Methylenetetrahydrofolate reductase gene rs1801133 polymorphism and essential hypertension risk from a comprehensive analysis

Essential hypertension (EH) is common and multifactorial disorders likely to be inuenced by multiple genes. The methylenetetrahydrofolate reductase (MTHFR) gene rs1801133 polymorphism is related to MTHFR enzyme activity and to plasma homocysteine concentration. In addition, variations in MTHFR functions likely play roles in the etiology of EH. So far, larger number of studies between MTHFR rs1801133 polymorphism and EH have provided controversial or inconclusive results. To better assess the purported relationship, we performed a comprehensive analysis of 50 publications. coronary heart disease (0.83, 0.78– 0.88), stroke (0.73, 0.68–0.77), and heart failure (0.72, 0.67–0.78)[4]. EH accounts for 90%-95% of all patients with hypertension, and about 20%-60% of its etiology is related to genetic factors[5]. and Hcy, and of plasma Hcy level[10, Above information indicated that MTHFR C677T or rs1801133 may related to EH development and It makes sense to demonstrate the association between this and EH risk, which may provide guidance for the prevention and diagnosis of EH in clinic.


Background
Essential hypertension (EH) is common and multifactorial disorders likely to be in uenced by multiple genes. The methylenetetrahydrofolate reductase (MTHFR) gene rs1801133 polymorphism is related to MTHFR enzyme activity and to plasma homocysteine concentration. In addition, variations in MTHFR functions likely play roles in the etiology of EH. So far, larger number of studies between MTHFR rs1801133 polymorphism and EH have provided controversial or inconclusive results. To better assess the purported relationship, we performed a comprehensive analysis of 50 publications.

Methods
Eligible studies were identi ed by searching the PubMed, Wanfang and CNKI databases. Odds ratios (ORs) with 95% con dence intervals (CIs) were estimated to assess this association.

Conclusions
Our study showed evidence that MTHFR rs1801133 null genotype may increase EH risk. Future studies with larger sample size are warranted to further evaluate this association in more detail.

Background
Essential hypertension (EH) has a high prevalence rate worldwide, which is considered as a complex interaction of diverse gene-gene or environmental conditions [1,2]. The present evidence-based treatment of EH is a critical intervention in reducing cardiovascular (CV) morbidity and mortality [3]. A contemporary meta-analysis of 123 studies with 613,815 hypertensive participants showed for every 10-mm Hg reduction in systolic blood pressure, there is a signi cant decreasing of the risk of major CV disease events (relative risk 0.80, 95% con dence interval [CI] 0.77-0.83), coronary heart disease (0.83, 0.78-0.88), stroke (0.73, 0.68-0.77), and heart failure (0.72, 0.67-0.78) [4]. EH accounts for 90%-95% of all patients with hypertension, and about 20%-60% of its etiology is related to genetic factors [5].
Because a high plasma concentration of homocysteine (Hcy) may predispose to atherosclerosis by injuring the vascular endothelium, which results in hypertension. Elevated Hcy has been identi ed as an independent risk factor for hypertension [6][7][8][9]. Methylenetetrahydrofolate reductase (MTHFR) plays an important role in the metabolism of Hcy. The C/T mutation of C667T site (rs1801133, a C to T transition at nucleotide position 677 in exon 4 generates an alanine to valine change at amino acid 222) of MTHFR gene may lead to the decrease of MTHFR activity and heat tolerance, which may lead to the metabolic damage of Hcy, and then to moderate increase of plasma Hcy level [10,11]. Above information indicated that MTHFR C677T or rs1801133 may related to EH development and susceptibility. It makes sense to demonstrate the association between this polymorphism and EH risk, which may provide guidance for the prevention and diagnosis of EH in clinic.
So far, numerous studies have reported the association between MTHFR rs1801133 polymorphism and EH risk, however, this relationship remains ambiguous and controversial. We performed a comprehensive analysis including 50 different case-control studies to achieve a convinced conclusion.

Materials And Methods
Identi cation of eligible studies The PubMed, Wanfang and CNKI databases (updated on Dec 30, 2020) were applied using following relative keywords: polymorphism/variant/mutation, hypertension/essential hypertension, and MTHFR/methylenetetrahydrofolate reductase. We included all studies that described the relationship between MTHFR rs1801133 polymorphism and EH susceptibility. All included studies should meet all of the following criteria: (1) association between MTHFR rs1801133 polymorphism and EH risk; (2) case-control study; (3) each genotype frequency is shown in Tables; (4) genotype distributions of control consistent with Hardy-Weinberg equilibrium (HWE) about control were more than 0.05.

Data extraction
We collected following information in our analysis: rst author's last name, year of publication, origin for ethnicity, sample size (cases/controls), study design, HWE of controls and genotype methods, number of genotype frequency in cases/controls.

Quality assessment
In our current meta-analysis, the quality was assessed using the Newcastle-Ottawa Scale (NOS) for cross-sectional study quality assessment tool. The methodological quality of each study (sampling strategy, response rate, and representativeness of the study), comparability, and outcome were checked using the NOS tool. Studies with a score of more than 7 out of 10 were considered as achieving good quality. This cut-off point was declared after reviewing relevant kinds of literature [12].

Statistical analysis
The extracted data were imported to Stata software (version 10.0, Corporation, College Station, Texas) for analysis. Odds ratios (ORs) with 95% con dence intervals (CIs) were used to measure the strength of the association [13,14]. Subgroup analysis strati ed by race was performed rst. Race was categorized as European, Asian, Mixed, China and non-China ve types. Source of control subgroups were performed on two classi cations: hospital-based (HB) and population-based (PB). For MTHFR rs1801133, we investigated the relationship between genetic variants and LC risk in ve different models (T-allele vs. Callele, TT vs. CC, TC vs. CC, TT+TC vs. CC and TT vs. TC+CC).
Heterogeneity evaluation within the included studies was assessed using Cochrane's Q test (Chi-square) and I 2 (%) statistics. A xed effect model will be applied when the effects are assumed to be homogenous (P>0.05, I 2 ≤50%); otherwise, the random effect model will be adopted (P<0.05, I 2 ≥50%). [15,16] If heterogeneity is existed, to explore the source of heterogeneity, subgroup analysis will be performed through the ethnicity, publication year, study design and genotype methods.
The presence of potential publication bias is determined through the Egger/Begg's test and presented graphically by a funnel plot [17]. In addition, the departure of frequencies of MTHFR polymorphism from expectation under HWE is assessed by χ 2 test in controls using the Pearson chi-square test [18]. Another, sensitivity analysis is conducted to assess the stability of the results. Finally, the power and sample size analysis of our meta-analysis was calculated by a program called PS: Power and Sample Size Calculation (http://biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize#Windows) [19].

Meta-regression
Considering the subgroup of publication year, ethnicity, source of control, genotype method as independent variables and the log as dependent variable, the random-effect meta-regression results were presented to de nite the source of publication bias [20].

Gene interaction network of MTHFR gene
In order to fully understand the role and potential and functional partners of MTHFR in EH, String online server (http://string-db.org/) uses the gene-gene interaction network of MTHFR gene [21,22].

Study selection and characteristics in our meta-analysis
We established databases according to the extracted information from each article. Using the keywords, we identi ed 353 articles from PubMed, 56 from CNKI and 362 from Wanfang databases. 547 articles were excluded after reading abstract section and 224 articles were left for full article evaluation. Among them, 38 article were about systematic analysis/meta-analysis/review; 24 just only were offered case group; 16 articles were duplicated with including other papers; 34 had no original numbers for case/control groups, only showed total numbers; 27 article were about H-type hypertension; 4 were related to aortic hypertension and 26 were hypertension-in-pregnancy ( Figure 1). After above review, 54 articles about 55 case-control studies were left, in which 5 case-control studies were not consistent with HWE and were excluded, nally, 50 case-control studies from 54 different literatures were included in our current analysis. All essential information was listed in Table 1. Table 1 showed rst author, publishing year, race, the numbers of cases and controls, HWE, genotype numbers in cases/controls, study design and genotype methods. So in our study, there are 10 European case-control studies, 36 Asian case-control studies and 4 Mixed. The T frequency in Asians was 45.55%, in Europeans was (47.88%), and in Mixed was (47.38), which did not exist statistically signi cant (P > 0.05). The distribution of genotypes in all the controls was agreement with HWE. In addition, we checked the Minor Allele Frequency (MAF) reported for the seven main worldwide populations in the 1000 Genomes Browser [23] (https://www.ncbi.nlm.nih.gov/snp/rs1801133): Global (0.335); European (0.345); East Asian (0.328); South Asian (0.167); African (0.123); African American (0.125); Asian (0.265) (Figure 2A). In order to observe the frequency of T-allele and Callele both in case and control groups, we analyzed and found the frequency between case and control was pretty much the same ( Figure 2B). Finally, we analyzed the trend of rs1801133 polymorphism from TCGA database, TT (AA) frequency was relatively low than other genotypes ( Figure 2C). This polymorphism is associated with coronary artery, rather than aorta artery left ventricle and tibial artery (https://www.gtexportal.org/home/) ( Figure 2D). Table 2 showed that the summary odds ratios of MTHFR based on10533 EH cases and 11743 matched controls, we observed increased association between the MTHFR rs1801133 polymorphism and EH in total population (for example: T-allele vs. C-allele: OR=1.37, 95%CI=1.24-1.52, P h <0.001, p<0.001, I-squared=82.1%, Figure 3A). Then, subgroup by ethnicity analysis, similar trend was also observed (T-allele vs. C-allele: OR=1.46, 95%CI=1.29-1.67, P h <0.001, p<0.001, I-squared=84.8% for Asian; OR=1.27, 95%CI=1.07-1.51, P h =0.008, p=0.007, I-squared=59.7% for European; OR=1.51, 95%CI=1.30-1.74, P h <0.001, p<0.001, I-squared=86.9% for China and OR=1.21, 95%CI=1.06-1.37, P h <0.001, p=0.003, I-squared=66.4% for non-China, Figure 3B). In order to analyze the source of control and nd the source of heterogeneity, HB and PB subgroups were calculated, signi cant increased relationships were shown (T-allele vs. Callele: OR=1.49, 95%CI=1.28-1.75, P h <0.001, p<0.001, I-squared=85.4% for HB; T-allele vs. C-allele: OR=1.27, 95%CI=1.12-1.44, P h <0.001, p<0.001, I-squared=77.2% for PB, Figure 3C). Different methods for detecting this polymorphism were applied in all including studies, we tried to nd whether positive results may be observed, nally, some signi cant ndings were found, such as PCR (T-allele vs. C-allele: OR=1.48, 95%CI=1.14-1.91, P h <0.001, p=0.003, I-squared=85.1%), PCR-RFLP (T-allele vs. C-allele: OR=1.49, 95%CI=1.30-1.72, P h <0.001, p<0.001, I-squared=65.1%) and HRM(T-allele vs. C-allele: OR=1.32, 95%CI=1.15-1.51, P h <0.001, p<0.001, I-squared=47.5%) ( Figure 3D, E).

Publication bias and sensitive analysis
The Begg's funnel plot and Egger's test were performed to access the publication bias of literature. Signi cantly obvious evidence of publication bias was detected in ve genetic model analysis (such as Figure 4 A, B about T-allele vs. C-allele) ( Table 3).
To delete studies which may in uence the power and stability of the whole study, the sensitive analysis was applied, nally, no sensitive case-control studies were found for this SNP in ve models (such as Figure 4C about T-allele vs. C-allele).

Meta-regression
The analysis showed signi cant relationship for allele model (T-allele vs. C-allele) for the ethnicity, source of control and genotype methods with a regression coe cient of 0.001, 0.004, 0.010 and 0.002, respectively, rather than publication year, which means the heterogeneity from rs1801133 polymorphism in EH may be from the ethnicity, source of control and genotype methods subgroups ( Figure 5A-E).

Discussion
The cause of hypertension is unknown, the risk factors include genetic factors, age and unhealthy lifestyle, among which 70-80% of hypertension is related to unhealthy lifestyle. As the risk factors for high blood pressure accumulate, the risk of high blood pressure increases [24,25].
Most people with hypertension do not have typical symptoms, it is easy to be ignored and can't go to see a doctor in time. Therefore, how to identify high-risk patients is a very meaningful work, which can predict the high-risk patients with high blood pressure as early as possible, timely monitor blood pressure, follow-up regularly, pay attention to improve unhealthy lifestyles. Once the blood pressure is rising, it can be immediate for detection, gain treatment timely, avoid complications, and ultimately reduce the incidence of hypertension and improve the quality of life of patients [3,26,27].
The detection of signi cant polymorphisms may be the suitable method to forecast the possible of individuals to the risk of hypertension. Our current study focused on EH, a common type of hypertensions, and included 10533 EH patients and 11743 health individuals. In the overall analysis, we found individuals carried TT or T-allele may increase EH risk than CC or C-allele (between 37% and 89%). Signi cant heterogeneity was indicated in all genetic models, so to search the source, we analyzed the associations in other subgroups, such as ethnicity, source of control and genotype method. In the same time, signi cant relationships were also observed from ethnicity, source of control and genotype method subgroups, which were proved that rs1801133 polymorphism was a risk factor for EH in further. In addition, we used the meta-regression to evaluate the source of heterogeneity, nally, which came from above three subgroups. The power of our study was 1, which suggested our conclusions was convince and persuasive.  [30]. Fourth, Yang et al. performed a meta-analysis of 27 studies including 5418 EH and 4997 controls and supported the evidence the carriers of the rs180113T allele were susceptible to EH susceptibility [31]. Above studies have some disadvantages: rst, the HWE in several studies were not consistent with more than 0.05, which may increase the heterogeneity and reduce the power of conclusions; second, each study omitted other related casecontrol studies from, our present study is a relative comprehensive analysis; third, some articles did not distinguish the types of hypertension, because different kinds of hypertension have different etiology, pathogenesis and genetic background, so it is better to focus on one kind; fourth, our analysis increased genotype subgroup and evaluation of power; fth, we analyzed gene-gene interactions between the related genes and MTHFR to make clear the potential function. However, the conclusions from our current study was similar with previous meta-analysis.
The bright spot from our study was the gene-gene interaction. We showed the signi cant ten genes. The average score is more than 0.9, the top three genes are MTR (0.995), TYMS (0.992) and MTHFD1 (0.989). MTHFR and MTR both participate the homocysteine metabolism, which regular different stages, MTHFR converts 5,10-methylene-THF into 5-methyl-THF, however, MTR catalyzes the demethylation of 5-methyl-THF to THF and the re-methylation of homocysteine to methionine [32,33]. MTR 2756 A/G polymorphism also was associated with hypertension risk[34]. Some limitations in our meta-analysis should be considered. Beginning, the heterogeneity was found in our study, which came from ethnicity, source of control and genotype methods, further studies should optimize the design of retrospective and prospective studies to overcome this de ciency. Second, EH is a complex disease including genetic and other factors (such as environment, diet, concomitant disease, etc.) [35], studies should analyze the gene-gene or gene environment interactions with larger sample studies. Third, further meta may include all kinds of hypertension, and analyze association for each kind and nd the different genetic background. Fourth, the speci c mechanism of rs1801133 polymorphism should be explored.
In present, our meta-analysis showed the evidence that MTHFR 677T null genotype was associated with increased EH risk. Therefore, further well-designed large studies are necessary. Also, we should focus on the mechanism of rs1801133T-allele in animal model to explain the complete chain of evidence for prevention of EH in the future.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this published article. Project (YP19ZB03). The funding body play no direct role in the design of the study, and collection, analysis, and interpretation of data, and in writing the manuscript.
Author's contributions YF and LW designed the study and drafted the manuscript; LW extracted, analyzed, interpreted the data, and collected the clinical data; LW and WZ performed the targeted sequencing, analyzed and interpreted the data; LW and WZ participated in the study coordination and revised the manuscript. All authors read and approved the nal version of the manuscript.    Figure 1 Flowchart illustrating the search strategy used to identify association studies for MTHFR gene rs1801133 polymorphisms and EH risk.