtained from all individuals.
Cases and controls were recruited simultaneously from the same clinical centres located in six provinces in China (Beijing, Chongqing, Shandong, Tianjin, Wuhan, and Xi'an). Two subtypes of ischaemic stroke were in the range of selection: cerebral thrombosis (thrombosis) and lacunar infarction (lacunar), whereas patients suffered from other types of stroke (transient ischaemic attack, subarachnoid haemorrhage, embolic brain infarction, brain tumours, cerebrovascular malformation) or had severe systemic diseases (such as collagenosis, endocrine or metabolic disease: inflammation, liver, neoplastic or renal disease, not including diabetes mellitus) were excluded from the study.
Stroke cases were determined by strict neurological examination: CT, MRI or both. The criterion of diagnosis was according to the International Classification of Disease (ninth revision). Study controls consist of community-based inhabitants and in-patients with minor illnesses from the departments of ophthalmology, gastroenterology, otorhinolaryngology and orthopedics. All controls were free of neurological diseases following the same exclusion criterion as cases. At each locality, people aged from 35 to 74 years old were recruited; afterwards, the population was grouped by gender and age (5-year range within each group). Once one case was enrolled, one control was randomly paired with it from the corresponding group.
At the beginning of the present study, 1300 cases and 1300 controls were recruited. After that, 141 subjects (68 controls, 53 thrombosis cases and 20 lacunar cases) were excluded due to the lack of materials integrity, such as missing body mass index, total plasma cholesterol, triglycerides, etc; 345 individuals (155 controls, 117 thrombosis cases, 73 lacunar cases) were removed from the study because of the failure of genotyping. A total of 990 cases consisted of 602 thrombosis cases and 388 lacunar infarction cases.
Traditional risk factors for stroke were also recorded: body mass index (BMI), total plasma cholesterol (TC), triglycerides (TG), blood pressure (SBP and DBP) and the presence of diabetes mellitus (fasting glucose ⩾7.8 mmol/l or with diabetes mellitus DM history).
Genomic DNA was extracted from peripheral white blood cells.10 A 206-bp fragment containing the GNB3 C825T was amplified by polymerase chain reaction, with the following primers: IndexTerm5′-CCTGACCCACTTGCCACCC-3′ and IndexTerm5′-GAAGTCGTCGTAGCCAGCGA-3′. The procedure started with a 4 min-denature step at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 65°C, 30 s at 72°C, ended with 10 min at 72°C. The 825C generates a BsedI restriction site, and digestion of the PCR product will engender 2 fragments (106 and 50 bp) which can be separated by 3.5% agarose gel electrophoresis. Stained with ethidium bromide, the digested products were analysed under UV light.
To examine the agreement of the GNB3 C825T genotype frequency, Hardy–Weinberg equilibrium expectation was tested with a χ2 goodness-of-fit test. Normal distribution of data was examined by the Kolmogorov–Smirnov normality test. Unpaired Student's t-test was used to inspect the difference of those numeric values that obeyed normal distribution between groups; otherwise the Mann–Whitney U-test was carried out. Relative risk analysis was performed with 2 × 2 cross-tabulation (crude OR) and a binary logistic regression model (adjusted OR). All statistics were performed with the SPSS 10.0 package. The criterion for statistical significance was P<0.05.
A total of 2114 subjects were embodied in this study. Distribution of C825T genotype was in the Hardy–Weinberg equilibrium, while no difference was observed between male and female subjects (Table 1).
Clinical features of study participants are shown in Table 2. The age is shown as mean±s.d., exhibiting 61.5±9.0 years in cases and 60.8±7.6 years in controls. Male occupied 63.4% in cases and 56.0% in controls (significantly different). Compared with controls, stroke cases had a higher prevalence of traditional vascular risk factors (higher BP, glucose and TG level, alcohol intake and incidence of DM, lower TC level).
C825T with stroke risk factors
T-allele frequency showed no difference between controls and any stroke category. According to previous studies on the biological effect of the GNB3 825T allele, one T allele was sufficient to express considerable levels of the more active splice variant. Thus, CT and TT genotypes were grouped together for further analysis.11, 12 Genotypes with some traditional stroke risk factors are shown as cross-tabulation excel in Table 3. As observed, there was no difference on T-allele frequency between hypertensive patients and normotensive individuals in controls and other stroke subtypes.
C825T with stroke and its subtypes
The SNP was not associated with unclassified ischaemic stroke in our study. When processing analysis between stroke subtypes and controls, the T allele was found to be positively associated with thrombosis in male subjects (OR=1.35, 95% CI, 1.01–1.82, P=0.046), whereas no association was discovered in lacunar cases in spite of grouping by gender (data not shown). Further adjusted for age, smoking, alcohol intake, TC, TG, BMI, hypertension and DM by multivariate logistic regression analysis, T allele was no longer associated with thrombosis in male subjects (Table 4). In an ungrouped analysis, smoking, DM, TG level and hypertension were strong risk factors for thrombosis, while TC level and BMI were protective factors (data not shown). This is consistent with what we observed in lacunar cases.
The present study is the first attempt to identify the association between the GNB3 825T allele with stroke and its subtypes in the Asian population. Numerous studies showed that hypertensive individuals suffered a higher risk stroke. Moreover, intensive trends of familial aggregation were observed in the incidence of both hypertension and stroke. The above suggests that the two diseases may share some common genetic pathways in their aetiology. As a candidate gene contributing to essential hypertension, GNB3 was highlighted in corresponding studies. It encodes a key component of G protein, which is highly expressed and broadly distributed.4 Most intracellular signal transmissions involve G protein-coupled receptors, and the GNB3 825T allele was discovered to be associated with increased G-protein activation (such as cardiac potassium channel activity and α2-adrenoceptor-mediated vasoconstriction). Meanwhile, T-allele carriers were reported to have enhanced chemokine-stimulated chemotaxis in lymphocytes, which also indicates the role of GNB3 825T in neuroregulative and humormodulatory process on vessel physiology4 (Table 5).
In previous investigations, GNB3 825T allele was considered to be an independent risk factor to essential hypertension in white subjects. However, we did not find such association between this SNP and hypertension in our study population (Chinese).8 We consider the various conclusions as the results of the difference in genetic background of study participants. The frequency of T allele varies in different ethnic groups (highest in blacks, lowest in white subjects, and intermediate in Asians).13 Studies tended to observe significant association of 825T allele with hypertension in the samples with a high prevalence of the 825C allele. Meanwhile, environmental factors can not be excluded. A hypothesis regards the 825T allele as a ‘thrifty’ allele. It made achievements in ancestral environments and became deleterious when sufficient improvements on nutrient conditions or certain dietetic habits were established. The Chinese have different dietary customs from Western people with a markedly high proportion of carbohydrates, which may help people escape from the ‘harm’ of the 825T allele.
Among all vascular morbidities, atherosclerosis contributes most to clinical stroke. As the T allele was reported to be associated with enhanced epinephrine-induced platelet aggregation, it may have a connection with the occurrence of atherosclerosis, which will help the allele to affect the pathogenesis of ischaemic stroke and haemorrhage stroke. Moreover, Brand et al14 reported the presence of higher haematocrit and erythrocyte count in 825T-allele carriers, which could lead to increased blood viscosity and easier formation of thrombus. This provides the 825T allele with a second pathway to affect the incidence of stroke independently.
Before the present study, Morrison et al15 detected its association with ischaemic stroke in a cohort study. This is not consistent with our result.8 The inconsistency may due to the sample size. In our study, 990 cases and 1124 controls were recruited, with strict selection criteria and definite diagnosis, and it overwhelmed the previous study. The large scale is enough to gain adequate statistical power and robust results.16 Additionally, participants varied on age (60.8 vs 53.9 in controls and 61.5 vs 56.5) and proportion of sex significantly between the two studies, which may contribute to varied conclusions. However, the difference on genotype frequency of study participants may be a more persuasive explanation. Morrison et al15 found an association of the GNB3 825T allele with ischaemic stroke in white subjects rather than in black subjects. We noticed a significant distinction on distribution of genotypes between the white group and the black one. The sample with low T-allele frequency again showed association with a certain phenotype. In our hypothesis, T allele is not an independent marker in stroke pathological pathways. Recent studies concluded that C825T was in strong linkage disequilibrium with several additional polymorphisms in the GNB3 gene, constituting two major haplotypes in the white population. As reported, Africans show greater genetic diversity than other races. The high diversity of gene loci may disturb the assembling of certain haplotype. Compared with black subjects, white subjects are more common to achieve certain haplotypes, which results in the expression of the splice variant Gβ3s and association with stroke, hypertension, etc. Such an interpretation may apply to our discovery in the Chinese population as well, for the frequency of T allele in Asians is much higher than in white subjects.
Moreover, gene–gene interaction should be considered. Yamamoto et al17 reported that the C825T polymorphism interacted with the angiotensin I-converting enzyme insertion/deletion polymorphism in the association with myocardial infarction. As a consequence, the association is not determined by 825T alone, and other genetic factors should be taken into account.
Our study also found systolic pressure, the presence of DM and high plasma level of triglycerides were strong independent risk factors to ischaemic stroke, and this is in accordanance with other studies identifying risk factors in stroke. Total plasma cholesterol may be a protective factor in ischaemic stroke (P=0.052). In a comparison on general index among different genotype groups, TT carriers had lower total plasma cholesterol level, indicating their higher risk to ischaemic stroke and its subtypes.
In conclusion, as a representative investigation in Asians, our study did not observe any association between the GNB3 825T allele and ischaemic stroke. Since pathogenic mechanisms differ among stroke subtypes, it is necessary to evaluate this SNP in haemorrhage stroke in subsequent studies.
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This work was sponsored by the Ministry of Science and Technology of China (973 Project) and was supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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Zhang, L., Zhang, H., Sun, K. et al. The 825C/T polymorphism of G-protein beta3 subunit gene and risk of ischaemic stroke. J Hum Hypertens 19, 709–714 (2005). https://doi.org/10.1038/sj.jhh.1001883
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