C677T (thermolabile alanine/valine) polymorphism in methylenetetrahydrofolate reductase (MTHFR): its frequency and impact on plasma homocysteine concentration in different European populations
Introduction
Coronary artery disease (CAD) is multifactorial in origin and is caused, in the general population, by interaction between an individual's genetic factors and his and her environment. In addition to the well known risk factors for CAD, such as low HDL cholesterol and elevated LDL cholesterol, triglycerides, fibrinogen, etc, a raised plasma homocysteine concentration has consistently, over the last twenty years, been identified as a risk factor for the development of vascular disease in a number of studies 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. The same effect on disease has been seen also more recently in two prospective studies 13, 17although the effect was not seen in a prospective study from Finland [18].
The determinants of plasma homocysteine concentration are both environmental as well as genetic [19]. The metabolism of homocysteine is dependent on vitamins such as folic acid, pyridoxal phosphate and cobalamine acting as co-substrates in the two interconnected metabolic pathways of transsulphuration and remethylation 20, 21. There are a number of enzymes in these pathways, and the two that have been studied most extensively are cystathionine beta synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR) 20, 21. Deleterious mutations in these enzymes cause severe hyperhomocysteinemia and the recessive syndrome of homocystinuria in the homozygous state 20, 21.
Biochemical studies of patients with CAD have identified a form of MTHFR with 50% of control activity at 37°C and marked thermolability at 46°C [22]. Engbersen et al. [23]showed the thermolabile variant to be a major cause of mildly elevated plasma homocysteine levels. A common missense mutation, which is a C to a T at bp 677 (C677T) of the cloned sequence and which causes an alanine (A) to valine (V) substitution has been described, with a frequency of the valine allele around 35% in the general population 24, 25. The A to V mutation has been shown to be responsible for the thermolabile enzyme 24, 26, 27and individuals homozygous for the valine variant had consistently higher plasma homocysteine concentration although the study was carried out on a small number of individuals [24]. Studies carried out more recently have confirmed the recessive nature of this aminoacid change and consistently shown the effect of this polymorphism on plasma concentration of homocysteine 25, 26, 27, 28, 29, though a small study where homocysteine was only measured on a subset of the individuals failed to observe this effect [30]. One other study [31], showed that in a group of individuals with plasma levels of homocysteine above 40 μmol/l, 73.1% were homozygous for the V allele compared to 10.2% of the controls strongly suggesting an important contribution of the V allele to plasma concentration of homocysteine. This same study showed also that in most cases this was reversible with folate [31]. There is some further evidence that individuals with this mutation may have higher requirement for folate to correct their hyperhomocysteinemia [25]and there are suggestions that plasma folate levels have to be low for this enzyme to be a determinant of plasma homocysteine 25, 28, 29.
Despite a convincing impact on plasma homocysteine levels, there are conflicting reports regarding the risk of vascular disease in those with the thermolabile MTHFR allele. Some studies have demonstrated an increased frequency of homozygotes for the variant 16, 22, 24, 32, 33, though other studies of similar design have failed to do so 30, 34, 35, 36, 37. The US physicians prospective study which demonstrated raised plasma homocysteine as a risk factor for the development of CAD [13], failed to show the thermolabile variant of MTHFR as an independent risk factor for MI [29], though the MTHFR polymorphism was associated with raised plasma homocysteine levels.
We describe here a study on the impact of the C677T, alanine to valine polymorphism in the MTHFR on plasma level of homocysteine in 12 different population based samples across Europe. Samples for homocysteine measurements and genotyping were obtained from participants in the second phase of the European Atherosclerosis Research Study (EARSII) [38], where young healthy male subjects with either a paternal history of MI or with no such history were recruited for metabolic studies.
Section snippets
Subjects
Subjects were participants from the EARS-II (European Atherosclerosis Research Study-II). The study was carried out in 1993. Male students aged between 18 and 28 years whose fathers had proven myocardial infarction before the age of 55 (cases) were recruited from 14 university student populations, from 12 European countries: Tallin, Estonia; Helsinki and Oulu, Finland, designated Baltic; Glasgow, Belfast and Bristol, UK, designated UK; Aarhus, Denmark; Hamburg, Germany; Ghent, Belgium; and
Results
Table 1 shows the physical and biochemical characteristics of the sample according to their status as cases or controls. There is no significant difference between the cases and the controls with respect of any of these parameters. There was no significant difference between the level of homocysteine according to smoking, alcohol consumption or physical activity (not shown).
As shown in Fig. 1. when plasma homocysteine levels were examined according to region of Europe, there was no difference
Discussion
We describe in this study the use of the MADGE system [42]for the detection of the C677T polymorphism in the MTHFR gene, that is responsible for the A to V change causing the thermolabile variant [24]. This type of economical, high throughput genotyping makes it feasible to genotype large numbers of individuals from population based samples. Using this system the entire sample of 785 EARSII subjects was genotyped within 5 working days, after optimization of the PCR and MADGE conditions, the
Acknowledgements
This work was supported by the European Commission contract BMH1-CT92-0206, and the British Heart Foundation, RG 16 and pG 95/189.
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