|Year : 2019 | Volume
| Issue : 3 | Page : 380-385
The relationship between plasma homocysteine levels and MTHFR gene variation, age, and sex in Northeast China
F Wang1, X Sui1, N Xu2, J Yang1, H Zhao1, X Fei1, Z Zhang1, Z Luo1, Y Xin1, B Qin3, X Zhao3, S Cao3, Y Zhang3, Z Yang3
1 Department of Geriatric Medicine, The First Affiliated Hospital of Jiamusi University, Heilongjiang, People's Republic of China
2 Department of Geriatric Medicine, The First Affiliated Hospital of Jiamusi University, Heilongjiang; Chinese Ministry of Health Key Laboratory of Geriatrics, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China
3 Chinese Ministry of Health Key Laboratory of Geriatrics, Beijing Hospital, National Center of Gerontology, Beijing, People's Republic of China
|Date of Acceptance||10-Dec-2018|
|Date of Web Publication||6-Mar-2019|
The First Affiliated Hospital of Jiamusi University, Department of Geriatric Medicine, No. 348, Dexiang Street, Jiamusi City, Heilongjiang Province
People's Republic of China
Dr. Z Yang
Chinese Ministry of Health Key Laboratory of Geriatrics, Beijing Hospital, National Center of Gerontology, No. 1 DaHua Road, Dong Dan, Beijing - 100730
People's Republic of China
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Hyperhomocysteinemia (HHcy) is the risk factor for cardiovascular disease and stroke. However, the impacts on the genetic variation of methylene tetrahydrofolate reductase (MTHFR) on plasma homocysteine levels in the Northeast Chinese population have not been studied. Therefore, this study was carried out to determine the relationship between HHcy and MTHFR gene variation, and whether it was influenced by age and sex of the population in Northeast China. Materials and Methods: A total of 466 subjects were randomly enrolled in this study. According to the homocysteine levels (Hcy ≥ 15 μmol/L) of the subjects, they were divided into hyperhomocysteine (HHcy = 206) and normal homocysteine (Hcy = 260). Polymerase chain reaction/high-resolution dissolution curve and homocysteine determination kit methods were used for genotype testing and homocysteine detection, respectively. Results: High plasma homocysteine levels are associated with MTHFR 677T and 1298A [P < 0.00, odds ratio (confidence interval) = 1.842 (1.418–2.394) >1], which is related to increasing age (Prange = 0.0005–0.0161), with the homocysteine levels of males higher than females (P < 0.0001). Conclusion: High plasma homocysteine levels were linked to the MTHFR gene mutation. In addition, plasma homocysteine levels increased significantly with age with male's homocysteine levels higher than that of females.
Keywords: Age, gender, homocysteine, MTHFR gene, Northeast China
|How to cite this article:|
Wang F, Sui X, Xu N, Yang J, Zhao H, Fei X, Zhang Z, Luo Z, Xin Y, Qin B, Zhao X, Cao S, Zhang Y, Yang Z. The relationship between plasma homocysteine levels and MTHFR gene variation, age, and sex in Northeast China. Niger J Clin Pract 2019;22:380-5
|How to cite this URL:|
Wang F, Sui X, Xu N, Yang J, Zhao H, Fei X, Zhang Z, Luo Z, Xin Y, Qin B, Zhao X, Cao S, Zhang Y, Yang Z. The relationship between plasma homocysteine levels and MTHFR gene variation, age, and sex in Northeast China. Niger J Clin Pract [serial online] 2019 [cited 2019 May 21];22:380-5. Available from: http://www.njcponline.com/text.asp?2019/22/3/380/253447
| Introduction|| |
Homocysteine (Hcy) is a natural homologous compound of amino acid cysteine, which only differs from the parent amino acid by a methylene groups in normal physiological conditions. Hcy is not obtained from diet but it is mainly synthesized during the methionine cycle. Hcy is an intermediate during the conversion of methionine to cysteine.
Hcy can synthesize methionine in the presence of group B vitamins and methylene tetrahydrofolate reductase. The interrelationships between methionine and Hcy is based on three reactions: (i) methionine cycle, (ii) methylation, and (iii) trans-sulfur pathway. Homocysteine is metabolized by the trans-sulfiding pathway to produce cysteine, which is used to synthesize proteins, glutathione, and coenzyme A. Animal experiments have shown that increased cholesterol and Hcy levels in diets rich in methionine can lead to atherosclerosis and cardiovascular disease (CVD), particularly if the diets are deficient in vitamin B6, B12, and folic acid. Several studies in recent years indicate that elevated levels of Hcy are positively correlated with arterial occlusive disease, particularly in the brain, heart, and kidney.,,
Hyperhomocysteine (HHcy) is caused by enzyme dysfunction associated with homocysteine metabolism. Plasma homocysteine levels of ≥15.0 μmol/L were referred to as homocysteinemia. A previous study has shown that when Hcy levels rise by 5 μmol/L, the risk of cerebrovascular disease and coronary heart disease increases by 59 and 32%, respectively. However, when the Hcy level was reduced by 3 mol/L, the risk of stroke and coronary heart disease decreased by 24 and 16%, respectively.
The mechanism of vascular injury caused by elevated Hcy may include the following: inhibition of endothelial growth and postinjury repair, induced endothelial dysfunction, promoting vascular remodeling, and inflammatory mononuclear differentiation. The common causes of elevated Hcy are heredity, folic acid, vitamin B6, B12 deficiency, chronic renal insufficiency, and lifestyle factors, but in developing countries such as in Northeast China, the data on genetic inheritance remain controversial and need further discussion.
Methylene tetrahydrofolate reductase (MTHFR) is encoded by the MTHFR gene. 5,10-Methylene tetrahydrofolate is transformed into 5-methyltetrahydrofolic acid by MTHFR, which is a necessary condition for the transformation of Hcy into methionine, and is a key enzyme in the methionine circulating pathway., Earlier researches have shown that in the MTHFR gene, there are more than 40 genetic sites that have been reported, including the C677T and A1298C mutations.,
MTHFR C677T is considered a risk factor for cancer, CVD, and cerebrovascular disease.,, The most common genetic reason of HHcy may be the conversion from C to T and A to C in C677T and A1298C, respectively. However, the frequency of the mutation and the activity of the enzyme in MTHFR (C677T and A1298C) are influenced by race and geographical locations., The frequency of C677T mutation, for instance, was found to be 7.8% among the population of Rajpur in India, but it was not found in the population of Kom, Meitei, and Paite in India.
This study explores the relationship between plasma homocysteine levels and MTHFR gene variation in Northeast China, and whether it is influenced by age and sex among the Han Chinese in Jiamusi district of Northeast China.
| Materials and Methods|| |
A total of 466 subjects were enrolled into the study through simple random sampling. All the participants are in Jiamusi district of Northeast China without intermarriage with other ethnic groups. The subjects were recruited from the first hospital of Jiamusi University from October in 2016 to March in 2017. Diagnostic criterion of hyperhomocysteine was a level of Hcy ≥15 μmol/L. This study was approved by the Ethics Committee of Jiamusi University and informed written consents from all the subjects were obtained.
| Experimental Method|| |
Experimental instruments and reagents
Genome DNA extraction kit, restriction endonuclease MIu I, polymerase chain reaction (PCR) primer (Beijing Chuangke Biological Co., Ltd), LC-green PLUS saturated fluorescent dye, high-resolution melting (HRM) LightScanner TMHR-196 (Idaho Inc., USA), Nanodrop ND-1000 spectrophotometer (Thermo, USA), PCR amplification device c-1000.
Sample collection and DNA preparation
Gender, age, and previous disease history of all subjects were recorded. Homocysteine, triglycerides, total cholesterol, high-density lipoprotein, low-density lipoprotein, and fasting blood glucose were measured in the morning after fasting. 5 mL of venous blood was collected in ethylenediaminetetraacetic acid anti-coagulation tube and DNA was extracted with DNA extraction kit. After quality control, the DNA concentration of all samples was calibrated with ddH2O between 90 and 120 ng/uL and saved at 4°C.
PCR reaction: The amplification condition was 95°C predenaturation for 3 min; Denaturing 20 s at 95°C, refitting 20 s at 64.1°C, extending for 4 s at 72°C, repeating steps 2–4 again for 45 cycles. Finally, extend at 72°C for 1 min, 95°C de-denature 1 min, and repeat steps 7–9 for two cycles. The last two cycles are the denaturing and refolding treatment before HRM analysis, which leads to the formation of heterologous double-strand DNA.
HRM analysis of PCR products: The PCR product was put into the 96-hole sweep panel specially designed for the LightScanner. After centrifugation for 5 min at 8000 rpm, the LightScanner was put in to collect the high temperature signal. The collected curve was analyzed with Light ScannerCall IT software.
Two-tailed t-test statistical tests were conducted in this study, and P < 0.05 was considered statistically significant. Statistical software SPSS 19.0 was used for data processing and statistical analysis. The Hardy-Weinberg balance test was used to validate the sample group representation. t- Test is used for comparison between groups of sample mean, and the classification variable is denoted by frequency. The relationship between C677T, A1298C mutation, clinical baseline data, and previous diseases were analyzed by Chi-square test and analysis of variance. The interaction of gene combinations (haplotype) in subjects with hyperhomocysteine and normal homocysteine were analyzed by SHEsis (http://analysis.bio-x.cn) online software. The three genotypes of C677T were CC, CT, and TT, respectively. The three genotypes of A1298C are AA, AC, and CC, respectively.
| Results|| |
The average age of subjects was (63.11 ± 8.53), and the males constituted 49.57%. Comparison of clinical baseline data between high homocysteine and normal homocysteine subjects is shown in [Table 1]. The table presents the comparison between the two groups in terms of gender (143/63 vs. 88/172, P = 0.00) and age (65.15 ± 9.03 vs. 62.37 ± 8.20, P = 0.04). The groups were further divided into the middle-age group (40–59 years), the young elderly (60–69 years), and the elderly (≥70 years), and the analysis results were still statistically significant (P = 0.00085). No significant difference was found in other clinical indicators such as fasting glucose and cholesterol (P > 0.05).
|Table 1: Comparison of clinical baseline data between high homocysteine and normal homocysteine group (HHcy group, Hcy group)|
Click here to view
There were 206 subjects in the high homocysteine (HHcy) group and 260 subjects in normal homocysteine (Hcy) group. Correlation analysis of genotype and allele frequency of MTHFR C677T and A1298C is shown in [Table 2] and [Table 3], respectively. The genotypes and alleles of MTHFR C677T and A1298C are analyzed independently: in the C677T, compared with the normal Hcy group, the TT genotype of the HHcy group was significantly higher (47.58 vs. 21.92%, P = 0.00), and the frequency of T allele was significantly higher than that of the normal Hcy group (69.17 vs. 50.58%, P = 0.00), and the difference was statistically significant.
|Table 2: Genotype and allele frequency distribution of MTHFR C677T gene polymorphism in the two groups (HHcy group and Hcy group)|
Click here to view
|Table 3: Genotype and allele frequency distribution of MTHFR A1298C gene polymorphism in the two groups (HHcy group and Hcy group)|
Click here to view
In A1298C, the CC genotype of HHcy group was significantly lower than that of normal Hcy group (1.94 vs. 8.85%, P = 0.003), and the C allele frequency was significantly lower than normal Hcy group and the A allele frequency was higher than normal Hcy group, respectively (80.10 vs. 73.46%; 19.90 vs. 26.54%, P = 0.02), and the difference was statistically significant.
The MTHFR C677T and A1298C gene polymorphisms were compared with the clinical baseline data and previous diseases of all the subjects in [Table 4] and [Table 5], respectively. In the C677T gene site, it is closely related to homocysteine (P = 0.00). In the A1298C gene site, it is related with homocysteine and age (Prange = 0.003–0.01); MTHFR C677T and A1298C had no significant correlation with hypertension, type 2 diabetes mellitus, hyperlipidemia, and hypercholesterolemia in this study (P > 0.05).
|Table 4: Comparison between MTHFR C677T gene polymorphisms and clinical baseline data and previous diseases of all the subjects|
Click here to view
|Table 5: Comparison between MTHFR A1298C gene polymorphisms and clinical baseline data and previous diseases of all the subjects|
Click here to view
The interaction of gene combinations (haplotype) in subjects with hyperhomocysteine is shown in [Table 6]. The results showed that when the HHcy group was compared with the Hcy group, 677C and 1298A had positive interaction effect [P = 0.003, odds ratio (OR) confidence interval (CI) = 0.634 (0.469–0.859) <1]; 677C and 1298C had positive interaction effect [P < 0.00, OR (CI) = 0.440 (0.298–0.648) <1]; 677T and 1298A had positive interaction effect [P < 0.00, OR (CI) = 1.842 (1.418–2.394) >1]; 677T and 1298C had no positive interaction [P = 0.055, OR (CI) = 1.586 (0.988–2.546) >1].
|Table 6: The interaction of gene combinations (haplotype) in subjects with hyperhomocysteine|
Click here to view
All subjects were grouped into middle-age (40–59 years), young older (60–69 years), and old people (≥70 years) to observe the relationship between age and Hcy, as shown in [Figure 1]a. The results showed that there was a statistically significant difference between the middle-age and elderly group (P = 0.0005). The comparison of Hcy in young older and old group was statistically significant (P = 0.0161).
|Figure 1: (a) The comparison between plasma homocysteine level and age. ***The comparison between the middle-age (40–59 years) and old people (70 years). *The comparison between the young older (60–69 years) and old people (70 years), Prange = 0.0005–0.0161. (b) The relationship between Hcy and gender (M: male, F: female). ****The comparison of Hcy level between males and females, P < 0.0001|
Click here to view
According to the gender of all the subjects, [Figure 1]b discusses the relationship between Hcy and gender. The results showed that the plasma homocysteine levels in males were higher than females, and the difference was statistically significant (P < 0.0001).
| Discussion|| |
The study observed that, in MTHFR C677T, the TT genotype was the most statistically significant and common among patients with HHcy, and HHcy was associated with TT genotype in MTHFR C677T (P = 0.00). Among those in the A1298C site, the most people with HHcy carriers were of the AA genotype, and the carriers with high homocysteine may be related to AA genotype (P = 0.003). In the interaction analysis of the two sites (C677T and A1298C), 677C and 1298C, 677C and 1298A may be protective factors against HHcy, while the interaction of 677T and1298A may be the pathogenic factor of HHcy. In addition, plasma homocysteine levels increased with age, particularly in the elderly, and were higher in men than in women.
Regarding the A1298C gene sites, the study by Zappacosa et al. showed that the Hcy of AA genotype was generally higher than that of CC type, and Jadeja et al. also showed that A1298C gene polymorphism was significantly correlated with HHcy., In this study, the number of people with AA genotype in the A1298C gene polymorphism is generally higher than that of CC type, and 1298A may be related to HHcy, and the results are the same as those of previous studies.
In China, the prevalence of HHcy is high in the inland regions, male and elderly, especially in the northern population, and the prevalence rate has increased in recent years. The difference between the sexes may be due to differences in hormone levels between men and women, which may lead to gender-related differences.
There was a clinical study on transsexual hormone management of transsexual people, which showed that in the 4 months of treatment of male and female transgenders with acetylene estradiol and anti-androgens, there was a significant decrease in Hcy level. On the contrary, the increase in plasma Hcy was caused by female transgenders receiving androgen therapy. In addition, compared with women, Chinese men have a higher proportion of alcohol consumption and smoking, which is related to the Hcy level. The possible mechanisms of the increase in HHcy associated with age include changes in renal function, decreased vitamin levels, and the damage of renal metabolism in Hcy.
Limitations of the study
This study did not measure folate levels, which may have some influence on the study.
| Conclusion|| |
High plasma Hcy levels are associated with the gene mutation of MTHFR and that elevated plasma Hcy levels are related with age and gender.
This research was carried out in accordance with the Declaration of Helsinki of the World Medical Association and received ethical approval from the Ethics Committee of Beijing Hospital, Ministry of Health, China.
Financial support and sponsorship
Conflict of interest
There are no conflicts of interest.
| References|| |
Kamat PK, Mallonee CJ, George AK, Tyagi SC, Tyagi N. Homocysteine, alcoholism and its potential epigenetic mechanism. Alcohol Clin Exp Res 2016;40:2474-81.
Zaric BL, Obradovic M, Bajic V, Haidara MA, jovanovic M, Isenovic ER. Homocysteine and hyperhomocysteinemia. Curr Med Chem 2018;25:1-12.
Finkelstein JD. Methionine metabolism in mammals. J Nutr Biochem 1990;1:228-37.
Martinov MV, Vitvitsky VM, Banerjee R, Ataullakhanov FI. The logic of the hepatic methionine metabolic cycle. Biochim Biophys Acta2010;1804:89-96.
Selhub J, Troen AM. Sulfur amino acids and atherosclerosis: A role for excess dietary methionine. Ann N
Y Acad Sci 2016;1363:18-25.
Brattstrom L, Lindgren A, Israelsson B, Malinow MR, Norrving B, Upson B, et al
. Hyperhomocysteinaemia in stroke: Prevalence, cause, and relationships to type of stroke and stroke risk factors. Eur J Clin Invest 1992;22:214-21.
Arnesen E, Refsum H, Bønaa KH, Uland PM, Førde OH, Nordrehaug JE. Serum total homocysteine and coronary heart disease. Int J Epidemiol 1995;24:704-9.
Katsiki N, Perez-Martinez P, Mikhailidis DP. Homocysteine and non-cardiac vascular disease. Curr Pharm Des 2017;23:3224-32.
Hankey GJ, Eikelboom JW. Homocysteine and vascular disease. Lancet 1999;354:407-13.
Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: Evidence on causality from a meta-analysis. BMJ 2002;325:1202.
Wang J, Ouyang N, Qu L, Lin T, Zhang X, Yu Y, et al
. Effect of MTHFR A1298C and MTRR A66G genetic mutations on homocysteine levels in the Chinese population: A systematic review and meta-analysis. J Transl Int Med 2017;5:220-9.
Shakir S, Ali N, Udin Z, Nazish H, Nabi M. Vitamin B6 and homocysteine levels in carbamazepine treated epilepsy of Khyber Pakhtunkhwa. Afr Health Sci 2017;17:559-65.
Richard E, Desviat LR, Ugarte M, Pérez B. Oxidative stress and apoptosis in homocystinuria patients with genetic remethylation defects. J Cell Biochem 2013;114:183-91.
Födinger M, Hörl WH, Sunder-Plassmann G. Molecular biology of 5, 10-methylenetetrahydrofolate reductase. J Nephrol 2000;13:20-33.
Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al
. A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111-3.
van der Put NM, Gabreëls F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes TK, et al
. A second common mutation in the methylenetetrahydrofolate reductase gene: An additional risk factor for neural-tube defects? Am J Hum Genet 1998;62:1044-51.
Sunder-Plassmann G, Födinger M. Genetic determinants of the homocysteine level. Kidney Int Suppl 2003;84:S141-4.
Sheweita SA, Baghdadi H, Allam AR. Role of genetic changes in the progression of cardiovascular diseases. Int J Biomed Sci 2011;7:238-48.
Mansoori N, Tripathi M, Luthra K, Alam R, Lakshmy R, Sharma S, et al
. MTHFR (677 and 1298) and IL-6-174 G/C genes in pathogenesis of Alzheimer's and vascular dementia and their epistatic interaction. Neurobiol Aging 2012;33:1003.e1-8.
Markan S, Sachdeva M, Sehrawat BS, Kumari S, Jain S, Khullar M. MTHFR 677 CT/MTHFR 1298 CC genotypes are associated with increased risk of hypertension in Indians. Mol Cell Biochem 2007;302:125-31.
Esfahani ST, Cogger EA, Caudill MA. Heterogeneity in the prevalence of methylenetetrahydrofolate reductase gene polymorphisms in women of different ethnic groups. J Am Diet Assoc 2003;103:200-7.
Yang B, Liu Y, Li Y, Fan S, Zhi X, Lu X, et al
. Geographical distribution of MTHFR C677T, A1298C and MTRR A66G Gene polymorphisms in China: Findings from 15357 adults of Han nationality. PLoS One 2013;8:e57917.
Saraswathy KN, Asghar M, Samtani R, Murry B, Mondal PR, Ghosh PK, et al
. Spectrum of MTHFR gene SNPs C677T and A1298C: A study among 23 population groups of India. Mol Biol Rep 2012;39:5025-31.
Zappacosta B, Graziano M, Persichilli S, Di Castelnuovo A, Mastroiacovo P, Iacoviello L. 5,10-Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms: Genotype frequency and association with homocysteine and folate levels in middle-southern Italian adults. Cell Biochem Funct 2014;32:1-4.
Jadeja SD, Mansuri MS, Singh M, Patel H, Marfatia YS, Begum R. Association of elevated homocysteine levels and Methylenetetrahydrofolate reductase (MTHFR) 1298 A > C polymorphism with Vitiligo susceptibility in Gujarat. J Dermatol Sci 2018;90:112-22.
Yang B, Fan S, Zhi X, Wang Y, Zheng Q, Sun G. Prevalence of hyperhomocysteinemia in China: A systematic review and meta-analysis. Nutrients 2015;7:74-90.
Giltay EJ, Hoogeveen EK, Elbers JM, Gooren LJ, Asscheman H, Stehouwer CD. Effects of sex steroids on plasma total homocysteine levels: A study in transsexual males and females. J Clin Endocrinol Metab 1998;83:550-3.
Hao L, Ma J, Stampfer MJ, Ren A, Tian Y, Tang Y, et al
. Geographical, seasonal and gender differences in folate status among Chinese adults. J Nutr 2003;133:3630-5.
Refsum H, Nurk E, Smith AD, Ueland PM, Gjesdal CG, Bjelland I, et al
. The Hordaland Homocysteine Study: A community-based study of homocysteine, its determinants, and associations with disease. J Nutr 2006;136:1731-40.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]