Table 1 Allele frequencies for HFE polymorphisms in 106 young ((cid:3) 55 years) and 35 elderly ((cid:4) 90 years) Sicilians

main genetic basis of hereditary haemochromatosis (HH). It destroys its ability to make up a heterodimer with an A;2 2- microglobulin. The defective protein fails to associate with the transferrin receptor and the complex cannot be transported to the surface of the duodenal crypt cells. As a consequence, in homozygous people two to three times the normal amount of iron is absorbed from food by the intestine.3,4,6,9 The disease is characterised by a progress- ive storage of iron in organs such as the liver, pancreas, pituitary gland and heart that results in end-organ dam- age and reduced life expectancy.10 Two other mutations, H63D (a histidine to aspartate at amino acid 63) and S65C (a serine to cysteine at amino acid 65), appear to be asso- ciated with milder forms of HH. In fact, a small number of patients with HH were identified as homozygous for H63D or as compound heterozygous H63D/C282Y and enrichment of S65C among HH chromosomes has been reported.6,9,11,12

C282Y* H63D S65C Wild Allele Wild Allele Wild Allele type type type <5 55 years 212 0 186 26 212 0 (100%) (0%) (88%) (12%) (100%) (0%) >9 90 years 59 11 59 11 70 0 (84%) (16%) (84%) (16%) (100%) (0%)

*P = 7.2 × 10−7 by Fisher exact test.

Table 2 Allele frequencies for C282Y HFE polymorphisms in 106 young ((cid:3) 55 years) and 35 elderly ((cid:4) 90 years) Sicilians analysed according to gender

Several studies have shown a high prevalence of the C282Y mutation in northern European populations, whereas in those of the Mediterranean basin the prevalence seems low and almost absent in Far-East countries.13–24 Besides, it usually occurs on the ancestral haplotype 7.1.4,12,20,25 Thus, it has been suggested that an estimated 60 to 70 generations ago, this mutation occurred in the HFE gene of a Celtic individual who is the ancestor of more than 5% of white subjects now carry- ing the allele.4,26 In contrast, the H63D substitution is not restricted to European populations, being found at allele frequencies of more than 5% in countries bordering the Mediterranean, in the Middle East, and in the Indian sub- continent, suggesting that the H63D substitution must have occurred earlier than the C282Y substitution.12,13,19,27 Accordingly, it does not occur on such a large ancestral haplotype as C282Y.12,27 A few studies have been perfor- med on the distribution of the S65C mutation.11,13,28

Men Women* Wild type Allele Wild type Allele <5 55 years 80 0 132 0 (100%) (0%) (100%) (0%) >9 90 years 12 2 47 9 (86%) (14%) (84%) (16%)

*P = 8.3 × 10−5 by Fisher exact test.

= subjects for C282Y were found both in elderly men (n = 2) and in elderly women (n 9), but by comparing the allele frequencies with those of young people significance (P = 7.2 × 10−7 by Fisher exact test) was attained only in women. However, it has to be taken into account that the small number of elderly men in the sample might not allow the identification of significant differences.

It has been claimed that the great expansion of Celtic people can be in part explained by the widespread pres- ence of this HFE gene mutation. This gave to hetero- zygous carriers selective advantages on the basis of improved survival during infancy, childhood, and preg- nancy, by leading to increased iron absorption and accumulation of larger body iron stores because ancient diet consisted mainly of iron-poor grains and cereals, whereas meat was highly uncommon.20 On the basis of this suggestion on the evolutionary significance of these mutations, we have evaluated their frequencies in a sam- ple of nonagenarian and centenarian Sicilians. Our hypothesis was to find an increase of elderly people het- erozygous for the mutations involved in iron sparing.

Table 3 reports on allele frequencies for H63D poly- morphisms analysed according to gender. No significant differences were observed, between the elderly and young people, both in men and in women.

Finally, in 20 young and in 20 elderly subjects we evaluated other less common HFE mutations (V53M, V59M, H63H, Q127H, E168Q, E168X, W169X and Q283P)29 and the recently described haemochromatosis related transferrin receptor 2 (TFR2) Y250X mutation,30 but none was found heterozygous (data not shown).

Results

Table 3 Allele frequencies for H63D HFE polymorphisms in 106 young ((cid:3) 55 years) and 35 elderly ((cid:4) 90 years) Sicilians analysed according to gender

Among the young individuals, none was heterozygous for the C282Y or for S65C mutation. Twenty-six were het- erozygous for H63D mutation. Among the elderly people, 11 were heterozygous for the C282Y mutation or for H63D mutation. None was heterozygous for the S65C mutation. No compound heterozygous individuals (C282Y/H63D) were found. A highly significant differ- ence (P = 7.2 × 10−7 by Fisher exact test) was observed in frequencies of C282Y alleles between young and oldest old subjects on the whole (Table 1).

Men Women Wild type Allele Wild type Allele <5 55 years 64 16 122 10 (80%) (20%) (92%) (8%) >9 90 years 14 0 45 11 (100%) (0%) (80%) (20%)

Table 2 reports on allele frequencies for C282Y morphisms analysed according to gender. Heterozygous

of randomly selected adults. Case-control studies might be subjected to a number of possible confounding factors, the total number of patients (elderly people in our study) and controls and the homogeneity of the population in terms of geographical origin among others. Although based on a relatively reduced number of controls, as reported above, our frequencies are in agreement with those obtained in southern Italy, suggesting the absence of population biases. In fact, both the elderly and young people were Sicilians, and the Sicilian population is a very homogeneous population because the last genetic admixtures there were several centuries ago, at the time of Arabian conquest.41

Discussion