Genotype S (143 C (158 Num- Fre- Num- Fre- individuals) individuals) ber quency ber Obs Exp Obs Exp XbaI-RFLP X– 164 573 (29) 196 620 X+ 122 427 (29) 120 380 X– X– 47 47.02 58 60.78 EcoRI-RFLP R+ 236 825 (22) 255 807 X– X+ 70 69.96 80 74.43 R– 50 175 (22) 61 193 X+ X+ 26 26.02 20 22.78 3¢ APOB-VNTR 23 – – 1 3 R+ R+ 95 97.37 104 102.89 29 – – 2 6 R+ R– 46 41.26 47 49.22 31 21 73 (15) 44 139 R– R– 2 4.37 7 5.89 33 11 38 (11) 17 54 APOB-VNTR 23.49 – – 1 0.07 35 75 262 (26) 79 250 29.31 – – 1 0.28 37 110 385 (29) 108 342 29.35 – – 1 0.50 39 14 49 (13) 12 38 31.31 – – 3 3.06 41 1 3 (3) 1 3 31.33 – – 3 2.37 43 – – 1 3 31.35 4 5.51 9 11.00 45 2 7 (5) 2 6 31.37 8 8.01 16 15.04 47 15 52 (13) 18 57 31.39 2 1.03 2 1.67 49 28 98 (18) 22 70 31.41 1 0.07 – – 51 7 24 (9) 5 16 31.45 – – 2 0.28 53 2 7 (5) 2 6 31.47 2 1.10 1 2.51 55 – – 2 6 31.49 3 2.06 2 3.06 31.51 – – 2 0.69 31.53 1 0.15 – – Table 3 The 3¢ APOB-VNTR absolute allele frequencies in 33.33 1 0.21 2 0.46 narians (S) and controls (C) subdivided according to sex (a) 33.35 3 2.88 5 4.25 origin (northern and southern Italy. N.I. and S. I., resptectively) 33.37 4 2.23 3 5.81 VNTR alleles are pooled into two classes according to the 33.39 1 0.54 1 0.65 of repeats 33.47 1 0.58 1 0.97 35.35 14 19.83 8 9.87 a Subdivision by sex 35.37 20 28.84 30 27.00 35.39 4 3.67 5 3.00 Allele S (286 alleles) C (316 alleles) 35.43 – – 1 0.25 35.45 1 0.52 – – Males Females Males 35.47 3 3.93 5 4.50 (102) (184) (154) (162) 35.49 10 7.34 4 5.50 35.51 2 1.84 1 1.25 ≤ 33 14 18 36 28 35.53 – – 1 0.50 > 33 88 166 118 134 35.55 – – 1 0.50 37.37 25 21.15 19 18.46 b Subdivision by origin 37.39 5 5.39 2 4.10 37.41 – – 1 0.34 Allele N.I. (160) S.I. (126) N.I. (136) S.I. 37.45 1 0.77 – – 37.47 5 5.77 6 6.15 ≤ 33 20 12 30 34 37.49 11 10.77 10 7.52 > 33 140 114 106 146 37.51 5 2.69 1 1.71 37.53 1 0.77 1 0.68 39.47 2 0.73 – – 39.49 – – 2 0.83 In order to verify the significance of the observed 47.47 – – 2 0.51 ference, VNTR alleles were pooled into two classes, 47.49 2 1.47 1 1.25 49.49 1 1.37 – – of which contained the alleles having repeat 49.51 – – 1 0.35 lower than or equal to 33 (alleles 23, 29, 31, 33). The 49.55 – – 1 0.14 were then analysed according to sex (Table 3a) or to

Locus Genotype S (143 C (158 individuals) individuals) Obs Exp Obs Exp XbaI-RFLP X– X– 47 47.02 58 60.78 X– X+ 70 69.96 80 74.43 X+ X+ 26 26.02 20 22.78 EcoRI-RFLP R+ R+ 95 97.37 104 102.89 R+ R– 46 41.26 47 49.22 R– R– 2 4.37 7 5.89 3¢ APOB-VNTR 23.49 – – 1 0.07 29.31 – – 1 0.28 29.35 – – 1 0.50 31.31 – – 3 3.06 31.33 – – 3 2.37 31.35 4 5.51 9 11.00 31.37 8 8.01 16 15.04 31.39 2 1.03 2 1.67 31.41 1 0.07 – – 31.45 – – 2 0.28 31.47 2 1.10 1 2.51 31.49 3 2.06 2 3.06 31.51 – – 2 0.69 31.53 1 0.15 – – 33.33 1 0.21 2 0.46 33.35 3 2.88 5 4.25 33.37 4 2.23 3 5.81 33.39 1 0.54 1 0.65 33.47 1 0.58 1 0.97 35.35 14 19.83 8 9.87 35.37 20 28.84 30 27.00 35.39 4 3.67 5 3.00 35.43 – – 1 0.25 35.45 1 0.52 – – 35.47 3 3.93 5 4.50 35.49 10 7.34 4 5.50 35.51 2 1.84 1 1.25 35.53 – – 1 0.50 35.55 – – 1 0.50 37.37 25 21.15 19 18.46 37.39 5 5.39 2 4.10 37.41 – – 1 0.34 37.45 1 0.77 – – 37.47 5 5.77 6 6.15 37.49 11 10.77 10 7.52 37.51 5 2.69 1 1.71 37.53 1 0.77 1 0.68 39.47 2 0.73 – – 39.49 – – 2 0.83 47.47 – – 2 0.51 47.49 2 1.47 1 1.25 49.49 1 1.37 – – 49.51 – – 1 0.35 49.55 – – 1 0.14

Table 3 The 3¢ APOB-VNTR absolute allele frequencies in cente- narians (S) and controls (C) subdivided according to sex (a) and origin (northern and southern Italy. N.I. and S. I., resptectively) (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

In order to verify the significance of the observed dif- ference, VNTR alleles were pooled into two classes, one of which contained the alleles having repeat number lower than or equal to 33 (alleles 23, 29, 31, 33). The data were then analysed according to sex (Table 3a) or to geo- graphic area (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 in the whole sample C (c 2 = 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.

Sample Haplotypes D D/Dmin c 2 P < 1 X– R+ X+ R+ X– R– X+ R– S Est 0.408 0.438 0.154 0.000 –0.075 100% 22.54 Exp 0.476 0.370 0.087 0.067 C Est 0.470 0.337 0.150 0.043 –0.030 41% 3.58 NS Exp 0.501 0.306 0.120 0.073 Sample Haplotypes D D/Dmax c 2 P < 1 R+ A R+ B R– A R– B S Est 0.793 0.032 0.015 0.160 0.127 90% 101.14 Exp 0.666 0.159 0.141 0.034 C Est 0.770 0.037 0.062 0.131 0.098 73% 70.15 Exp 0.672 0.135 0.161 0.032 Sample Haplotypes D D/Dmin c 2 P < 1 X– W X– Z X+ W X+ Z S Est 0.027 0.546 0.200 0.226 –0.103 79% 34.45 Exp 0.130 0.443 0.097 0.330 C Est 0.032 0.588 0.126 0.254 –0.066 67% 21.87 Exp 0.098 0.522 0.060 0.320

and D linkage values for pairs of markers at XbaI and EcoRI polymorphic restriction sites (codons 2488 and 4154, respec- tively, of the APOB gene). (Est values estimated by maximum likelihood, Exp values expected under random association, S centenarians, C controls)

Table 6 Haplotype frequencies

(P = 0.003 vs the whole control; P = 0.06 vs control fe- males; P = 0.001 vs control males), since centenarian males did not differ from the other samples (P = 0.18 vs the whole control sample; P = 0.55 vs control females; P = 0.08 vs control males).