Sequence Variation of the Methylene Tetrahydrofolate Reductase Gene (677C>T and 1298 A>C) and Traditional Risk Factors in a South Indian Population

Abstract

Methylene tetrahydrofolate reductase (MTHFR) plays a significant role in the metabolism of methionine. MTHFR deficiency is an autosomal recessive trait that could be a significant risk factor for a number of defects, for example, vascular events, due to lower dietary folate intake among South Indians. To find the incidence of 677 C>T and 1298 A>C in MTHFR gene single nucleotide polymorphisms (SNPs) among the south Indian population, polymerase chain reaction and restriction fragment length polymorphism were employed among 152 patients with myocardial infarction and 167 controls. The MTHFR 677CT genotype was found among 35 (22.4%) cases and 08 (4.8%) controls, the MTHFR 677CC allele was found among 115 (73.7%) cases and 159 (94.6%) controls. Also, the analysis of the MTHFR 1298A>C SNP identified the MTHFR 1298CC genotype among 16 (10.3%) cases and 01 (0.6%) control, the MTHFR 1298AC genotype was found in 56 (35.9%) cases and 27 (16.2%) controls, and the MTHFR 1298AA genotype was observed in 80 (51.3%) cases and 139 (82.6%) controls. The C vs. A allele also showed significantly higher frequency among the patients in comparison with the controls (p<0.0001). The results of this study indicate that the MTHFR A1298C SNP is more prevalent among south Indians compared with the MTHFR C677T SNP, suggesting a possible role of MTHFR A1298C in the pathogenesis of heart diseases.

Introduction

The methylene tetrahydrofolate reductase (MTHFR) gene, comprised of 11 exons, has been localized on chromosome 1p36.3. Eighteen mutations have been reported so far in the MTHFR gene, the most common being C677T and A1298C mis-sense mutations, which have been reported to induce a milder form of MTHFR deficiency. The C677T transition in exon 4 was extensively studied in the Western countries; it decreases enzyme activity because of thermolability leading to dissociation of dimer into monomers and loss of flavin adenine dinucleotide (FAD)-binding capacity (Frosst et al., 1995). The other frequent MTHFR mutation in exon 7, that is, A1298C trans version, is not associated with thermolability of the enzyme nor with impact on plasma total homocysteine and it has not yet been studied in detail. The compound heterozygosity for 677CT/1298AC will have a similar clinical impact as of C677T homozygosity. An individual with a 677TT genotype is always reported to have 1298AA genotype and vice versa (Yamanda et al., 2001).

MTHFR is a flavoprotein that catalyzes the reduction of 5,10-methylenetetrahydrofolate (5,10-CH₂-H₄ folate) to 5,methyltetrahydrofolate (5,CH₃-H₄ folate), the major circulatory form of folate and the methyl donor for homocysteine remethylation, thereby regulating fasting plasma homocysteine levels. Hyperhomocysteinemia is an established risk factor for coronary artery disease and is associated with increased mortality in patients with confirmed coronary artery disease. A common C to T transition at nucleotide 677 of the MTHFR gene coding sequence leads to the substitution of ala-222 by valine, and an A to C transition at nucleotide 1298 (A1298C) results in the replacement of Glu-429 by alanine; these are the most frequent genetic causes for mild hyperhomocysteinemia (Frosst et al., 1995; Van der Put et al., 1998). Controversial studies to date have investigated the allelic frequency of both A1298C and C677T single nucleotide polymorphisms (SNPs). MTHFR polymorphism among Asians was studied only among Japanese and Sri Lankans. Limited data are available for other Asian populations, especially among Indians (Nair et al., 2002). No data exist on the south Indian population, which prompted this study of SNPs in MTHFR and their impact on homocysteine.

Hence, with this background, the present study was designed to study the MTHFR (A1298C and C677T) genotypes among the south Indian population and to compare the frequencies of the genotypes between patients with myocardial infarction (MI) and normal individuals.

Materials and Methods

Study group

The present study was carried out among 156 patients with MI (male: female=124: 32) with an age range of 23-74 years who were admitted to the cardiac intensive care unit, Apollo Health City, Hyderabad, Andhra Pradesh, India, during the period from April 2006 to June 2010. All the patients admitted with acute coronary syndrome underwent a coronary angiogram. On the basis of electro cardiogram changes, elevated cardiac markers, and clinical history, they were confirmed as MI cases by the cardiologists. All these 156 patients were without previous history of coronary artery disease. On the other hand, 167 healthy age- and sex-matched controls (Male: Female=132: 35) were included in the study for a comparative analysis. All the healthy controls were collected from the blood bank of the same hospital, and some samples were from the public who were friends, who did have any history of MI among them and in their family (which was confirmed by the laboratory basic analysis). Clinical data and family history of each patient were documented using a pretested form designed for the purpose. All the objectives of the study were explained clearly to the subjects; a consent of signature in a prescribed format before them participating in this study was sought before their inclusion and exclusion criteria. The demographic data included (i) age at the time of a first event, (ii) family history of cardiac events, (iii) social habits such as smoking, alcohol consumption, (iv) diet, (v) parameters such as diabetic status, blood pressure, lipid profile, and (vi) three-generation pedigree.

DNA isolation

Genomic DNA was prepared from peripheral lymphocytes by salt precipitation (Miller et al., 1988).

677 C>T polymorphism

The method described by Frosst et al. (1995) was used for detection of 677 C>T polymorphism. A length of 198 base pairs on exon 4 of MTHFR gene was amplified in the thermal cycler and a three-step polymerase chain reaction (PCR) with an initial denaturation at 94°C for 2 min followed by cycling at 94°C for 30 s, annealing temperature at 57°C for 45 s at the appropriate temperature, 72°C for 1 min for about 35 cycles, and a final extension at 72°C for 5 min was carried out. The amplified fragment was digested with restriction enzyme HinfI according to the manufacturer's instructions (Bangalore Genei). The digested products were sized by electrophoresis on a ten-percent nondenaturing polyacrylamide gel, and genotype was determined by examination using 0.2% ethidium bromide. Wild-type (677CC) shows a single fragment of 198 bp. The presence of the ‘T’ allele introduces a cut among heterozygous (677 CT), and three fragments at 198, 175, and 23 bp were seen. The homozygous (677 TT) have two fragments of 175 bp and 23bp (Frosst et al., 1995).

1298 A>C mutation was analyzed by the method described by Radha Ramadevi et al. (2004). PCR amplification results in a fragment of 163 bp on exon 7 of MTHFR gene using the thermal cycler and a three-step PCR with an initial denaturation at 94°C for 2 min followed by cycling at 94°C for 30 s, annealing temperature of 60°C for 45 s at the appropriate temperature, 72°C for 1 min for about 35 cycles, and a final extension of 72°C for 5 min was carried out. The amplified fragment was digested with restriction enzyme MboII according to the manufacturer's instructions (Fermentas Life Sciences). The digested products were sized on a twenty-percent non-denaturing polyacrylamide gel. The gel was stained with 0.2% ethidium bromide and viewed under a UV transilluminator. Wild-type (1298 AA) produced fragments of 56, 30/31, 28, and 18 bp. Heterozygous (1298 AC) produced fragments of 84, 56, 30/31, 28, and 18 bp, whereas homozygous for 1298 polymorphism (1298 CC) produced fragments of 84, 30/31, and 18 bp (Radha Ramadevi et al., 2004).

Statistical analysis

The demographic characteristics, biochemical, and gene polymorphisms were analyzed using different statistical tools such as student's t-test, chi square (χ²), and odds ratio (OR). Statistical computations were calculated using SPSS 10.0 windows software. Hardy-Weinberg law of equilibrium was in good agreement tested for genotypic and allelic frequencies. The association of the sequence variations in patients and controls was examined using OR and χ² tests with confidence interval (CI) 95%. p<0.05 was considered significant.

Results

Epidemiology (Table 1)

The mean±SD age of patients and controls were 47.94±13.76 and 47.46±13.57, respectively. The sex ratio of males and females in patients was 124:32 and 132:35 among controls. The mean body mass index in patients was higher (27.17±4.3) than the controls (24.2±3.36). The percentage of individuals with diabetes in patients was 95 (61%), and the controls were selected without any such history, as they are the risk factors for MI. Higher frequency of smokers was observed among the patients: 106 (68%) than the controls: 38 (22.8%). The frequency level of alcoholics in patients was 76 (48.7%) and in controls it was 19 (11.3%), which is also higher among the patients than the controls. The percentage of individuals with family history of MI in patients was 67 (42.9%), and the controls were selected without any such history, as they are the risk factors for MI.

Table 1.

Demographic Profile of the Patient and the Control Groups

Variables	Cases (n=156) mean±SD	Controls (n=167) mean±SD	p value^a
Age (years)	47.94±13.7	47.4±13.5	0.74
BMI (Kg/m²)^b	27.17±4.3	24.2±3.3	<0.0001
SBP (mmHg)^c	126.6±21.8	109.2±17.9	<0.0001
TSC (mg/dl)^d	203.4±92.5	171.4±16.4	<0.0001
LDL (mg/dl)^e	164.5±42.1	88.9±9.5	<0.0001
HDL (mg/dl)^e	29.0±2.9	42.9±4.2	<0.0001
TGL (mg/dl)^e	171.3±25.2	133.4±5.9	<0.0001
Smoking status (%)	106 (68%)	38 (28%)	<0.0001
Alcoholic consumption (%)	76 (46.7%)	19 (11.3%)	<0.0001
Diabetes mellitus (%)	95 (61%)	Nil
Family history of MI (%)	67(42.9%)	Nil

Student's t-test (for continuous variables) and the chi-square test (for discrete variables) were used to compare the values for patient and control groups.

Body mass Index (BMI) was defined as per the Guidelines of National Institute of Health.

Hypertension was defined as a systolic blood pressure mm Hg at the time of admission to the hospital.

Hypercholesterolemia was defined as a total serum cholesterol level mg per deciliter at the time of admission to the hospital.

LDL, HDL, and TGL were defined as mg per deciliter at the time of admission to the hospital.

Plus-minus values are means±SD; for the case and controls, the value refers to the age at onset of myocardial infarction (MI) or unstable angina.

SBP, systolic blood pressure; TSC, total serum cholesterol; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TGL, triglycerides.

Biochemical markers (Table 1)

The Mean±SD of total cholesterol in patients was higher (203.4±92.5) than the controls (171.45±16.4), and the difference between them was highly significant (p<0.0001). The Mean±SD of triglycerides (TGL) were significantly (p<0.0001) elevated in patients (171.4±25.3) than in the controls (133.4±5.95). The lower Mean±SD of high-density lipoprotein (HDL) levels was observed in the patients (29.0±2.9) than in the controls (42.9±4.2) and differed significantly between them (p<0.0001). The Mean±SD of low-density lipoprotein (LDL) in patients was 164.5±42.1 and in controls it was 88.9±9.5. The LDL was significantly higher in patients when compared with controls (p<0.0001).

Sequence variations of the MTHFR

Among 152 MI cases and 167 controls, we found that the MTHFR 677TT genotype was absent only in the control group. The MTHFR 677CT genotype among cases was 35/152 (22.4%) and 08/167 (4.8%) in controls, and the MTHFR 677CC allele was observed among 115/152 (73.7%) cases and 159/167 (95%) controls (Table 2). Heterozygosity for the MTHFR 677CT allele was higher in patients than the controls, indicating that the individuals who are heterozygous for this polymorphism may be at an increased risk for developing an MI.

Table 2.

Distribution of MTHFR (C677T) Genotypes and Allelic Frequencies of the Total Study Group

	MTHFR (C677T) genotypes				Allelic frequencies
Study group	CC	CT	TT	Total	C	T	Total
Controls n (%)	159 (94.6)	08 (4.8)	00 (0)	167	326 (0.976)	08 (0.024)	334
Patients n (%)	115 (76)	35 (23)	02 (1)	152	265 (0.872)	39 (0.128)	304

The total number of patients and controls for individual risk factors may not equal in cases of 156 patients (only 152 case patients were genotyped) and in controls of 167, respectively, as data were missing for some subjects. For the case patients, age refers to the age at onset of MI or unstable angina and not the current age.

Analysis was also done for the MTHFR1298A>C SNP. The results identified MTHFR 1298CC genotype in 16/152 (11%) cases and 01/167 (0.6%) controls. The MTHFR 1298AC genotype was 56/152 (37%) in cases and 27/167 (16.2%) in controls, and the MTHFR 1298AA genotype was observed in 80/152 (51.3%) cases and 139/167 (83%) controls (Table 3).

Table 3.

Distribution of MTHFR (A1298C) Genotypes and Allelic Frequencies of the Total Study Group

	MTHFR (A1298C) genotypes				Allelic frequencies
Study group	AA	AC	CC	Total	A	C	Total
Controls n (%)	139 (82.6)	27 (16.2)	01 (0.6)	167	305 (0.913)	29 (0.087)	334
Patients n (%)	80 (53)	56 (36)	16 (11)	152	216 (0.711)	88 (0.289)	304

The total number of patients and controls for individual risk factors may not equal in cases of 156 patients (only 152 case patients were genotyped) and in controls of 167, respectively, data were missing for some subjects. For the case patients, age refers to the age at onset of MI or unstable angina and not the current age.

The occurrence of the MTHFR 1298CC allele was higher in cases than the control group, suggesting that the homozygous genotype might have a role toward MI. The CC vs. AA genotype showed statistical significance between the patients and the controls (χ²=21.59, OR; 27.8, p<0.00001). The CC vs. AA+AC genotype showed statistical significance in patients compared with controls (χ²=15.5, OR; 19.53, p<0.001). The C vs. A allele also showed significantly higher frequency in patients in comparison with controls (χ²=43.57, OR; 4.2, p<0.00001) (Table 4).

Table 4.

Comparison of Genotypic and Allelic Frequencies of MTHFR 1298 Gene in Patients with MI and Controls

			CI 95%
Genotypes and alleles (patients vs. controls)	Chi-square (χ²)	Odds ratio	L.limit	U.limit	p value
CC vs. AA	21.59	27.8	3.61	213.5	<0.00001
CC vs. AC	4.92	7.7	0.97	61.2	0.02
CC vs. AA + AC	15.5	19.53	2.55	149.1	0.0002
C vs. A	43.57	4.2	2.72	6.74	<0.00001

CI, confidence interval.

The overall frequencies for ‘C’ and ‘T’ alleles for C677T were 0.976 and 0.024 in controls, whereas it was 0.872 and 0.128 in cases. The overall frequencies for ‘A’ and ‘C’ alleles of A1298C SNP was 0.913 and 0.087 in controls, and 0.711 and 0.289 in cases (Tables 2 and 3). The ‘T’ allele frequency was higher in a Mexican population, and the frequency of the 1298C allele was 0.33 in an Italian study.

Discussion

MTHFR catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate and is responsible for the remethylation of homocysteine to methionine. Mutation in the MTHFR gene is associated with an increased plasma homocysteine, an established risk factor for cardiovascular disease (CVD) (Genest and Malinow, 1992; Frosst et al., 1995). This meta-analysis demonstrates a significant risk for the TT genotype relative to the wild type (only three studies demonstrated significance) (Kang et al., 1991; Morita et al., 1997; Ou et al., 1998). No individual or combined studies showed a correlation of the TT genotype with stroke.

In an earlier study, it has been indicated that an individual with a 677TT genotype always has 1298AA genotype and vice versa, thus concluding that these two alleles are always in trans-configuration (Van der Put et al., 1998). Most of our data are in accordance with this genotypic structure, except among two cases with 677CT/298CC genotype, which has also been reported by Hanson et al. (Hanson et al., 2001; Rosenberg et al., 2002). The exceptional genotype of 677CT/1298CC could be rare and presumably due to crossing over. The C677T mutation in MTHFR gene was found to cause a mild-to-moderate homocysteinemia. This is due to a dissociation of the active dimer into monomers and loss of FAD-binding capacity of the enzyme, thus leading to hypomethylation. Gender difference was observed in homocysteine in normal and heterozygous states. Women were found to have low homocysteine than men.

This possible difference may be due to the protective effect of estrogen and other physiological factors among women (Dimitrova et al., 2002). Elevation is more pronounced in the homozygous TT genotype, but this may reflect the lack of TT homozygous male cases. Gender differences in plasma homocysteine in homozygous mutants cannot be commented on. For adequate functioning of the MTHFR enzyme among mutants, both in heterozygous and homozygous conditions, folate supplementation is required. Low folate intake and defective folate absorption will result in hyperhomocysteinemia, which will be more pronounced in mutants for C677T. TT homozygous or compound heterozygous women with low circulating folate have increased risk of birth defects, resulting in increased mortality and morbidity. Multi-disease associations, including cancer, have also been reported in individuals with negative folate status and a mutation in the MTHFR gene.

Finally, it can concluded that hyperhomocysteinemia is a multifactorial character, involving genetic and environmental factors. Apart from mutations in the genes involved in the metabolic pathway, nutritional deficiencies, namely, folic acid, B6, and B12, also are causative agents for hyperhomocysteinemia. Hence, one can reduce homocysteine levels to some extent and decrease the risk for associated disorders by correcting these deficiencies by supplementation. These results need to be confirmed in larger studies. The results of our study suggest that the individuals who are heterozygous for the 677CT SNP may be at an increased risk for developing MI; and since the distribution of MTHFR 1298CC is higher in patients than in the controls, south Indians with MTHFR homozygous 1298CC genotype may be at an increased risk for MI.

Footnotes

Acknowledgments

This study was supported by the Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Hyderabad, and Apollo Hospitals Educational and Research Foundation, Apollo Health City, Hyderabad. The authors thank all the individuals who participated in the study and the cardiac intensive care unit team of Apollo Health City for their excellent support.

Disclosure Statement

No competing financial interests exist.

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