Effect of MTHFR C677T gene polymorphism on folic acid intervention in Chinese hypertensive patients：a multicentered, randomized, double-blind, clinical trial

doi:10.21203/rs.3.rs-1527911/v1

Download PDF

Research Article

Effect of MTHFR C677T gene polymorphism on folic acid intervention in Chinese hypertensive patients：a multicentered, randomized, double-blind, clinical trial

https://doi.org/10.21203/rs.3.rs-1527911/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Objectives To describe the changes in serum folate concentration and assess the influence of individual methylenetetrahydrofolate reductase (MTHFR) C677T on the changes of serum folate concentration that resulted from 8 weeks of supplementation with folic acid in Chinese hypertensive patients.

Methods We conducted a multicenter, randomized, double-blind controlled clinical trial of different dosages folate supplementation in Chinese hypertensive patients.A total of 1657 patients with mild or moderate essential hypertension were randomly assigned to one of eight treatment groups corresponding to eight different doses of folic acid, ranging from 0 mg to 2.4 mg,once daily for 8 weeks. Individual serum folate levels were measured at baseline, and at 4 and 8 weeks posttreatment.

Results compared with patients with 677CC genotype, those with TT genotype had significantly lower folate concentrations at baseline.After 4 or 8 weeks of treatment, increases in serum folate were seen across all genotypes and FA dosage groups. At 8 weeks posttreatment ,there was no statistically significant difference in folate levels between groups according genotypes,and patients with CC genotype showed an attenuated response compared with those with TT genotype.( CC,0.45 vs. TT,0.56 ,P< 0.05).

Conclusion We demonstrated that MTHFR C677T polymorphisms affected folate levels at the baseline and the therapeutic response of folic acid intervention. Patients with TT genotype benefited more after folic acid supplementation in Chinese hypertensive population.

Hypertension

Folic acid

China

Folate (vitamin B9) is an essential water-soluble nutrient which cannot be synthesised or stored by humans. Physiologic folate is found naturally in some foods such as vegetables and fruits, and has lower bioavailability^{[1, 2]}. Due to the poor stability of folic acid^[3] and chinese traditional dietary habits, this can lead to the destruction of large amounts of natural folic acid during cooking. So, it is difficult to ensure adequate folate intake solely through diet^{[2, 4]}.From 1998 the United States of America (USA) and Canada implemented mandatory folic acid fortification of enriched cereal grain products to improve folate status. China, however, has not adopted a folic acid fortification policy, leading to a relatively low levels of folic acid in the Chinese population. A previous study on folic acid levels in Chinese^[5] showed that folic acid levels in men and women in China were 4.3 ng / ml and 6.3 ng / ml, respectively. At the same time, the folic acid level of the American population was about 14.2 ng / ml^[6], and the folic acid level of the Chinese population was far lower than that of the American population. Previous studies have demonstrated that folate is associated with numerous diseases, including neural tube defects^{[7, 8]} and cardiovascular disease^[9–11], hearing disorder^[12] and cognitive impairment^[13]. Therefore, it is very important to maintain the best folate status in vivo by supplementing folic acid. This is a simple, effective and economic method to reduce the risk of related diseases and relieve the economic burden of diseases.

Methylenetetrahydrofolate reductase (MTHFR) is a regulatory enzyme involved in the homocysteine and folate metabolic pathways. MTHFR converts 5, 10-methylene-THF into 5-methyl-THF(1-carbon metabolism), the dominant circulating form of folate, and is a methyl donor for the remethylation of homocysteine to methionine^{[14, 15]}. The MTHFR C677T polymorphism could affect enzyme activity. At the MTHFR C677T locus, compared with the wild-type (CC) genotype carriers, heterozygous variant (CT) have 30% lower enzyme activity and homozygous variant (TT) carriers have 60% lower enzyme activity^[16]. Those with the TT genotype have lower enzyme activity, Which may be the cause of decreased folate and increased levels of plasma homocysteine^[17].The distribution of the TT genotype varies by region and population.The frequency of MTHFR 677 TT genotype in western population was about 10%,while that in northern Chinese women of childbearing age reached 19.8%^[18]. However,a hypertensive adult study has shown a higher TT genotype frequency (about 25%)^{[19, 20]}.

Epidemiological studies have shown that the higher folate intake,the lower the risk of hypertension^[21]. In addition, Homocysteine (Hcy) is an independent risk factor for cardiovascular diseases. Previous studies have shown that hyperhomocysteinemia (HHcy) and hypertension often coexist and HHcy accounts for about 75% of chinese hypertension patients^[22].Synergistic effects of hypertension and homocysteine increase the risk of cardiovascular disease^[23]. Interesting,folate treatment can decrease homocysteine levels and the the risk of hypertension.Therefore,it is especially important for hypertensive patients of China (a region with low folate intake and without universal folic acid fortification) to supplement folic acid.To our knowledge,no clinical trial, large-scale and multi-dose folic acid supplementation, has been done to date to carried out in Chinese hypertensive patients,a population at high-risk of stroke and cardiovascular diseases.This study aims to describe the changes in serum folate concentration and assess the influence of MTHFR genotypes in response to different dosages.

Patients and ethics

This was a large randomized, double-blind, controlled clinical trial based on Chinese hypertensive population (Trial Number:ChiCRT1800016135). We recruited 1677 subjects in Lianyungang, Anqing and Wuyuan in 2018. The inclusion of research objects has the following criteria :

(a) Aged 45–75 years; (b) Patients with confirmed primary hypertension, including those currently taking antihypertensive drugs; For those who did not take antihypertensive drugs within two weeks, two consecutive examinations were performed at least one day interval, and two sitting blood pressure (average three measurements) met the following criteria : systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg. (c) Women of childbearing age agreed to use a reliable contraceptive method during the experiment. (d) Clinicians judge that enalapril folic acid tablets are suitable for treatment. Participants were excluded if they had a history of cardiovascular diseases, had chronic illness including severe chronic heart failure or serious mental disorders.

This study was approved by the Ethics Committee of the Institute of Biomedicine, Anhui Medical University, Hefei, China. Participants read and signed informed consent forms that described all study procedures prior to enrollment.

Study Design

This study included three phases : a screening period (2–10 days), a lead-in period (2 weeks), and a double-blind treatment period (8 weeks). During the screening period, patients were asked to register and file, and to complete relevant examinations and questionnaires. During the lead-in period, patients were asked to take an oral daily dose of 10 mg of enalapri for 2 weeks. The purpose of this phase is to examine the patient's tolerance to enalapril,eliminate the effects of previous drug treatment, complete the baseline assessment and confirm that the patient was still fully compliant with inclusion criteria. Patients were assigned a code and tested for the MTHFR C677T genotype during the lead-in period. Eligible participants were stratified by sex and methylenetetrahydrofolate reductase C677T genotype(CC, CT, TT), then each was randomly assigned to one of eight folic acid dose treatment groups. This study was divided into eight treatment groups :

( 1 ) 0 mg folic acid group : enalapril ( 10 mg )

( 2 ) 0.4 mg folic acid group : enalapril folic acid tablets ( 10 mg : 0.4 mg )

( 3) 0.6 mg folic acid group : enalapril folic acid tablets ( 10 mg : 0.4 mg ) + folic acid ( 0.2 mg )

( 4 ) 0.8 mg folic acid group : enalapril folic acid tablets ( 10 mg : 0.8 mg )

( 5 ) 1.2 mg folic acid group : enalapril folic acid tablets ( 10 mg : 0.8 mg ) + folic acid ( 0.4 mg )

( 6 ) 1.6 mg folic acid group : enalapril folic acid tablets ( 10 mg : 0.8 mg / tablet ) + folic acid ( 0.8 mg )

( 7 ) 2.0 mg folic acid group : enalapril folic acid tablets ( 10 mg : 0.8 mg / tablet ) + folic acid ( 1.2 mg )

( 8 ) 2.4 mg folic acid group : enalapril folic acid tablets ( 10 mg : 0.8 mg / tablet ) + folic acid ( 1.6 mg )

The study drug should be taken orally, once a day. The double-blind treatment period lasted 8 weeks. During each research stage, related drugs that affect the evaluation of efficacy should not be taken. The patients were followed up at the end of the 4th and 8th weeks of treatment, and their blood pressure and blood samples were collected. This trial has been registered with Trial Number: ChiCTR1800016135.

Blood Sample Collection And Laboratory Measurement Methods

After fasting for 10–12 hours, venous blood was taken from patients.Plasma or serum samples were separated within 15 min after collection, and analyzed within 30 min or stored at − 80°C .

Measurement of plasma homocysteine levels :Agilent 1100 liquid chromatograph, Hypersil C18 column (4.6×250mm, 5µm); mobile phase: 0.07mol/L acetate buffer (containing 2% methanol); flow rate 0.8ml/min; injection volume 15µl; column temperature 25°C; analysis time: 15 minutes; fluorescence detection conditions: excitation wavelength 390nm, emission wavelength 470nm. Good linearity and recovery (~ 100%) were obtained using an internal standard method (N-acetylcysteine (Fluka), internal standard); within-assay variation was 3.5% and between-assay variation was 4.2%.

Measurement of folic acid level : The instrument was Becman Conlter Access Immuno Assay System (Beckman-Coulter Canada, Inc, Mississauga, Canada), and the detection method was electrochemiluminescence method. The intra-assay variation was 2.3%, and the inter-assay variation was 7.3%.

Genotype determination : The MTHFR C677T polymorphisms were genotyped by the PCR-restriction fragment length polymorphism method in the laboratory .

Statistical Analysis

Due to the positive skewed distribution of folic acid concentration, the natural logarithms of these variables are used as continuous variables for all statistical analysis. One-way analysis of variance for continuous variables and chi-square test (or Fisher’s exact test) for categorical variables were applied to compare the the difference in population characteristics among the three genotypes. Wilcoxon signed-rank test was used to compare the serum folic acid concentrations at different time points before and after treatment. Changes in folate levels after 4 or 8 weeks of treatment were expressed as differences between baseline and folate levels at 4 or 8 weeks of treatment, which was the main result.Multiple regression model was used to compare the response to folic acid supplementation for different genotypes. Statistical analysis was carried out using the Empowerstats 2.0.

A total of 1657 patients were recruited for this study. This analysis excluded 119 patients who were ineligible to participate in the trial. Accordingly, a total of 1538 patients were included in our final analysis. The flow of participants through the study is shown in Fig. 1. The frequency of CC, CT, and TT genotype in Chinese hypertensive patients was 30.8, 45.9, and 23.3%,respectively. This population had no significant deviations of genotype distributions from the expected Hardy-Weinberg equilibrium.

Table 1 describes the characteristics of study subjects at baseline. Subjects were enrolled in the current study with data on sex, age, body mass index (BMI), baseline systolic blood pressure (SBP), diastolic blood pressure (DBP), MTHFR genotypes, current smoking status, current drinking status, and laboratory results based on the 8 various dosages of folic acid supplementation. No statistical differences were observed between groups. The folate levels of patients with CC, CT and TT genotype were 12.3ng/ml, 11.9ng/ml and 8.5ng/ml, respectively. Patients with TT genotype had significantly lower folate levels than patients with CC or CT genotype. After eight weeks of folic acid treatment, the folic acid levels of patients with CC, CT and TT genotype were 40.3ng/ml, 42.9 ng/ml and 40.0ng/ml ( P = 0.784 ), respectively. There was no statistical difference in folate among genotypes.

As shown in Table 2. Elevated folate levels were observed across all genotypes and folate dose groups after 4 or 8 weeks of folic acid treatment.

As shown in Table 3. We used multiple linear regression models to characterize the relationship between changes of serum folate concentrations and the doses of folic acid supplementation. After 4 or 8 weeks of folic acid treatment, serum levels of folate were positively associated with folate dosages.No significant difference in the response of gene polymorphisms to folic acid supplementation (P = 0.893) was observed at week 4.However, there was a significant difference in response to folic acid supplementation across genotypes (P = 0.043) and the change of serum folic acid concentration in patients with TT genotype is more sensitive than those with CC genotype(TT:0.56 vs. CC:0.45) for every increase in 1 mg/day of folic acid dose at 8 weeks,adjusted for age, sex, BMI,smoking status, drinking status and other factors.

Table 1

Baseline characteristic of participants
Characteristic	Treatment group								P value
Characteristic	0 mg	0.4 mg	0.6 mg	0.8 mg	1.2 mg	1.6 mg	2.0 mg	2.4 mg	P value
N	214	215	164	211	156	206	160	212
Male, No.(%)	95 (44.4)	101 (47.0)	80 (48.8)	98 (46.4)	72 (46.2)	94 (45.6)	85 (53.1)	100 (47.2)	0.844
Age, year	65.5 ± 8.1	63.9 ± 7.7	65.0 ± 7.7	64.9 ± 7.9	64.7 ± 8.1	64.6 ± 7.4	64.0 ± 9.1	64.8 ± 9.0	0.580
BMI, kg/m2	24.9 ± 6.9	25.0 ± 3.5	25.3 ± 11.5	24.9 ± 3.5	24.3 ± 3.5	24.4 ± 3.4	24.7 ± 3.2	24.6 ± 3.4	0.695
SBP at baseline,mmHg	153.8 ± 17.6	152.2 ± 17.4	152.9 ± 18.9	152.4 ± 15.9	154.6 ± 17.4	154.2 ± 16.7	154.2 ± 18.5	152.1 ± 17.5	0.721
DBP at baseline,mmHg	90.0 ± 10.1	90.2 ± 10.9	90.0 ± 11.8	89.9 ± 9.6	89.7 ± 10.0	90.6 ± 10.5	90.5 ± 11.1	90.1 ± 11.0	0.990
Current smoking, No. (%)	44 (20.6)	49 (22.8)	28 (17.1)	56 (26.5)	36 (23.1)	44 (21.4)	36 (22.5)	46 (21.7)	0.615
Current drinking, No. (%)	48 (22.5)	59 (27.4)	34 (20.7)	51 (24.2)	33 (21.2)	45 (21.8)	33 (20.6)	46 (21.7)	0.757
Laboratory results									0.861
tHcy, µmol/L	14.4 (11.8–17.7)	14.2 (12.3–17.2)	14.6 (11.8–17.7)	14.0 (12.1–17.7)	13.2 (11.1–17.4)	14.5 (11.8–17.6)	14.2 (11.9–17.6)	14.5 (12.2–17.7)	0.869
Folate, ng/ml	12.4 (7.7–16.9)	10.4 (7.1–16.6)	11.2 (8.0-16.1)	10.3 (7.1–14.5)	12.4 (8.4–17.7)	10.9 (7.7–16.4)	11.2 (8.3–17.3)	11.1 (7.3–16.5)	0.335
5-METHF, ng/ml	7.1 (5.0-10.2)	6.8 (4.7–9.5)	8.0 (5.7–11.9)	6.7 (4.8–9.5)	8.5 (6.0-12.3)	7.6 (5.4–10.7)	8.2 (6.0-12.4)	6.7 (4.8–10.5)	< 0.001
MTHFR genotypes, No.(%)
CC	64 (29.9)	63 (29.3)	53 (32.3)	66 (31.3)	52 (33.3)	56 (27.2)	47 (29.4)	73 (34.4)	0.856
CT	104 (48.6)	104 (48.4)	78 (47.6)	96 (45.5)	67 (42.9)	95 (46.1)	78 (48.8)	84 (39.6)
TT	46 (21.5)	48 (22.3)	33 (20.1)	49 (23.2)	37 (23.7)	55 (26.7)	35 (21.9)	55 (25.9)
Center,No.(%)									0.238
Anqing	42 (19.6)	39 (18.1)	32 (19.5)	42 (19.9)	30 (19.2)	42 (20.4)	28 (17.5)	48 (22.6)
Lianyungang	73 (34.1)	78 (36.3)	47 (28.7)	77 (36.5)	43 (27.6)	74 (35.9)	39 (24.4)	71 (33.5)
Wuyuan	99 (46.3)	98 (45.6)	85 (51.8)	92 (43.6)	83 (53.2)	90 (43.7)	93 (58.1)	93 (43.9)

Table 2

Folic acid levels in different dose groups at different times by MTHFR C677T genotypes
	Treatment group
	0 mg	0.4 mg	0.6 mg	0.8 mg	1.2 mg	1.6 mg	2.0 mg	2.4 mg
Baseline
CC	13.8 (10.0-18.4)	13.2 (8.0-18.1)	11.8 (7.4–17.8)	11.4 (7.7–14.9)	14.4 (8.0–19.0)	12.6 (8.9–17.0)	13.1 (9.3–17.4)	12.2 (7.4–15.1)
CT	12.6 (7.5–17.2)	10.9 (7.4–17.1)	12.2 (8.5–16.5)	11.3 (7.2–14.8)	13.1 (9.5–18.3)	11.8 (8.2–17.5)	11.6 (8.9–18.7)	12.1 (8.5–18.2)
TT	10.5 (6.8–12.7)	7.8 (5.7–11.4)	8.8 (6.9–12.3)	8.0 (5.4–10.0)	11.1 (8.1–13.5)	8.4 (5.9–13.8)	9.0 (6.2–12.3)	8.0 (5.9–11.2)
Week 4
CC	11.2 (7.8–15.1)	20.0 (16.7–41.1)	34.8 (18.2–50.9)	41.4 (20.7–60.5)	48.1 (23.3-126.8)	55.5 (27.5-158.2)	58.8 (22.4–148.0)	107.8 (27.3-232.7)
CT	9.5 (6.4–12.2)	19.8 (14.8–35.2)	37.0 (19.3–51.3)	40.3 (20.0-59.8)	44.8 (18.6-103.8)	42.0 (19.9-132.5)	74.2 (28.5-216.8)	62.4 (23.8-189.9)
TT	7.8 (5.5–10.0)	25.2 (14.7–38.7)	30.3 (18.2–46.4)	27.5 (16.2–48.3)	61.8 (23.9-102.2)	55.1 (18.9-132.4)	76.1 (18.7–165.0)	45.5 (18.6-126.9)
Week 8
CC	12.0 (7.9–16.5)	30.1 (19.6–40.7)	40.9 (27.0-55.3)	48.0 (24.0-80.8)	89.3 (39.0-136.5)	129.6 (31.9-195.5)	161.2 (39.9-272.3)	129.7 (35.7-283.2)
CT	9.8 (7.2–14.3)	26.2 (17.3–39.7)	42.0 (27.4–59.5)	52.3 (27.4–91.8)	63.7 (29.8-146.9)	107.2 (23.7-179.7)	193.6 (67.7-343.3)	176.8 (37.7-355.2)
TT	8.7 (7.0-11.5)	20.7 (16.6–40.2)	31.9 (21.3–48.1)	40.5 (22.4–66.1)	94.0 (31.2-167.4)	68.2 (21.4-229.6)	150.7 (37.0-315.4)	153.1 (52.7-317.8)

Table 3

Response of serum folate to different doses and duration of folic acid supplementation by methylenetetrahydrofolate reductase C677T genotypes
	N	FA.4w.Change	4W	P for interaction	FA.8w.Change	8W	P for interaction
	N	ng/mL	β(SE)P	P for interaction	ng/mL	β(SE)P	P for interaction
Linear equation
	1538	16.8 (4.4–57.2)	0.37(0.02),<0.001		27.9 (7.2-111.1)	0.51(0.02),<0.001
CC	474	17.9 (5.3–74.0)	0.39(0.04),<0.001	0.893	28.7 (7.4–112.0)	0.45(0.04),<0.001	0.043
CT	706	14.7 (3.5–48.9)	0.38(0.03),<0.001		27.5 (5.8-110.3)	0.54(0.03),<0.001
TT	358	19.6 (4.9–57.9)	0.35(0.04),<0.001		28.4 (8.6-112.2)	0.56(0.04),<0.001

Adjusted for age, sex, body mass index, smoking status, drinking status, baseline systolic blood pressure,baseline diastolic blood pressure, baseline glomerular filtration rate,baseline homocysteine, center, baseline total cholesterol, baseline triglyceride, baseline high density lipoprotein, baseline glucose,baseline folate,baseline riboflavin,baseline vitamin b6.

In the past, Several clinical studies on folic acid supplementation were carried out in Western countries(fortification of foods with folic acid became mandatory).However,suboptimal results might be observed in populations where mandatory folic acid food fortification has already been implemented.Our study was conducted in China (a region not fortified with folic acid) to avoid the effects of mandatory folic acid supplementation.This study is a clinical trial study with the largest sample size and the largest number of folic acid dose groups in the Chinese hypertensive population.We found that MTHFR C677T genotype was associated with the baseline folic acid status. After 8 weeks of folic acid intervention, the therapeutic effect of folic acid intervention was influenced by the MTHFR C677T gene polymorphism.

The benefits of folic acid have been proven, and folic acid supplementation has been used to increase serum folate levels in some regions.Therefore, it is very important to supplement folic acid to improve serum folate status.We found that serum folate levels increased regardless of the supplemental dose of folic acid.A data from the Framingham progeny study cohort^[24] showed that folic acid consumption in middle-aged and elderly people increased the mean serum folic acid concentration. This is consistent with our results.A previous clinical trial on folic acid supplementation in Chinese hypertensive populations^[20], supplemented with 0.4 mg and 0.8 mg of folic acid for 8 weeks, respectively. The study found that supplementation with 0.8 mg of folic acid for 8 weeks resulted in a plateau in folic acid levels.However, the highest dose designed in this study is only 0.8 mg, and the conclusions drawn are not rigorous. And we designed a higher folic acid dose group. We found that the higher the supplemental folic acid dose, the higher the serum folic acid level. There was a clear dose-response relationship between the dose of folic acid supplementation and the concentration of folic acid, with no threshold for the concentration.

We found that the dose of folic acid supplementation was always positively correlated with the change in serum folic acid concentration regardless of whether the folic acid intervention was performed for 4 weeks or 8 weeks.We found that the dose of folic acid supplementation was always positively correlated with the change in serum folic acid concentration regardless of whether the folic acid intervention was performed for 4 weeks or 8 weeks. Moreover, the response of folic acid intervention for 8 weeks (β = 0.51) was more sensitive than the response of folic acid intervention for 4 weeks (β = 0.37) in the Chinese hypertensive population. The same results were obtained in different genotypes.This shows that the time of folic acid supplementation will influence the effect of folic acid intervention. The longer the folic acid intervention time, the higher the serum folic acid concentration of patients ^{[20, 25]}, and the better the effect.

The folate level of Chinese is much lower than that of some western countries, and the mutation frequency of MTHFR is higher in Chinese hypertensive population. Many studies have shown that the MTHFR C677T variant is closely related to a variety of diseases ^[26–28], including depression, stroke, and cancer. A meta-analysis showed ^[29] that the effect was greater in low-folate regions such as Asia than in folate-fortified regions (United States, Australia, and New Zealand).Therefore, a study of folic acid supplementation on MTHFR genotypes in Chinese hypertensive populations was carried out to provide new insights for maintaining beneficial folic acid status and disease prevention in Chinese hypertensive populations.MTHFR gene polymorphisms are associated with folate status.At baseline, folate levels in patients with CC and CT genotypes were significantly higher than those with TT genotype.MTHFR is a key enzyme in the folate metabolism process by converting 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate.Reduced activity of methylenetetrahydrofolate reductase due to the mutated T allele, which is responsible for low folate levels in patients with the TT genotype^[30–32].

ANDERSON CAM found that in the 0.4 mg folic acid supplementation group, the folic acid response of individuals with TT genotype was significantly larger than those with cc^[33]. Similar findings were found in the plasma folic acid response of de BREE A in the Dutch population^[34].However, both studies were conducted in areas fortified with folic acid.Another trial of folic acid supplementation in women of childbearing age in northern China showed that that the MTHFR 677 TT genotype was an independent predictor of lower folate concentrations, regardless of the FA dose.However, the subjects are young women, and the characteristics of the hypertensive population are too different.This study found that gene polymorphisms have a modified effect on the response to folic acid intervention. After 8 weeks of folic acid supplementation in Chinese hypertensive population, the response of TT genotype (β = 0.56) was than that of CC genotype (β = 0.45).Previous studies have shown ^[35] that the molecular mechanism of clinical repair of enzyme variants is to overcome the binding defects of Km mutants by increasing the levels of cofactors or substrates, or to act as chemical partners to improve the stability of mutant enzyme variants.In this study, folic acid supplementation for 8 weeks enhanced the activity of mutant enzymes and response to folic acid supplementation in patients with tt genotype, which made individuals with tt genotype more beneficial.In this study, 8 weeks of folic acid supplementation enhanced the enzyme activity and response to folic acid supplementation in individuals with TT genotypes, which made them more beneficial.

The benefits of folic acid are not limited to reducing hcy levels, but also soften blood vessels, improve cognitive function, etc. However, there is still a lack of attention to folic acid in China. There are about 245 million hypertensive people in China, and it is still growing every year, and the mutation frequency of MTHFR C677T in the Chinese hypertensive population is as high as about 25%, which means that the MTHFR C677T mutation is more harmful to the Chinese hypertensive population. Therefore, it has important public health significance to explore the therapeutic effect of folic acid intervention on MTHFR gene polymorphism in Chinese hypertensive population.

Our advantage is that this study used a large sample, multicenter, randomized double-blind design. The sample size of this study ensures statistical power.In addition, all bioassays in this study were performed in a central laboratory, using uniform standards to reduce bias.Of course, this study also has some shortcomings. First, we did not investigate the dietary intake of the study subjects, although we asked each subject to strictly control their diet and not consume other nutritional supplements. Second, only an 8-week trial was conducted in this study, and a longer study trial is still needed to draw more rigorous conclusions.

In conclusion, we found that MTHFR gene polymorphisms not only affected baseline folic acid levels, but also modified folic acid interventions in a Chinese hypertensive population.

Acknowledgements

We express our appreciation to the participants of this study.

Authors’ Contributions: JianF Zou:analysis and interpretation of data, drafting of the manuscript; Yan Zhang, XiuL Shi, GuiM Chen, and GenF Tang: study concept and design, administrative, technical, or material support; Lei Tang, interpretation of data, revised the manuscript for important intellectual content; Xu Zhang, YaP Xu,RuS Zhang, Rui Wang, Qian Wu: provided a critical review of the content of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding: This study was supported by the National Natural Science Foundation of China(81773534).

Ethics approval and consent to participate

Consent for publication

Not applicable.

Availability of data and materials

The datasets generated during and/or analyzed during the current study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Chan YM, Bailey R, O'Connor DL. Folate. Adv Nutr. 2013 Jan 1;4(1):123-5. doi: 10.3945/an.112.003392. PMID: 23319130; PMCID: PMC3648733.
McNulty H, Pentieva K. Folate bioavailability. Proc Nutr Soc. 2004 Nov;63(4):529 – 36. doi: 10.1079/pns2004383. PMID: 15831124.
Gazzali AM, Lobry M, Colombeau L, Acherar S, Azaïs H, Mordon S, Arnoux P, Baros F, Vanderesse R, Frochot C. Stability of folic acid under several parameters. Eur J Pharm Sci. 2016 Oct 10;93:419 – 30. doi: 10.1016/j.ejps.2016.08.045. Epub 2016 Aug 27. PMID: 27575880.
McNulty H, Scott JM. Intake and status of folate and related B-vitamins: considerations and challenges in achieving optimal status. Br J Nutr. 2008 Jun;99 Suppl 3:S48-54. doi: 10.1017/S0007114508006855. PMID: 18598588.
Hao L, Ma J, Stampfer MJ, Ren A, Tian Y, Tang Y, Willett WC, Li Z. Geographical, seasonal and gender differences in folate status among Chinese adults. J Nutr. 2003 Nov;133(11):3630-5. doi: 10.1093/jn/133.11.3630. PMID: 14608086.
Centers for Disease Control and Prevention (CDC). Spina bifida and anencephaly before and after folic acid mandate–United States, 1995–1996 and 1999–2000. MMWR Morb Mortal Wkly Rep. 2004 May 7;53(17):362–5. PMID: 15129193.
Czeizel AE, Dudás I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med. 1992 Dec 24;327(26):1832-5. doi: 10.1056/NEJM199212243272602. PMID: 1307234.
Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. MRC Vitamin Study Research Group. Lancet. 1991 Jul 20;338(8760):131-7. PMID: 1677062.
Wang X, Qin X, Demirtas H, Li J, Mao G, Huo Y, Sun N, Liu L, Xu X. Efficacy of folic acid supplementation in stroke prevention: a meta-analysis. Lancet. 2007 Jun 2;369(9576):1876–1882. doi: 10.1016/S0140-6736(07)60854-X. PMID: 17544768.
Huo Y, Li J, Qin X, Huang Y, Wang X, Gottesman RF, Tang G, Wang B, Chen D, He M, Fu J, Cai Y, Shi X, Zhang Y, Cui Y, Sun N, Li X, Cheng X, Wang J, Yang X, Yang T, Xiao C, Zhao G, Dong Q, Zhu D, Wang X, Ge J, Zhao L, Hu D, Liu L, Hou FF; CSPPT Investigators. Efficacy of folic acid therapy in primary prevention of stroke among adults with hypertension in China: the CSPPT randomized clinical trial. JAMA. 2015 Apr 7;313(13):1325-35. doi: 10.1001/jama.2015.2274. PMID: 25771069.
Zhao M, Wu G, Li Y, Wang X, Hou FF, Xu X, Qin X, Cai Y. Meta-analysis of folic acid efficacy trials in stroke prevention: Insight into effect modifiers. Neurology. 2017 May 9;88(19):1830–1838. doi: 10.1212/WNL.0000000000003909. Epub 2017 Apr 12. PMID: 28404799.
Durga J, Verhoef P, Anteunis LJ, Schouten E, Kok FJ. Effects of folic acid supplementation on hearing in older adults: a randomized, controlled trial. Ann Intern Med. 2007 Jan 2;146(1):1–9. doi: 10.7326/0003-4819-146-1-200701020-00003. PMID: 17200216.
Ravaglia G, Forti P, Maioli F, Martelli M, Servadei L, Brunetti N, Porcellini E, Licastro F. Homocysteine and folate as risk factors for dementia and Alzheimer disease. Am J Clin Nutr. 2005 Sep;82(3):636 – 43. doi: 10.1093/ajcn.82.3.636. PMID: 16155278.
Lim U, Wang SS, Hartge P, Cozen W, Kelemen LE, Chanock S, Davis S, Blair A, Schenk M, Rothman N, Lan Q. Gene-nutrient interactions among determinants of folate and one-carbon metabolism on the risk of non-Hodgkin lymphoma: NCI-SEER case-control study. Blood. 2007 Apr 1;109(7):3050-9. doi: 10.1182/blood-2006-07-034330. PMID: 17119116; PMCID: PMC1852210.
Wang L, Shangguan S, Chang S, Yu X, Wang Z, Lu X, Wu L, Zhang T. Determining the association between methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and genomic DNA methylation level: A meta-analysis. Birth Defects Res A Clin Mol Teratol. 2016 Aug;106(8):667–74. doi: 10.1002/bdra.23511. Epub 2016 May 13. PMID: 27173682.
Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995 May;10(1):111-3. doi: 10.1038/ng0595-111. PMID: 7647779.
Hustad S, Midttun Ø, Schneede J, Vollset SE, Grotmol T, Ueland PM. The methylenetetrahydrofolate reductase 677C–>T polymorphism as a modulator of a B vitamin network with major effects on homocysteine metabolism. Am J Hum Genet. 2007 May;80(5):846–55. doi: 10.1086/513520. Epub 2007 Mar 13. PMID: 17436239; PMCID: PMC1852731.
Wilcken B, Bamforth F, Li Z, Zhu H, Ritvanen A, Renlund M, Stoll C, Alembik Y, Dott B, Czeizel AE, Gelman-Kohan Z, Scarano G, Bianca S, Ettore G, Tenconi R, Bellato S, Scala I, Mutchinick OM, López MA, de Walle H, Hofstra R, Joutchenko L, Kavteladze L, Bermejo E, Martínez-Frías ML, Gallagher M, Erickson JD, Vollset SE, Mastroiacovo P, Andria G, Botto LD. Geographical and ethnic variation of the 677C > T allele of 5,10 methylenetetrahydrofolate reductase (MTHFR): findings from over 7000 newborns from 16 areas world wide. J Med Genet. 2003 Aug;40(8):619 – 25. doi: 10.1136/jmg.40.8.619. Erratum in: J Med Genet. 2004 May;41(5):400. Redlund, M [corrected to Renlund, M]. PMID: 12920077; PMCID: PMC1735571.
Qin X, Li J, Cui Y, Liu Z, Zhao Z, Ge J, Guan D, Hu J, Wang Y, Zhang F, Xu X, Wang X, Xu X, Huo Y. MTHFR C677T and MTR A2756G polymorphisms and the homocysteine lowering efficacy of different doses of folic acid in hypertensive Chinese adults. Nutr J. 2012 Jan 10;11:2. doi: 10.1186/1475-2891-11-2. PMID: 22230384; PMCID: PMC3274435.
Qin X, Li J, Cui Y, Liu Z, Zhao Z, Ge J, Guan D, Hu J, Wang Y, Zhang F, Xu X, Wang X, Xu X, Huo Y. Effect of folic acid intervention on the change of serum folate level in hypertensive Chinese adults: do methylenetetrahydrofolate reductase and methionine synthase gene polymorphisms affect therapeutic responses? Pharmacogenet Genomics. 2012 Jun;22(6):421–8. doi: 10.1097/FPC.0b013e32834ac5e8. PMID: 21869730.
Forman JP, Rimm EB, Stampfer MJ, Curhan GC. Folate intake and the risk of incident hypertension among US women. JAMA. 2005 Jan 19;293(3):320-9. doi: 10.1001/jama.293.3.320. PMID: 15657325.
Li JP, Huo Y, Liu P. [Efficacy and safety of Enalapril-Folate acid tablets in lowering blood pressure and plasma homocysteine]. Beijing Da Xue Xue Bao Yi Xue Ban. 2007 Dec 18;39(6):614-8. Chinese. PMID: 18087553.
Pezzini A, Grassi M, Del Zotto E, Assanelli D, Archetti S, Negrini R, Caimi L, Padovani A. Interaction of homocysteine and conventional predisposing factors on risk of ischaemic stroke in young people: consistency in phenotype-disease analysis and genotype-disease analysis. J Neurol Neurosurg Psychiatry. 2006 Oct;77(10):1150–6. doi: 10.1136/jnnp.2005.076083. Epub 2006 Apr 19. PMID: 16624841; PMCID: PMC2077547.
Jacques PF, Selhub J, Bostom AG, Wilson PW, Rosenberg IH. The effect of folic acid fortification on plasma folate and total homocysteine concentrations. N Engl J Med. 1999 May 13;340(19):1449-54. doi: 10.1056/NEJM199905133401901. PMID: 10320382.
Crider KS, Zhu JH, Hao L, Yang QH, Yang TP, Gindler J, Maneval DR, Quinlivan EP, Li Z, Bailey LB, Berry RJ. MTHFR 677C->T genotype is associated with folate and homocysteine concentrations in a large, population-based, double-blind trial of folic acid supplementation. Am J Clin Nutr. 2011 Jun;93(6):1365–72. doi: 10.3945/ajcn.110.004671. Epub 2011 Apr 20. PMID: 21508090.
Zhi X, Yang B, Fan S, Wang Y, Wei J, Zheng Q, Sun G. Gender-specific interactions of MTHFR C677T and MTRR A66G polymorphisms with overweight/obesity on serum lipid levels in a Chinese Han population. Lipids Health Dis. 2016 Oct 28;15(1):185. doi: 10.1186/s12944-016-0354-9. PMID: 27793164; PMCID: PMC5084372.
Li Z, Yadav U, Mahemuti A, Tang BP, Upur H. Association of MTHFR genetic polymorphisms with venous thromboembolism in Uyghur population in Xinjiang, China. Int J Clin Exp Med. 2015 Oct 15;8(10):17703–11. PMID: 26770360; PMCID: PMC4694260.
Waseem M, Hussain SR, Kumar S, Serajuddin M, Mahdi F, Sonkar SK, Bansal C, Ahmad MK. Association of MTHFR (C677T) Gene Polymorphism With Breast Cancer in North India. Biomark Cancer. 2016 Sep 27;8:111–117. doi: 10.4137/BIC.S40446. PMID: 27721657; PMCID: PMC5040218.
Holmes MV, Newcombe P, Hubacek JA, Sofat R, Ricketts SL, Cooper J, Breteler MM, Bautista LE, Sharma P, Whittaker JC, Smeeth L, Fowkes FG, Algra A, Shmeleva V, Szolnoki Z, Roest M, Linnebank M, Zacho J, Nalls MA, Singleton AB, Ferrucci L, Hardy J, Worrall BB, Rich SS, Matarin M, Norman PE, Flicker L, Almeida OP, van Bockxmeer FM, Shimokata H, Khaw KT, Wareham NJ, Bobak M, Sterne JA, Smith GD, Talmud PJ, van Duijn C, Humphries SE, Price JF, Ebrahim S, Lawlor DA, Hankey GJ, Meschia JF, Sandhu MS, Hingorani AD, Casas JP. Effect modification by population dietary folate on the association between MTHFR genotype, homocysteine, and stroke risk: a meta-analysis of genetic studies and randomised trials. Lancet. 2011 Aug 13;378(9791):584–94. doi: 10.1016/S0140-6736(11)60872-6. Epub 2011 Jul 29. PMID: 21803414; PMCID: PMC3156981.
van der Put NM, Steegers-Theunissen RP, Frosst P, Trijbels FJ, Eskes TK, van den Heuvel LP, Mariman EC, den Heyer M, Rozen R, Blom HJ. Mutated methylenetetrahydrofolate reductase as a risk factor for spina bifida. Lancet. 1995 Oct 21;346(8982):1070-1. doi: 10.1016/s0140-6736(95)91743-8. PMID: 7564788.
Ma J, Stampfer MJ, Hennekens CH, Frosst P, Selhub J, Horsford J, Malinow MR, Willett WC, Rozen R. Methylenetetrahydrofolate reductase polymorphism, plasma folate, homocysteine, and risk of myocardial infarction in US physicians. Circulation. 1996 Nov 15;94(10):2410-6. doi: 10.1161/01.cir.94.10.2410. PMID: 8921781.
Nelen WL, Blom HJ, Thomas CM, Steegers EA, Boers GH, Eskes TK. Methylenetetrahydrofolate reductase polymorphism affects the change in homocysteine and folate concentrations resulting from low dose folic acid supplementation in women with unexplained recurrent miscarriages. J Nutr. 1998 Aug;128(8):1336-41. doi: 10.1093/jn/128.8.1336. PMID: 9687553.
Anderson CA, Beresford SA, McLerran D, Lampe JW, Deeb S, Feng Z, Motulsky AG. Response of serum and red blood cell folate concentrations to folic acid supplementation depends on methylenetetrahydrofolate reductase C677T genotype: results from a crossover trial. Mol Nutr Food Res. 2013 Apr;57(4):637–44. doi: 10.1002/mnfr.201200108. Epub 2013 Mar 4. PMID: 23456769; PMCID: PMC4132693.
de Bree A, Verschuren WM, Bjørke-Monsen AL, van der Put NM, Heil SG, Trijbels FJ, Blom HJ. Effect of the methylenetetrahydrofolate reductase 677C–>T mutation on the relations among folate intake and plasma folate and homocysteine concentrations in a general population sample. Am J Clin Nutr. 2003 Mar;77(3):687 – 93. doi: 10.1093/ajcn/77.3.687. PMID: 12600862.
Marini NJ, Gin J, Ziegle J, Keho KH, Ginzinger D, Gilbert DA, Rine J. The prevalence of folate-remedial MTHFR enzyme variants in humans. Proc Natl Acad Sci U S A. 2008 Jun 10;105(23):8055-60. doi: 10.1073/pnas.0802813105. Epub 2008 Jun 3. PMID: 18523009; PMCID: PMC2430358.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Effect of MTHFR C677T gene polymorphism on folic acid intervention in Chinese hypertensive patients：a multicentered, randomized, double-blind, clinical trial

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Patients and ethics

Study Design

Blood Sample Collection And Laboratory Measurement Methods

Statistical Analysis

Results

Discussion

Declarations

References

Additional Declarations

Status:

Version 1