presence of homozygosity for C677T was shown to result in slightly higher tHcy and this effect was amplified in subjects with total plasma folate levels below the median value [9].

The question of whether homozygosity for C677T itself is a risk factor for vascular disease has been addressed in many studies by comparing genotype frequencies in control and patient populations. These studies have revealed strikingly wide differences of frequencies of homozygosity for C677T ranging from 4% in a control population from Brazil [10] to 23% in an Italian control group [11]. So far a number of studies have indeed shown an increased fre- quency of the homozygosity rate in vascular disease patient groups [10,12–16] and Kluijtmans et al. [15] have calcu- lated an odds ratio of 1·2 for risk of vascular disease in the presence of homozygosity for C677T, based on eight case- control studies. In contrast the majority of studies includ- ing cerebrovascular disease [17,18], deep-vein thrombosis [11,19,20], peripheral arterial disease [21] and cardiovas- cular disease [20–34] show no higher frequency in vascular disease, pointing to an opposite conclusion at least in some populations. If the genotype, homozygosity for the muta- tion as frequent cause of hyperhomocysteinemia, is indeed a genetic risk factor for cardiovascular disease, associated with premature death, its frequency is expected to decrease with increasing age in a population of healthy controls. Brattstro¨m found no evidence for the C677T mutation predisposing for disease or premature death in a meta- analysis including 1388 elderly and 1415 younger subjects [35]. On the other hand Faure-Delanef described a trend towards a lower frequency of the mutation in elderly [36] whereas Matsushita found a significant difference in fre- quency between younger and older healthy groups [37]. The emerging picture is one of a high frequency of the polymorphism in all populations, a clear association between the occurrence of the mutation and tHcy levels [38,39] which is exacerbated when folate tends towards lower levels, but so far no conclusive evidence that the mutation itself is a risk factor for vascular disease.

Methods

Venous blood samples for DNA isolation were collected and stored as whole blood at ¹708C or lymphocytes were isolated immediately and stored as dry pellets at ¹708C. Genomic DNA was isolated from 200mL EDTA blood or  lymphocytes with the QIAamp blood kit (Qiagen, Basel, Switzerland) as described by the manufacturer’s protocol. DNA extracts from 362 subjects were screened for the thermolabile MTHFR variant using the restriction frag- ment length polymorphism–polymerase chain reaction (RFLP–PCR). PCR amplification using the primer pair as proposed by Frosst [8] generates a 198-bp fragment. As the C677T mutation creates a recognition site for the restriction enzyme HinfI, digestion of the amplified frag- ment with the restriction enzyme HinfI (Promega, Catalys AG, Wallisellen, Switzerland) results in a 175-bp and 23-bp fragment if the mutation is present. Digestion was performed for 16h and the restriction digests were sepa- rated on a 3% agarose gel containing ethidium bromide for visualisation (Fig. 1). In each run a H O control was 2 included to check for unspecific reactions. To check the PCR reaction, the resulting PCR product was sequenced on one occasion to confirm a correct match with the published sequence.

In this study we established the frequency of the C677T mutation in healthy Swiss subjects and patients with vascular disease and evaluated the relationship between the muta- tion and plasma Hcy. Furthermore, we compared the frequency of the mutation as a function of age between the different study groups.

Fasting tHcy and MeTHF concentrations were assayed in EDTA plasma. Sample collection conditions and deter- mination of the parameters by HPLC with fluorescence detection were performed as described previously [40].

Statistics

Subjects and methods

Compliance of genotype distributions to the Hardy–Wein- berg equilibrium was evaluated by x2 analysis. To look for differences between the various groups, unpaired t-test for measured variables and x2-test for contingency tables were used. P <0·05 were considered to be statistically signifi- cant. multifactorial analysis was used to test for ANOVA differences in tHcy levels between the different genotypes, with age, gender and estimated creatinine clearance [41] as covariates. Values are expressed as the mean 6SD. Statis- tical analysis was performed with the software packages Statview and Student 1·0. SYSTAT

Patients and control subjects

Volunteers were recruited at the University Hospital Basel. All subjects gave written informed consent and the study was approved by the Ethics Committee of the institution. This Swiss population included 224 healthy subjects and 138 hospitalised patients with a history of vascular disease. The healthy subjects were divided into young (#65 years) and elderly (>65 years) subjects. The patients included 75

 

Figure 1 Methylenetetrahydrofolate reductase genotyping by PCR and restriction analysis. Normal homozygotes (¹/¹) lacking the C677T mutation are identified by the presence of a single 198-bp fragment, whereas C677T homozygotes (þ/þ)

show a single 175-bp fragment. Heterozygotes for the C677T mutation (þ/¹) are characterised by the presence of both the 198-bp and the 175-bp fragment.

Results

tendency towards significance was confirmed when con- sidering the number of carriers against noncarriers of the C677T mutation between these control groups (P ¼ 0·03).

The prevalence of the three C677T genotypes and overall allele frequencies in young and elderly controls as well as the two patient groups are shown in Table 1.