FGB Gene − 148C>T Polymorphism Is Associated with Increased Risk of Ischemic Stroke in a Chinese Population: A Meta-Analysis Based on 18 Case-Control Studies

Abstract

Background: Several published articles investigated the relationship between a polymorphism −148C>T in the β-fibrinogen gene (FGB) and risk of ischemic stroke, and did not reach the same conclusion. To shed light on these inconclusive findings, we performed a meta-analysis of studies relating the FGB genetic polymorphism (−148C>T) to the risk of ischemic stroke. Methods: We identified articles by searching PubMed, EMBASE, Chinese National Knowledge Infrastructure databases (CNKI), and Wanfang database in China and by reviewing the references of retrieved articles. We included studies that reported odds ratio (OR) with 95% confidence interval (CI) for the association between the FGB −148C>T polymorphism and stroke risk. Data from eligible studies were extracted for meta-analysis. Stroke risk associated with FGB −148C>T polymorphism was estimated by pooled ORs and 95% CIs. The software Review Manager (version 5.2) was utilized for meta-analysis. Publication bias was tested by funnel plot. Results: Eighteen independent case-control studies containing 2159 ischemic stroke patients and 3222 control subjects were included. Our results showed that −148C>T polymorphism in the FBG gene was associated with increased risk of ischemic stroke ([TT+CT] vs. CC: OR=1.40, 95% CI [1.20-1.45], p<0.0001; T vs. C: OR=1.35, 95% CI [1.18-1.56], p<0.0001, respectively] by a meta-analysis. Conclusion: The results of our meta-analysis suggested that the−148C>T polymorphism in the FGB gene is a susceptibility marker of ischemic stroke.

Introduction

Ischemic stroke, after ischemic heart disease, is the commonest cause of mortality worldwide and caused about 5.7 million deaths in 2005 (WHO, 2000). China is the world's most populous country and stroke is an enormous healthcare burden, representing the second leading cause of death and occurring in ∼2 million people each year (Wei et al., 2010). Prospective studies with large samples have suggested that the plasma fibrinogen level is an independent risk factor for ischemic stroke (Wilhelmsen et al., 1984; Maresca et al., 1999). Elevated plasma fibrinogen levels can be affected by genetic factors. Fibrinogen is encoded by three separate genes located in a 50-kb cluster on the long arm of chromosome 4, which encode for the α, β, and γ chains (Kant et al., 1985). The rate limiting step in fibrinogen formation is the synthesis of the β-polypeptide chain regulated by a β-fibrinogen promoter (Roy et al., 1990). Polymorphisms of the β-fibrinogen gene (FGB), including the β −148 C>T polymorphism, have been shown to be related to the elevation of the plasma fibrinogen level (Xu et al., 2008; Guo et al., 2009; Yuan et al., 2009). Several studies have suggested that the FGB −148C>T polymorphism is associated with increased risk of ischemic stroke (Fu et al., 2005; Gao et al., 2006; Liang et al., 2006; Ma et al., 2006; Chen et al., 2007). However, the relatively small sample size of a single study may not have enough power to detect slight effects of −148C>T polymorphism on stroke; meta-analysis may provide more credible evidence by systematically summarizing the existing data. Although a previous meta-analysis (Chen et al., 2007) has reported on the relationship between FBG gene polymorphism and stroke, it involved a small sample size and had lower power. In addition, there are several new articles that have been published since then. Therefore, we have collected almost all published case-control studies to perform a meta-analysis to further explore the relationship between FGB gene −148C>T polymorphism and ischemic stroke.

Materials and Methods

Literature search and selection

We carried out a publication search in PubMed, EMBASE, Chinese National Knowledge Infrastructure [CNKI], and Wanfang database in China, with the following search terms: [“fibrinogen” or “β-fibrinogen” or “FGB”] and [“cerebral infarction” or“stroke” or “brain infarction” or “cerebrovascular disease”] and [“SNP” or “polymorphism” or “mutation” or “genetics”] by two independent investigators. Publication language was restricted to English and Chinese, and the subjects were limited to Chinese in our search. By means of online retrieval and literature review, references obtained using the above-mentioned databases were reviewed again to ensure that no relevant studies were missed.

Selection criteria

Inclusion criteria were as follows: (1) independently published case-control or cohort studies on the relationship between FBG gene polymorphism and stroke; (2) with comprehensive statistical indicators directly or indirectly: odds ratio (OR) or relative risk values and 95% confidence interval (CI); and (3) similar themes and methods, that is, case-control or cohort studies about the relationship of the FGB gene polymorphism and stroke. Articles were excluded if relevant data were not available or there was heterogeneity of gene polymorphism in the control population. For the heterogeneity test method, we utilized the Q-test and I² test of the RevMan 5.2 software.

Data extraction

Two reviewers independently evaluated the research design, enrolled patients, observation results of the literature, and selected trials according to the above-mentioned inclusion criteria. Inconsistencies were resolved through discussion. We used the Cochrane Handbook 5.2 quality evaluation criteria to assess the methodological quality of included studies such as study subjects and impact factors, the source of the cases and controls, matching age and gender. To determine the quality of data by the quality of ultimately determined literature, the useless ones were excluded, such as studies that have been reported repeatedly and those with poor quality or less information and having special selection of laboratory sample; relevant data were extracted from included articles. Because there is C or T allele in the −148C>T site, the genotype in this locus of the population was identified as CC, CT, or TT. For each study that met our criteria, the following information was collected: first author, year of publication, country of origin, number of cases and controls, genotype distribution, genotyping methods, and allele frequency.

Statistical analysis

For each study, we first examined whether the genotype distribution in controls was consistent with the Hardy-Weinberg equilibrium (HWE) by a chi-square test. Meta-analysis was performed using RevMan 5.2 software provided by the Cochrane Collaboration. We used the Q-test and I² test to examine the heterogeneity between each study. We used OR for efficacy analysis statistics. Using the heterogeneity test, if p>0.05, we selected the fixed-effects model, and if p<0.05, we selected the random-effects model to merge the OR. p<0.05 was considered as a significant difference. Analysis of sensitivity includes the difference of point estimation and CIs of the combined effects value of different models to observe whether it changes the result; poor quality articles were excluded or reanalyzed according to the quality evaluation criteria to determine whether it changed the findings. To test the publication bias, we used the RevMan 5.2 statistical software to make the funnel plot.

Results

Literature search

As shown in Figure 1, 476 studies were preliminarily detected, which includes 414 Chinese articles and 62 English articles; 404 articles were excluded because of duplicate publication and nonclinical-based research literature. Seventy-two studies appeared to be potentially relevant for inclusion in our study. Fifty-one studies were further excluded because they did not detect the −148C>T genotype. Therefore, 21 full-text articles were reviewed. Three studies were further excluded for no control population. Therefore, a total of 18 articles met the inclusion criteria (Liu et al., 2002, 2004; Lv et al., 2003; Zhang et al., 2003; Zhao et al., 2003; Qian et al., 2004; Fu et al., 2005, 2006; Pan et al., 2005; Song et al., 2005; Xu et al., 2005; Lu et al., 2007; Liu and Zhao, 2008; Yuan et al., 2009, 2010; Cui et al., 2010; He et al., 2010; Guo et al., 2009).

FIG. 1.

Flow diagram of study identification.

Study characteristics

The characteristics of included studies are summarized in Table 1. The 18 included studies were published between 2000 and 2010 and comprised a total of 2159 ischemic stroke cases and 3222 control subjects. All the subjects included in these studies were Chinese. A classic polymerase chain reaction assay was performed in all of these 18 studies. The genotyping method in all these studies was polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and the genotype distributions among the controls of all studies were in agreement with HWE.

Table 1.

The Characteristics of Included Studies

						−148C>T (n)
Author	Year	Country	Genotyping methods	Groups	n	CC	CT	TT
Cui	2010	China	PCR-RFLP	Case	92	36	48	8
				Control	98	56	37	5
Fu	2006	China	PCR-RFLP	Case	132	75	50	7
				Control	171	102	58	11
He	2010	China	PCR-RFLP	Case	92	36	48	8
				Control	80	56	37	5
Liu	2004	China	PCR-RFLP	Case	90	43	39	8
				Control	102	64	34	4
Liu	2008	China	PCR-RFLP	Case	220	105	85	30
				Control	140	84	49	7
Liu	2002	China	PCR-RFLP	Case	96	62	30	4
				Control	273	177	86	10
Lu	2007	China	PCR-RFLP	Case	148	72	63	13
				Control	130	79	45	6
Lv	2003	China	PCR-RFLP	Case	151	70	65	16
				Control	113	62	47	4
Ma	2006	China	PCR-RFLP	Case	151	85	63	3
				Control	101	70	30	1
Pan	2005	China	PCR-RFLP	Case	69	41	26	2
				Control	60	41	18	1
Qian	2004	China	PCR-RFLP	Case	102	66	30	6
				Control	90	43	39	8
Song	2005	China	PCR-RFLP	Case	88	38	42	8
				Control	80	47	27	6
Song	2006	China	PCR-RFLP	Case	135	53	56	26
				Control	120	65	43	11
Xu	2005	China	PCR-RFLP	Case	90	52	32	6
				Control	60	47	17	2
Yuan	2010	China	PCR-RFLP	Case	160	101	47	12
				Control	162	112	42	8
Yuan	2009	China	PCR-RFLP	Case	144	64	46	4
				Control	1277	820	387	70
Zhang	2003	China	PCR-RFLP	Case	48	25	18	5
				Control	52	39	11	2
Zhao	2003	China	PCR-RFLP	Case	151	70	65	16
				Control	113	62	47	4

PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism.

Meta-analysis

The association between −148C>T polymorphism and susceptibility to stroke was analyzed in 18 independent studies with 2159 stroke patients and 3222 control subjects. Results of the meta-analysis are shown in Figures 2 and 3. These two figures showed the result of meta-analysis of studies on the correlation between ischemic stroke and FGB −148C>T polymorphism in 18 case-control studies. The systematic reviews of the included studies can be seen from Figures 2 and 3, which include the number of case and control groups, weight, OR value, and 95% CI.

FIG. 2.

Forest plot of cerebral infarction and −148C>T polymorphism (TT+CT vs. CC), the horizontal lines correspond to the study-specific odds ratio (OR) and 95% confidence interval (CI), respectively. The area of the squares reflects the study-specific weight. The diamond represents the pooled results of OR and 95% CI.

FIG. 3.

Forest plot of cerebral infarction and −148 C>T polymorphism (T vs. C), the horizontal lines correspond to the study-specific OR and 95% CI, respectively. The area of the squares reflects the study-specific weight. The diamond represents the pooled results of OR and 95% CI. In this analysis, the fixed-effects model was used.

The heterogeneity test of the various studies revealed heterogeneous results (p=0.04, I²=40%; p=0.01, I²=48%); therefore, we used the random-effects model in the analysis. Overall, the association of FGB −148 TT/CT genotype with a higher risk of ischemic stroke was observed (OR=1.35, 95% CI [1.18-1.56]; p<0.0001) (Fig. 2). In addition, we found the −148 T allele carriers to be associated with the increased risk of ischemic stroke (OR=1.40, 95% CI [1.20-1.65]; p<0.0001) (Fig. 3).

Test of sensitivity

For the sensitivity analysis, we deleted one single study from the overall pooled analysis each time to check the influence of the removed data set to the overall ORs. The pooled ORs and 95% CIs were not significantly altered when any part of the study was omitted, which indicated that any single study had little impact on the overall ORs.

Publication bias

RevMan 5.2 software was used to analyze the publication bias; the funnel plot (Fig. 4) showed that the points are evenly distributed and symmetrical, and most of the points are within the 95% CI. Also, the shape of funnel plots showed no obvious asymmetry and the result of Egger's test did not show statistical evidence for bias either (p=0.13; p=0.16, respectively). It indicates that there is no publication bias, and the result of the study is credible.

FIG. 4.

Begg's funnel plot for publication bias tests. Each point represents a separate study for the indicated association. LogOR represents natural logarithm of OR. Vertical line represents the mean effects size. (A) (TT+CT) versus CC. (B) T versus C.

Discussion

In this meta-analysis, we found that the polymorphism −148C>T in the FGB gene was significantly associated with ischemic stroke in a Chinese population by the pooled results from 18 published studies. The results demonstrated that the −148T carriers had an increased 35% risk for developing ischemic stroke.

It is well known that elevated plasma fibrinogen levels can be affected by environmental and genetic factors. Polymorphisms of the FGB, especially involved in the rate-limiting steps of the formation of the β-chain, have been shown to be closely related to elevation of the plasma fibrinogen level and ischemic stroke (Liu et al., 2002; Fu et al., 2005; Gao et al., 2006; Liang et al., 2006; Ma et al., 2006; Chen et al., 2007; Xu et al., 2008; Guo et al., 2009; Yuan et al., 2009, 2010). In the present study, we combined the results of 18 studies to pool analyze the relationship between −148C>T polymorphism and ischemic stroke. The result showed that there was 40% increased risk of stroke for the variant genotypes (CT+TT) compared with the wild CC homozygotes. Thus, the allele T might be a genetic risk factor to increase the susceptibility to stroke at the protein and genetic levels.

The characteristic of meta-analysis is to combine comparable studies to increase the sample size and statistical power and draw a more compelling result. However, meta-analysis confounds factors such as publication bias, method of sampling, different genetic backgrounds of subjects, different protocols, and quality of analysis. In the present study, we did not find publication bias, all the subjects were Chinese, and the genotypes in all studies were detected with genetic DNA from blood samples using PCR-RFLP genotyping methods. All the studies checked genotypes for quality control. Genotype distribution of controls in all studies was consistent with HWE.

In addition, sensitivity analysis also showed that omission of any single study did not have significant impact on the combined ORs. This made the results of this meta-study more reliable to some extent.

However, there remained some limitations in this meta-analysis. Although the genotyping methods used in all the studies were the same, other clinical factors such as age, sex, and different chemotherapies in each study might lead to bias. Determining whether or not these factors influence the results of this meta-analysis would need further investigation.

Conclusion

Our study suggested that −148C>T polymorphism in the FGB gene was associated with a significantly increased risk of ischemic stroke. Larger well-designed epidemiological studies with ethnically diverse populations and functional evaluations are warranted to confirm our findings.

Footnotes

Acknowledgment

This work was supported financially by the Project of Science & Technology of Xinxiang Medical College (2007YJAO2).

Author Disclosure Statement

No competing financial interests exist.

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