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Prevalence of methylenetetrahydrofolate reductase C677T and A1298C polymorphisms in Taiwanese patients with Type 2 diabetic mellitus
Clinical Biochemistry 44 (2011) 1370–1374
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Prevalence of methylenetetrahydrofolate reductase C677T and A1298C polymorphisms in Taiwanese patients with Type 2 diabetic mellitus Yih-Hsin Chang a, b, 1, Wen-Mei Fu c, 1, Yu-Hui Wu d, Chih-Jung Yeh e, Chien-Ning Huang f, g, Ming-Yuh Shiau h,⁎ a
Institute of Biotechnology in Medicine, National Yang-Ming University, Taipei, Taiwan, ROC School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, ROC Department of Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC d Department of Clinical Laboratory, Lee General Hospital, Taichung, Taiwan, ROC e School of Public Health, Chung Shan Medical University, Taichung, Taiwan, ROC f Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC g Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC h Hungkuang University, Taichung, Taiwan, ROC b c
a r t i c l e
i n f o
Article history: Received 11 May 2011 Received in revised form 23 August 2011 Accepted 16 September 2011 Available online 8 October 2011 Keywords: Homocysteine Methylenetetrahydrofolate reductase Type 2 diabetes mellitus
a b s t r a c t Objectives: Deficiency and/or decreased activity of methyltetrahydrofolate reductase (MTHFR) resulted from MTHFR variants are associated with hyperhomocysteinemia, an independent risk factor for vasculopathies in diabetic patients. The aim of this study was to examine MTHFR genotypes between healthy and type 2 diabetes mellitus (T2DM) subjects. Design and methods: MTHFR C677T and A1298C genotypes were analyzed in 56 T2DM and 62 healthy subjects by PCR-RFLP. Association between MTHFR genotypes and T2DM as well as the lipid/glucose metabolic indexes among T2DM subjects was statistically analyzed. Results: No significance in the distribution of MTHFR genotypes between healthy and T2DM subjects is found. Besides, no significant associations between lipid/glucose metabolic indexes with MTHFR genotypes among diabetic patients are observed. Conclusions: These data indicate the previous observation that MTHFR polymorphisms may play some roles in the pathogenesis and complications of T2DM in Caucasians are unlikely to be applied in Taiwanese patients. © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction According to the statistics from the Department of Health, diabetes mellitus has been ranked as the forth mortality in Taiwanese population. Diabetic prevalence in Taiwanese population has been rapidly increasing during the past decade. The most important underlying factors for this high diabetic mortality are the irreversible complications resulted from the chronic process of atherosclerosis including thrombosis, myocardial infarction and stroke. It is well known that patients with type 1 or type 2 diabetes mellitus (T2DM) have an increased risk of cardiovascular events and that the complete arterial vasculature can be compromised. In T2DM patients, the risk of cardiovascular events is 2–4 fold higher than that in the non-diabetic population. Therefore, management of cardiovascular disease and their risk factors is extremely important in diabetic patients.
⁎ Corresponding author at: Hungkuang University, Taichung 433, Taiwan, ROC. Fax: + 886 4 26522280. E-mail address: [email protected] (M.-Y. Shiau). 1 These authors contributed equally as the first authors.
Elevated homocysteine (Hcy) levels are associated with endothelial dysfunction [1], insulin resistance [2], prothrombotic state [3], macroangiopathy [4,5] and nephropathy [6,7] in diabetic patients. Several studies have demonstrated that elevated Hcy levels (hyperhomocysteinemia, HHcy) predict the risk of death or coronary events in T2DM patients [8–10]. The enzyme methylenetetrahydrofolate reductase (MTHFR) participates in the Hcy metabolism by methylating Hcy to methionine. A frequently occurring MTHFR mutation at nucleotide 677 (C677T) of the coding region, which results in the substitution of valine for alanine at position 226 of the amino acid, is linked with HHcy [11–14]. Another replacement at nucleotide 1298 (A1298C) which leads to a substitution of alanine for glutamine also results in decreased MTHFR activity and the subsequent HHcy [15,16]. Taking the above evidence regarding the correlations between diabetes and HHcy as well as between MTHFR mutations and Hcy levels together, MTHFR gene mutations are associated with the predisposition to cardiovascular diseases in diabetic populations. The aim of this study was to examine the MTHFR C677T and A1298C polymorphisms in Taiwanese patients with T2DM. The allele frequencies of the C677T and A1298C mutations of MTHFR gene were investigated in the Taiwanese diabetic population. The putative
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Y.-H. Chang et al. / Clinical Biochemistry 44 (2011) 1370–1374
association between the biochemical manifestations and the MTHFR genotypes in our diabetic population was also examined. Subjects and methods Subjects To determine the relationship between diabetic clinical manifestations and MTHFR gene polymorphisms, C677T and A1298C genotypes were examined among T2DM patients. Fifty-six subjects (20 men and 36 women; mean age 54.8 ± 8.7 years) were recruited from the outpatient diabetes clinic of Chung Shan Medical University Hospital. Information regarding biochemical parameters, diabetic duration, medications, other illnesses and the presence of diabetic complications was collected and filed for further analysis. Sixty-two age- and gendermatched (19 males and 43 females; mean age 51.5 ± 11.0 years) nondiabetic healthy control subjects were also recruited. All study subjects were of Taiwanese ethnic origin. The procedures followed were in accordance with the Helsinki Declaration. Informed consent was obtained from all participating adult subjects and from parents or legal guardians for minors or incapacitated adults, together with the manner in which informed consent was obtained. Biochemical measurements Participants were phlebotomized after an overnight fast (10 to 14 h), and blood samples were taken. Plasma Hcy levels were assessed at base-line in both patients and controls. All whole blood specimens for Hcy analysis were drawn into vacutainers containing EDTA, and immediately refrigerated at 4 °C. Within 2 h of collection, blood samples were centrifuged, plasma was separated and stored at −20 °C until use. Total plasma Hcy was determined by automated fluorescence Polarization Immunoassay [17]. Glycemic control was assessed before examination by measuring HbA1c levels on HPLC Variant System (Bio-Rad, Hercules, CA, USA). In addition, data of total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides (TG), fasting glucose, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were filed for further analysis (Table 1). Genetic analysis Genomic DNA was extracted from peripheral blood mononuclear cells using GenMaker commercial kit (GeneMaker Technology, Taiwan). Amplification of C677T and A1298C fragments was performed in a
Table 1 Demographic information and blood chemistry parameters of study subjects. Study subjects
Male/Female (n) Age (years) BMI (kg/m2) Glucose (70–110 mg/dL) TG (20–200 mg/dL) TC (110–220 mg/mL) HDL (> 35 mg/mL) LDL (b 160 mg/mL) HbA1c (3.4–6.1%) Hcy (4.45–12.1 μmol/L) SBP (120–140 mm Hg) DBP (70–90 mm Hg)
p Value
Control subjects (n = 62)
T2DM subjects (n = 56)
23/39 51.5 ± 11.0 24.8 ± 4.0 98.8 ± 10.2 116.8 ± 75.0 194.6 ± 38.0 55.8 ± 16.9 119.8 ± 32.6 5.6 ± 0.5 8.9 ± 2.2 124.4 ± 19.2 80.9 ± 12.0
20/36 54.8 ± 8.7 25.6 ± 4.2 193.6 ± 82.0 155.2 ± 124.7 197.2 ± 46.0 50.8 ± 13.4 123.7 ± 40.2 8.7 ± 2.8 9.9 ± 3.1 132.8 ± 16.7 82.1 ± 10.3
0.87 0.74 0.36 b 0.05 b 0.05 0.73 0.75 0.56 b 0.05 b 0.05 b 0.05 0.56
Numbers in the parentheses indicated the corresponding normal reference range of each biochemical test. Data were presented as mean ± standard deviation.
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volume of 30 μL containing 200 ng of template DNA, 10 mmol/L Tris– HCl (pH9.0), 50 mM KCl, 3 mM MgCl2, 0.01% gelatin, 0.1% Triton X-100, 125 μM dNTP, 0.5 μM of each primer (C677T: 5′-TGAAGGAGAAGGTGTCTGCGGGA-3′ and 5′-GGACGGTGCGGTGAGAGTG-3′ with annealing condition 59 °C, 1 min; A1298C: 5′-CTTTGGGGAGCTGAAGGACTACTAC-3′ and 5′-CACTTTGTGACCATTCCGGTTTG-3′ with annealing condition 51 °C, 1 min) and 1 unit GeneTaq DNA polymerase (GeneMaker Technology, Taiwan). The parameters for thermocycling were as followings: an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 60 s; annealing at the respective condition as described above; extension at 72 °C for 30 s; and followed by final extension at 72 °C for 5 min. C677T and A1298C genotypes of the amplified PCR products were characterized by electrophoresis on 12% polyacrylamide gels after the fragments were digested by Hinf I and Mbo II (MBI fermentas; Vilna, Lithuania), respectively.
Statistical analyses Statistical analyses were performed as described [15]. In brief, allele frequencies were calculated by allele counting. Concordance of genotype frequencies with Hardy-Weinberg equilibrium was tested by chi-square test. The baseline values of these groups were compared using the unpaired t test. Statistical tests were performed using statistical software (SPSS; Chicago, IL).
Results Characteristics of the patients' population Data regarding biochemical parameters of recruited subjects, including the healthy control individuals and diabetic patients, were listed in Table 1. The age and gender between the control and diabetic subjects were matched. Significant differences were observed in blood glucose (193.6± 82.0 vs 98.8 ± 10.2, p b 0.05), TG (155.2± 124.7 vs 116.8 ± 75.0, p b 0.05) and HbA1c (8.7 ± 2.8 vs 5.6 ± 0.5, p b 0.05) under fasting conditions between patients and controls. Although Hcy levels in the diabetic population were within the normal range, diabetic subjects had significant higher Hcy levels than the control subjects (9.9± 3.1 vs 8.9± 2.2, p b 0.05). SBP in diabetic patients was also significantly higher than control subjects (132.8 ± 16.7 vs 124.4 ± 19.2, p b 0.05).
Distribution of MTHFR genotypes among the study subjects Among the 62 non-diabetic control individuals, 36 (58.1%) and 26 (41.9%) subjects carried homologous T/T and C-allele containing genotype (including 23 C/T [37.1%] and 3 C/C [4.8%]), respectively; while the corresponding number in T2DM patients was 30 (53.6%) and 26 (46.4%, including 25 C/T [44.6%] and 1 C/C [1.8%]). The prevalence of 677 C and T allele in control individuals was 76.6% and 23.4%, respectively; and that in T2DM counterpart was 75.9% and 24.1%. No significant difference in the distribution of MTHFR C677T genotypes (p = 0.38) and alleles (p = 0.90) between diabetic patients and control subjects was observed (Table 2). In terms of the MTHFR A1298C polymorphisms, 28 (45.2%) and 34 (54.8%) carried homologous AA and C-allele containing genotype (including 33 A/C [53.2%] and 1 C/C [1.6%]), respectively; while 21 (37.5%) and 35 (62.5%, including 32 A/C [57.1%] and 3 C/C [5.4%]) T2DM patients carried the corresponding genotype. The prevalence of 1298 A and C allele in control individuals was 71.8% and 28.2%, respectively; and that in T2DM patients was 66.1% and 33.9%. No significant difference of MTHFR A1298C genotypes (p = 0.26) and alleles (p = 0.21) distribution between diabetic patients and control individuals was found.
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Table 2 Genotypic frequencies of MTHFR polymorphisms among study subjects. Genotype/allele
C677T T/T C/T and C/C T allele C allele A1298C A/A A/C and C/C A allele C allele
Study subjects
p Value
Control subjects (n = 62)
T2DM subjects (n = 56)
36 26 95 29
(58.1%) (41.9%) (76.6%) (23.4%)
30 (53.9%) 26 (46.4%) 85 (75.9%) 27 (24.1%)
0.38
28 34 89 35
(45.2%) (54.8%) (71.8%) (28.2%)
21 (37.5%) 35 (62.5%) 74 (66.1%) 38 (33.9%)
0.26
0.90
0.21
of 1298 A and C allele in T2DM male individuals was 67.5% and 32.5%, respectively; and that in female patients was 65.3% and 34.7%. No significant difference in the distribution of MTHFR A1298C genotypes (p = 0.67) and alleles (p = 0.80) between control and diabetic subjects of different gender was found. Association of MTHFR genotypes and biochemical parameters among diabetic patients The association between diabetic subjects' biochemical parameters, including lipid/glucose metabolic indexes, was statistically analyzed. No significant association between the above biochemical parameters and MTHFR genotypes was found (Table 4). Discussion
MTHFR genotypes and gender In diabetic patients, Hcy increases significantly with age and males have higher Hcy levels than females [18]. Therefore, the association between gender and MTHFR genotypes was subsequently analyzed (Table 3). Among the non-diabetic control individuals, 12 (63.2%) and 7 (36.8%) males carried T/T and C/T, respectively (no C/C genotype was found); while 24 (55.8%) and 19 (44.2%, including 16 C/T [37.2%] and 3 C/C [7.0%]) female carried the corresponding genotype. Among the T2DM patients, 12 (60.0%) and 8 (40.0%) males carried T/T and heterologous C/T genotype, respectively (no C/C genotype was found); and the frequency of T/T and C-containing genotype (including 17 C/T [47.2%] and 1 C/C [2.8%]) in 36 females was half-half respectively. The prevalence of T and C allele in control male individuals was 81.6% and 18.4%, respectively; and that in control female subjects was 74.4% and 25.6%. The prevalence of T and C allele in male T2DM patients was 80.0% and 20.0%, respectively; and that in female patients was 73.6% and 26.4%. No significant difference in the distribution of MTHFR C677T genotypes (p=0.79) and alleles (p=0.72) between control and diabetic patients of different gender was found. In terms of the A1298C polymorphisms, 8 (42.1%) and 11 (57.9%) control males carried MTHFR 1298 A/A and A/C genotype, respectively (no C/C genotype was found); while 20 (46.5%) and 23 (53.5%, including 22 A/C [51.2%] and 1 C/C [2.3%]) female control subjects carried the corresponding genotype. In T2DM patients, 9 (45.0%) and 11 (55.0%) males carried A/A and C-containing genotype (including 9 A/C [45.0%] and 2 C/C [10.0%]), respectively; while 12 (33.3%) and 24 (66.7%, including 23 A/C [63.9%] and 1 C/C [2.8%]) female carried the corresponding genotype. The prevalence of 1298 A and C allele in control male individuals was 71.0% and 29.0%, respectively; and that in control female subjects was 72.1% and 27.9%. The prevalence
Table 3 Genotypic frequencies of MTHFR polymorphisms among study subjects stratified by gender difference. Genotype/allele
C677T T/T C/T and C/C T allele C allele A1298C A/A A/C and C/C A allele C allele
Control subjects
T2DM subjects
p Value
Male (n = 19)
Female (n = 43)
Male (n = 20)
Female (n = 36)
12 (63.2%) 7 (36.8%) 31 (81.6%) 7 (18.4%)
24 (55.8%) 19 (44.2%) 64 (74.4%) 22 (25.6%)
12 (60.0%) 8 (40.0%) 32 (80.0%) 8 (20.0%)
18 (50.0%) 18 (50.0%) 53 (73.6%) 19 (26.4%)
0.79
8 (42.1%) 11 (57.9%) 27 (71.0%) 11 (29.0%)
20 (46.5%) 23 (53.5%) 62 (72.1%) 24 (27.9%)
9 (45.0%) 11 (55.0%) 27 (67.5%) 13 (32.5%)
12 (33.3%) 24 (66.7%) 47 (65.3%) 25 (34.7%)
0.67
0.72
0.80
Gene encoding MTHFR is located on chromosome 1p36.3. Mutations in MTHFR are considered as a model of Hcy metabolic disorders due to its main role in the remethylation process of Hcy metabolism [18,19]. MTHFR C677T and A1298C are 2 common genetic polymorphisms which lead to reduced MTHFR activity [20]. Individuals with insufficient MTHFR activity by carrying either one of these 2 variants have lower 5-methyltetrahydrofolate and higher Hcy levels [21]. HHcy and MTHFR polymorphisms, particularly C677T, are recognized as independent risk factors for cardiovascular diseases [12]. Elevated plasma Hcy levels impair functions of the endothelium which in turn results in an uncontrolled proliferation of smooth muscle cells and consequently accelerate the atherogenic process. The association of HHcy with cardiovascular diseases is particularly evident in T2DM patients [5]. Approximately 35% of diabetic patients demonstrate HHcy with prevalence of 43% and 18% in male and female, respectively [22,23]. In diabetic patients, Hcy increases significantly with age and males have higher Hcy levels than females while diabetic duration does not affect Hcy levels [24]. Insulin resistance is reported to be positively associated with Hcy levels [2], whereas other study concludes that T2DM patients with hyper- or normo-homocysteinemia have similar degree of insulin resistance [4]. Likewise, while some reports reveal that diabetic patients have higher Hcy levels [1,25–27], others find similar [28–30] or even lower [9,34,35]. Taken together, no definitive correlation between plasma Hcy levels and insulin resistance can be concluded by these conflicting results. Other diabetes-related factors such as HbA1c and insulin dependency do not have a significant influence on Hcy levels [4,28,31]. Fiorina et al. reported that potential interaction between Hcy and glucose intolerance is a risk factor for atherosclerosis [32]. In addition to cardiovascular diseases, HHcy is also associated with other diabetic complications, including renal dysfunction [6,7] and retinopathy [33]. Nevertheless, Hcy levels in uncomplicated diabetic patients are usually parallel to those in healthy control subjects [34]. Our results show that the distribution of MTHFR genotypes between healthy and T2DM subjects is not significantly different. In addition, no significant associations between lipid/glucose metabolic indexes with MTHFR genotypes among diabetic patients are observed. These data indicate that the conclusion which MTHFR polymorphisms may play some roles in the pathogenesis and complications in Caucasians T2DM patients is unlikely to be applied in Taiwanese patients. Several possible factors could contribute to the lack of association between the MTHFR genotypes with T2DM and diabetic related indexes in our study. First of all, ethnic variations in MTHFR genotypes may contribute to our observation. A given population may have elements in its genetic reservoir that are protective against certain disease despite of the high prevalence of disease-susceptible alleles. Such a protective effect is found in the Japanese population in which the C677T mutation is associated with lower blood pressure, a protective factor for cerebral vascular disease [35]. Therefore, the possibility of differential protective
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Table 4 Association between MTHFR genetic polymorphisms and biochemical parameters in T2DM patients. C677T genotype
2
BMI (kg/m ) Glucose (mg/dL) TG (mg/dL) TC (mg/mL) HDL (mg/mL) LDL (mg/mL) HbA1c (%) Hcy (μmol/L) SBP (mm Hg) DBP (mm Hg)
A1298C genotype
T/T (n = 30)
C/T and C/C (n = 26)
p Value
A/A (n = 21)
A/C and C/C (n = 35)
p Value
25.4 ± 3.4 207.2 ± 93.9 170.5 ± 159.7 200.3 ± 40.6 50.2 ± 14.5 126.3 ± 37.7 9.1 ± 2.8 10.1 ± 3.3 133.3 ± 14.0 84.5 ± 10.4
25.6 ± 4.2 178.0 ± 64.0 137.5 ± 63.4 193.7 ± 52.2 51.4 ± 12.3 120.7 ± 43.5 8.1 ± 2.7 9.8 ± 3.6 132.2 ± 19.7 79.3 ± 9.6
0.74 0.19 0.33 0.60 0.74 0.60 0.21 0.72 0.80 0.06
25.1 ± 4.2 193.2 ± 99.2 130.9 ± 58.8 175.7 ± 56.4 54.2 ± 13.7 121.3 ± 44.6 8.2 ± 2.1 8.9 ± 2.5 129.4 ± 13.1 81.0 ± 9.2
25.8 ± 4.2 193.9 ± 71.3 169.7 ± 150.1 180.1 ± 52.5 48.0 ± 12.6 119.7 ± 36.4 8.7 ± 2.9 9.7 ± 2.9 133.6 ± 17.2 83.9 ± 11.9
0.52 0.98 0.26 0.77 0.90 0.14 0.54 0.41 0.40 0.40
Numbers in the parentheses indicated the corresponding normal reference range of each biochemical test. Data were presented as mean ± standard deviation.
genetic factors related to a particular ethnicity may lead to the conflicting results among studies. Although many reports show that MTHFR mutation is related to increased Hcy, the association of MTHFR mutation with a variety of vascular disorders is still controversial. Additionally, linkage disequilibrium of the mutant allele with a nearby noncausative polymorphism may underlie the findings. Therefore, the possibility of linkage disequilibrium of the variant alleles with a nearby non-causative polymorphism in our population cannot be ruled out. Secondly, differences in Hcy metabolism among population may exist. For example, Africans metabolize Hcy more effectively than Caucasians [36]. The rarity of gene frequency predisposing to HHcy in Finland may contribute to the lack of association between Hcy and cardiovascular diseases in their population [37]. In summary, our results show that no significant difference in the distribution of MTHFR genotypes between healthy and T2DM subjects is found. Additionally, no significant associations between lipid/glucose metabolic indexes with MTHFR genotypes among diabetic patients are observed. These data indicate the previous observation that MTHFR polymorphisms may play some roles in the pathogenesis and complications of Caucasians T2DM patients are unlikely to be applied in Taiwanese patients. Acknowledgments The work was supported by grants from Chung Shan Medical University Hospital (CSH-96-29), Taiwan, Republic of China. References [1] Doupis J, Eleftheriadou I, Kokkinos A, et al. Acute hyperhomocysteinemia impairs endothelium function in subjects with type 2 diabetes mellitus. Exp Clin Endocrinol Diabetes 2010;118:453–8. [2] Shimomura T, Anan F, Umeno Y, et al. Hyperhomocysteinaemia is a significant risk factor for white matter lesions in Japanese type 2 diabetic patients. Eur J Neurol 2008;15:289–94. [3] Aso Y, Yoshida N, Okumura K, et al. Coagulation and inflammation in overt diabetic nephropathy: association with hyperhomocysteinemia. Clin Chim Acta 2004;348:139–45. [4] Buysschaert M, Dramais AS, Wallemacq PE, et al. Hyperhomocysteinemia in type 2 diabetes: relationship to macroangiopathy, nephropathy, and insulin resistance. Diabetes Care 2000;23:1816–22. [5] Sun J, Xu Y, Zhu Y, et al. Methylenetetrahydrofolate reductase gene polymorphism, homocysteine and risk of macroangiopathy in type 2 diabetes mellitus. J Endocrinol Invest 2006;29:814–20. [6] Mtiraoui N, Ezzidi I, Chaieb M, et al. MTHFR C677T and A1298C gene polymorphisms and hyperhomocysteinemia as risk factors of diabetic nephropathy in type 2 diabetes patients. Diabetes Res Clin Pract 2007;75:99–106. [7] Ukinc K, Ersoz HO, Karahan C, et al. Methyltetrahydrofolate reductase C677T gene mutation and hyperhomocysteinemia as a novel risk factor for diabetic nephropathy. Endocrine 2009;36:255–61. [8] Kalin A, Alatas O, Colak O. Relation of plasma homocysteine levels to atherosclerotic vascular disease and inflammation markers in type 2 diabetic patients. Eur J Endocrinol 2008;158:47–52.
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Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Prevalence of methylenetetrahydrofolate reductase C677T and A1298C polymorphisms in Taiwanese patients with Type 2 diabetic mellitus Yih-Hsin Chang a, b, 1, Wen-Mei Fu c, 1, Yu-Hui Wu d, Chih-Jung Yeh e, Chien-Ning Huang f, g, Ming-Yuh Shiau h,⁎ a
Institute of Biotechnology in Medicine, National Yang-Ming University, Taipei, Taiwan, ROC School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan, ROC Department of Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC d Department of Clinical Laboratory, Lee General Hospital, Taichung, Taiwan, ROC e School of Public Health, Chung Shan Medical University, Taichung, Taiwan, ROC f Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC g Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, ROC h Hungkuang University, Taichung, Taiwan, ROC b c
a r t i c l e
i n f o
Article history: Received 11 May 2011 Received in revised form 23 August 2011 Accepted 16 September 2011 Available online 8 October 2011 Keywords: Homocysteine Methylenetetrahydrofolate reductase Type 2 diabetes mellitus
a b s t r a c t Objectives: Deficiency and/or decreased activity of methyltetrahydrofolate reductase (MTHFR) resulted from MTHFR variants are associated with hyperhomocysteinemia, an independent risk factor for vasculopathies in diabetic patients. The aim of this study was to examine MTHFR genotypes between healthy and type 2 diabetes mellitus (T2DM) subjects. Design and methods: MTHFR C677T and A1298C genotypes were analyzed in 56 T2DM and 62 healthy subjects by PCR-RFLP. Association between MTHFR genotypes and T2DM as well as the lipid/glucose metabolic indexes among T2DM subjects was statistically analyzed. Results: No significance in the distribution of MTHFR genotypes between healthy and T2DM subjects is found. Besides, no significant associations between lipid/glucose metabolic indexes with MTHFR genotypes among diabetic patients are observed. Conclusions: These data indicate the previous observation that MTHFR polymorphisms may play some roles in the pathogenesis and complications of T2DM in Caucasians are unlikely to be applied in Taiwanese patients. © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction According to the statistics from the Department of Health, diabetes mellitus has been ranked as the forth mortality in Taiwanese population. Diabetic prevalence in Taiwanese population has been rapidly increasing during the past decade. The most important underlying factors for this high diabetic mortality are the irreversible complications resulted from the chronic process of atherosclerosis including thrombosis, myocardial infarction and stroke. It is well known that patients with type 1 or type 2 diabetes mellitus (T2DM) have an increased risk of cardiovascular events and that the complete arterial vasculature can be compromised. In T2DM patients, the risk of cardiovascular events is 2–4 fold higher than that in the non-diabetic population. Therefore, management of cardiovascular disease and their risk factors is extremely important in diabetic patients.
⁎ Corresponding author at: Hungkuang University, Taichung 433, Taiwan, ROC. Fax: + 886 4 26522280. E-mail address: [email protected] (M.-Y. Shiau). 1 These authors contributed equally as the first authors.
Elevated homocysteine (Hcy) levels are associated with endothelial dysfunction [1], insulin resistance [2], prothrombotic state [3], macroangiopathy [4,5] and nephropathy [6,7] in diabetic patients. Several studies have demonstrated that elevated Hcy levels (hyperhomocysteinemia, HHcy) predict the risk of death or coronary events in T2DM patients [8–10]. The enzyme methylenetetrahydrofolate reductase (MTHFR) participates in the Hcy metabolism by methylating Hcy to methionine. A frequently occurring MTHFR mutation at nucleotide 677 (C677T) of the coding region, which results in the substitution of valine for alanine at position 226 of the amino acid, is linked with HHcy [11–14]. Another replacement at nucleotide 1298 (A1298C) which leads to a substitution of alanine for glutamine also results in decreased MTHFR activity and the subsequent HHcy [15,16]. Taking the above evidence regarding the correlations between diabetes and HHcy as well as between MTHFR mutations and Hcy levels together, MTHFR gene mutations are associated with the predisposition to cardiovascular diseases in diabetic populations. The aim of this study was to examine the MTHFR C677T and A1298C polymorphisms in Taiwanese patients with T2DM. The allele frequencies of the C677T and A1298C mutations of MTHFR gene were investigated in the Taiwanese diabetic population. The putative
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Y.-H. Chang et al. / Clinical Biochemistry 44 (2011) 1370–1374
association between the biochemical manifestations and the MTHFR genotypes in our diabetic population was also examined. Subjects and methods Subjects To determine the relationship between diabetic clinical manifestations and MTHFR gene polymorphisms, C677T and A1298C genotypes were examined among T2DM patients. Fifty-six subjects (20 men and 36 women; mean age 54.8 ± 8.7 years) were recruited from the outpatient diabetes clinic of Chung Shan Medical University Hospital. Information regarding biochemical parameters, diabetic duration, medications, other illnesses and the presence of diabetic complications was collected and filed for further analysis. Sixty-two age- and gendermatched (19 males and 43 females; mean age 51.5 ± 11.0 years) nondiabetic healthy control subjects were also recruited. All study subjects were of Taiwanese ethnic origin. The procedures followed were in accordance with the Helsinki Declaration. Informed consent was obtained from all participating adult subjects and from parents or legal guardians for minors or incapacitated adults, together with the manner in which informed consent was obtained. Biochemical measurements Participants were phlebotomized after an overnight fast (10 to 14 h), and blood samples were taken. Plasma Hcy levels were assessed at base-line in both patients and controls. All whole blood specimens for Hcy analysis were drawn into vacutainers containing EDTA, and immediately refrigerated at 4 °C. Within 2 h of collection, blood samples were centrifuged, plasma was separated and stored at −20 °C until use. Total plasma Hcy was determined by automated fluorescence Polarization Immunoassay [17]. Glycemic control was assessed before examination by measuring HbA1c levels on HPLC Variant System (Bio-Rad, Hercules, CA, USA). In addition, data of total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides (TG), fasting glucose, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were filed for further analysis (Table 1). Genetic analysis Genomic DNA was extracted from peripheral blood mononuclear cells using GenMaker commercial kit (GeneMaker Technology, Taiwan). Amplification of C677T and A1298C fragments was performed in a
Table 1 Demographic information and blood chemistry parameters of study subjects. Study subjects
Male/Female (n) Age (years) BMI (kg/m2) Glucose (70–110 mg/dL) TG (20–200 mg/dL) TC (110–220 mg/mL) HDL (> 35 mg/mL) LDL (b 160 mg/mL) HbA1c (3.4–6.1%) Hcy (4.45–12.1 μmol/L) SBP (120–140 mm Hg) DBP (70–90 mm Hg)
p Value
Control subjects (n = 62)
T2DM subjects (n = 56)
23/39 51.5 ± 11.0 24.8 ± 4.0 98.8 ± 10.2 116.8 ± 75.0 194.6 ± 38.0 55.8 ± 16.9 119.8 ± 32.6 5.6 ± 0.5 8.9 ± 2.2 124.4 ± 19.2 80.9 ± 12.0
20/36 54.8 ± 8.7 25.6 ± 4.2 193.6 ± 82.0 155.2 ± 124.7 197.2 ± 46.0 50.8 ± 13.4 123.7 ± 40.2 8.7 ± 2.8 9.9 ± 3.1 132.8 ± 16.7 82.1 ± 10.3
0.87 0.74 0.36 b 0.05 b 0.05 0.73 0.75 0.56 b 0.05 b 0.05 b 0.05 0.56
Numbers in the parentheses indicated the corresponding normal reference range of each biochemical test. Data were presented as mean ± standard deviation.
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volume of 30 μL containing 200 ng of template DNA, 10 mmol/L Tris– HCl (pH9.0), 50 mM KCl, 3 mM MgCl2, 0.01% gelatin, 0.1% Triton X-100, 125 μM dNTP, 0.5 μM of each primer (C677T: 5′-TGAAGGAGAAGGTGTCTGCGGGA-3′ and 5′-GGACGGTGCGGTGAGAGTG-3′ with annealing condition 59 °C, 1 min; A1298C: 5′-CTTTGGGGAGCTGAAGGACTACTAC-3′ and 5′-CACTTTGTGACCATTCCGGTTTG-3′ with annealing condition 51 °C, 1 min) and 1 unit GeneTaq DNA polymerase (GeneMaker Technology, Taiwan). The parameters for thermocycling were as followings: an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 60 s; annealing at the respective condition as described above; extension at 72 °C for 30 s; and followed by final extension at 72 °C for 5 min. C677T and A1298C genotypes of the amplified PCR products were characterized by electrophoresis on 12% polyacrylamide gels after the fragments were digested by Hinf I and Mbo II (MBI fermentas; Vilna, Lithuania), respectively.
Statistical analyses Statistical analyses were performed as described [15]. In brief, allele frequencies were calculated by allele counting. Concordance of genotype frequencies with Hardy-Weinberg equilibrium was tested by chi-square test. The baseline values of these groups were compared using the unpaired t test. Statistical tests were performed using statistical software (SPSS; Chicago, IL).
Results Characteristics of the patients' population Data regarding biochemical parameters of recruited subjects, including the healthy control individuals and diabetic patients, were listed in Table 1. The age and gender between the control and diabetic subjects were matched. Significant differences were observed in blood glucose (193.6± 82.0 vs 98.8 ± 10.2, p b 0.05), TG (155.2± 124.7 vs 116.8 ± 75.0, p b 0.05) and HbA1c (8.7 ± 2.8 vs 5.6 ± 0.5, p b 0.05) under fasting conditions between patients and controls. Although Hcy levels in the diabetic population were within the normal range, diabetic subjects had significant higher Hcy levels than the control subjects (9.9± 3.1 vs 8.9± 2.2, p b 0.05). SBP in diabetic patients was also significantly higher than control subjects (132.8 ± 16.7 vs 124.4 ± 19.2, p b 0.05).
Distribution of MTHFR genotypes among the study subjects Among the 62 non-diabetic control individuals, 36 (58.1%) and 26 (41.9%) subjects carried homologous T/T and C-allele containing genotype (including 23 C/T [37.1%] and 3 C/C [4.8%]), respectively; while the corresponding number in T2DM patients was 30 (53.6%) and 26 (46.4%, including 25 C/T [44.6%] and 1 C/C [1.8%]). The prevalence of 677 C and T allele in control individuals was 76.6% and 23.4%, respectively; and that in T2DM counterpart was 75.9% and 24.1%. No significant difference in the distribution of MTHFR C677T genotypes (p = 0.38) and alleles (p = 0.90) between diabetic patients and control subjects was observed (Table 2). In terms of the MTHFR A1298C polymorphisms, 28 (45.2%) and 34 (54.8%) carried homologous AA and C-allele containing genotype (including 33 A/C [53.2%] and 1 C/C [1.6%]), respectively; while 21 (37.5%) and 35 (62.5%, including 32 A/C [57.1%] and 3 C/C [5.4%]) T2DM patients carried the corresponding genotype. The prevalence of 1298 A and C allele in control individuals was 71.8% and 28.2%, respectively; and that in T2DM patients was 66.1% and 33.9%. No significant difference of MTHFR A1298C genotypes (p = 0.26) and alleles (p = 0.21) distribution between diabetic patients and control individuals was found.
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Table 2 Genotypic frequencies of MTHFR polymorphisms among study subjects. Genotype/allele
C677T T/T C/T and C/C T allele C allele A1298C A/A A/C and C/C A allele C allele
Study subjects
p Value
Control subjects (n = 62)
T2DM subjects (n = 56)
36 26 95 29
(58.1%) (41.9%) (76.6%) (23.4%)
30 (53.9%) 26 (46.4%) 85 (75.9%) 27 (24.1%)
0.38
28 34 89 35
(45.2%) (54.8%) (71.8%) (28.2%)
21 (37.5%) 35 (62.5%) 74 (66.1%) 38 (33.9%)
0.26
0.90
0.21
of 1298 A and C allele in T2DM male individuals was 67.5% and 32.5%, respectively; and that in female patients was 65.3% and 34.7%. No significant difference in the distribution of MTHFR A1298C genotypes (p = 0.67) and alleles (p = 0.80) between control and diabetic subjects of different gender was found. Association of MTHFR genotypes and biochemical parameters among diabetic patients The association between diabetic subjects' biochemical parameters, including lipid/glucose metabolic indexes, was statistically analyzed. No significant association between the above biochemical parameters and MTHFR genotypes was found (Table 4). Discussion
MTHFR genotypes and gender In diabetic patients, Hcy increases significantly with age and males have higher Hcy levels than females [18]. Therefore, the association between gender and MTHFR genotypes was subsequently analyzed (Table 3). Among the non-diabetic control individuals, 12 (63.2%) and 7 (36.8%) males carried T/T and C/T, respectively (no C/C genotype was found); while 24 (55.8%) and 19 (44.2%, including 16 C/T [37.2%] and 3 C/C [7.0%]) female carried the corresponding genotype. Among the T2DM patients, 12 (60.0%) and 8 (40.0%) males carried T/T and heterologous C/T genotype, respectively (no C/C genotype was found); and the frequency of T/T and C-containing genotype (including 17 C/T [47.2%] and 1 C/C [2.8%]) in 36 females was half-half respectively. The prevalence of T and C allele in control male individuals was 81.6% and 18.4%, respectively; and that in control female subjects was 74.4% and 25.6%. The prevalence of T and C allele in male T2DM patients was 80.0% and 20.0%, respectively; and that in female patients was 73.6% and 26.4%. No significant difference in the distribution of MTHFR C677T genotypes (p=0.79) and alleles (p=0.72) between control and diabetic patients of different gender was found. In terms of the A1298C polymorphisms, 8 (42.1%) and 11 (57.9%) control males carried MTHFR 1298 A/A and A/C genotype, respectively (no C/C genotype was found); while 20 (46.5%) and 23 (53.5%, including 22 A/C [51.2%] and 1 C/C [2.3%]) female control subjects carried the corresponding genotype. In T2DM patients, 9 (45.0%) and 11 (55.0%) males carried A/A and C-containing genotype (including 9 A/C [45.0%] and 2 C/C [10.0%]), respectively; while 12 (33.3%) and 24 (66.7%, including 23 A/C [63.9%] and 1 C/C [2.8%]) female carried the corresponding genotype. The prevalence of 1298 A and C allele in control male individuals was 71.0% and 29.0%, respectively; and that in control female subjects was 72.1% and 27.9%. The prevalence
Table 3 Genotypic frequencies of MTHFR polymorphisms among study subjects stratified by gender difference. Genotype/allele
C677T T/T C/T and C/C T allele C allele A1298C A/A A/C and C/C A allele C allele
Control subjects
T2DM subjects
p Value
Male (n = 19)
Female (n = 43)
Male (n = 20)
Female (n = 36)
12 (63.2%) 7 (36.8%) 31 (81.6%) 7 (18.4%)
24 (55.8%) 19 (44.2%) 64 (74.4%) 22 (25.6%)
12 (60.0%) 8 (40.0%) 32 (80.0%) 8 (20.0%)
18 (50.0%) 18 (50.0%) 53 (73.6%) 19 (26.4%)
0.79
8 (42.1%) 11 (57.9%) 27 (71.0%) 11 (29.0%)
20 (46.5%) 23 (53.5%) 62 (72.1%) 24 (27.9%)
9 (45.0%) 11 (55.0%) 27 (67.5%) 13 (32.5%)
12 (33.3%) 24 (66.7%) 47 (65.3%) 25 (34.7%)
0.67
0.72
0.80
Gene encoding MTHFR is located on chromosome 1p36.3. Mutations in MTHFR are considered as a model of Hcy metabolic disorders due to its main role in the remethylation process of Hcy metabolism [18,19]. MTHFR C677T and A1298C are 2 common genetic polymorphisms which lead to reduced MTHFR activity [20]. Individuals with insufficient MTHFR activity by carrying either one of these 2 variants have lower 5-methyltetrahydrofolate and higher Hcy levels [21]. HHcy and MTHFR polymorphisms, particularly C677T, are recognized as independent risk factors for cardiovascular diseases [12]. Elevated plasma Hcy levels impair functions of the endothelium which in turn results in an uncontrolled proliferation of smooth muscle cells and consequently accelerate the atherogenic process. The association of HHcy with cardiovascular diseases is particularly evident in T2DM patients [5]. Approximately 35% of diabetic patients demonstrate HHcy with prevalence of 43% and 18% in male and female, respectively [22,23]. In diabetic patients, Hcy increases significantly with age and males have higher Hcy levels than females while diabetic duration does not affect Hcy levels [24]. Insulin resistance is reported to be positively associated with Hcy levels [2], whereas other study concludes that T2DM patients with hyper- or normo-homocysteinemia have similar degree of insulin resistance [4]. Likewise, while some reports reveal that diabetic patients have higher Hcy levels [1,25–27], others find similar [28–30] or even lower [9,34,35]. Taken together, no definitive correlation between plasma Hcy levels and insulin resistance can be concluded by these conflicting results. Other diabetes-related factors such as HbA1c and insulin dependency do not have a significant influence on Hcy levels [4,28,31]. Fiorina et al. reported that potential interaction between Hcy and glucose intolerance is a risk factor for atherosclerosis [32]. In addition to cardiovascular diseases, HHcy is also associated with other diabetic complications, including renal dysfunction [6,7] and retinopathy [33]. Nevertheless, Hcy levels in uncomplicated diabetic patients are usually parallel to those in healthy control subjects [34]. Our results show that the distribution of MTHFR genotypes between healthy and T2DM subjects is not significantly different. In addition, no significant associations between lipid/glucose metabolic indexes with MTHFR genotypes among diabetic patients are observed. These data indicate that the conclusion which MTHFR polymorphisms may play some roles in the pathogenesis and complications in Caucasians T2DM patients is unlikely to be applied in Taiwanese patients. Several possible factors could contribute to the lack of association between the MTHFR genotypes with T2DM and diabetic related indexes in our study. First of all, ethnic variations in MTHFR genotypes may contribute to our observation. A given population may have elements in its genetic reservoir that are protective against certain disease despite of the high prevalence of disease-susceptible alleles. Such a protective effect is found in the Japanese population in which the C677T mutation is associated with lower blood pressure, a protective factor for cerebral vascular disease [35]. Therefore, the possibility of differential protective
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Table 4 Association between MTHFR genetic polymorphisms and biochemical parameters in T2DM patients. C677T genotype
2
BMI (kg/m ) Glucose (mg/dL) TG (mg/dL) TC (mg/mL) HDL (mg/mL) LDL (mg/mL) HbA1c (%) Hcy (μmol/L) SBP (mm Hg) DBP (mm Hg)
A1298C genotype
T/T (n = 30)
C/T and C/C (n = 26)
p Value
A/A (n = 21)
A/C and C/C (n = 35)
p Value
25.4 ± 3.4 207.2 ± 93.9 170.5 ± 159.7 200.3 ± 40.6 50.2 ± 14.5 126.3 ± 37.7 9.1 ± 2.8 10.1 ± 3.3 133.3 ± 14.0 84.5 ± 10.4
25.6 ± 4.2 178.0 ± 64.0 137.5 ± 63.4 193.7 ± 52.2 51.4 ± 12.3 120.7 ± 43.5 8.1 ± 2.7 9.8 ± 3.6 132.2 ± 19.7 79.3 ± 9.6
0.74 0.19 0.33 0.60 0.74 0.60 0.21 0.72 0.80 0.06
25.1 ± 4.2 193.2 ± 99.2 130.9 ± 58.8 175.7 ± 56.4 54.2 ± 13.7 121.3 ± 44.6 8.2 ± 2.1 8.9 ± 2.5 129.4 ± 13.1 81.0 ± 9.2
25.8 ± 4.2 193.9 ± 71.3 169.7 ± 150.1 180.1 ± 52.5 48.0 ± 12.6 119.7 ± 36.4 8.7 ± 2.9 9.7 ± 2.9 133.6 ± 17.2 83.9 ± 11.9
0.52 0.98 0.26 0.77 0.90 0.14 0.54 0.41 0.40 0.40
Numbers in the parentheses indicated the corresponding normal reference range of each biochemical test. Data were presented as mean ± standard deviation.
genetic factors related to a particular ethnicity may lead to the conflicting results among studies. Although many reports show that MTHFR mutation is related to increased Hcy, the association of MTHFR mutation with a variety of vascular disorders is still controversial. Additionally, linkage disequilibrium of the mutant allele with a nearby noncausative polymorphism may underlie the findings. Therefore, the possibility of linkage disequilibrium of the variant alleles with a nearby non-causative polymorphism in our population cannot be ruled out. Secondly, differences in Hcy metabolism among population may exist. For example, Africans metabolize Hcy more effectively than Caucasians [36]. The rarity of gene frequency predisposing to HHcy in Finland may contribute to the lack of association between Hcy and cardiovascular diseases in their population [37]. In summary, our results show that no significant difference in the distribution of MTHFR genotypes between healthy and T2DM subjects is found. Additionally, no significant associations between lipid/glucose metabolic indexes with MTHFR genotypes among diabetic patients are observed. These data indicate the previous observation that MTHFR polymorphisms may play some roles in the pathogenesis and complications of Caucasians T2DM patients are unlikely to be applied in Taiwanese patients. Acknowledgments The work was supported by grants from Chung Shan Medical University Hospital (CSH-96-29), Taiwan, Republic of China. References [1] Doupis J, Eleftheriadou I, Kokkinos A, et al. Acute hyperhomocysteinemia impairs endothelium function in subjects with type 2 diabetes mellitus. Exp Clin Endocrinol Diabetes 2010;118:453–8. [2] Shimomura T, Anan F, Umeno Y, et al. Hyperhomocysteinaemia is a significant risk factor for white matter lesions in Japanese type 2 diabetic patients. Eur J Neurol 2008;15:289–94. [3] Aso Y, Yoshida N, Okumura K, et al. Coagulation and inflammation in overt diabetic nephropathy: association with hyperhomocysteinemia. Clin Chim Acta 2004;348:139–45. [4] Buysschaert M, Dramais AS, Wallemacq PE, et al. Hyperhomocysteinemia in type 2 diabetes: relationship to macroangiopathy, nephropathy, and insulin resistance. Diabetes Care 2000;23:1816–22. [5] Sun J, Xu Y, Zhu Y, et al. Methylenetetrahydrofolate reductase gene polymorphism, homocysteine and risk of macroangiopathy in type 2 diabetes mellitus. J Endocrinol Invest 2006;29:814–20. [6] Mtiraoui N, Ezzidi I, Chaieb M, et al. MTHFR C677T and A1298C gene polymorphisms and hyperhomocysteinemia as risk factors of diabetic nephropathy in type 2 diabetes patients. Diabetes Res Clin Pract 2007;75:99–106. [7] Ukinc K, Ersoz HO, Karahan C, et al. Methyltetrahydrofolate reductase C677T gene mutation and hyperhomocysteinemia as a novel risk factor for diabetic nephropathy. Endocrine 2009;36:255–61. [8] Kalin A, Alatas O, Colak O. Relation of plasma homocysteine levels to atherosclerotic vascular disease and inflammation markers in type 2 diabetic patients. Eur J Endocrinol 2008;158:47–52.
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