Figure 2 Statistical comparison (unpaired t-test) of (a) homocysteine plasma concentrations in homozygous carriers of the mutation and subjects without the mutation in young (P¼0·09) and elderly (P¼0·15) control subjects and CAD (P¼0·12) and PAOD (P¼0·05) patients, and (b) MeTHF plasma concentrations in homozygous carriers of the mutation and subjects without the mutation in young control subjects (P¼0·42) and CAD patients (P¼0·14).

the observation [1,12,47–50] that Hcy values were signifi- cantly higher in both patient groups and elderly controls compared with the younger control group.

difference in frequency between younger and older groups. The meta-analysis by Brattstro¨m revealed no significant difference in genotype between young and elderly [35], but here again the C677T homozygotes were under repre- sented in the older group.

A statistically significant difference of tHcy values was found between the different genotypes in the PAOD patients but not in the other groups. This absence of a correlation between MTHFR genotype and tHcy levels in three of the groups, could be attributed to a sufficient folate intake that largely compensates for the enzyme disturbance [9,27,29,31,33,45,51]. This lack of correlation between genotype and tHcy levels raises the question of whether the apparently contradictory findings in the relationship between thermolabile MTHFR genotype and vascular disease can be attributed to differences in folate status only, and are due to a lack of a measurable influence of the thermolabile MTHFR genotype and Hcy in health and disease.

When the allele frequencies were considered, there was a significantly lower presence of the C677T allele in the elderly control group compared with younger controls and with CAD. This difference was less marked when the elderly controls were compared with the PAOD group. In this context the mean ages of the old control group (76 years 6 16) and the PAOD group (74 years 610) are very similar, and different from the other groups. This age- associated decrease in the prevalence of the mutation may indicate a shift in favour of the wild type allele through aging, indicating a lower life expectancy for individuals homozygous for the mutation. Whether this lower life expectancy is caused by vascular disease remains open, as we found no direct association between the thermolabile MTHFR genotype and disease. In this regard it is note- worthy that the PAOD group patients were of similar age to the elderly control group and also revealed a reduced frequency of the þ/þ genotype. The difference in allele frequency compared to the young controls seems therefore to be influenced by age rather than by disease status.

Several investigators showed an increased frequency of the C677T mutation in patients in a wide range of vascular diseases [10,12,14,16,46]. In our study, comparison of healthy and patient populations revealed no statistically significant difference in the C677T genotype distribution. This lack of association between the thermolabile MTHFR genotype and the disease confirms the results reported in many other studies [20,22–25,29,33,34,45].

These findings are in apparent contrast to earlier studies but these were not specially designed to evaluate the influence of age [12,22,45]. Most studies have focused on the comparison of age-matched controls and patients [25] or compared mutation frequencies in groups with no or with only a small age difference [12,23,27,45]. These studies including the meta-analysis of Brattstro¨m con- cluded that no differences exist when considering age [12,22–24,45]. In our study the average difference between younger and elderly controls of 37 years is much greater than in most studies. This age difference is similar to that reported by Faure-Delanef [36], and Matsushita [37] who also reported a trend toward a lower frequency of the MTHFR C677T þ/þ genotype in old age groups. A secular shift could possibly explain the difference seen between young and old controls.

In conclusion, tHcy concentrations are not consistently related to the mutant allele; at least in subjects with adequate folate intake. Therefore the possibility that the reduced frequency of the C677Tallele with increasing age is not directly related to Hcy induced vascular morbidity or mortality needs to be evaluated. Finally this study rein- forces the lack of a link between the presence of homo- zygosity for the mutation and the occurrence of vascular disease shown in the majority of previous studies.

Acknowledgement

This study was supported in part by a grant from the Swiss National Science Foundation (No. 32–45988·95).

Furthermore Anderson et al. [23] observed no increased risk for myocardial infarction in subjects above 60 years of age with the þ/þ genotype in contrast to subjects below 60 in whom increased risk was found. This could indicate a lower frequency of the þ/þ genotype in elderly subjects also in this study. Although none of these findings showed high statistical significance, age-dependency may have contrib- uted also to the results of Deloughery et al. and van Bockxmeer et al. [21,45] where a lower incidence of the þ/þ genotype was observed in the older patient group compared to younger controls. Thus the data available so far comparing older and younger groups allow no definite conclusion to be made and further studies focusing on the question of whether the homozygous status for C677T has an impact on life expectancy, in particular prospective studies, are needed.

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