Relationship between CRP gene polymorphisms and ischemic stroke risk: A systematic review and meta-analysis

Abstract Ischemic stroke (IS), usually caused due to an abrupt blockage of an artery, is the leading cause of disability and the second leading cause of death worldwide. The association of the C-reactive protein (CRP) gene (s3093059 T/C and rs1205 C/T) polymorphisms and IS susceptibility has been widely studied, but the results remain inconsistent. Our study aimed to assess the association between CRP gene (s3093059 T/C and rs1205 C/T) polymorphisms and IS risk. PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure, and WanFang databases were searched up to April 2022 to identify eligible studies. The Newcastle-Ottawa scale (NOS) score was calculated to assess study quality. The odd ratios (ORs) with a 95% confidence interval (CI) were calculated to assess the association between CRP gene (rs3093059 T/C and rs1205 C/T) polymorphisms and IS risk. Eighteen case–control studies with 6339 cases and 29580 controls were identified. We found that CRP (s3093059 T/C and rs1205 C/T) polymorphism was not significantly associated with the risk of IS in any genetic model (recessive model: OR 1.00, 95% CI 0.79–1.26; OR 1.06, 95% CI 0.90–1.25). When stratified analysis by country, genotype method, source of controls, and NOS score, still no statistically significant association was found. Our study indicated that the CRP (rs3093059 T/C and rs1205 C/T) polymorphisms were not associated with the susceptibility to IS.


Introduction
Ischemic stroke (IS) is the more common type and is regarded as the leading cause of death, physical disability, and cognitive decline worldwide [1]. With the global population aged 65 and over growing faster than all other age groups, the incidence of stroke is also increasing. Accordingly, early accurate identification of modifiable risk factors and management of the people potentially at high risk of stroke are of great significance. There is strong evidence of a connection between the chronically activated and sustained inflammatory states and a variety of diseases including cancer [2], neurodegenerative disease [3], and cardiovascular and cerebrovascular disease [4]. The inflammatory processes have been observed in atherosclerotic initiation, plaque rupture, platelet activation, and coagulation system activation, which all contribute to the occurrence of IS [5]. Thus, inflammatory factor C-reactive protein (CRP), as one of the underlying circulating inflammatory markers, was identified to be a reactant in an acute phase of IS. In addition, elevated CRP levels are also generally associated with poor outcomes in acute IS patients [6]. However, the controversies have shown differences with respect to the risk prediction of IS because of genetic factors that influence CRP levels [7]. For example, some studies suggested that there was a positive relationship between elevated CRP and atherosclerosis as a precursor to IS [8]. Also, some showed that high-sensitivity CRP was not associated with IS and atherosclerotic changes [9,10]. Therefore, we speculated that the concentration of CRP in plasma depends on the CRP gene polymorphism. So far, approximately 30 single nucleotide polymorphisms (SNPs) of the CRP gene have been confirmed [11]. Their gene variability could be considered a predictive genetic marker for IS, so investigating the relationship between crucial binding sites SNPs and IS susceptibility may have diagnostic and prognostic implications.
Understanding the relationship between gene polymorphism and disease may help inform the design of pharmacotherapies by using multiple silico techniques [12][13][14]. A number of studies have been conducted to investigate the potential associations between common polymorphisms (rs2794521 (717G>A), rs3091244 (286CT>A), rs1800947 (1,059G>C), rs1130864 (1,444C>T)) in CRP gene and IS risk [15,16]. However, there is still a lack of summary conclusions about the association of CRP rs3093059 (757T>C) and rs1205 C/T polymorphism (2147C>T) with IS risk, though these SNPs polymorphism has also been proposed as possible biomarkers to predict IS risk in some researches. Therefore, we aimed to evaluate whether these two sites' polymorphisms are associated with the risk of IS through a meta-analysis using all available data.

Materials and methods
This meta-analysis was conducted based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [17].

Literature search
A systematic literature search was conducted by using the combination of the following terms: "CRP,", "CRP" "rs3093059," "rs1205," "polymorphism," "variant," and "IS" based on PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure, and WanFang databases before April 2022. There were no language restrictions during the literature search. Additionally, the references of relevant articles were manually searched for potential studies.

Inclusion and Exclusion Criteria
The criteria for including studies in this meta-analysis were as follows: (1) the design of the study was case-control or cohort studies; (2) studies assessed the association between CRE gene polymorphism (rs3093059 or rs1205) and IS risk; (3) studies provided available genotype distribution in cases and controls; (4) the genotype distribution of control group conformed to Hardy-Weinberg equilibrium (HWE). The exclusion criteria were (1) studies that reported incomplete data or without data in cases and controls group; (2) duplicate data; and (3) review, case reports, or animal experiments.

Data extraction and quality assessment
Two authors independently extracted the following information from included studies: first author's name, year of publication, country, ethnicity, genotype methods, genotype counts in cases groups and control groups, HWE results for control groups, and Newcastle-Ottawa scale (NOS) assessment. The NOS was calculated for the quality assessment of included studies. Discrepancies were resolved by consensus.

Statistical analyses
Meta-analyses were performed using the STATA version 12.0 (Stata Corporation, College Station, TX, USA), with a value of p < 0.05 which was considered statistically significant. To estimate a summary effect size for IS risk, the odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by using the command "metan" based on five genetic models: allelic model, heterozygous model, homozygous model, dominant model, and recessive model. The significance of the pooled OR was determined by Z-test. Between-study heterogeneities were evaluated with I 2 statistic and Cochran's Chi-squarebased Q test. A fixed-effect model (Mantel-Haenszel method) was used when I 2 was ≤ 50%. Otherwise, analyses would be performed with random-effect models (Mantel-Haenszel method). HWE was tested by Chisquare test in controls. Sensitivity analysis was used to verify the stabilities of synthetic results. Publication bias was assessed using Begg's funnel plots and Egger's regression by "metafunnel" and "metabias" commands. We also conducted subgroup analyses by country, genotype method, source of controls, and NOS score. Trimand-fill method was performed to adjust OR value when publication bias was found.

CRP rs3093059 T/C polymorphism and IS risk
The main results for the association between CRP rs3093059 T/C polymorphism and IS risk are summarized in Table 2 (Figure 2). Moreover, the synthesized result suggested a null association between the rs3093059 T/C polymorphism and IS risk in the subgroup analysis according to source of controls, genotype method, and NOS score ( Table 2). Significant between-study heterogeneities were observed in some genetic models; thus, to confirm the robustness of the meta-analysis, sensitivity analyses were necessary to be carried out. As shown in Figure 3, none of the studies affected the pooled result in the dominant genetic model, which suggested that our results were statistically robust. In addition, publication bias usually makes it difficult to have confidence in any reported differences. Thus, Begg's and Egger's linear regression tests were used to visualize publication bias, and the results of Begg's test and Egger's test suggested a statistically significant publication bias in heterozygous and allelic genetic models ( Table 2). Therefore, we conducted the trim-and-fill method to make the OR value more reliable.

CRP rs1205 C/T polymorphism and IS risk
The main results for the association between CRP rs1205 C/T polymorphism and IS risk are summarized in  Figure 4]). The synthesized result suggested a null association between the CRP rs1205 C/T polymorphism and IS risk in the subgroup analysis according to country, genotype method, source of controls, and NOS score ( Table 3). Significant between-study heterogeneity was observed in some genetic models; thus, to confirm the robustness of the meta-analysis, sensitivity analyses were necessary to be carried out. As shown in Figure 5, none of the studies affected the pooled result, which

Discussion
This meta-analysis included the literature published in recent years about the association between CRP polymorphisms (rs3093059, rs1205) and IS susceptibility. The pooled results revealed that CRP polymorphisms (rs3093059, rs1205) might not associate with IS risk. CRP, a glycoprotein released by the liver, has been regarded as an essential mediator and a hallmark of the acute-phase response to inflammation and is recommended for use in risk assessment in IS patients [35]. CRP is an evolutionarily conserved protein with a unique pentameric structure and binds to ligands in a calciumdependent manner [36]. Once binding to ligands, CRP interacts with the classical complement pathway and Fcγ receptors to participate in the activation of the innate immune system [37,38]. The gene of CRP is located on chromosome 1 and has only two exons and a single intron, including 204-amino acid peptides in the coding regions of exons and 280-base pairs in the domain of the intron [39]. Due to the existence of single-nucleotide polymorphisms (SNPs), CRP genetic variants and individual variations in the inflammatory response are significant. Many researchers have proved that the altered serum level of CRP should be attributed to the CRP gene variations on chromosomes 1q21 to 1q23 [40]. A couple of sequence variations at this locus have been shown to modulate plasma CRP levels and the risk of IS [41]. So the SNPs of CRP are a vital factor in the development of IS.
As an inflammation-associated protein, CRP can be divided into two structurally and functionally independent forms: (1) net anti-inflammatory, serum-associated native pentameric CRP, and (2) pro-inflammatory tissueassociated, monomeric CRP (mCRP) [37,42]. Several studies proved that a dramatic increase in the expression of mCRP had been observed in blood vessels of damaged brain regions in IS patients [43][44][45]. Krupinski and his colleagues found a higher expression of mCRP within microvessels with unstable plaques while normal-looking arteries, and stable fibrous lesions contained a significantly lower expression [44]. It suggested that mCRP may have a pathological role in developing unstable atherosclerosis and/or increased risk of plaque thrombosis, which could lead to the occurrence of IS. mCRP increases the activation of the inflammation both in vitro and in vivo, getting deposited chronically within the brain after IS, and may play a role in perpetuating neuroinflammation after brain injury [35]. Of course, there are still some opposite opinions. The function of CRP in the development of IS should be studied further.
Though the clear mechanism is ambiguous, CRP is closely related to the occurrence, development, and outcome of IS. The results of clinical studies show that CRP levels increase in the first 48 h after onset, are still elevated at 7 days and remain high for 3-6 months after IS [46,47]. CRP levels correlate with IS severity and can be a marker of IS etiology, with higher CRP in more severe cardioembolic or large artery disease stroke than in stroke caused by small artery disease [47][48][49]. Numbers of clinical studies use CRP as a biomarker to predict the occurrence of IS, and try to explore the relationship between SNPs of CRP and IS. The effect of CRP SNPs such as rs1800947, rs1417938, rs1130864, and rs3093077 on circulating protein level and the outcome has been assessed in a cohort of in-patients with cardiovascular diseases (e.g., IS) by Schulz et al. They found that both CRP level ≥5 mg/L and SNP rs1800947 of the CRP gene were independent risk factors for further adverse vascular events among patients with cardiovascular diseases within a 3-year follow-up [50]. A study of clinical samples by Williams et al. found that SNPs at rs3093068, rs16842599, and rs11265260 loci of CRP were associated with the occurrence and recurrence of IS [51]. Manuela and his colleagues consider that CRP levels after a minor first cerebrovascular event (transient ischemic attack or lacunar stroke) can contribute to identifying patients at high risk of a second ischemic event. Rs3093059 is located in the promoter region of the CRP gene. The mutation of this site would provide convenience for LHX2 binding to promote expression [52]. C alleles at rs3093059 were positively associated with increased CRP elevation in IS patients, which is inconsonant with our results. After multivariate    adjustment, rs3093059 was found to be associated with decreased IS risk in the Chinese population [24]. Also, no association was detected between CRP gene polymorphisms and IS risk in the Swedish [41] population and Indian population [53]. It suggested that whether Rs3093059 can be judged as a risk factor in IS cases may be related to the population and environment. The rs1205 was located in the untranslated region of the CRP gene region. It was reported that CRP rs1205 polymorphism is associated with elevated CRP levels in Aortic stenosis patients and cardioembolic stroke [54]. However, it was found a negative association in our study is attributed to the type of stroke and the underlying condition of the patient. Circulating levels of CRP could be influenced by age, obesity, sex, smoking, diabetes, and use of medications summarily [55].
To our knowledge, this study is the first meta-analysis to focus on CRP polymorphisms (rs3093059 T/C and rs1205 C/T) and IS risk and proved CRP rs3093059 T/C and rs1205 C/T polymorphisms have little association with the risk of IS. Based on the aforementioned analysis, our study still has some limitations and shortages. On the one hand, the data are still relatively small and may not provide sufficient power to estimate the association between CRP gene polymorphisms and IS risk. Few studies have investigated the association between the CRP gene and patients' stroke subtypes and patient-based characteristics, which has to be confirmed in more populations. On the other hand, as a type of retrospective study, a meta-analysis may encounter recall or selection bias, possibly influencing the reliability of our study results. Therefore, more studies with larger sample sizes are needed to accurately provide a more representative conclusion.

Conclusion
The current meta-analysis result suggests that CRP (rs3093059 T/C and rs1205 C/T) polymorphisms might not be associated with the risk of IS. In addition, CRP genetic variant might be associated with multiple internal and external factors, which suggests that further efforts are needed to dissect subgroups and patients' overall physical condition. However, large sample size and well-designed studies within different ethnic are needed to confirm the findings of our study Funding information: Authors state no funding involved.
Author contributions: Z.C. and J.Y. conceived the study. Z.C., F.J., and M.Y. searched the databases and extracted the data. ZC and FJ performed the data analysis. Z.C., F.J., and J.Y. wrote the paper. All the authors read and approved the final manuscript.

Conflict of interest: Authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.