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(Circulation. 1996;94:2154-2158.)
© 1996 American Heart Association, Inc.

Articles

Homocysteine and Risk of Premature Coronary Heart Disease

Evidence for a Common Gene Mutation

Paula M. Gallagher, MSc; Raymond Meleady, MRCPI; Denis C. Shields, PhD; Kok Soon Tan, MRCP; Dorothy McMaster, PhD; Rima Rozen, PhD; Alun Evans, MD; Ian M. Graham, FRCPI; Alexander S. Whitehead, DPhil

the Department of Genetics, Trinity College, Dublin 2, Ireland (P.M.G., D.C.S., A.S.W.); Department of Cardiology, Adelaide Hospital, Trinity College, Dublin (R.M., I.M.G.); Department of Epidemiology and Preventive Medicine, Royal College of Surgeons, Dublin, Ireland (I.M.G.); Department of Medicine, Toa Payoh Hospital, Singapore (K.S.T.); Department of Epidemiology and Public Health, The Queen's University of Belfast, Northern Ireland (D.M., A.E.); and Departments of Human Genetics, Pediatrics, and Biology, McGill University, Montreal, Quebec, Canada (R.R).

Correspondence to Prof A.S. Whitehead, Department of Genetics, Trinity College, Dublin 2, Ireland.

	Abstract

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Background Plasma homocysteine levels are modulated by nutritional and genetic factors, among which is the enzyme methylenetetrahydrofolate reductase (MTHFR). A common defective (thermolabile) variant of this enzyme is causally associated with elevated plasma homocysteine, itself an independent risk factor for coronary heart disease.

Methods and Results To examine the hypothesis that the allele (T) that codes for the thermolabile defect increases the risk of coronary heart disease, we studied 111 patients with clinical and objective investigational evidence of coronary heart disease and 105 control subjects. The frequencies of the thermolabile defect (T) in patients and control subjects were measured, and the prevalence of elevated plasma total homocysteine according to genotype was assessed. The frequency of the defective allele was higher in patients than in control subjects with an OR of 1.6 (95% CI, 1.1 to 2.4; P=.02). The OR in the coronary heart disease group for the homozygous TT genotype was 2.9 (95% CI, 1.2 to 7.2; P=.02); 17% of patients and 7% of control subjects had the TT genotype. Plasma total homocysteine levels were significantly associated with disease status, a relationship that matched the strength of the association between disease and homozygous inheritance of the defective enzyme.

Conclusions Homozygotes for the defective allele (T) are at increased risk of premature coronary heart disease. MTHFR, which modulates basal plasma homocysteine concentration, is folate dependent, and dietary supplementation or fortification with folic acid may reduce plasma homocysteine levels and consequent coronary risk in a significant proportion of the general population.

Key Words: coronary heart disease • homocysteine • MTHFR • mutation

	Introduction

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An elevated plasma level of the amino acid Hcy is a significant and independent risk factor for the development of coronary heart disease.^{1 2 3 4 5 6 7 8 9} Plasma Hcy levels are controlled by the action of a number of enzymes, the functions of which are dependent on nutritional factors such as vitamins B12 and B6 and folic acid¹⁰ (see the Figure). Several inherited enzyme defects that give rise to greatly elevated plasma Hcy levels have been described. The first to be extensively studied was defective CBS activity.¹¹ Subjects who are homozygous for dysfunctional CBS alleles have greatly elevated plasma Hcy levels and consequently homocystinuria, one aspect of which is the development of early and aggressive atherothrombotic vascular disease.¹⁰ The considerably rarer severe deficiency of the remethylation enzyme MTHFR in the homozygous state is also associated with greatly elevated plasma Hcy levels and premature vascular disease.¹² Observing the common metabolic feature of these two disorders, McCully¹³ formulated the hypothesis that Hcy is a significant etiologic factor in atherothrombotic vascular disease.

View larger version (15K): [in this window] [in a new window]   Figure 1. Transsulfuration and remethylation of Hcy. B6 indicates vitamin B6; B12, vitamin B12.

Severely defective CBS or MTHFR alleles are too infrequent to account for more than a small proportion of the association between modest elevations in the plasma Hcy level and coronary heart disease.¹⁴ This observation, together with the well-documented inverse relationship between plasma Hcy level and plasma and red cell folate, plasma vitamin B12, and vitamin B6,¹⁵ stimulated an examination of the relationship between nutritional determinants of plasma Hcy and vascular disease¹⁶ (the Figure). More recently, however, it has been established that some individuals have a thermolabile form of the MTHFR enzyme that has <30% residual activity after heat inactivation at 46°C for 5 minutes compared with >50% residual activity in subjects without the defective allele and that this variant may be associated with increased risk of coronary heart disease.^{17 18 19} The thermolabile form of MTHFR was subsequently shown to be a major cause of mildly elevated plasma Hcy levels.²⁰ Taken together, these findings suggest a direct relationship between thermolabile MTHFR, elevated plasma Hcy, and coronary heart disease. The underlying genetic defect was recently defined as a C-to-T missense mutation at nucleotide 677, which substitutes a valine for a highly conserved alanine residue.²¹ This finding has provided the opportunity for us to assess the significance of both the thermolabile MTHFR genotype and an elevated plasma Hcy level on coronary heart disease risk. The present study also examines the contribution to coronary heart disease risk of the CBS mutation G307S, the most prevalent defective allele among Irish patients with homocystinuria.²²

	Methods

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Subjects
The study population comprised 111 patients of both sexes <55 years of age from Dublin and Belfast for whom stored blood samples were available and 105 control subjects. The mean age of control subjects (48 years) was similar to that of patients (49 years), but there were more women in the control group (25%) compared with the patient group (10%). Most blood samples were from subjects recruited for the EC Concerted Action project Homocysteinemia and Vascular Disease.¹⁶ The remainder (30 patients and 13 control subjects), on whom data on plasma tHcy comprising protein-bound plus free homocysteine moieties are incomplete, were from an earlier study in which subjects were enrolled by use of similar intake criteria¹ but in whom postload free rather than fasting tHcy levels had been measured. Patients were included if they had both clinical evidence of coronary heart disease, namely angina or myocardial infarction, and objective investigational evidence, ie, a twofold increase in cardiac enzymes with ST-T changes or pathological Q waves, or at least 70% obstruction of one major coronary vessel at angiography. Sixty-two patients with coronary heart disease were recruited in Dublin; 49 were recruited in Belfast. Because of suspected effects of acute illness on Hcy levels that have since been confirmed,²³ biochemical measurements were made at least 3 months after any such event. Control subjects were included if there was no historical, clinical, or biochemical evidence of coronary heart or systemic disease. Of these, 61 subjects came from Dublin and were recruited from an industrial medical screening program of employees with a socioeconomic background similar to that of patients, while the 44 Belfast control subjects were randomly selected from the same area of the community as the patients by use of general practitioner lists available from the Central Services Agency of Northern Ireland. The study was approved by the ethics committee of each center, and informed consent was obtained from all subjects.

Collected data included age, sex, family history, cardiovascular risk factors, and clinical and investigative details. After an overnight fast, blood was drawn from participants for estimation of plasma tHcy. A standardized methionine loading test (100 mg/kg)²⁴ was performed, and blood was taken 6 hours later for determination of post–methionine load tHcy level. Samples were centrifuged and stored at -70°C. Plasma tHcy was measured by high-performance liquid chromatography in the laboratory of Profs Per Ueland and Helga Refsum, University of Bergen, Norway, with a previously described method.²⁵

Mutation Analysis
Crude cell lysates suitable for PCR analysis were prepared from blood after removal of plasma and lysis of red blood cells.²² Analysis of both patients and control subjects for the C-to-T mutation at nucleotide 677 in the MTHFR gene that specifies the alanine-to-valine amino acid substitution was carried out by use of the method of Frosst et al.¹⁹ With primers flanking the mutation site, PCR was performed over 40 cycles of 1-minute denaturation at 94°C, 1-minute annealing at 55°C, and 1-minute extension at 72°C. The product of the mutant gene carries a HinfI restriction site that can be identified after digestion and electrophoresis with 10% polyacrylamide gels and ethidium bromide staining.

Identification of the G307S mutation in exon 8 of the CBS gene in patients and control subjects was also performed.²² Briefly, PCR was performed on cell lysates with flanking primers and the reaction conditions outlined above. PCR products were then digested with Pvu II, which cuts in the presence of the mutation. Electrophoresis on a 10% polyacrylamide gel followed by ethidium bromide staining allowed detection of mutant alleles.

Statistical Analysis
Given the artificial and likely transient nature of an elevated post–methionine load tHcy level, the present study primarily analyzed fasting plasma tHcy, which is at least as good a predictor of vascular disease as the postload level.²⁶ Elevated fasting and postload plasma tHcy levels were defined as 12.07 µmol/L and 37.99 µmol/L, respectively. These cutoff points represent the top quintiles of the fasting and postload plasma tHcy distributions in the control population of the larger multicenter EC Concerted Action project study.¹⁶

Logistic regression was used to calculate the OR and associated 95% CI for the frequency of the thermolabile allele T and of the homozygous thermolabile genotype TT among patients and control subjects. Additional analyses adjusted for age and sex were performed. All probability values presented are for two-tailed tests.

	Results

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Analysis of 111 coronary heart disease patients and 105 control subjects for the alanine-to-valine amino acid substitution in the MTHFR gene¹⁹ and for the G307S mutation in the CBS gene²² was performed. Table 1 shows the distribution of each MTHFR genotype (TT homozygotes, TN heterozygotes, and NN nonthermolabile homozygotes) in patients and control subjects, and Table 2 shows the prevalence of elevated fasting and post–methionine load plasma tHcy level for each genotype in the subjects for whom the tHcy level was determined. Table 3 shows the ORs with the associated 95% CIs for associations between disease status, genotype, and plasma tHcy level.

View this table: [in this window] [in a new window]   Table 1. Distributions of MTHFR Genotype in Patients and Control Subjects

View this table: [in this window] [in a new window]   Table 2. Frequencies of Elevated Fasting and Postload Plasma tHcy Levels for Each Genotype in Patients and Control Subjects

View this table: [in this window] [in a new window]   Table 3. OR and 95% CI for Associations Between CHD Status, Elevated tHcy Level, and Genotype

The frequencies of the MTHFR thermolabile allele were comparable in Belfast control subjects (0.30) and Dublin control subjects (0.27) (P=.76 by Fisher's exact test). Although the frequencies of the T allele and TT homozygous state differ for populations from different ethnic backgrounds,²⁷ the frequencies observed in our control population are similar to those observed in Irish control groups used in other disease association studies (unpublished results). T allele distributions did not differ significantly according to age and sex. Log-transformed fasting tHcy levels were approximately normally distributed among the 71 patients for whom it was assayed; among the 92 control subjects on whom tHcy data were available, 1 TT individual had exceptionally high levels of both fasting (38.2 µmol/L) and postload (76.6 µmol/L) plasma tHcy. To avoid bias by this individual, analysis was carried out by use of the classification of high and low tHcy as defined in "Methods."

The frequency of the T allele was significantly higher in patients than control subjects (OR, 1.6; P=.02). It is evident from Table 1 that the association of the T allele with disease is mainly due to an increased frequency of the TT homozygote among the patients. Thus, the OR in the disease group compared with control subjects for the TT genotype was 2.9 (P=.02) and remained unchanged when adjusted for age and sex (P=.03). When the TT genotypes were excluded from consideration, the TN heterozygote genotype was not significantly associated with disease (OR, 1.3; P=.39). This is consistent with a recessive mode of action of the thermolabile enzyme on coronary heart disease risk.

None of the patients carried the G307S mutation of the CBS gene. Two of the control subjects were G307S CBS heterozygotes; their MTHFR genotypes were TN and NN. These two individuals had a mean fasting plasma tHcy level (11.4 µmol/L) that was not significantly greater than the control mean (10.8 µmol/L; SD=4.3); their mean plasma tHcy level after methionine loading (65.2 µmol/L) was considerably higher than the control mean (35.7 µmol/L; SD=10.1).

There was a significant association between disease status and elevated fasting tHcy level (OR, 2.0; P=.04). Elevated fasting tHcy levels themselves showed a dependence on the TT genotype in patients (OR, 11.3; P=.001) and in control subjects (OR, 4.1; P=.08). Compared with fasting tHcy, postload tHcy levels showed a weaker association with disease status (OR, 1.7; P=.12). When the thermolabile homozygotes were excluded from analysis, there was no longer a significant association between disease status and elevated tHcy, but the OR was only partly reduced (Table 3), suggesting that TT status may account for only a proportion of the association between disease and elevated fasting tHcy.

	Discussion

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Over the past decade, a consensus has emerged that elevated plasma Hcy is an independent risk factor for coronary heart disease.^{1 2 3 4 5 6 7 8 9} The underlying contribution of genetic factors, however, has remained ill defined. Initially, the clinical reports of early and aggressive vascular disease among those individuals with homozygous defective CBS mutations and greatly elevated plasma Hcy levels led to an assessment of vascular risk among heterozygotes who have more mildly elevated plasma Hcy levels.²⁸ However, an increased coronary risk in CBS heterozygotes has not been demonstrated.^{28 29 30} Four concurrent studies^{1 7 8 9} of subjects with elevated plasma Hcy in numbers that, based on frequency calculations,¹⁴ could not be accounted for by defective CBS established a strong and independent relationship between elevated plasma Hcy and coronary heart disease. Others demonstrated an inverse relationship between plasma Hcy level and plasma vitamin B12, vitamin B6, and red-cell folate level.^{15 16} In one study,³¹ plasma folate accounted for coronary heart disease risk in subjects with elevated plasma Hcy level, suggesting that any underlying genetic defects would likely involve enzymes that are dependent on adequate levels of folate. MTHFR is such a folate-dependent enzyme and is essential for the remethylation of Hcy.

There is evidence that the thermolabile form of MTHFR may be an independent risk factor for coronary heart disease.^{17 18 19 20} This defective enzyme causes a mild elevation in plasma Hcy level.²⁰ Frosst et al¹⁹ identified a single common mutation of the MTHFR gene that causes reduced activity and thermolability of the enzyme; homozygosity for this results in significantly elevated plasma Hcy levels. Furthermore, they confirmed that this genetic variant is responsible for the phenotypically thermolabile enzyme by performing functional analysis of mutant protein produced by in vitro expression of a mutant cDNA. The present study investigates the relationship between elevated plasma tHcy, coronary heart disease status, and homozygosity for the alanine-to-valine thermolabile mutation in the MTHFR gene and heterozygosity for the G307S mutation in the CBS gene.

We have shown that the frequency of the homozygous thermolabile genotype is significantly higher in patients with premature coronary heart disease than in control subjects. No CBS G307S mutant alleles were found in the coronary heart disease group, whereas two heterozygotes were found in the control group. This number matches the estimated heterozygote frequency of 1 in 110 in the Irish population, indicating that heterozygous CBS deficiency is unlikely to confer attributable risk for coronary heart disease in more than 1% to 2% of the population.¹⁴ Each of the two CBS heterozygotes had fasting tHcy levels similar to control subjects but had significantly elevated post–methionine load levels. This observation is consistent with the role of CBS in the transsulfuration of excess plasma concentrations of Hcy. However, the control of the basal plasma Hcy level through remethylation is probably etiologically more important for atherogenesis than the capacity to effectively reduce transient postload or postprandial Hcy levels, which are regulated principally by CBS.³² Because MTHFR operates in the remethylation of basal plasma Hcy to methionine, defective MTHFR activity may be of greater etiologic significance than defective CBS. This may explain the poor association observed between heterozygosity for dysfunctional CBS alleles and coronary heart disease status.^{28 29 30}

Elevated fasting tHcy levels were positively correlated with homozygosity for the MTHFR T allele in patients and control subjects together and in control subjects alone. A significant association between fasting tHcy and disease also was observed, supporting the hypothesis that the effect of homozygosity for the thermolabile mutation on coronary heart disease may be mediated primarily through raised basal plasma tHcy levels. Postload tHcy levels showed significantly weaker associations with both genotype and disease. However, the thermolabile homozygous status may not account for all the relationship between tHcy and coronary heart disease; any additional Hcy-associated risk may have an environmental or alternative genetic origin.

In conclusion, this study demonstrates that homozygosity for a common variant in the MTHFR gene, which confers thermolability and reduced activity on the enzyme, is significantly more prevalent in coronary heart disease patients than control subjects. The presence of this mutation also is causally associated with significantly elevated plasma tHcy levels. Given this and other evidence suggesting a causal relationship between elevated plasma Hcy and coronary heart disease,^{1 2 3 4 5 6 7 8 9} we propose that homozygosity for this common mutation significantly increases the risk of coronary heart disease. Although evaluation of coronary risk may in the future include MTHFR genotyping, the high frequency of TT homozygotes in the population suggests that public health recommendations regarding dietary supplementation with folic acid, which may at least partly overcome the enzyme defect,³³ may be more appropriate and less expensive. At present, there are insufficient data to fully justify such recommendations, and randomized, controlled trials are required to quantify the effect of lowering plasma Hcy levels on coronary heart disease risk.^{34 35}

	Selected Abbreviations and Acronyms

 

CBS = cystathionine ß-synthase Hcy = homocysteine MTHFR = methylenetetrahydrofolate reductase PCR = polymerase chain reaction tHcy = total Hcy

	Acknowledgments

 
This work was supported by a project grant from the Irish Heart Foundation, by a unit grant from the Health Research Board/Irish Heart Foundation, and by the Medical Research Council of Canada and the Northern Ireland Chest, Heart and Stroke Association. Data analysis was supported by Wellcome Trust (equipment grant number 039618/Z/93/Z). We also wish to acknowledge the EC Concerted Action Project Homocysteinaemia and Vascular Disease (contract number PL931708), which formed an integral part of this work.

Received February 20, 1996; revision received April 30, 1996; accepted May 7, 1996.

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