Methylenetetrahydrofolate Reductase (MTHFR) C677T gene polymorphism is associated with decreased MTHFR enzyme activity that leads to hyperhomocysteinemia and hypertension. The MTHFR C677T polymorphism is also associated with lower affinity and faster unbinding of flavin adenine dinucleotide, a riboflavin-derived cofactor for MTHFR enzyme. Natuna is a district which once reported has the highest prevalence of hypertension in Indonesia, based on a national basic health research. Therefore, this study aims to investigate the association between MTHFR C677T polymorphism and riboflavin intake with hypertension in the Natuna district population. An analytic cross-sectional population study was conducted in the region. Data of demographic, physical examination, and food recall, as well as MTHFR genotyping from blood samples of study subjects were collected. The subjects were 268 adults, among which 128 (45.9%) had hypertension, 49 (17.5%) had mutant allele of MTHFR genotype (CT or TT), and 158 (56.4%) had low daily riboflavin intake. There was association between MTHFR C677T polymorphism and hypertension [OR 3.387 (1.724 – 6.654), p = 0.001]. Post-hoc analysis showed that group of mutant MTHFR C677T genotype with low riboflavin intake showed strong association with hypertension [OR 19.320 (4.498–82.980), p<0.01]. Meanwhile, group with mutant MTHFR C677T genotype and normal riboflavin intake did not show a significant association with hypertension. As a conclusion, there was association between interaction of riboflavin intake and MTHFR C677T polymorphism with hypertension. This suggests possibility of riboflavin supplementation in population with high prevalence of hypertension especially those with MTHFR C677T gene polymorphism
Research Article
Interaction between Polymorphism of MTHFR C677 Gene and Riboflavin Intake as the Risk of Hypertension in a Region with High Prevalence
https://doi.org/10.21203/rs.3.rs-2010845/v1
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Hypertension
MTHFR C677T
Natuna
Polymorphism
and Riboflavin
Hypertension is known as the major risk factor for cardiovascular disease and death globally [1], [2]. The risk factors for developing hypertension vary including genetics and nutrition. Hyperhomocysteinemia (Hhcy) is associated with this disease due to its ability to induce vascular smooth muscle damage and endothelial cell dysfunction [3]–[5]. Homocysteine metabolism requires the optimal functioning of Methylenetetrahydrofolate Reductase (MTHFR) enzyme. However, MTHFR C677T gene polymorphism is associated with decreased enzyme activity that leads to Hhcy [5]–[9]. A meta-analytic study reported that MTHFR polymorphism is associated with hypertension in Caucasians and eastern Asians, [10].
Recent study from our institution by Candrasatria et al. reported a significant association between MTHFR C6777T gene polymorphism and hypertension in adult population at another district in Indonesia [11]. Despite multiple reports on the association of MTHFR C677T polymorphism, the result remained inconsistent across different populations.[8] Those findings indicate that external factor plays a role in modifying the association between MTHFR C677T polymorphism and hypertension. MTHFR C677T polymorphism was associated with lower affinity and faster unbinding of flavin adenine dinucleotide (FAD), a riboflavin-derived cofactor for MTHFR enzyme [12], [13]. Indonesia, the fourth most populous country in the world has an increasing hypertension prevalence with the highest cases being found in Natuna district in 2007, which was 53,3% by blood pressure examination [1], [2]. Therefore, this study aims to evaluate the association of riboflavin and MTHFR C677T polymorphism with hypertension in Natuna district.
Study population
The samples were obtained from the adult population during community service programme in Natuna district of Indonesia. The inclusion criteria were: (1) age ≥18 years, and (2) willing to participate in the study, while exclusion criteria were individual with chronic kidney disease and other secondary cause of hypertension. Clinical evaluation and laboratory blood tests were conducted to select the study subjects. Ethical approval was obtained from the Ethics Committee at National Cardiovascular Centre Harapan Kita (NCCHK). Also, all the patients or caregivers provided written informed consents.
Hypertension and riboflavin intake status determination
Hypertension status was defined as a known history of anti-hypertensive medication (ACE-I, ARB, CCB, BB, and diuretic) in patients. Meanwhile, for those without such history, hypertension status was defined as systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg from 3 consecutive measurements. Blood pressure measurement and classification followed the 2020 International Society of Hypertension Global Hypertension Practice Guidelines. Dietitians obtained daily riboflavin intake status by interviewing subjects using food recall questionnaires. The subjects were asked about dietary intake including menu, ingredient, processing method, and a portion of the food/drink consumed. The data from the food frequency questionnaires were converted to riboflavin intake data according to Tabel Komposisi Pangan tahun 2017, a national guideline for food composition in Indonesia. Low riboflavin intake was defined as daily riboflavin intake ≤ 1.3 mg/day in males and ≤1.1 mg/day in females, according to previous study.[5]
Blood sample collection and DNA genotyping
Venous blood samples were collected in EDTA and citrate tubes from which standard biochemical parameters such as urea, creatinine, blood glucose, and lipid profile were obtained. In addition, MTHFR C677T genotyping was conducted by amplifying DNA fragment with the Taqman method using Applied Biosystems® 7500 Real-Time PCR Systems (rs ID of rs1801133), and the genotype was determined to be normal (CC) or mutant (CT and TT). Genotyping was conductid according to current genotyping guideline.
Statistical Analysis
Continuous data with a normal distribution were presented as means ± standard deviation, and compared using the independent T-Test or Mann-Whitney. But categorical data were presented as numbers and percentages and also compared by the Chi-square and Yates Correction or Fisher’s exact test. Post-hoc analysis was utilized to determine the association of hypertension across 4 groups according to MTHFRC677T genotype and riboflavin intake status. A two-tailed 
-value < 0.05 indicated statistical significance, while the analyses were carried out by IBM SPSS Statistics 22.0 (SPSS inc, Chicago, IL, and the USA).
Baseline characteristics
Out of the 550 subjects screened in the community service program in Natuna, 268 met the inclusion and exclusion criteria for further statistical analysis. Mean age was 56.61 ± 10.80 years, mean daily riboflavin intake was 1.40 ± 1.95 mg/day with 145 (54.1%) subjects having low daily intake, and 128 (47.8%) had hypertension. Apart from the MTHFR C677T polymorphism, age was the only variable found to be significantly associated with the disease (p= 0.019) (Table 1). Multivariate analysis showed age and central obesity was significantly associated with hypertension (Table 2).
TABLE 1
Baseline characteristics
|
Variabel |
Non-hypertension |
Hypertension |
P value |
|
N |
140 |
128 |
|
|
Male (%) Female (%) |
90 (33,6) 50 (18,7) |
83 (31,0) 45 (16,8) |
0,924 |
|
Age (years) |
55,13 ± 10,80 |
58,22 ± 10,61 |
0,019 |
|
Obesity (%) |
73 (52,1) |
74 (57,8) |
0,352 |
|
Central Obesity (%) |
84 (60,0) |
88 (68,8) |
0,136 |
|
Diabetes Mellitus Type 2 (%) |
21 (15,0) |
21 (16,4) |
0,752 |
|
Smoking (%) |
27 (19,3) |
21 (16,4) |
0,776 |
|
Daily riboflavin intake (mg/day) |
1,21 (0,10 – 14,31) |
1,03 (0,02 – 27,28) |
0,136 |
|
Low riboflavin intake (%) |
70 (50,0) |
75 (58,6) |
0,158 |
TABLE 2
Multivariate analysis.
|
Variables |
|
Model 1 |
|
Model 2 |
||
|
B coefficient |
P value |
OR (CI 95%) |
B coefficient |
P value |
OR (CI 95%) |
|
|
MTHFR C677T Polymorphism |
1.200 |
0.001 |
3.320 (1.687–6.535) |
1.207 |
0.001 |
3.344 (1.675–6.679) |
|
Riboflavin intake |
0.302 |
0.223 |
1.352 (0.824–2.219) |
0.342 |
0.185 |
1.408 (0.849–2.335) |
|
Age |
- |
- |
- |
0.032 |
0.009 |
1.033 (1.008-1.058) |
|
Central Obesity |
- |
- |
- |
0.546 |
0.047 |
1.726 (1.006–2.961) |
|
Constant |
-0.464 |
|
|
-2.657 |
|
|
Correlation between MTHFR C677T polymorphism and hypertension
MTHFR C677T genotyping showed that of the 268 subjects, 219 (81.7%) had CC, 47 (17.5%) had CT and 2 (0.8%) had TT genotypes, respectively. Then the association between MTHFR C677T polymorphism [normal (CC) vs mutant (CT and TT)] and hypertension was compared. Chi-square analysis showed the polymorphism was associated with hypertension [p<0.001, OR (CI95%) = 3.387 (1.724 – 6.654)] (Table 3). Multivariate analysis showed significant association between MTHFR C677T polymorphism and hypertension (p=0.001 for model 1, and p=0.001 for model 2). (Table 2)
TABLE 3
Correlation between MTHFR C677T polymorphism and hypertension
|
Variables |
Genotype (%) |
|
Allele frequency (%) |
|
|
CC |
CT |
TT |
Mutant (CT +TT) Normal (CC) |
|
|
Hypertension |
93 (34.7) |
34 (12.7) |
1 (0.4) |
35 (13.1%) 93 (34.7%) |
|
Non-hypertension |
126 (47.0) |
13 (4.9) |
1 (0.4) |
14 (5.2%) 126 (47.0%) |
|
P value |
reference |
<0.001 |
0.749 |
<0.001 |
|
OR (CI 95%) |
- |
3,45 (1,72-6,92) |
1,578 (0,01-25,89) |
3.387 (1.724 – 6.654) |
Correlation between riboflavin intake status and MTHFR C677T polymorphism with hypertension
Study population was divided into 4 groups according to MTHFR C677T genotype (mutant or normal) and riboflavin intake status (low or normal). Post-hoc analysis showed that group with low riboflavin intake and mutant MTHFR C677T genotype had significant association with hypertension [p<0,001; OR (CI) = 19.320 (4.498–82.980)], but not in the other 3 groups. Chi-square analysis was conducted to evalute this association across different MTHFR C677T genotype (CC vs CT vs TT). A stronger association was discovered in CT genotype group [p=0.0001; OR (CI) = 25.667 (4.480147.064)]. Statistical analysis to evaluate this association was not conducted in the TT genotype group because it only consisted of 2 subjects with normal riboflavin intake (table 4).
TABLE 4
Correlation between riboflavin intake status and MTHFR C677T polymorphism with hypertension
|
Group |
Hypertension (128) |
Control (140) |
P value |
OR (CI 95%) |
|
Normal genotype with normal riboflavin intake |
46 (35.9%) |
58 (41.4%) |
0.426 |
0.793 (0.484 – 1.299) |
|
Mutant genotype with normal riboflavin intake |
7 (5.5%) |
12 (8.6%) |
0.453 |
0.617 (0.235 – 1.619) |
|
Normal genotype with low riboflavin intake |
47 (36.7%) |
68 (48.6%) |
0.067 |
0.614 (0.377 – 1.002) |
|
Mutant genotype with low riboflavin intake |
28 (21.9%) |
2 (1.4%) |
< 0.001 |
19.320 (4.498 – 82.980) |
The present study revealed substantial number (18.3%) of study subjects having MTHFR C677T gene polymorphism with at least single mutant allele. This polymorphism has significant association to hypertension. We also revealed that group with MTHR gene polymorphism and having low riboflavin intake had strong association with hypertension, but not in the group with normal riboflavin intake. This is among the first study to report interaction of MTHFR gene polymorphism and riboflavin intake with risk of hypertension.
MTHFR C677T polymorphism is known as a risk factor for hypertension which is pathophysiologically based on the interaction between MTHFR enzyme and homocysteine. However, previous studies stated that riboflavin status modified the gene-homocysteine interaction leading to lower blood pressure [5], [8], [12], [14]. Being a district with the highest hypertension prevalence in Indonesia, the association between riboflavin intake and MTHFR C677T with the development of this disease in Natuna district needs to be evaluated.
This study revealed that MTHFR C677T polymorphism was associated with hypertension in Natuna population [p=0.001, OR (CI95%) = 3.387 (1.724 – 6.654)], correlating to a previous similar study [5]–[9]. Generally, transition from C to T in location 677 of the MTHFR gene causes alanine to valine replacement leading to thermolabile MTHFR enzyme. This change contributes to loss of the enzyme’s affinity to its cofactor, FAD [13], [15]. Without the cofactor’s aid, the enzyme activity decreases leading to hyperhomocisteinemia (Hhcy) and finally, hypertension. MTHFR is one of the enzymes involved in one carbon metabolism, which catalyse 5,10 methylene-tetrahydrofolate (THF) conversion to 5 methyl-THF. The conversion of homocysteine into methionine requires simultaneous 5 methyl-THF conversion to THF. Therefore, the decreasing enzyme activity in MTHFR C677T polymorphism inhibits the conversion process [16].
Despite multiple studies reporting the association MTHFR C677T polymorphism with hypertension, the result remained inconsistent across different populations [17]–[19]. There are some reports on insignificant association between MTHFR C677T polymorphism and hypertension [19]–[26]. The different results indicate that external factor plays a role in modifying the association between MTHFR C677T polymorphism and hypertension. Candrasatria et al reported an association between MTHFR C677T polymorphism and hypertension in Bogor district, Indonesia, which correlates to this present study that also has a similar result, in the same country with similar geographic characteristic. However, there are limited evaluations of the geographical influence on MTHFR gene and hypertension. Nonetheless, some studies have shown one risk factor that is pathophysiologically relevant to influence the association between MTHFR C677T gene and hypertension, which is riboflavin intake.
This study showed that group with low riboflavin intake and mutant MTHFR C677T genotype had significant association with hypertension [p<0,001; OR (CI) = 19.320 (4.498–82.980)], but not in the other populations. This is concordant to a previous report on the significant effect of riboflavin in lowering hcy level in the blood [27]. Hhcy was also found in individuals with mutant MTHFR C677T gene and low riboflavin intake [5], [28]. MTHFR cofactor (FAD) derived from riboflavin. In vitro study stated mutant MTHFR C677T gene is associated with faster cofactor unbinding and change in the enzyme’s active site that contributes to less effective binding with FAD [16], [29]. Increasing riboflavin level was thought to be the counter mechanism to handle this abnormality. Rooney et al. reported that riboflavin supplementation led to lower hcy in TT genotype, but not in CC. By lowering the hcy level, there is possibility for hypertension not to develop. Also, Mcnulty et al, Rooney et al, Horigan et al, and Wilson et al, stated hypertension in individuals with TT genotype were responsive to riboflavin supplementation, but not in those with CT or CC [5], [14], [27], [30]. To best of our knowledge, there has been no study reporting significant effectiveness of riboflavin supplementation in lowering blood pressure in individuals with heterozygous MTHFR C677T polymorphism. But this present study reported a strong association between riboflavin intake and hypertension in CT genotype group [p=0.0001; OR (CI 95%) = 25.667 (4.480-147.064)]. However, statistical analysis to evaluate this association was not conducted in TT genotype group because it only consisted of 2 subjects with a normal riboflavin intake. Study showed that activity of the MTHFR enzyme in subjects with CT genotype (47.7%) is higher than that of TT genotype (24.8%) [31]. The result showed riboflavin intake was associated with hypertension even in individuals with CT genotype that had better enzyme activity than the TT genotype individuals.
Data from this study suggested importance of riboflavin as a possible interventional agent to reduce the risk of hypertension especially in those with MTHFR gene polymorphism. Riboflavin is well known in the public as vitamin B2. It has also been used widely as a food supplement. Futher study is certainly required whether supplementation of Riboflavin may reduce the risk of hypertension in the population.
Study Limitation
This study did not analyse the homocysteine level of the subjects, thus the mechanism underlying strong association between riboflavin intake and MTHFR C677T polymorphism still needs to be defined. The riboflavin status was determined by food recall evaluation, which is not supported with data on level of riboflavin in the blood. However, we believe food recall method may provide information on chronic status of riboflavin level in the blood, which is more relevant than a one-time riboflavin level measurement in the blood, given the fact that hypertension is also chronic disorder.
Individual with low riboflavin intake and mutant MTHFR C677T genotype had strong association with hypertension but not in the group with normal riboflavin intake. There is association between riboflavin intake and MTHFR C677T polymorphism interaction with hypertension. This suggests possibility of riboflavin supplementation in population with high prevalence of hypertension especially those with MTHFR C677T gene polymorphism
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Ethical approval was obtained from the Ethics Committee at National Cardiovascular Centre Harapan Kita (NCCHK) in accordance with the Declaration of Helsinki. Ethical clearance number was LB.02.01 / VII / 522 / KEP 015 / 2021. Also, all the patients or caregivers provided written informed consents.
CONSENT FOR PUBLICATION
Not applicable
COMPETING INTERESTS
The authors declare no conflict of interests regarding this paper’s publication.
FUNDING
This article is fully self-funded from the beginning of the data collection to final review of the article.
AUTHORS' CONTRIBUTIONS
Renan Sukmawan, Praise Jeremiah Hendrik, Rissa Ummy Setiani, Rita Zahara and Taofan, wrote the main manuscript text; Praise Jeremiah Hendrik, Beta Canina Harlyjoy, Dian Zamroni, and Bambang Widyantoro collected the data; Renan Sukmawan, Praise Jeremiah Hendrik, and Taofan conceived and designed the analysis; Rita Zahara, Dian Zamroni, Beta Canina Harlyjoy, and Bambang Widyantoro performed the analysis. All authors reviewed the manuscript.
ACKNOWLEDGEMENTS
The authors acknowledge the support from Research and Development Center of National Cardiovascular Center Harapan Kita for providing the MTHFR C677T genotyping for this study. Special thanks also given to dr. Surya Dharma for his contribution in reviewing and helping the data analysis of this study.
DATA AVAILIBILITY
The datasets supporting the conclusions of this article are included within the article.
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No competing interests reported.