Methylenetetrahydrofolate reductase gene polymorphism and its association with coronary artery disease

ABSTRACT CONTEXT AND OBJECTIVE: Obstructive coronary artery disease (CAD) is characterized by the deposition of atherosclerotic plaque on the coronary artery wall. Its manifestations depend on interactions between environmental and genetic risk factors. The aim of this work was to analyze the frequency of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in patients with CAD and its association with plasma homocysteine levels. Risk factors for CAD were also evaluated. DESIGN AND SETTING: Retrospective with blind quantitative analysis, at Hospital de Base, Faculdade de Medicina de São José do Rio Preto. METHODS: One hundred and twenty-seven individuals were studied. All completed a questionnaire to analyze risk factors for CAD. MTHFR polymorphism was investigated by restriction fragment length analysis and correlated with the number of affected arteries and degree of arterial obstruction determined by coronary cineangiography, and with plasma homocysteine levels measured by liquid chromatography/sequential mass spectrometry. RESULTS: Smoking (p = 0.02) and high-density lipoprotein cholesterol (p = 0.01) were associated with CAD. The C allele was the most prevalent in patients (0.61) and controls (0.66). There was no correlation between MTHFR/C677T polymorphism and plasma homocysteine levels. However, in patients with the TT genotype there was a correlation with the prevalence of coronary obstruction greater than 95% (p = 0.02) and the presence of two affected arteries (p = 0.04). CONCLUSIONS: The TT genotype is associated with coronary artery obstruction greater than 95% and the presence of two affected arteries. This confirms the relationship between genetic variants in specific patient subgroups and cardiovascular diseases.


INTRODUCTION
Obstructive coronary artery disease (CAD) is characterized by the deposition of atherosclerotic plaque on the coronary artery wall.This chronic disease is frequently asymptomatic, but acute events can occur as a result of instability of the atherosclerotic plaque, with consequent arterial thrombosis that results in myocardial infarction. 1 Its manifestation depends on the interactions between environmental and genetic risk factors. 2 Individual susceptibility to this disease has been associated with functional allelic variations.][4] The gene that codes for the methylenetetrahydrofolate reductase enzyme (MTHFR), which is involved in metabolizing homocysteine (Hcy), is of great interest in clinical practice. 2,5,68][9] The serum concentration of Hcy is high in 40% of patients suffering from coronary cerebral or peripheral artery diseases, while in control groups only 15% of individuals present with elevated levels. 9he mechanism for the vascular lesions induced by hyperhomocysteinemia remains unclear.Experimental evidence suggests that Hcy may be involved in atherogenesis and thrombogenesis, leading to hyperplasia of cell tissue and fi brosis.Additionally, it facilitates the vascular oxidative process, alters the co agulation system and reduces the vasomotor regulation of the endothelium. 6,7,10,11moderate increase in Hcy is more generally associated with the cytosine-to-thymine substitution at nucleotide 677 (C677T) of the MTHFR gene, thereby leading to an alanine-for-valine substitution.This alteration is associated with increased enzyme thermolability, causing a 50% reduction in its normal activity. 7,11

OBJECTIVE
This study had the aim of analyzing the frequency of C677T polymorphism in the MTHFR gene in patients with obstructive coronary artery lesions, in comparison with individuals without angiographic evidence of the disease.In addition, it evaluated the association between this polymorphism and the number of affected arteries and degree of arterial obstruction and plasma Hcy levels.Risk factors for CAD were also evaluated.

METHODS
For this retrospective study, after obtaining informed consent, we recruited 127 Caucasian individuals (83 men and 44 women; mean age = 60 ± 12 years).Although in Brazil there is widespread miscegenation, we considered as Caucasians those individuals who had no other ethnic group in the three generations preceding them. 4All of them underwent coronary cineangiography because of clinical indications relating to angina or positive induced ischemia tests.Cineangiography identifi ed 91 individuals with CAD (65 men and 26 women; mean age 60.3 ± 12 years) and 36 control subjects, without signs of the disease (22 men and 14 women; mean age 60.4 ± 12 years).The disease was defi ned and identifi ed in patients according to current criteria 1 .The coronary cineangiography results were analyzed by two independent observers in a quantitative blind analysis in the Hemodynamics and CONTEXT AND OBJECTIVE: Obstructive coronary artery disease (CAD) is characterized by the deposition of atherosclerotic plaque on the coronary artery wall.Its manifestations depend on interactions between environmental and genetic risk factors.The aim of this work was to analyze the frequency of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism in patients with CAD and its association with plasma homocysteine levels.Risk factors for CAD were also evaluated.

DESIGN AND SETTING:
Retrospective with blind quantitative analysis, at Hospital de Base, Faculdade de Medicina de São José do Rio Preto.

METHODS:
One hundred and twenty-seven individuals were studied.All completed a questionnaire to analyze risk factors for CAD.MTHFR polymorphism was investigated by restriction fragment length analysis and correlated with the number of affected arteries and degree of arterial obstruction determined by coronary cineangiography, and with plasma homocysteine levels measured by liquid chromatography/sequential mass spectrometry.

RESULTS:
Smoking (p = 0.02) and high-density lipoprotein cholesterol (p = 0.01) were associated with CAD.The C allele was the most prevalent in patients (0.61) and controls (0.66).There was no correlation between MTHFR/C677T polymorphism and plasma homocysteine levels.However, in patients with the TT genotype there was a correlation with the prevalence of coronary obstruction greater than 95% (p = 0.02) and the presence of two affected arteries (p = 0.04).

CONCLUSIONS:
The TT genotype is associated with coronary artery obstruction greater than 95% and the presence of two affected arteries.This confi rms the relationship between genetic variants in specifi c patient subgroups and cardiovascular diseases.Interventional Cardiology Section of Faculdade de Medicina de São José do Rio Preto (Famerp), São José do Rio Preto, São Paulo, Brazil.Individuals who had undergone heart surgery and those with coronary prosthesis were excluded from the study.
All participants completed a questionnaire to analyze risk factors such as diabetes (patients who were using antidiabetic medication or presented with blood sugar levels greater than 126 mg/dl), hypertension (patients who were using specifi c medications or had blood pressures higher than 140/90 mmHg), sedentary patients (patients who did not do regular, controlled physical exercise), alcoho lism (patients who were consuming alcohol at a defi ned frequency, without ta king into account the quantity) and smoking (minimum of fi ve cigarettes per day).Blood samples were obtained after 12 hours of fasting, to analyze the lipid profi le.The analysis included the serum levels of total cholesterol (TC) and the fractions of lowdensity lipoprotein cholesterol (LDLc), highdensity lipoprotein cholesterol (HDLc), very low-density lipoprotein cholesterol (VLDLc) and triglycerides (TG), measured using the enzymatic colorimetric assay method and Friedewald's formula to calculate LDLc.Measurement of Hcy was performed by liquid chromatography/sequential mass spectrometry.Genomic DNA was obtained form leukocytes, 12 followed by its amplifi cation by means of the polymerase chain reaction (PCR) using the primers described by Bova et al. 9 and restriction enzyme digestion with HinfI.The digested product was submitted to electrophoresis on 9.6% polyacrylamide gel and the digestion products were viewed by staining with silver nitrate.
The data obtained were statistically assessed using the chi-squared, Fisher's exact, Kruskal-Wallis or Mann-Whitney tests, depending on the type of variable analyzed, by means of the Graphpad computer software, and using logistic regression.An alpha error of 5% was considered acceptable, and therefore p-values less than or equal to 0.05 were considered signifi cant. 13n accordance with the guidelines on human research from the Brazilian Health Ministry, approval was obtained for this study from the Research Ethics Committee of Famerp, São José do Rio Preto, State of São Paulo, and from the National Research Commission in Brasilia, Federal District.HDLc (p = 0.01).No associations were seen between CAD and diabetes (p = 0.1359), hypertension (p = 0.1065) or C677T polymorphism (p = 0.56), in the individuals studied.

Table 1 shows characteristics of patients
The mean values relating to the lipoprotein profi le showed slightly higher levels of TC and LDLc in patients (193 ± 61 mg/dl and 124 ± 50 mg/dl respectively) than in controls (187 ± 39 mg/dl and 119 ± 31 mg/dl).Even so, both groups presented with levels within the reference ranges.On the other hand, the lower mean values for HDLc were similar in the two groups (38 ± 11 mg/dl and 38 ± 7 mg/dl, respectively).
Analysis of the allelic and genotypic frequencies of C677T polymorphism (Table 2) demonstrated that the most prevalent allele type and controls and their lipid profi le.Prior diseases identifi ed in the CAD group (91 patients) included high blood pressure in 81% of the individuals and diabetes in 25%.In the control subjects the rates were 60% and 8%, respectively.Seventy-one percent of the patients reported they were smokers; hence there was no signifi cant difference in comparison with the controls (55%; p = 0.13).Serum levels for the lipid profi le remained within the reference range in both groups except for HDLc, for which levels of less than 40 mg/dl were signifi cantly more frequent in the patients (53%) than in the control subjects (30%, p = 0.03).
Logistic regression indicated associations between CAD and smoking (p = 0.02) and Sao Paulo Med J. 2007;125(1):4-8.was the C allele, in both patients (0.61) and controls (0.66).The most frequent genotype was the CT genotype, in both groups (65% and 55%, respectively), followed by the CC genotype (28% and 39%, respectively).There was no signifi cant difference between the groups with regard to the allelic and genotypic frequencies.
A degree of arterial obstruction greater than 95% and presence of two damaged arte ries seemed to be associated with the TT genotype, in comparison with the other genotypes, u sing Fisher's exact test (p = 0.029, Table 3; and p = 0.04, Table 4).Twenty-four patients (26%) presented obstruction in one artery, 38 (42%) in two arteries and 29 (32%) in three arteries.The artery affected was the anterior descen ding artery in 84 cases (92%), followed by the right coronary artery in 71 cases (78%) and the circumfl ex artery in 50 cases (55%).
Analysis of mean Hcy concentrations demonstrated higher levels in patients (20.53 µmol/l) than in controls (16.29 µmol/l) but the difference was not statistically signifi cant (p = 0.25); in relation to C677T polymorphism the mean Hcy levels were higher in patients with the CT genotype (23.95 µmol/l) and TT genotype (15.63 µmol/l) than in the controls (16.39 µmol/l and 3.15 µmol/l, respectively) but again without statistical signifi cance (Table 5).In addition, no associations were observed between the presence of the T allele and the number of patients with raised Hcy levels in either the patient or control group.Table 4  similar prevalences for the T allele in patients (0.39) and controls (0.34), this fi nding corroborates with the literature. 23The TT genotype, with a prevalence of 7% in patients, was associated with a degree of obstruction greater than 95%, which is in agreement with a study on CAD patients by Morita et al., 20 in 1997.There has also been reference to associations between the T allele and peripheral vascular disease, but with decreased frequencies (0.31 in patients and 0.27 in controls). 16Nevertheless, patients with atherosclerosis of the carotid artery have presented with signifi cantly higher frequencies of the T allele (0.47) than in control subjects (0.27; p < 0.02). 9tudies have shown that there is a relationship between the C677T genetic variant, 21,23 Hcy level and myocardial infarction.Higher plasma Hcy concentrations have particularly been detected in patients with CAD and the TT genotype. 23ome studies have been conducted on Brazilian patients at risk of vascular thrombosis.High Hcy levels were detected in patients with the MTHFR 677TT genotype; however, this was not associated with increased risk of the disease. 4,24,25oncentrations greater than or equal to 15.6 µgmol/l have been associated with greater risk of myocardial infarction than in individuals with levels less than 10.0 µgmol/l (odds ratio = 2.3; 95% confi dence interval range from 0.94 to 5.64). 23tudies have also revealed a relationship between the TT genotype and decreased Hcy levels in patients with daily folate supplementation of approximately 400 µg together with vitamins B 12 and B 6. 8 Schwartz et al. 23 investigated the relationships between the risk of myocardial infarction and serum folate levels, vitamin B 12 , Hcy and C677T polymorphism.In that study, women with folate levels greater than or equal to 8.39 nmol/l presented with decreased prevalence of myocardial infarction in comparison with women whose folate levels were less than 5.27 nmol/l (odds ratio = 0.54; 95% confi dence interval range from 0.23 to 1.28).However, there were no signifi cant differences with regard to vitamin B 12 levels (odds ratio = 0.90, 95% confi dence interval range from 0.31 to 2.29).Thus, even though folic acid, vitamin B 6 and vitamin B 12 supplementation can reduce Hcy levels, 26 such intake is neglected by the general population and by healthcare professionals.
Although the present study did not demonstrate any associations between Hcy, CAD and C677T polymorphism, the higher mean Hcy levels in patients with the T allele suggest

DISCUSSION
In this study, MTHFR/C677T polymorphism did not differentiate between patients and controls.Moreover, CAD was mainly observed in patients with decreased HDLc levels and those who were smokers (logistic regression), which is in agreement with other studies. 14,15On the other hand, the similar levels of TC, LDLc, VLDLc and TG in the two groups found in the present study are discordant with other published studies. 16,17he other risk factors and clinical signs, including high blood pressure, lack of exercise, ventricular involvement and alcoholism were also similar between the groups.It is possible that the selection of controls (with negative angiography for coronary artery obstruction) in the cardiology outpatient clinic may have contributed towards the very high prevalences of hypertension (60%) and angina (86%). 18677T polymorphism of the MTHFR gene has been designated a strong candidate for increased risk of vascular disease because of its infl uence on Hcy levels, which are an independent risk factor for atherosclerosis, 2,7,[9][10][11]16,19 such that the T allele is signifi cantly more frequent in patients with CAD (0.42) than in controls (0.33; p = 0.0001). 20 Hoever, individual and meta-analytical studies on approximately 6000 patients have not confi rmed this hypothesis.7,21,22 Moreover, although the present study showed REFERENCES that signifi cant results might be found in larger cohorts.Thus, further studies are needed, using larger series to analyze the real risk exerted by the presence of C677T polymorphism and its relationship with risk factors for CAD.
One diffi culty in this study was the ethnic characterization.A great number of individuals were excluded because they were unable to unequivocally affi rm their ethnic origin.Another diffi culty was the cost relating to Hcy measurements, which was resolved through collaboration with the Thonson Laboratory of the Chemistry Department of the University of Campinas.
Because of the importance of studies on different populations and with greater sample sizes, the authors intend to continue investigating the relationship between CAD and the MTHFR gene, as well as other genes related to folate metabolism (methionine reductase and methionine synthetase).Furthermore, the importance of B 6 and B 12 vitamin intake and folic acid intake will be studied in an attempt to analyze the infl uence of these variables.

CONCLUSIONS
The present study identifi ed associations between the TT genotype and coronary artery obstruction of greater than 95% and presence of two injured arteries.Additionally, the relationship between genetic variants in specifi c patient subgroups and cardiovascular diseases was confi rmed.However, the most important contribution of the present study was the fi nding of an association between C677T polymorphism of the MFHFR gene and the severity of the disease.

.
Distribution of the genotypic frequencies of methylenetetrahydrofolate reductase/ C677T (MTHFR/C677T) polymorphism, in relation to the number of arteries affected

Table 1 .
Characterization of patients with coronary artery disease and controls who underwent angiography, and the respective statistical signifi cance between the two groups (p-value) TC = total cholesterol; LDLc = low-density lipoprotein cholesterol fraction; HDLc = high-density lipoprotein cholesterol fraction; VLDLc = very low-density lipoprotein cholesterol fraction; TG = triglycerides.* p < 0.05.

Table 2 .
Distribution of the allelic and genotypic frequencies of C677T polymorphism in 127 individuals who underwent angiography * absolute frequency.

Table 3 .
Coronary artery involvement in patients, in relation to C677T polymorphism, and the respective statistical signifi cance (p-value) * p < 0.05.

Table 5 .
Analysis of homocysteine (Hcy) levels between patients and controls, in relation to C677T polymorphism, and the respective statistical signifi cance (p-value) * Analysis not performed, because the control group had only one individual with the TT genotype.