On the basis of these considerations, we decided to verify whether VNTR markers at candidate loci were able to reveal significant differences between gene pools from centenarians and controls. The first loci chosen for analysisContinuity correction was used to verify whether there were significant differences between allele frequencies in cases and controls. A possible association between APOB alleles and longevity has been investigated in French samples (the restriction fragment length polymorphism XbaI-RFLP 2488 and an insertion/deletion polymorphism in the signal peptide region, Schachter et al. 1994) and in Finnish samples (XbaI-RFLP and EcoRI-RFLP, Kervinen et al. 1994; XbaI-RFLP, Louhija et al. 1994). However, a significant association between longevity and the APOB locus was observed for only one marker (EcoRI-RFLP) in one sample group (Kervinen et al. 1994). The methods described in Nei (1987) were used to estimate haplotypic frequencies, linkage disequilibrium D, and to test the null hypothesis D = 0. They were applied directly to diallelic polymorphisms while the multiallelic VNTR polymorphism was previously recoded as diallelic by grouping VNTR alleles into two classes. The classes were defined as follows: firstly, the estimates of the frequencies of all haplotypes were computed by maximizing the likelihood of phenotypic data (De Benedictis et al. 1996); then the linkage disequilibrium of each haplotype was evaluated; finally, two APOB-VNTR allele classes were defined, one including the alleles for which the linkage disequilibrium was positive, the other those for which the linkage disequilibrium was negative. The TPO gene codes for thyroid peroxidase, the key enzyme in thyroid metabolism. This gene was included in our analysis because of the unusual thyroid functions observed in centenarians (Mariotti et al. 1992). The TPO-VNTR polymorphism (Bikker et al. 1992; Rose et al. 1993) is caused by a variable number of 50-bp repeats in intron 10 of the TPO gene (2pterp24). More than 20 alleles (from 4 to 34 repeats; nomenclature according to Rose et al. 1993) are known, with the highest frequency peak at 15 repeats. The polymorphic information content (PIC) is higher than 80%. Linkage disequilibrium was found between the TPO-VNTR polymorphism and the AcyI-RFLP (212 bp upstream of exon 10 of the same gene), but the TPO-VNTR (2pterp24) and APOB-VNTR (2p24p23) polymorphisms are in equilibrium (De Benedictis et al. 1996).

Results
The APOB locus
The XbaI-RFLP, EcoRI-RFLP and APOB-VNTR genotype and allele frequencies in centenarians (S) and controls (C) are reported in Tables 1 and 2, respectively. In both samples and for each polymorphism, the observed genotypes were in agreement (P > 0.05) with those expected at Hardy-Weinberg equilibrium.

Samples
The PIC values for the VNTR polymorphism were 0.731 in sample S and 0.762 in sample C, with 11 and 15 alleles, respectively. The observed heterozygosity was not statistically different between cases and controls (0.713 ±0.038 and 0.785 ±0.033 in S and C, respectively; P = 0.19). The intragroup homogeneity, computed as the proportion of mismatches between genotypes within each group, was 0.09 and 0.07 in cases and controls, respectively.

The sample of centenarians was composed of 143 healthy unrelated subjects from northern and southern Italy (80 and 63 subjects, respectively). Fifty-one were men and 92 were women. The criterion for inclusion in the study was to be at least 100 years old on the day of blood collection. The oldest subject was 110 years old. The average age was 102 years, with a standard deviation of 1.8 years.

The control group was composed of 158 healthy unrelated subjects randomly collected from northern and southern Italy (68 and 90 individuals, respectively).

Table 2: XbaI-RFLP, EcoRI-RFLP and APOB-VNTR allele numbers and frequencies (per 1000) in centenarians (S) and controls (C). Standard errors in parentheses [X+/X and R+/R presence/absence of XbaI and EcoRI restriction sites; VNTR allele designations in numbers of repeats (Boerwinkle et al. 1989)]

Table 1: XbaI-RFLP (0.83: In order to verify the significance of the observed difference, VNTR alleles were pooled into two classes, one of which contained the alleles having a repeat number lower than or equal to 33 (alleles 23, 29, 31, 33). The data were then analyzed according to sex (Table 3a) or to geographic area (northern and southern Italy, N.I. and S.I., respectively) (Table 3b) of centenarians (S) and controls (C). The data in Table 3a show that there is no significant difference within S (P = 0.41) or C (P = 0.23), while the frequency of alleles with low repeat number was lower in the whole sample S than in the whole sample C (χ2 = 8.54 with 1 df, P = 0.003). The difference between the S and C allele pools was chiefly due to centenarian females. Neither the XbaI-RFLP (P = 0.28) nor the EcoRI-RFLP (P = 0.64) were able to discriminate between the S and C gene pools. On the contrary, the frequency of VNTR alleles with fewer than 35 repeats appeared to be lower in cases than in controls.

Table 3: The APOB-VNTR absolute allele frequencies in centenarians (S) and controls (C) subdivided according to sex (a) and origin (northern and southern Italy, N.I., and S.I., respectively) (b). VNTR alleles are pooled into two classes according to the number of repeats.

a Subdivision by sex 
Allele S (286 alleles) C (316 alleles) 
Males Females Males Females 
(102) (184) (154) (162) 
33 14 18 36 28 
> 33 88 166 118 134 

b Subdivision by origin 
Allele N.I. (160) S.I. (126) N.I. (136) S.I. (180) 
33 20 12 30 34 
> 33 140 114 106 146