remaining VNTR alleles (preferentially associated with the R– allele). In this way, the multiallelic VNTR system was recoded as diallelic (Table 5) and the statistical sig- nificance of D was then tested. In both S and C samples, D was different from 0 (c 2 = 101.4 with 1 df; P < 0.001 in S; c 2 = 70.15 with 1 df; P < 0.001 in C); however, the as- sociations R + A and R– B were greater in S than in C (D/Dmax = 90% in sample S; D/Dmax = 73% in sample C).

As to geographic area, the data in Table 3b show that there is no difference within S (P = 0.55) or C (P = 0.58), while both northern and southern Italian centenarians dif- fered from the matched controls (S vs C in northern Italy: P = 0.04; S vs C in southern Italy: P = 0.04).

By applying the above procedure to the analysis of linkage disequilibrium between the Xba-RFLP and ¢ 3 APOB-VNTR, the data shown in Table 6 were obtained. The same allelic associations were favoured in the two samples (X+ with VNTR alleles 37 and 39 , pooled in class W; X– with the remaining alleles, pooled in class Z) and, in both samples, the disequilibrium between the poly- morphic systems was significantly different from 0 (c 2 = 34.45 with 1 df; P < 0.001 in sample S; c 2 = 21.87 with 1 df; P < 0.001 in sample C). In this case too, the associa- tions X + W and X – Z were greater in S than in C (D/ Dmin = 79% in sample S; D/Dmin = 67% in sample C).

case and control gene pools. Let us consider firstly the analysis of linkage disequilibrium between the XbaI- RFLP and EcoRI-RFLP (Table 4). In sample S, the link- age disequilibrium value (D) is significantly different from 0 (c 2 = 22.54 with 1 df; P < 0.001). On the contrary, in sample C the value of D does not reach statistical sig- nificance (c 2 = 3.58 with 1 df; P > 0.05). Furthermore the haplotype X + R–, although at low frequency, is present in C but absent from the S allele pool.

Now let us consider the analysis of linkage disequilib- ¢ rium between the EcoRI-RFLP and 3 APOB-VNTR. A preliminary evaluation of haplotypic frequencies was car- ried out by the maximum-likelihood method. It enabled us to group the VNTR alleles into two classes: the A class consisting of VNTR alleles with repeat number lower than 41 (preferentially associated with the R + allele in both S and C samples), and the B class consisting of the

The TPO locus

No significant difference was found between the VNTR allele pools at the TPO locus in the S and C samples. Twenty-two and 21 alleles were observed in samples S and C, respectively (from 5 to 28 repeat number) sharing

might be related rather to VNTR alleles than to gene alleles.

the same unimodal distribution (major peak at 15 repeat number).

At a speculative level it is interesting to compare our data with those obtained in other sample populations. Since the 3¢ APOB-VNTR polymorphism is used in foren- sic science, a number of frequency data are available. Cau- casians share the same bimodal frequency distribution (peaks at 37 and 49 repeats), but if we look at the fre- quency of alleles with a repeat number lower than 35, variability is observed between populations. For instance, the frequency of these alleles estimated in other Italian or Mediterranean groups (D’Aloja et al. 1992; Cucurachi et al. 1994; De Benedictis et al. 1994; Domenici et al. 1994; Pelotti et al. 1994) is in accord with the value found in our control group (about 20%), while in a Swedish sample tested by Renges et al. (1992) this value was significantly lower (about 7%). Obviously the difference could be due to ethnic components, but it could also be due to environ- mental factors, such as diet, that keep the frequency of these alleles low in the Swedish gene pool. Interestingly, in a sample of Swedish patients affected by coronary heart disease analysed in the same study (Renges et al. 1992), the frequency of alleles with repeat number lower than 35 was significantly higher in cases than in controls (17% vs ¢ 7%). It therefore seems that 3 APOB-VNTR alleles with a low number of repeats are unfavourable to long life ex- pectancy because of their association with coronary heart disease; the low incidence of these alleles found in healthy centenarians agrees with this hypothesis.

Discussion

lele pools at two multiallelic loci that were candidates for longevity (APOB and TPO) between centenarians (S) and controls (C). The aims of the study were: (i) to identify a possible gene/longevity association; (ii) to verify whether DNA multiallelic markers could be more informative than single diallelic polymorphisms in revealing this associa- tion.

The Italian Multicentric Study on Centenarians re- ported that the estimated total number of centenarians liv- ing in Italy on 31 December 1993 was 4004 (Motta et al. 1995). The S sample tested in our study (143 subjects who were at least 100 years old on the day of blood collection) therefore represents about 3% of all centenarians living in Italy; furthermore, it was made up of subjects from vari- ous socio-economic conditions, and was collected in both northern and southern Italy. Moreover, in order to stress possible subtle differences with respect to the control group, only healthy centenarians were included in our analysis.