Relationship between PPAR‐γ gene polymorphisms and ischemic stroke risk: A meta‐analysis

Abstract Background Published researches have suggested some associations between PPAR‐γ and ischemic stroke (IS) development. This meta‐analysis was conducted to evaluate the association between PPAR‐γ gene polymorphisms and IS risk. Materials and methods A systematic search was conducted in PubMed, Embase, Web of Science, China National Knowledge Infrastructure, and WanFang databases. The pooled association of odd ratios (ORs) and its 95% confidence interval (CI) was calculated to assess the IS risk of PPAR‐γ rs1801282 C/G and rs3856806 C/T polymorphisms. Furthermore, the heterogeneity test, cumulative analyses, sensitivity analyses, and publication bias were conducted. Result Sixteen publications with 3786 cases and 5343 controls were identified. Overall findings indicated that rs1801282 C/G polymorphism may be associated with an increased risk for IS (GG vs. CC: OR = 2.17 95%CI = 1.09–4.35, p = .03, I 2 = 0%; GG vs. CC+CG: OR = 2.15, 95%CI = 1.07–4.32, p = .03, I 2 = 0%). The similar results were also found in the subgroup analysis. In addition, no significant association was observed between rs3856806 C/T polymorphism and IS risk. Conclusion In conclusion, our study showed that PPAR‐γ rs1801282 C/G polymorphism probably plays an important role in IS occurrence. The result should be verified with more studies in the future.

hypertension, genetic background, and unhealthy lifestyle habits have additionally been suggested to be associated with IS development.
Increasing evidence suggests the existence of a strong polygenic inheritance factor for IS etiology, including abnormal gene expression and genetic mutations.
Peroxisome proliferator-activated receptor-γ (PPAR-γ) belongs to the nuclear receptor superfamily, which mainly mediates liganddependent transcriptional activation and repression. PPAR-γ plays a key role in the regulation of adipocyte differentiation, lipid metabolism, and insulin sensitivity in vivo, with PPAR-γ2 exhibiting a higher activity in adipocytes than other types. Accumulating evidence indicates that PPAR-γ participates in the regulation of adipocyte differentiation, lipid metabolism, and the concentration of lipids and cholesterol. Dyslipidemia is a well-established risk factor for cardiovascular diseases, which triggers the development of atherosclerosis and increases the risk of IS risk. PPAR-γ also plays an important role in regulating inflammatory processes, participates in vascular endothelial cell repair, and inhibits thrombosis formation (D. Li et al., 2005). A recent study demonstrated that PPAR-γ agonists exert an active anti-inflammatory effect in IS models and protect against thrombosis by downregulating the activation of nuclear factor-κB and decreasing the expression of proinflammatory cell adhesion molecules (Jin et al., 2015).
The PPAR-γ gene is located on chromosome 3p25, is comprised of 100Kb DNA bases, and consists of nine exons. The rs1801282 C/G and rs3856806 C/T polymorphisms are the most common single nucleotide polymorphism (SNP) loci in the PPAR-γ gene, and have been extensively studied in different ethnic groups and found to be associated with the risk of several metabolic diseases, including diabetes and its complications. This mutation may change the transcriptional activity and subsequently alter the protein synthesis of PPAR-γ (Masugi et al., 2000).
This leads to abnormal lipid metabolism and subcellular metabolism in arterial foam cells, resulting in the formation of atherosclerosis and an altered risk of stroke (Grbić et al., 2018). Furthermore, PPAR-γ agonists have been extensively studied as potential neuroprotective agents in recent decades (Kinouchi et al., 2018;Shimazu et al., 2005).
In 2005, Shen and Ha (2005) conducted the first case-control study and found no significant association between the rs1801282 C/G polymorphism and IS risk in a Chinese population. However, study results that followed Shen et al. on genetic associations of this SNP with IS risk have been controversial and inconclusive. Therefore, this metaanalysis was conducted to amalgamate the results of existing studies in order to elucidate the association of rs1801282 C/G and rs3856806 C/T polymorphisms with IS risk.

MATERIALS AND METHODS
This meta-analysis was conducted according to the online guidance of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. All collected data were extracted from published studies, and no ethical issue was involved (Moher et al., 2009).

Inclusion and exclusion criteria
The criteria for inclusion of identified studies for the purpose of this meta-analysis were as follows: (1) case-control studies on the association between PPAR-γ rs1801282 C/G, rs3856806 C/T polymorphisms, and IS risk; (2) studies supplying sufficient information on the genotypes in both case and control groups with respect to evaluate the odd ratios (ORs) and 95% confidence intervals (CIs); (3) studies published either in English or Chinese; (4) the polymorphism locus was calculated with at least three studies; and the largest or latest data with more adequate information were collected when duplicate publications or overlapping data were presented. The exclusion criteria were as follows: (1) review articles, case reports, and animal experiments; (2) biological fundamental research; (3) studies without sufficient data; and (4) duplicate or overlapping data on the same theme.

Data extraction and quality evaluation
Two authors (Cheng and Zhou) independently reviewed the included studies and extracted the relevant information: the first author' name, year of publication, study country, ethnicity difference, control design, genotyping method, sample sizes of the cases and controls, frequency of the genotype distribution in the cases and control groups, the degree of Hardy-Weinberg equilibrium (HWE) in the control group, minor allele frequency, and Newcastle-Ottawa scale (NOS) evaluation. The NOS was adopted to evaluate the quality of the included studies. The scores ranged from 0 (worst) to 11 (best) ( Table 1). Studies with a score of eight points or higher indicate a good research quality.

Statistical analysis
The ORs and 95% CIs were calculated to examine the statistical power between PPAR-γ polymorphisms and IS risk using five genetic models: allelic model (

Study characteristics
According to the search strategy utilizing a variety of literature 13 studies were based on Asian descendants, and three studies were based on European descendants. Four genotype methods were used among these studies, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), TaqMan, multilocus genotyping assay, and SNaPshot Multiplex Kit. All of the included characteristics are summarized in Table 1.

PPAR-γ rs1801282 C/G polymorphism and IS risk
Ten case-control studies involving a total of 2804 patients and 4457 controls were identified for the PPAR-γ2 rs1801282 C/G polymor-

PPAR-γ rs3856806 C/T polymorphism and IS risk
Seven case-control studies, involving 1873 patients and 1769 controls, were identified for the meta-analysis of the association between PPAR-γ rs3856806 C/T polymorphism and IS risk. Our results suggest that there is no significant association between the rs3856806 TA B L E 2 Summary odd ratios (ORs) and 95% confidence interval (CI) of peroxisome proliferator-activated receptor-γ (PPAR-γ) polymorphism and ischemic stroke risk

DISCUSSION
According to the World Health Organization (WHO), stroke is one of the leading causes of mortality and disability worldwide and is responsible for approximately 6.1million deaths in 2019 (World Health Organization, 2020). IS frequently results from the occlusion of cerebral blood vessels, usually caused by thrombosis with blood clot formation or embolism with intracranial stenosis, accounting for approximately 85% of all stroke incidences . With an increase in the aging population, the number of IS patients has been increasing in recent years and is predicted to continue, which brings a substantial economic and social burden to the public health system.
Hypertension, hyperlipidemia, hyperglycemia, atherosclerosis, and arterial stenosis have been demonstrated to be the main causes of IS.
In addition, smoking, drinking, unhealthy lifestyle habits, and psychosocial stress have additionally been suggested as high-risk factors for the development of IS. Thrombosis caused by atherosclerosis and inflammatory injury of the vascular endothelium is one of the most direct causes of IS and is significantly associated with aberrant expression or dysfunction of the PPAR family. PPAR-γ is a member of the nuclear receptor superfamily and a ligand-activated transcription factor which is widely expressed in adipose cells and tissues and plays a critical role in the regulation of adipogenesis metabolism, insulin sensitivity,
In the past decades, PPAR-γ agonists such as cyclooxygenase-2, interleukin-1β (IL-1β), and tumor necrosis factor-α, which are known to decrease the level of inflammation in many IS models, have been shown to attenuate proinflammatory mediators during IS development. In contrast, murine stroke models with PPAR-γ knocked out also present a stronger proinflammatory response with a higher risk of secondary intracerebral hemorrhage (Gliem et al., 2015). SNPs are one of the most prevalent forms of gene mutations which can influence gene expression and protein activity. In terms of the PPAR-γ gene, rs1801282 C/G and rs3856806 C/T polymorphisms are the most common SNP loci, which have been reported to be involved in various disease susceptibilities in the past decades. In 1997, the rs1801282 C/G polymorphism locus was first reported by Yen et al. (1997). This mutation comprises an exchange of cytosine for guanine, resulting in the substitution of alanine for proline at codon 12 of exon B.
The Ala allele reduces the binding affinity to the cognate promoter element as well as promoter activity and transcription of PPAR-γ (Deeb et al., 1998). In 2005, Shen and Ha (2005) conducted the first casecontrol study focusing on the PPAR-γ gene rs1801282 C/G polymorphism and IS risk in a Chinese population; however, they did not find any significant association between this mutation and IS. Since then, subsequent series of studies have been conducted, and some inconsistent or contradictory results have been reported. Lee et al. (2006) found a potential protective trend of the rs1801282 C/G genotype in IS patients with type 2 diabetes in Korean people (OR = 0.43, p = .025).

F I G U R E 4
Sensitivity analysis when each study was removed with publication year on peroxisome proliferator-activated receptor-γ (PPAR-γ) rs1801282 C/G polymorphism and ischemic stroke susceptibility (a: GG vs. CC model; b: GG vs. CC+CG model) Tong et al. (2016) additionally reported a protective effect against IS formation in a Chinese population. In contrast, another study by Wang et al. (2019) demonstrated that the G allele presented a significantly higher frequency of the rs1801282 polymorphism than that in the control group and carried a 1.844-fold increased risk of IS (OR = 1.844, p < .001). However, most studies have suggested that there is no significant correlation between this mutation and IS risk (Bazina et al., 2015;L. Huang et al., 2007;Zafarmand et al., 2008). Ethnic differences, small sample sizes of studies, and differences in scientific research approaches and qualities of data may be the main reasons for the inconsistent conclusions among previous studies.
Meta-analysis is a useful statistical method that combines quantitative methods with synthetic data from published studies and draws conclusions on the same theme. For the PPAR-γ gene, the rs1801282 C/G polymorphism could result in a missense mutation and lead to an amino acid change from proline to alanine. This alteration could reduce the transcriptional activity and regulate the lipoprotein lipase activity, thereby affecting the removal of triglycerides, which leads to atherosclerosis due to dyslipidemia (Schneider et al., 2002). Therefore, we conducted this meta-analysis with 10 case-control studies to assess the precise association between the PPAR-γ rs1801282 polymorphism and IS susceptibility. Overall, the synthesized data suggested that there were significant associations between the rs1801282 polymorphism and IS susceptibility in the homozygous and recessive models. Additionally, ethnic stratification indicated that there was a significant increase in IS risk in Asians with the above-  (Sun, 2010;Wei et al., 2013). In the current meta-analysis, we found no statistically significant association between this polymorphism and IS risk in the general analysis. Moreover, a similar result of no association was observed in the ethnic and other subgroups. The current results indicate that the single-site mutation is not sufficient to affect the susceptibility to IS.
To our knowledge, this study is the first meta-analysis to focus on PPAR-γ gene polymorphisms (rs1801282 C/G and rs3856806 C/T) and IS risk. Some advantages exist in this study that help to improve the stability and credibility of the results: (1) a comprehensive and scientific retrieval strategy was used to collect complete information; (2) the current study included a large sample size with all published studies to date; (3) rigorous and accurate methods, including the NOS evaluation, HWE test, cumulative/sensitivity analyses, and publication bias were used one at a time; and (4) subgroup analyses in two polymorphism loci were conducted to explore potential associations. Inevitably, there are certain limitations that need to be addressed: (1) only two polymorphism loci were examined in this study separately, and the interaction mechanism between loci and heterozygous/homozygous state was not involved; (2) we incorporated studies that mainly originated from Asian and European countries, and the number of collected studies or participants from each study was insufficient, which may lead to uncertainty and variations in results; (3) This study was based on unadjusted data, and other environmental exposure factors, such as serum triglyceride, blood pressure, and body mass index were not involved, which limits our understanding of the underlying mechanisms; and (4) some moderate heterogeneity was observed in genetic models among two polymorphism loci, which might affect the reliability and accuracy of the results.

CONCLUSION
In summary, this meta-analysis suggests that the PPAR-γ rs1801282 C/G polymorphism may be associated with IS risk in Asians. More studies with larger sample sizes in different ethnic backgrounds are needed to either refute or verify the current conclusions in the future.

This study was supported by the Health Commission of Hubei Province
Scientific Research project (No. WJ2021M049).

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
All data collected or analyzed for this study are available from the corresponding author upon reasonable request to any researchers.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/brb3.2434