Association of C‐reactive protein gene polymorphisms with the risk of ischemic stroke: A systematic review and meta‐analysis

Abstract Background and purpose The heterogeneous, complex condition known as ischemic stroke (IS) is brought on by the interaction of a number of risk factors and genetic variables. The association between C‐reactive protein (CRP) gene polymorphisms and IS has, however, been the subject of inconsistent findings. Therefore, we conducted a meta‐analysis to comprehensively address possible associations of CRP genes with the risk of IS. Methods A comprehensive literature search for all the published articles was performed in electronic databases including PubMed, EMBASE, Cochrane Library, and Google Scholar from January 1, 1950 to June 30, 2022. Odds ratio (OR) with 95% Confidence interval (CIs) along with fixed/random effect models were used to calculate summary estimates. Results Twelve case‐control studies totalling 3880 IS cases and 5233 controls were included for the association of CRP gene polymorphisms (rs1800947, rs1130864, rs3093059, rs2794521, and rs1205). Across all genotyping models, we discovered that rs1130864, rs3093059, rs2794521, and rs1205SNPs were not substantially related to IS risk. A trend for significant association for rs1800947 under dominant (OR = 1.19; 95% CI = 0.97 to 1.48), recessive (OR = 1.49; 95% CI = 0.71 to 3.14) and allelic model (OR = 1.21; 95% CI = 0.99 to 1.48) was observed. However, protective association for rs1130864 under dominant (OR = 0.80; 95% CI = 0.70 to 0.91) and rs3093059 under allelic model (OR = 0.18; 95% CI = 0.14 to 0.22) was found. Conclusion Our thorough study revealed that the CRP gene variants rs1800947, rs1130864, rs3093059, rs2794521, and rs1205 could not be related to the risk of ischemic stroke. However, additional research must focus on the rs1800947 polymorphisms in a particular group.


INTRODUCTION
The second most prevalent cause of mortality worldwide is a stroke. (Feigin et al., 2014) About 80% of all strokes are ischemic strokes, which are a diverse, complex condition brought on by one or more risk factors such age, hypertension, diabetes mellitus, hyperlipemia, smoking, and obesity. (Nath et al., 2022) These risk factors, however, only account for a portion of the condition. (Appunni et al., 2022;Flossmann et al., 2004) Animal models have revealed the significant genetic component of stroke in twin and family-based association studies. (Boehme et al., 2017) Atherosclerosis, a significant contributor to ischemic stroke, is mostly mediated by inflammation. (Anrather & Iadecola, 2016) C-Reactive protein (CRP) is a crucial inflammatory measure that plays a significant part in determining the prognosis of IS. (VanGilder et al., 2014;Yu et al., 2019) According to reports, elevated serum CRP levels enhance the risk of IS (Di Napoli et al., 2005;Mengozzi et al., 2020;Ridker et al., 1995;Smith et al., 2006). The genetic variants within the CRP gene, which is present on chromosomes 1q21 to 1q23, should be considered a key contributor to the altered serum level of CRP. CRP genotype may more precisely reflect the baseline and long-term of exposure to CRP than blood CRP levels, which only indicate the level of inflammation at a specific time point. However, the association between CRP single nucleotide polymorphisms (SNPs) (rs1800947, rs1130864, rs3093059, rs2794521, and rs1205) and ischemic stroke is still debatable. SNPs 2667 [rs1800947] is located in exon 2; 3872 [rs1205] located in the 3-flanking region at position +1846; SNP 790 (rs3093058) located at position −757 in the 5-promoter region of CRP gene (Hage & Szalai, 2007). It is well known that the rs1130864 variant is found in the 3′ untranslated region at position +1444 of the CRP gene and that its predominant allele has been associated with lower levels of circulating CRP. (Komurcu-Bayrak et al., 2009;Kong et al., 2012;Miller & Zee et al., 2005;Ridker et al., 1998) The correlations between IS and variations in the eNOS, ACE, ANRIL, MTHFR, and IL-10 genes have been identified in previous metaanalyses. (Alhazzani et al., 2018;Kumar et al., 2020;Kumar et al., 2017;Rui et al., 2020;Wei et al., 2017) Additionally, a growing body of research on CRP candidate genes has produced some contradictory findings. Das et al., 2014;Du et al., 2015 Jun, 3;Flex et al., 2004;Ladenvall et al., 2006;Li et al., 2020;Morita et al., 2006;Shen et al., 2013;Wu et al., 2017;Zhou et al., 2017;Zhu et al., 2019) In order to fully address potential association of CRP genes with the pathogenesis of IS, we set out to conduct a meta-analysis.

Search strategy
The systematic literature review was performed using the guidelines of the Preferred Reporting Items for Systematic Reviews and Metaanalyses (PRISMA) statement. (Moher et al., 2015) A comprehensive search for all the published articles was performed in electronic databases including PubMed, EMBASE, Cochrane Library, and Google Scholar from 01 st January 1950 to 30 th June 2022. The search criteria include the following terms: ("Ischemic stroke" OR "cerebral vascular accident" OR "CVA" OR "atherothrombotic cerebral infarction" OR "cerebral stroke") AND ("CRP" OR "C Reactive Protein") AND ("rs1800947", OR "rs1130864", "rs3093059", "rs2794521" OR "rs1205") AND ("Gene" OR "Genotype). The literature search covered all English-language articles. Additionally, the reference list of retrieved studies, review articles and previous meta-analyses, were manually searched for collecting more relevant studies, which were not found while performing the electronic search. Studies involving human subjects were considered in our meta-analysis. When there were duplicates, the source with the most detailed information was utilised.

Eligibility criteria
Studies that matched the following criteria and focused on the association between CRP gene polymorphism and risk of ischemic stroke were included: Inclusion criteria: (a) Case-control studies investigating for the association of CRP gene polymorphisms rs1800947 and/or rs1130864 and/or rs3093059 and/or rs2794521 and/or rs1205 with risk of IS.; (b) The number of controls and cases in each group were reported; and (c) The odds ratio (OR) and 95% confidence interval (CI) or the data that might be used to calculate the OR and CI were used to describe the genotype and allele distribution.

F I G U R E 1
Flow diagram for the selection of studies and specific reasons for exclusion from the present meta-analysis.

Exclusion criteria:
(a) Letters, editorials, case studies, reviews, meta-analyses, commentary, studies involving animals or the environment, and (b) non-English studies.

Risk of bias in individual studies
The risk of bias was assessed by New-castle Ottawa Scale (NOS) for quality assessment of all the included studies in the meta-analysis. (Stang, 2010) NOS Scale considers participant selection, comparability, and exposure, to assess the case-control study's quality. Nine points was the maximum score. Studies rated eight or higher were regarded as being of excellent quality. Publication bias was assessed by using the funnel plot analysis. The asymmetry of the funnel plot was determined by using the Begg's and Egger's regression test. (Begg & Mazumdar, 1994;Egger et al., 1997)

Data extraction
We gathered following information: the first author's name, the publication year, the nation, the method for measuring genotype, the sample size, the proportion of male and female participants, the mean age, and the genotype frequencies of the case and control groups. Separately reviewing the publications and gathering data, the two authors settled any disagreements by consensus or with the help of a third author.

Statistical analysis
To determine the strength of the association between CRP gene polymorphisms and risk of IS, Odds Ratio (OR) with 95% confidence interval

Trial sequential analysis
Trial sequential analysis (TSA) was conducted by following the guidelines mentioned by Meng et al. (Meng et al., 2018) To determine the necessary sample size, we chose a type I error significance of 5%. We then constructed the TSA monitoring boundaries using TSA software Version 0.9.5.10 Beta.   The NOS scores varied from 7 to 9, and 67% (8/12) of the studies were of excellent quality, indicating that the methodology was adequate. Table S1 is the PRISMA 2020 checklist used to guide the reporting of this systematic review and meta-analysis. The PRISMA checklist is a widely recognized tool for ensuring the completeness and transparency of reporting in systematic reviews and meta-analyses. The checklist includes 27 items covering the different aspects of a systematic review and meta-analysis, such as the study design, search strategy, study selection process, data extraction, and risk of bias assessment.

TA B L E 1
Characteristic of the included studies in the meta-analysis for the between CRP gene polymorphism with the risk of ischemic stroke.

S. No
Author

Association between CRP (rs1800947) gene polymorphism and IS risk
Seven studies Das et al., 2014;Flex et al., 2004;Ladenvall et al., 2006;Li et al., 2020;Morita et al., 2006;Zhu et al., 2019) were included for the association between rs1800947 gene polymorphisms and IS risk. Across all genotyping models, a trend for the significant association for rs1800947 under dominant (OR = 1.19; 95% CI = 0.97 to 1.48; I 2 = 34.7%), recessive (OR = 1.49; 95% CI = 0.71 to 3.14; I 2 = 37.9%] and allelic model (OR = 1.21; 95% CI = 0.99 to 1.48; I 2 = 41.9%) was observed (Figure 2). A fixed-effect model was applied to pool the data from the included studies as a lower degree of het-erogeneity was observed. The summary estimates for the association between CRP gene polymorphism with the risk of ischemic stroke are represented in Table 3.

Association between CRP (rs1130864) gene polymorphism and IS risk
Five studies (Andersson et al., 2009;Du et al., 2015 Jun, 3;Ladenvall et al., 2006;Li et al., 2020;Morita et al., 2006) reported the data for the association between rs1130864 gene polymorphisms and IS

Trial sequential analysis
The results of TSA are shown in Figure 7, the required sample size is 21,728 samples, and the cumulative z-curve crossed the trial F I G U R E 6 Forest plots of the association between rs1205 SNP with ischemic stroke risk under (a) dominant model; (b) recessive model; and (c) allelic model. sequential monitoring boundary before reaching the required sample size, which means that our conclusions are robust with this sufficient evidence.

Sensitivity analyses
Furthermore, we performed sensitivity analyses to assess the influence of each individual study on the pooled ORs by sequential omission of individual included studies. However, the corresponding pooled ORs were not significantly altered by removing any of the studies ( Figure S1a-e). Therefore, the sensitivity analysis confirmed that the results of this meta-analysis were statistically reliable and robust.

Publication bias
Funnel plot and the Egger's test were performed to assess the publication bias arising from the literature included in our meta-analysis. No obvious asymmetry was observed for the included studies according to the visual assessment of the funnel plot. In addition, there was no statistical evidence of publication bias among the studies using Egger's regression test (p-value: 0.20 for rs1800947; 0.24 for rs1130864; 0.24 for rs3093059; 0.34 for rs2794521; and < 0.12 for rs1205 SNPs; Figure S2a-e).

Trial sequential analysis
The result of TSA is shown in Figure 7, the required sample size is 5591 samples, and the cumulative z-curve does not cross the trial sequential monitoring boundary before reaching the required sample size, which means that more studies with large sample size are required in order to reach to the conclusive findings.

DISCUSSION
The largest cause of long-term impairment and mortality world-  Zhou et al., 2016) In the current meta-analysis, we could not discover any association between the IS risk associated with the polymorphisms rs2794521, and rs1205. The 3′ untranslated region of the CRP gene contained the polymorphism rs1130864, which has been discovered to be related to the susceptibility to a number of inflammatory disorders. (Delongui et al., 2017;Schulz et al., 2016) Earlier, the mendelian randomization, however, did not clearly demonstrate that an increased CRP concentration was causally linked to an increased risk of IS. (Zhang et al., 2020) Additionally, other studies has shown no connection between the rs1130864 polymorphism and the chance of developing IS. respectively, reported the connection between CRP gene polymorphisms and IS susceptibility. One of which showed no significant associations between these two polymorphisms (rs1800947 and rs1205) in CRP genes and IS (González-Giraldo et al., 2016), one showed scant evidence to support a role for CRP gene rs2794521 polymorphisms in ischemic stroke predisposition, (Liu et al., 2015) and one could provide the first proof that genetic variants (rs1130864 and rs2794521) within the CRP locus are associated with ischemic stroke. (Chen et al., 2014) According to the best of our knowledge, this is the first thorough meta-analysis and systematic review analyzing the association of five SNPs in the CRP gene with IS. Although conducted comprehensively, following limitations were present in our meta-analysis: (i) certain casecontrol studies did not pass the Hardy-Weinberg equilibrium test, suggesting that there was a bias in the selection of the control group; (ii) the studies included in our systematic review and meta-analysis varied in terms of ethnicity, age and environmental factors and sources of control subjects; (iii) we did not check for the false discovery rate which might arise from the multiple comparisons made in the meta-analysis; (iv) due to the fact that among matched groups, the other covariates corrected were not the same, whereas within unmatched groups, some studies may have had other covariates adjusted for them while others may have had none at all and lastly; and (v) significant heterogeneity was present in some of the comparisons made in our meta-analysis.
However, we used a random-effect model throughout to account for the between study heterogeneity and also assessed the quality of each included study.

CONCLUSION
Our thorough study revealed that the CRP gene variants rs1800947, rs1130864, rs3093059, rs2794521, and rs1205 could not be related to the risk of ischemic stroke. However, additional research must focus on the rs1800947 polymorphisms in a particular group.

AUTHOR CONTRIBUTIONS
All authors contributed to the study conception and design. W.C., X.Z., L.J., and L.W. independently assessed the quality of included studies.
All authors read and approved the final draft.