Association between eNOS rs1799983 polymorphism and hypertension: a meta-analysis involving 14,185 cases and 13,407 controls

Background Essential hypertension is a complex disease determined by the interaction of genetic and environmental factors, eNOS is considered to be one of the susceptible genes for hypertension. Our study aimed to evaluate the association between eNOS rs1799983 polymorphism and hypertension, and to provide evidence for the etiology of hypertension. Methods Case–control studies of eNOS rs1799983 polymorphism and hypertension were included by searching PubMed, Embase, Web of Science, Medline, Scopus, WanFang datebase, Vip datebase, and CNKI database according to PRISMA guideline. Eligible data were extracted and pooled, and were analyzed using R software based on five different genetic models. Results A total of 60 eligible articles involving 14,185 cases and 13,407 controls were finally selected. We found significant association between eNOS rs1799983 polymorphism and hypertension under any genetic model (T vs G: OR = 1.44, 95% CI 1.26–1.63; GT vs GG: OR 1.34, 95% CI 1.18–1.52; TT vs GG: OR 1.80, 95% CI 1.41–2.31; GT + TT vs GG: OR 1.42, 95% CI 1.25–1.63; TT vs GG + GT: OR 1.68, 95% CI 1.35–2.08; GT vs GG + TT: OR 1.24, 95% CI 1.11–1.40). Conclusions We found that eNOS rs1799983 polymorphism is associated with the increased risk of hypertension under any genetic model. Moreover, investigations of gene–gene and gene-environment interactions are needed to give more insight into the association between eNOS rs1799983 polymorphism and hypertension. Supplementary Information The online version contains supplementary material available at 10.1186/s12872-021-02192-2.


Background
Essential hypertension (EH) is a complex disease determined by the interaction of genetic and environmental factors, and EH is regarded as a predisposing risk factor for many diseases, such as myocardial infarction, stroke, and chronic renal failure [1]. So far, the pathogenesis underlying hypertension is still unclear in spite of the in-depth research being conducted on the mechanism of EH. However, increasing evidence supports the theory that genetic factors are a determinant of hypertension to a large extent [2], thus it is pivotal to identify susceptible genes for prevention, diagnosis, and treatment of hypertension [3]. Genes (eNOS) encoding endothelial nitric oxide synthase is considered to be one of the susceptible genes for hypertension because its enhanced production or enzyme bioavailability can lead to constitutive release of nitric oxide (NO) in endothelial cells, which is involved in blood pressure (BP) regulation [4].
Previous studies have shown that eNOS plays a critical role in regulating vascular tone and blood pressure. For example, overexpression of eNOS gene in transgenic mice leads to a significant decrease in blood pressure [5]. In addition, it was found that inhibition of eNOS gene in healthy individuals is associated with decreased levels of NO release and increased blood pressure [6].
The eNOS gene at 7q35-36 spans 21 kb, with 26 exons and 25 introns. There are about 10 polymorphic loci distributed in the promoter, exon, and intron of the eNOS gene. In these loci, the common mutation that leads to amino acid substitutions in mature proteins is G894T or Glu298Asp (rs1799983) mutations, in which base substitution of G to T will result in glutamic acid (Glu) being replaced at exon 7 by aspartic acid (Asp) at position 298 of the corresponding amino acid [7]. This genetic mutation reduces the production of NO and subsequently affects the development of EH [8].
A large number of articles have studied the association between eNOS rs1799983 polymorphism and hypertension; however, these results are still inconsistent. Recently, it is noted that new studies [9][10][11][12] on this theme have been published since the last meta-analysis published in 2017 [13]. Therefore, we included these newly published studies and conducted a further metaanalysis to investigate whether eNOS rs1799983 polymorphism is associated with hypertension.

Literature search strategy
This meta-analysis was performed according to the statements in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting standard [14]. Systematic literature search was performed in PubMed, Embase, Web of Science, Medline, Scopus, WanFang datebase, Vip datebase, and CNKI database up to October 30, 2020. Various combinations of terms used for searching were ("endothelial nitric oxide synthase" OR "nitric oxide synthase type III" OR "eNOS" OR "NOS3") AND ("polymorphism" OR ''variant" OR "mutation") AND ("hypertension" OR "high blood pressure"). Moreover, we also retrieved and scrutinized related articles from the reference lists of literatures to replenish literatures that had not been identified in the initial search. A detailed form of the search strategy used in datebases was displayed in Additional file 1: Table S1.

Inclusion/exclusion criteria
Studies included had to meet the following criteria: (1) case-control studies; (2) patients with essential hypertension were defined as cases, healthy subjects without hypertension were defined as controls; (3) evaluation of the association between eNOS rs1799983 polymorphism and hypertension. The exclusion criteria satisfied the followings: (1) case reports, review articles or crosssectional studies; (2) duplicate articles; (3) secondary hypertension or gestational hypertension; (4) lack of sufficient information on genotype or allele frequencies.

Data extraction and quality assessment
For each eligible study, the following data were extracted: name of first author, year of publication, region and ethnicity of study population, sample size, and numbers of eNOS genotype or allele in cases and controls. Hardy-Weinberg equilibrium (HWE) among the controls was calculated.
Quality of the included studies was evaluated using the Newcastle-Ottawa scale (NOS) [15] that has a "star" rating system consisting of selection, comparability, and exposure. The highest score of this rating system is 9 points. Moreover, the data extraction and quality assessment were performed by two investigators (Jikang Shi and Yanbo Guo) independently, and conflicts were resolved by discussing with the third investigator (Sainan Liu) if the results of two investigators didn't reach an agreement.

Statistical analysis
HWE was evaluated for control groups of each study using Goodness of fit Chi-square test, and P < 0.05 was considered as a significant deviation from HWE. The associations between eNOS rs1799983 polymorphisms and hypertension in this meta-analysis were measured based on five different genetic models including six comparisons: allelic model (T vs G), codominant model (GT vs GG and TT vs GG), dominant model (GT + TT vs GG), recessive model (TT vs GG + GT), overdominant model (GT vs GG + TT). Odds ratios (OR) and 95% confidence intervals (95% CI) were used to assess the strength of association between eNOS rs1799983 polymorphisms and hypertension. Q-statistic and I 2 -statistic were used to evaluate heterogeneity, random-effect models (DerSimonian and Laird methods) were used when heterogeneity existed (I 2 ≥ 50% considered heterogeneity existed in between-study in this meta-analysis); otherwise, fixed-effect models (Mantel and Haenszel methods) were used. Subgroup analyses were performed by region, ethnicity, and HWE to detect main sources of heterogeneity and observe differences of the association in different groups. Sensitivity analysis was conducted to evaluate stability of our results by omitting each study at each time. Publication bias was estimated using funnel plots, and quantified by the Egger's tests (P < 0.05 considered statistically significant publication bias) [16]. All data management and statistical analyses were performed using R Studio (Version 1.1.383) (RStudio, Inc., MA, USA) for Windows.

Trial sequential analysis (TSA)
The risk of random error in traditional meta-analysis may increase because of the dispersed data and repeated significance testing [17,18]. TSA was used to reduce the risk of type I error by adjusting threshold for statistical significance and to evaluate the required information size (RIS) and statistical reliability [19]. In our meta-analysis, trial sequential analysis software (TSA, version 0.9; Copenhagen Trial Unit, Copenhagen, Denmark, 2011) were performed, and additional studies were not needed when Z-curve crossed the trial sequential monitoring boundary or RIS has reached; otherwise, further studies were needed.

Study characteristics
A total of 60 eligible articles involving 14,185 cases and 13,407 controls were finally selected after strict screening on the basis of the inclusion and exclusion criteria, the protocol of literature search and selection is shown in Fig. 1, and the main characteristics and genotype distribution of the eligible studies are listed in Table 1.

Association between eNOS rs1799983 polymorphism and hypertension
There were significant heterogeneities between eNOS rs1799983 polymorphism and hypertension in the five different genetic models, and thus random-effects model was used for all analyses. We found significant association between eNOS rs1799983 polymorphism and the risk of hypertension under any genetic model (T  (Fig. 2).

Subgroup analysis
We performed subgroup analysis by region and ethnicity because gene polymorphism may be associated with variations in region and ethnicity. For region, there is only difference for the association between eNOS rs1799983 polymorphism and hypertension under overdominant model, when GT was compared with GG + TT, the association with risk of hypertension was identified in China (OR 1.29; 95% CI 1.12-1.49), and the association between eNOS rs1799983 polymorphism with risk of hypertension was found in any region under other genetic models. With regard to ethnicity, we found the association between eNOS rs1799983 polymorphism with risk of hypertension was significant in Asian population under all genetic models (T vs G: OR

Sensitivity analysis and publication bias
To examine the influence of individual study on the overall results, sensitivity analysis was performed by excluding a single study at each time in our meta-analysis. The results of sensitivity analysis showed that the corresponding pooled ORs and 95% CIs under any model of inheritance were not substantially altered after excluding any single study, suggesting that results of our meta-analysis were relatively stable and credible (Additional file 2: Figure S1).
Publication bias was evaluated by funnel plots and quantified by Egger's tests. The funnel plots for recessive model (TT vs GG + GT) seemed symmetrical, and the results of Egger's tests showed that there was no publication bias (P = 0.102); however, the funnel plots were asymmetrical in other genetic models for the association between eNOS rs1799983 polymorphism with hypertension, and the results of Egger's tests showed that there were publication bias (T vs G: P = 0.026; GT vs GG: P = 0.023; TT vs GG: P = 0.032; GT + TT vs GG: P = 0.011; GT vs GG + TT: P = 0.038) (Additional file 3: Figure S2).

Trial sequential analysis (TSA)
For the association between eNOS rs1799983 polymorphism with hypertension under codominant model (GT vs GG), codominant model (TT vs GG), and dominant model (GT + TT vs GG), the Z-curve crossed trial sequential monitoring boundary, although the sample size did not reach the RIS (Fig. 3B-D). However, for the association between eNOS rs1799983 polymorphism with hypertension under allelic model (T vs G), recessive model (TT vs GG + GT), and overdominant model (GT vs GG + TT), the Z-curve crossed trial sequential monitoring boundary, and the sample sizes were also more than the RIS (Fig. 3A, E, F). Therefore, concrete evidence indicates that further studies are not necessary for the association between eNOS rs1799983 polymorphism with hypertension.

Discussion
In the meta-analysis, we collected related articles comprehensively to investigate the association between eNOS rs1799983 polymorphism and hypertension. Our results suggest that there is an association between eNOS rs1799983 polymorphism and risk of hypertension under any genetic model (T vs G, GT vs GG, TT vs GG, GT + TT vs GG, TT vs GG + GT, and GT vs GG + TT), especially among Asian population. Moreover, with respect to contrast of TT versus GG and TT versus GG + GT, the TT genotype is associated with the increased risk of hypertension not only in Asian population but also in other population.
Nine meta-analyses on association between eNOS rs1799983 polymorphism and hypertension have been   risk of hypertension. In addition, Li et al. studied the association between T allele of eNOS rs1799983 polymorphism and hypertension, and Liu et al. studied the association between GT + TT genotype of eNOS rs1799983 polymorphism and hypertension, and their results are also consistent with our results. Pereira et al. [24] studied the association between GT + TT genotype of eNOS rs1799983 polymorphism and hypertension, and consistent with the discoveries of Pereira et al., we also identified the heterogeneity and publication bias in the meta-analysis, they may exist owing to the gene-environment interactions. Niu et al. [25] only studied the association between T allele of eNOS rs1799983 polymorphism and hypertension, we all found the T allele of eNOS rs1799983 polymorphism was a risk factor of hypertension, especially among Asian population. Moreover, of the nine metaanalyses, the results of Takeuchi [26] and Zintzaras [27] were negative, they found that there was no association between eNOS rs1799983 polymorphism and hypertension, the reason they had this negative results may be a small size, or interaction of polymorphisms within haplotypes, which is a major determinant of disease susceptibility, not the individual polymorphism [28].
For the meta-analysis of Xie et al. [13], the last metaanalysis published in 2017, their results showed there is no association between TT genotype and hypertension when TT genotype was compared with GG + GT genotype, but TT genotype was associated with the increased risk of hypertension in our meta-analysis. In addition, our result of TSA also demonstrated that the Z-curve crossed trial sequential monitoring boundary, and the sample sizes were also more than the RIS. Therefore, it is adequate to draw a conclusion that TT genotype is associated with the increased risk of hypertension.
The meta-analysis may report false positive results for the risk of type I errors, and these results are usually caused by publication bias, heterogeneity between studies, or poor study quality. However, a limited number of trials may not provide enough information, resulting in incorrect estimates [29]. Thus, we conducted TSA to reduce the risk of type I errors and evaluated whether further studies are necessary by calculating the required information size. In our metaanalysis, either the sample size was greater than the required information size or the Z-curve crossed trial sequential monitoring boundary, indicating that the results of our meta-analysis are reliable and sufficient to draw conclusions on the association between eNOS rs1799983 polymorphism and hypertension.
The vasodilator effect of NO that is produced by eNOS is very important for maintenance of vascular function [30], and the G894T polymorphism (Glu-298Asp or rs1799983) at exon 7 of the eNOS gene is associated with reduced eNOS expression, activity and subsequently reduced NO production, could be a potential candidate marker for hypertension development [31,32]. Moreover, clinical studies have showed that vascular responsiveness is altered in subjects with this variant owing to an increased vasoconstrictive response to phenylephrine for the subjects with Asp298 [33], and several clinical and experimental studies also indicate that alteration of NO metabolism plays a key role in the occurrence and conventional therapy of hypertension [34][35][36].Therefore, it is necessary to   identify the association between eNOS rs1799983 polymorphism and hypertension. Our study has some limitations. First, there is heterogeneity in our article, and the main sources of heterogeneity remain unclear. Second, publication bias was found in the association between eNOS rs1799983 polymorphism and hypertension under any genetic model except the recessive model, because negative articles are unpublished. Third, our research cannot prove the existence of causality, but only an association because of the design of case-control. Despite the above limitations, our research also has some advantages. First of all, we have collected the latest articles extensively, which provides more statistical power to draw effective conclusions on this issue. Secondly, the results of sensitivity analysis show that our conclusion is stable and reliable. Third, to our knowledge, this is the first TSA to evaluate the association between eNOS rs1799983 polymorphism and hypertension, which further offers reliable evidence to reach the conclusion.

Conclusion
In conclusion, eNOS rs1799983 polymorphism is associated with increased risk of hypertension under any genetic model. Moreover, investigations of gene-gene and gene-environment interactions are needed to give more insight into the association between eNOS rs1799983 polymorphism and hypertension.