2. Materials and methods

there may be a survival difference for very old women, among carriers and non-carriers of alleles involved in iron sparing. It is well known that heterozygotes for mutations in HFE have a higher iron content (Burt et al., 1998; Steinberg et al., 2001), but it has until now been assumed that this was without significant influence on morbidity and mortality rates. Our study does not shed light on which mechanisms are behind this longer life expectancy. However, it is tempting to suggest that the difference is owing to accumulation of iron, but this should be further investigated. In the previous report on Sicilian population, considering the historical and social contest in which the generation of women under study lived, we have suggested that the possession of iron sparing alleles significantly increases women possibility to reach longevity. For instance, in Sicily as well in Sardinia and in other pre-industrial countries, a lot of pregnancies and an iron-poor diet, consisted mainly in grains, vegetables and fruits, were yet the rule for the women born at the beginning of last century (Shorter, 1982). Future research needs to elucidate this point by analysing other populations.

Genomic DNA samples for this study were obtained through AKEA (the name ‘AKEA’ is derived from an expression in the Sardinian language that means ‘health and life for 100 years!’) study of Sardinian centenarians (Deiana et al., 1999). At the time of the present study, DNA samples for HFE typing were available from 57 centenarians (age range from 100 to 105; 17 men and 40 women) and from 60 controls (60 years old; 21 men and 39 women). Samples were typed for C282Y, H63D and S65C alleles using polymerase chain reaction and sequence specific primers as previously reported (Lio et al., 2002). HFE allelic and genotypic frequencies were compared by Monte-Carlo x2-test implemented in SPSS package (SPSS, Chicago, Illinois).

3. Results

Among the young individuals, none was heterozygous for the C282Y mutation. Fifteen subjects were hetero- zygous for H63D mutation and one for S65C. Among the centenarians, none was heterozygous for the C282Y mutation, whereas 25 were heterozygous for H63D mutation and four for the S65C mutation. No signifi- cant differences were observed in frequencies of the different alleles between young and centenarians both on the whole (Table 1) and when the data were analysed according to gender (data not shown). However, there was a trend for an increased frequency of H63D mutation in centenarians. In particular, the frequency of H63D allele was higher in centenarian women than in control women (24 vs. 17%, i.e. 19/80 vs. 13/78).

It is seemingly puzzling that HFE mutations have been suggested to be involved in unsuccessful ageing too. In fact, these mutations have been suggested to be involved in Alzheimer disease, in chronic liver diseases, and coronary heart disease (Bathum et al., 2001; Balistreri et al., 2002; Candore et al., 2002). However, it is not surprising that HFE mutations are associated both with longevity and diseases affecting life span. In fact, available data from genetics of longevity (Caruso et al., 2000, 2001) show that longevity may be associated with alleles with increased risk to a variety of diseases in the younger phases of life (antagonistic pleiotropy). This

Table 1

Allele frequencies for HFE polymorphisms in 60 controls ((cid:1)60 years) and 57 centenarians ((cid:1)100 years) from Sardinia / /

C282Y H63D S65C Wild type Allele Wild type Allele Wild type Allele (cid:1)60 years 120 (100%) 0 (0%) 99 (82.5%) 21 (17.5%) 119 (99.2%) 1 (0.8%) / (cid:1)100 years 114 (100%) 0 (0%) 87 (76.3%) 27 (23.7%) 110 (96.5%) 4 (3.5%) /

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