Association of Methylenetetrahydrofolate Reductase C677T and Cystathionine β-Synthase Polymorphisms in Cardiovascular Disease in the Algerian Population

Abstract

Aim: Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) and cystathionine β-synthase (CBS) genes, involved in the intracellular metabolism of homcysteine, can result in hyperhomocysteinemia. The objective of this study was to evaluate prevalence estimates of MTHFR C677T and the CBS insertion of 68-bp (844ins68) polymorphisms among individuals with cardiovascular disease (CVD). Methods: In total, 131 patients (61 men and 70 women) were hospitalized in the Cardiology Department in CHU of Sétif, Algeria. The control group included 147 apparently healthy adults (82 women and 65 men). The genetic analysis of the MTHFR C677T polymorphism was performed by real-time polymerase chain reaction on a Light Cycler; the CBS genotype was analyzed by polymerase chain reaction in a thermal cycler. Results: The frequency of the TT genotype was 16.1% in the patient group and 14.3% in the control group. The CT genotype constituted 43.5% and 40.1% in the patient group and the control group, respectively. There was no significant difference in the occurrence of the TT genotype between the studied groups. The frequency of C677T/MTHFR in male and female patients was 16.4% and 15.7% for the TT genotype, respectively. There was no significant difference in T allele frequencies between sexes. However, the frequency of C677T homozygotes in the patients was higher in men with CVD than that in corresponding control subjects (40.2% vs. 29.2%), but the difference was not statistically significant. The coexistence of the MTHFR 677TT genotype and the common CBS 844ins68 variant was lower among patients. Conclusions: The MTHFR C677T and CBS 844ins68 variants tested in this study, individually or combined, are not associated with CVD in the Algerian population.

Introduction

Methylenetetrahydrofolate reductase (MTHFR) and cystathionine β-synthase (CBS) deficiency are two well-defined inborn errors of homocysteine metabolism (Blom, 1998).

A common gene variant of MTHFR (EC 1.7.99.5) is the enzyme that catalyses the conversion of 5,10-methylenetetrahydrofolate to 5-methyltertrahydrofolate, a cosubstrate for homocysteine remethylation to methionine (Yamada et al., 2001; Gellekink et al., 2005; Loscalzo, 2006), which is the most frequent genetic cause of mild hyperhomocysteinemia. The MTHFR gene is located on chromosome 1 at 1p36.3; so far, 14 rare mutations of the MTHFR gene have been associated with severe MTHFR deficiency. The MTHFR 677C-T polymorphism was identified in 1995 (Frosst et al., 1995). The T allele causes an alanine-to-valine amino acid substitution (Ala222Val) within the catalytic domain of the enzyme, which results in the production of a thermolabile enzyme (Frosst et al., 1995) with decreased activity, with TT homozygotes having ∼35%-50% reduction in enzyme activity compared to control values (Weisberg et al., 1998; Kluijtmans et al. 2003; Gellekink et al., 2005).

The prevalence of MTHFR C677T is rather heterogeneous among different ethnic groups and ranges from 2% to 54.5% in various populations (Pepe et al., 1998). The homozygous mutant TT genotype for the MTHFR C677T polymorphism typically affects about 10% of individuals worldwide but can be as high as 26% (southern Italy) and 32% (Mexico) in some areas (Wilcken et al., 2003).

The C677T mutation should be a risk factor for cardiovascular disease (CVD) (Kang et al., 1991, 1993; Gallagher et al., 1996; Izumi et al., 1996; Kluijtmans et al., 1996). Individuals with the homozygous mutant (TT) genotype have a significantly higher (14%-21%) risk of heart disease (McNulty et al., 2008). However, analysis of the odds ratio (OR) among countries shows a large geographical variation in the association between this polymorphism and heart disease risk (Klerk et al., 2002; Lewis et al., 2005). On the other hand, some studies (Adams et al., 1996; DeFranchis et al., 1996; Ma et al., 1996; Schmitz et al., 1996; Wilcken et al., 1996; Akar et al., 1998) did not find this mutation to be associated with an increased risk for coronary artery disease. Further, a meta-analysis pointed at a moderate increase in plasma homocysteine and the risk of CVD in patients with MTHFR mutation (Wald et al., 2002).

On the other hand, 677C>T homozygozity has been associated with an increased risk of neural tube defects (NTD), and with other congenital anomalies (Botto and Yang, 2000) in various studies but not in others. Consequently, our results with Algerian NTD mothers did not show a significant association for any group, suggesting that the thermolabile variant C677T in the MTHFR gene is not a risk factor for a mother to have NTD offspring (Houcher et al., 2009).

Degradation of homocysteine via cystathionine to cysteine in the transsulfuration pathway is initiated by the pyridoxal phosphate-dependent CBS. CBS activity of heterozygotes in the range for CBS deficiency has been reported in cardiovascular patients with moderate hyperhomocysteinaemia (Clarke et al., 1991). These enzyme data have not been confirmed by others. It has been reported that vascular disease patients homozygous for CBS have not an increased risk compared to the control subjects (Mudd et al., 1981; Gallagher et al., 1996; Kluijtmans et al., 1996).

A previous study reported that coexistence of the MTHFR 677TT genotype and the common CBS 844ins68 variant was significantly higher among patients (10.7%) with idiopathic thrombosis than among control subjects (1.2%) (Gaustadnes et al., 2000).

In the present study, we aimed to determine the prevalence of the MTHFR C677T polymorphism as well as the CBS 844ins68 variant in the Algerian population, and evaluated their impact on CVD individuals and their relatives.

Patients and Methods

Study population

This research was carried out as a prospective study on 131 patients (61 men and 70 women) hospitalized in the Cardiology Department in CHU of Sétif, Algeria. The physical examination was performed by a cardiologist and an internist. The patients suffered from coronary heart disease, with clinical evidence of angial or myocardial infarction. All subjects resided in Sétif or the surrounding areas. A group of 147 apparently healthy adults (82 women and 65 men) constituted the control group. Blood samples for the study were obtained from the hospital of Sétif and transferred to Ankara, Turkey. Written consent was obtained from all participants.

Sample collection and DNA extraction

Peripheral blood samples were collected by venipuncture, collected in test tubes that contained EDTA as an anticoagulant, and maintained frozen at −20°C until the extraction of DNA and genotyping. DNA was extracted using the conventional phenol-chloroform method. After hemolysis of blood in hypotonic solution, DNA was isolated by a simple proteinase K treatment at 65°C in the presence of sodium dodecyl sulfate, followed by ammonium acetate precipitation of debris and ethanol precipitation of DNA.

Analysis of polymorphisms

The genetic analysis of the MTHFR C677T polymorphism was performed by real-time polymerase chain reaction (PCR) via a melting curve analysis performed on a Light Cycler (Roche Molecular Biochemicals) in borosilicate capillaries with an MTHFR C677T polymorphism detection kit (Roche Molecular Biochemicals). The MTHFR genotype was detected by an analysis of the melting peaks of the run of the real-time PCR. The presence of just 1 melting peak at 63.0°C indicates a wild-type genotype, 2 melting peaks at 54.5°C and 63.0°C indicate a heterozygous mutant, and 1 melting peak at 54.5°C indicates a homozygous mutant.

The CBS genotype was analyzed by PCR in a thermal cycler (Biometra) using 5 μL of 10 × PCR Buffer, 25 mM MgCl₂, 10 mM of deoxynucleoside triphosphate mix, and 10 pmol of each primer. PCR for CBS ex 8,68-bp insertion was carried out with the primers 5′-CTG GCC TTG AGC CCT GAA-3′ (sense), derived from intron 7, and 5′-GGC CGG GCT CTG GAC T-3′ (antisense), derived from intron 8 and 5 U of Taq polymerase (Fermentas), in a total reaction volume of 50 μL. The PCR conditions were as follows: denaturation at 94°C for 1 min, annealing at 57°C for 1 min, and extension at 72°C for 1 min by 35 cycles, followed by a final extention to 72°C (7 min). A 8 μL aliquot of PCR product was electrophoresed in a 2% agarose gel stained with ethidium bromide and observed under UV light in a transilluminator. Two fragments, 184 and 252 bp, were observed for insertion mutation.

Statistical analysis

Allele frequencies were deduced from genotype distribution. Genotype and allele frequencies between cases and control subjects were compared by a χ²-test. Statistical significance was accepted at p < 0.05. The OR as well as their 95% confidence intervals were computed to assess strength of association, if any, between different genotypes.

Results

A total of 131 patients were included in this prospective study: 70 women and 61 men, aged 20-96 years (56 ± 17 years). Overall, the thermolabile MTHFR mutation (C677T) was seen in 57/131 patients (43.5%), whereas the CBS 844ins68 variant was documented in 14/87 (16.1%). Previously, the reported MTHFR genotype frequency in the healthy Algerian population has shown 14.3% of subjects to be homozygous for the mutated allele (TT), 45.6% homozygous for the wild-type allele (CC), and 40.1% heterozygous (CT) (Bourouba et al., 2009).

The observed frequencies of the various genotypes and alleles of 677 C → T polymorphisms in the MTHFR gene are shown in Tables 1-2. The overall frequency of the T and C allele was 34.4% and 65.6%, respectively. Comparing our patients to this reported distribution, we found that the T allele was not different (OR = 0.86 [0.61-1.21]; χ² = 0.71) (Table 1). Further, these frequencies were not significantly different from those observed in a sample of the control population.

Table 1.

Methylenetetrahydrofolate Reductase C677T Polymorphism Genotype and Allele Frequency and Combination of Variant Methylenetetrahydrofolate Reductase Genotype with Cystathionine β-Synthase 844ins68 Mutation in Patients with Cardiovascular Disease

Genotype/allele	Patients (n = 131)	Control (n = 147)	OR (95% CI)
Genotypes
CC	53 (40.4)	67 (45.6)	1
CT	57 (43.5)	59 (40.1)	0.82 (0.49-1.37)
TT	21 (16.1)	21 (14.3)	0.79 (0.39-1.6)
Alleles
C	163 (62.2)	193 (65.6)	1
T	99 (37.8)	101 (34.4)	0.86 (0.61-1.21)
Combined genotype distribution of MTHFR and CBS844ins68 mutation

	CBS (N)	CBS (INS)
CC	37 (36.6)	3 (2.9)
CT	39 (38.6)	8 (7.9)
TT	11 (10.9)	3 (2.9)

Values in parentheses denote genotype frequencies (columns 2 and 3) or 95% CI (column 4).

CI, confidence interval; OR, odds ratio; N, normal; INS, insertion; MTHFR, methylenetetrahydrofolate reductase; CBS, cystathionine β-synthase.

The frequencies of C677T/MTHFR in female patients were 44.3% for CC, 40.0% for CT, and 15.7% for TT genotypes, whereas those in male patients were 36.0% for CC, 47.6% for CT, and 16.4% for TT genotypes. There was no significant difference in T allele frequencies between sexes (Table 2). The frequency of 677T homozygotes in the patient group was higher in men with CVD than that in corresponding control subjects (40.2% vs. 29.2%), but the difference was not statistically significant (χ² = 3.33) (Table 2).

Table 2.

Genotype Distribution and Allele Frequency of the C677T Polymorphism in the Methylenetetrahydrofolate Reductase Gene by Sex in Patients with Cardiovascular Disease and Control Subjects

	Men			Women
Genotype/allele	Patients (n = 61)	Control subjects (n = 65)	OR (95% CI) ^a	Patients (n = 70)	Control subjects (n = 82)	OR (95% CI)
Genotypes
CC	22 (36.0)	34 (52.3)	1	31 (44.3)	33 (40.2)	1
CT	29 (47.6)	24 (36.9)	0.54 (0.25-1.16)	28 (40.0)	35 (42.7)	1.17 (0.58-2.35)
TT	10 (16.4)	7 (20.8)	0.45 (0.15-1.36)	11 (15.7)	14 (17.1)	1.2 (0.47-3.04)
Alleles
C	73 (59.8)	92 (70.8)	1	90 (64.3)	101 (61.6)	1
T	49 (40.2)	38 (29.2)	0.62 (0.37-1.05)	50 (35.7)	63 (38.4)	1.12 (0.7-1.79)

Values in parentheses denote genotype frequencies (columns 2, 3, 5, and 6) or 95% CI (columns 4 and 7). Differences by sex across MTHFR genotypes were nonsignificant.

CI, confidence interval.

The homozygous CBS 844ins68 mutation was also found in 14/101 (13.8%) patients. Data from analysis of individuals carrying the two risk alleles are presented in Table 1. In the 14 patients, 3 had two risk factors (MTHFR 677TT and CBS 844ins68); 8 had two risk factors (MTHFR 677CT and CBS 844ins68), and 3 patients had a single underlying risk factor, CBS 844ins68 mutation alone. We find no evidence for a significant association between MTHFR and CBS with CVD subjects (Table 1).

Discussion

No previous investigations concerning the prevalence of the MTHFR C677T and CBS 844ins68 variants have been performed in CVD in the Algerian population. Further, little is known about the association of these two variants with CVD. However, one study concluded that hyperhomocysteinemia, which may result from mutations in the MTHFR gene, is independently associated in healthy Algerian population (Abdessemed, 2009).

The homozygous mutant TT genotype for the MTHFR C677T polymorphism typically affects about 10% of individuals worldwide but can be as high as 14% (Algeria) (Bourouba et al., 2009), 26% (south Italy), and 32% (Mexico) in some areas (Wilcken et al., 2003).

Frosst et al. (1995) reported that MTHFR C677T mutation is an important risk factor for vascular disease. Since then, there have been conflicting reports on the relationship between the mutation and CVD. In the current study, there was no significant difference between the patient group and the control group regarding the distribution of the TT genotype. Since Frosst et al. (1995) suggested the C677T mutation as a possible reason for the occurrence of vascular complications, several groups have evaluated this hypothesis in cardiovascular patients from different ethnic groups (Mogk et al., 2000; Esfahani et al., 2003; Qi et al., 2003). However, many studies did not show an association between the MTHFR mutation and subsequent development of CVDs (Brugada and Marian 1997; Folsom et al., 1998; Thogersen et al., 2001; Lewis et al., 2005). Our results are in keeping with the results of these studies.

In a meta-analysis by Brattström et al. (1998), the prevalence of the TT genotype ranged between 5.4% and 16.0% in the different groups of control subjects and between 6.5% and 29.7% in the different groups of patients with CVD. In our study, the TT genotype was present in 16.1% of the patients and in 14.3% of the control subjects. Consequently, the allele frequency was almost identical in patients and control subjects (37.8% vs. 34.4%). Nonetheless, the TT genotype was found less frequently in patients than in control subjects in men (16.4% vs. 20.8%) and women (15.7% vs. 17.1%). The prevalence between the two study groups was not statistically significant. This indicates that the presence of this MTHFR C677T variant does not predispose individuals to CVD, at least in our population. Some studies have reached a similar conclusion (Brattström et al., 1998; El-Sammak et al., 2004; Zee et al., 2007). However, other studies, conducted on individuals from different ethnic groups, have concluded that there is a strong association between the presence of this MTHFR variant and the occurrence of CVD (Kang et al., 1988; Wald et al., 2002; Cronin et al., 2005).

Whether the MTHFR polymorphism is an independent contributor to cardiovascular risk has been debated recently. Many studies of the general population showed that the C677T mutation is a major cause of mild hyperhomocysteinaemia, but not a factor affecting CVD risk (Brattström et al., 1998; Folsom, 1998; Abu-Amero et al., 2003; Guéant-Rodriguez et al., 2005). Then, it is not surprising that the MTHFR C677T polymorphism increases homocysteine levels, but is not by itself a cardiovascular risk factor (Aucella et al., 2005; Rassoul et al., 2008). Folate and B-vitamins are important cofactors in the metabolism of homocysteine. It has been suggested that the MTHFR T allele carries higher CVD risk in the setting of low dietary intake of folate or B-vitamins (Klerk et al., 2002). Belkahla et al. (2008) found a relationship between MTHFR TT and coronary stenosis risk, especially with low folatemia.

Previously, in our population, dietary folate intake was low compared with reported values for adult populations in developed countries (Houcher et al., 2003). Our results highlight the relevance of the vitamin status and particularly of folate levels in the modulation of fasting total homocysteine levels in the patients with CVD (Houcher et al., 2007). Potential interactions of gene-diet and gene-homocysteine concentration in relation to incident CVD were examined, because dietary factors have been postulated to explain the differing associations of MTHFR 677C>T genotype status with CVD by geographical regions (Lewis et al., 2005).

Franco et al. (1998) described the prevalence of the heterozygote genotype for CBS 844ins68 in the European (13.5%), African (37.7%), and Asian (0%) populations. Gaustadnes et al. (2000) related that coexistence of the MTHFR 677TT genotype and the common CBS 844ins68 variant was significantly higher among patients (10.7%) than in control subjects (1.2%). When we analyzed our results, this coexistence was lower among patients (2.9%), indicating that this genotype combination is not a cardiovascular risk factor. However, MTHFR and CBS 844ins68 are important risk factors along with protein C deficiency (Pathare et al., 2006). Consequently, it was suggested that specific CBS alleles are a risk factor for the development of vascular disease and that genetic information could be predictive of individual response to folic acid supplementation (Kruger et al., 2000).

In conclusion, neither the C677T variant nor the CBS 844ins68 variant, either individually or combined, can be used as an independent genetic risk factor for CVD in the Algerian population. The apparent discrepancies may be possibly explained by differences in the sample populations such as ethnic origin and dietary intake in conjunction with other genetic predisposition factors.

Footnotes

Acknowledgments

This study was supported by Ankara University (Turkey). We extend our special thanks to the personnel of cardiovascular sections in Sétif University Hospital (Algeria) and the patients who participated in this study.

Disclosure Statement

No competing financial interests exist.

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