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.

The C677T substitution was found to be common in all groups with an overall allele frequency of 27·3–38·5%. The frequencies of this mutant allele in the patients and in the combined control group were similar (P >0·3). A signifi- cant difference was found when comparing allele frequen- cies both between the elderly control group and the young control group (P¼ 0·01; mutant allele 1·4 times more pre- valent), or the CAD group (P ¼ 0·04; mutant allele again 1·4 more prevalent). No difference of allele frequencies was found between the elderly group and the PAOD group. The MTHFR genotypes followed the Hardy–Weinberg equili- brium in all groups except for the PAOD group.

The frequency of the homozygous mutant genotype in the healthy Swiss population was 16%. No statistically significant difference in genotype distribution was found when comparing gender, when comparing the four differ- ent groups, or when cases (PAOD and CAD) and controls (young and elderly) were compared with each other. As folate values were not available in all groups, subjects were divided into those with tHcy levels above or below the median values. A tHcy level above the median might serve as a marker for low folate status and allow testing of the genotype–cardiovascular link. No differences of the genotype distributions between high and low tHcy groups were found, confirming the lack of such a link in our study. A P-value of 0·056 was calculated for the genotype distri- bution between the young and elderly control group. This

Age, tHcy and MeTHF concentrations of the different groups partially reported elsewhere [40] are summarised in Table 2. tHcy levels were significantly different between all groups (0·001 < P <0·02), and showed a clear increase with age. Although the mean age of the elderly healthy group

Table 1 Prevalence of C677T mutation (þ) of the MTHFR gene in the different groups

C677T genotype [n] (%) Group n ¹/¹ þ/¹ þ/þ Allele frequency in percentage (95% CI) Young controls 118 46 (39%) 53 (45%) 19 (16%) 38·5 (32·3–44·7) Elderly controls 106 57 (54%) 40 (38%) 9 (8%) 27·3 (21·4–33·4) CAD 75 30 (40%) 34 (45%) 11 (15%) 37·3 (29·6–45·1) PAOD 63 24 (38%) 36 (57%) 3 (5%) 33·3 (25·1–41·6)

CAD, coronary artery disease; PAOD, peripheral arterial occlusive disease.

Table 2 Patient characteristics, homocysteine and methyltetrahydrofolate values in healthy subjects and vascular disease patients

Age* Homocysteine** n Years (range) Male/female mmolL¹1 (range) nmolL¹1 (range) healthy 118 39611 (21–64) 69 (49) 9·062·5 (3·3–17·3) 24·1614·5 (8·5–74·7) healthy 106 76616 (68–95) 58 (48) 13·9610·9 (4·4–109) n. d. patients 75 55612 (21–79) 59 (16) 10·664·1 (5–33·3) 19·969·2 (5·2–54·8) patients 63 74610 (49–93) 29 (34) 18·6610·2 (5·1–64·8) n. d.

CAD, coronary artery disease; PAOD, peripheral arterial occlusive disease. *P¼0·1 between elderly healthy and PAOD patients; P<0·0001 between other groups. **At least P<0·02 between all groups. ***P¼0·05 between young controls and CAD patients n. d., not done.

gender and estimated creatinine clearance (estimated crea- tinine clearance was calculated as {[150–age (y)]· weight (kg)/serum creatinine (mmolL¹1)} adjusted for gender: · 0·9 for females and· 1·1 for males [41]) revealed a statis- tically significant difference between the þ/¹ and þ/þ genotype (P¼ 0·01) and between the ¹/¹ and þ/þ genotype (P¼ 0·05) within the PAOD group. Otherwise no difference was found for tHcy values.

and the PAOD patients was comparable, the Hcy increase was even more pronounced in the latter group compared to the young controls. The difference of the MeTHF values between the two groups in which MeTHF was determined was statistically significant (P ¼0·05).

The values of Hcy and MeTHF are shown in Fig. 2. Comparison of tHcy between different genotype groups within the different study groups, after adjusting for age,

In a similar manner the homozygous subjects were compared with the other subjects, both ¹/¹ and þ/¹ grouped together. A statistically significant difference was found for tHcy values between homozygous subjects and these with the other genotypes within the PAOD group (P ¼ 0·01) and within the CAD group (P¼ 0·04). No statistically significant difference was found between sub- jects with the þ/þ genotype and these with the ¹/¹ and þ/¹ genotypes combined together within the control groups. In a similar way comparison of subjects carrying the mutation (either þ/¹ or þ/þ genotype) and subjects without the mutation (¹/¹), revealed no significant differ- ence (P¼ 0·057) for tHcy concentrations.

Homocysteine (umol L") &MeTHF (nmol L™)12010080604020    80° °de-/-+/-+/+ -/-+/- +/+ -/- +/- +/+ -/-+/-+/+Control Elderly CAD PAODcontrol° °°° °a3 &a -l— tl— tltControl CAD

Discussion

In this study the prevalence of the MTHFR C677T muta- tion, a possible risk factor for vascular disease, was inves- tigated in the Swiss population, focusing on elderly and vascular disease patients. The prevalence of the homozy- gous genotype (16%) found in our control population was towards the higher level found in other populations of Caucasian origin, including Dutch (5%) [14,42], Irish (6%) [43], Canadian (17%) [9] and Americans (12%) [8], although a much higher prevalence (30%) was found by Abbate in Italy’s Tuscany region [44].

An important finding in our study is the lower presence of the C677T allele in the elderly control group compared with the younger controls. Although some unrecognised differences between the sample cohorts cannot be com- pletely excluded, these results confirm the findings of Faure-Delanef [36] who described a trend towards a lower frequency of homozygotes for the C677T mutation in elderly people. Matsushita [37] found a significant