eNOS rs2070744 polymorphism might influence predisposition to hemorrhagic cerebral vascular diseases in East Asians: A meta‐analysis

Abstract Introduction Endothelial nitric oxide synthase (eNOS) polymorphisms might influence predisposition to hemorrhagic cerebral vascular diseases, but the results of already published studies regarding relationship between eNOS polymorphisms and hemorrhagic cerebral vascular diseases were still controversial. Methods The authors performed this meta‐analysis to estimate relationship between eNOS polymorphisms and hemorrhagic cerebral vascular diseases in a larger pooled population by combing the results of already published related studies. The authors searched Pubmed, Embase, Web of Science, and CNKI for already published studies. Results Eighteen already published studies were pooled analyzed in this meta‐analysis. The pooled meta‐analyses results showed that eNOS rs2070744 polymorphism was significantly associated with predisposition to hemorrhagic cerebral vascular diseases in East Asians (dominant comparison: OR = 0.77, p = .01; overdominant comparison: OR = 1.24, p = .04; allele comparison: OR = 0.78, p = .006) Nevertheless, the pooled meta‐analyses did not reveal any positive results for eNOS rs1799983 and rs869109213 polymorphisms. Conclusions This meta‐analysis suggested that eNOS rs2070744 polymorphism, but not rs1799983 and rs869109213 polymorphisms, might influence predisposition to hemorrhagic cerebral vascular diseases in East Asians.


| INTRODUC TI ON
Hemorrhagic cerebral vascular disease, characterized by structural or functional abnormalities of cerebral vessels, and associated spontaneous bleeding into brain tissue, is one of the leading causes of death and disability all over the world (Global Burden of Disease Study 2013Collaborators, 2015Caceres & Goldstein, 2012). Although its definite etiologies and pathogenesis mechanisms are still unclear, accumulating evidence suggested that genetic architecture greatly influence its development. First, the incidences of hemorrhagic cerebral vascular diseases in different populations differed significantly (Backhaus et al., 2015;Krishnan et al., 2018), and genetic background was probably the underlying reason of this phenomenon. Second, previous association studies also detected numerous predisposing gene loci of hemorrhagic cerebral vascular diseases in different populations (Carpenter, Singh, Gandhi, & Prestigiacomo, 2016;Chen, Chang, & Chen, 2018). However, the etiologies and pathogenesis mechanisms of hemorrhagic cerebral vascular diseases are extremely sophisticated, and genetic factors that contribute to the development of hemorrhagic cerebral vascular diseases still need intensively explorations.
Endothelial nitric oxide synthase (eNOS) is essential for maintaining vascular homeostasis and plays vital roles in modulating vascular endothelial function (Besler et al., 2011;Tsutsui, Shimokawa, Morishita, Nakashima, & Yanagihara, 2006). Therefore, if a polymorphism can impact gene expression or protein structure of eNOS, it is likely that this polymorphism might lead to severe vascular endothelial dysfunction and influence predisposition to hemorrhagic cerebral vascular diseases.
In the last two decades, investigators across the world extensively explored relationship between eNOS polymorphisms and hemorrhagic cerebral vascular diseases, especially for intracranial aneurysm (IA) and its associated aneurysmal subarachnoid hemorrhage (aSAH), yet the relationship between eNOS polymorphisms and hemorrhagic cerebral vascular diseases is still controversial.
Thus, we performed this meta-analysis to get a more statistically reliable conclusion regarding relationship between eNOS polymorphisms and hemorrhagic cerebral vascular diseases by pooling the results of already published relevant studies.

| MATERIAL S AND ME THODS
The PRISMA guideline was followed by the authors when conducting this meta-analysis (Moher, Liberati, Tetzlaff, Altman, & PRISMA group, 2009).

| Literature search and inclusion criteria
Literature searching of Pubmed, Web of Science, Embase, and CNKI was performed by the authors using the following terms: (endothelial nitric oxide synthase or nitric oxide synthase type III or eNOS or NOS3) and (polymorphism or variant or variation or mutation or SNP or genome-wide association study or genetic association study or genotype or allele) and (hemorrhagic cerebral vascular disease or hemorrhagic cerebrovascular disease or cerebral hemorrhage or basal ganglia hemorrhage or putaminal hemorrhage or subarachnoid hemorrhage or cerebrum hemorrhage or brain hemorrhage or intracranial hemorrhage or intracranial aneurysm or cerebral aneurysm). When searching CNKI, the authors translated searching terms into Chinese. The authors also checked the references of obtained articles manually for additional related studies.
Eligible studies must meet all of three inclusion criteria: I. formally published case-control studies evaluating relationship be-  The authors used Newcastle-Ottawa scale (NOS) to assess the quality of included studies (Stang, 2010). The NOS's score range is from zero to nine, and the methodology quality of an article was considered to be good when it got a score of more than seven.

| Data extraction and quality assessment
Data extraction and quality assessment of included studies were performed by the first author and the second author separately.
The authors wrote to the corresponding authors for additional data when they failed to extract necessary information from included studies. In case of disagreement between the first author and the second author, the two authors would consult the corresponding author for suggestions, and a consensus must be reached among three authors.

| Statistical analyses
The authors used Review Manager to pool meta-analyses results to pool the results if I 2 is larger than 50%. Otherwise, the authors used Mantel-Haenszel method (fixed-effect model) to pool the results. The authors also conducted subgroup analyses by ethnicity.
The authors examined stabilities of pooled results through omitting one study each time and pooling the results of the other studies. The authors examined publication biases through funnel plots.

| Characteristics of included studies
One hundred and sixty articles were retrieved by the authors through our literature searching strategy. The authors assessed thirty-six articles for eligibility after omitting unrelated or repeated reports. Twelve reviews and six case series were further excluded by the authors. Totally eighteen studies were finally pooled in our meta-analyses ( Figure 1). Extracted data of eligible studies were summarized in Table 1.

| Meta-analyses of eNOS rs1799983 polymorphism and hemorrhagic cerebral vascular diseases
Thirteen studies involving 1792 cases and 1936 control subjects were eligible for estimation of relationship between eNOS rs1799983 polymorphism and hemorrhagic cerebral vascular diseases. The pooled meta-analyses did not reveal any significant associations for eNOS rs1799983 polymorphism and hemorrhagic cerebral vascular diseases (see Table 2).

| Meta-analyses of eNOS rs869109213 polymorphism and hemorrhagic cerebral vascular diseases
Nine studies involving 1,443 cases and 1741 control subjects were eligible for estimation of relationship between eNOS rs869109213 polymorphism and hemorrhagic cerebral vascular diseases. The pooled meta-analyses did not reveal any significant associations for eNOS rs869109213 polymorphism and hemorrhagic cerebral vascular diseases (see Table 2).

| Sensitivity analyses
Stabilities of pooled meta-analyses results were examined by omitting one study each time and pooling the results of the other studies.
The trends of associations remained unchanged in sensitivity analyses, indicating that our pooled meta-analyses results were statistically stable.

First author (year)
The values in bold represent there is statistically significant differences between cases and controls.

| D ISCUSS I ON
Endothelial nitric oxide synthase, which is primarily found in the vascular endothelium, can continuously generate NO, and thus, it is essential for maintaining basal vascular tone and normal cerebral blood flow, and its dysregulation has also been found to be closely related to vascular endothelial dysfunction (Rudic et al., 1998;Faraci & Brian 1994). Since vascular endothelial dysfunction is clearly implicated in vascular diseases, eNOS gene polymorphisms were also intensively studied regarding their relationship with predisposition to various types of cerebral vascular diseases. There were already abundant meta-analyses regarding eNOS gene polymorphisms and ischemic cerebral vascular diseases (Shyu et al., 2017;Wei et al., 2017). So in this meta-analysis, we focused on hemorrhagic cerebral vascular diseases. The pooled meta-analyses results demonstrated that eNOS rs2070744 polymorphism might influence susceptibility to hemorrhagic cerebral vascular diseases in East Asians, but we failed to find any significant associations for eNOS rs1799983 and rs869109213 polymorphisms. The trends of associations remained unchanged in sensitivity analyses, suggesting that our pooled meta-analyses results were quite statistically stable.
Few points need to be considered when interpreting our findings. First, previous experimental studies demonstrated that all investigated polymorphisms were correlated with altered gene expression or protein structure of eNOS (AlFadhli, 2013;Dosenko, Zagoriy, Haytovich, Gordok, & Moibenko, 2006;Elakkad, Abou-Aisha, Hassanein, & Gad, 2017). To be brief, the rs2070744 polymorphism is associated with a thymine to cytosine mutation at coden-786 in the 5′-flanking region of the eNOS gene, which can significantly reduce eNOS gene expression. The rs1799983 polymorphism is associated with a Glu-to-Asp change at nucleotide 298 in exon 7 that is demonstrated to be associated with a trend of reduction of eNOS enzyme activity, and rs869109213 polymorphism, which can result in a variable number of tandem repeats (27 bp VNTRs) in intron 4, is also associated with altered eNOS enzyme activity. Thus, it is likely that these eNOS variations might influence normal functioning of eNOS, lead to vascular endothelial dysfunction, and influence predisposition to hemorrhagic cerebral vascular diseases. In this meta-analysis, only rs2070744 polymorphism was found to be significantly associated with predisposition to hemorrhagic cerebral vascular diseases. Nevertheless, we noticed that the trends of associations for rs1799983 and rs869109213 polymorphisms were actually similar to rs2070744 polymorphism. Therefore, maybe our pooled meta-analyses were still not statistically sufficient to detect the real associations between eNOS polymorphisms and hemorrhagic cerebral vascular diseases, and future studies in larger populations are still needed so as to get a more statistically robust conclusion. Second, the etiologies and pathogenesis mechanisms of hemorrhagic cerebral vascular diseases are extremely sophisticated. In this meta-analysis, we noticed that almost all eligible studies were about IA and its associated aSAH, whereas only a few studies were about hypertensive cerebral hemorrhage. Nevertheless, considering that the majority of hemorrhagic cerebral vascular diseases should be related to hypertensive disorders. Future studies should concentrate more on elucidating the relationship between eNOS polymorphisms and hemorrhagic cerebral vascular diseases of other etiologies, particularly hypertension. Third, we aimed to analyze all eNOS polymorphisms at the beginning. However, we did not find sufficient eligible studies to support meta-analyses of other eNOS polymorphisms, so we have to focus on only three polymorphisms in this meta-analysis.
Like all meta-analyses, a few limitations of our pooled meta-analyses have to be acknowledged. First, our pooled meta-analyses results were derived from pooling unadjusted findings of eligible studies since the authors did not have raw data (Xie, Shi, & Liu, 2017). Second, environmental factors might also influence relationship between eNOS polymorphisms and hemorrhagic cerebral vascular diseases. However, most investigators only focused on genetic associations in their works, so genetic-environmental interactions could not be explored in this meta-analysis (Wang et al., 2018). Third, we did not consider gray literatures (data that are not formally published in scientific books or journals, which include working papers, theses and dissertations, market reports, government documents, and conference posters). Therefore, despite that funnel plots were overall symmetrical, we still could not rule out the possibility that our pooled results might be affected by potential publication biases (Suvatha et al., 2018).
So to conclude, this meta-analysis demonstrated that eNOS rs2070744 polymorphism might influence predisposition to hemorrhagic cerebral vascular diseases in East Asians. These results also suggested that eNOS might involve in the development of hemorrhagic cerebral vascular diseases, and it may serve as a potential therapeutic target for hemorrhagic cerebral vascular diseases.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R S ' CO NTR I B UTI O N S
Qiuling Wang and Minfeng Zhou designed this meta-analysis. Qiuling Wang and Hongri Sun searched literatures. Xiaoguang Qi analyzed the data. Qiuling Wang and Minfeng Zhou wrote the manuscript. All authors approved the final manuscript as submitted.

E TH I C A L A PPROVA L
This article does not contain any studies with human participants or animals performed by any of the authors; thus, ethical approval is not required.