The XbaI and EcoRI restriction analyses at codons 2488 and 4154, respectively, of the APOB gene were carried out on PCR- amplified DNA fragments as previously described (De Benedictis et al. 1993). In both cases, allelic designations refer to the presence (allele +) or absence (allele –) of the restriction site.

Statistical evaluations

Allele frequencies were computed by counting genes from the ob- served genotypes. Departures from Hardy-Weinberg equilibrium of the genotype distributions were tested for significance by the c 2 test; for VNTR markers the three statistics suggested by Chakraborty et al. (1991) were applied. The PIC was estimated ac- cording to Botstein et al. (1980). The probability of random match- ing (Pm) between genotypes was computed as the sum of the squares of all possible genotype frequencies at the VNTR locus under study. Chi-square tests, with continuity correction, were 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 re- striction fragment length polymorphism XbaI-RFLP 2488 and an insertion/deletion polymorphism in the signal pep- tide region, Schachter et al. 1994) and in Finnish samples (XbaI-RFLP and EcoRI-RFLP , Kervinen et al. 2488 4154 1994; XbaI-RFLP , Louhija et al. 1994). However, a 2488 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 poly- morphisms, while the multiallelic VNTR polymorphism was pre- viously 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; fi- nally, two 3¢ APOB-VNTR allele classes were defined, one includ- ing 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 ob- served 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 (2pter–p24). More than 20 al- leles (from 4 to 34 repeats; nomenclature according to Rose et al. 1993) are known, with the highest frequency peak at 15 repeats. The PIC is higher than 80%. Linkage disequilibrium was found between the TPO-VNTR poly- morphism and the AcyI-RFLP (212 bp upstream of exon 10 of the same gene), but the TPO-VNTR (2pter–p24) ¢ and 3 APOB-VNTR (2p24–p23) polymorphisms are in equilibrium (De Benedictis et al. 1996).

Results

The APOB locus

¢ The XbaI-RFLP, EcoRI-RFLP and 3 APOB-VNTR geno- type and allele frequencies in centenarians (S) and con- trols (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 ex- pected 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 Pm 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 unre- lated subjects from northern and southern Italy (80 and 63 sub- jects, 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 1.8 years standard devi- ation (SD).

The control group was composed of 158 healthy unrelated sub- jects randomly collected from northern and southern Italy (68 and

Table 2 XbaI-RFLP, EcoRI-RFLP and 3¢ APOB-VNTR allele numbers and frequencies (· 1000) in centenarians (S) and controls (C). Standard errors in parentheses [X+/X– and R+/R– presence/ab- sence of XbaI and EcoRI restriction sites; VNTR allele designa- tions in numbers of repeats (Boerwinkle et al. 1989)]

Table 1 XbaI-RFLP (restriction fragment length polymorphism), EcoRI-RFLP and 3¢ APOB-VNTR (variable number of tandem re- peats) observed genotypes with values expected at Hardy-Wein- berg equilibrium in centenarians (S) and controls (C). [X+/X– and R+/R– presence/absence of XbaI and EcoRI restriction sites; VNTR allele designations in numbers of repeats (Boerwinkle et al. 1989)]

Locus Allele S (286 alleles) C (316 alleles) Num- Fre- Num- Fre- ber quency ber quency XbaI-RFLP X– 164 573 (29) 196 620 (27) X+ 122 427 (29) 120 380 (27) EcoRI-RFLP R+ 236 825 (22) 255 807 (22) R– 50 175 (22) 61 193 (22) 3¢ APOB-VNTR 23 – – 1 3 (3) 29 – – 2 6 (4) 31 21 73 (15) 44 139 (19) 33 11 38 (11) 17 54 (13) 35 75 262 (26) 79 250 (24) 37 110 385 (29) 108 342 (27) 39 14 49 (13) 12 38 (11) 41 1 3 (3) 1 3 (3) 43 – – 1 3 (37) 45 2 7 (5) 2 6 (4) 47 15 52 (13) 18 57 (13) 49 28 98 (18) 22 70 (14) 51 7 24 (9) 5 16 (7) 53 2 7 (5) 2 6 (4) 55 – – 2 6 (4)