The association between genotypes and plasma parameters was analyzed (Tables 4 and 5). In adults, remarkable associations were found: Hinf((cid:27):(cid:27)) ho- mozygotes with lower LDL-C and apo B concentra- tions, and Fnu(1:1) homozygotes with lower HDL-C and higher TG and apo B concentrations. In centenari- ans, the same associations were present as a trend; further, the Hinf((cid:27):(cid:27)) homozygotes had a signifi- cantly higher Lp(a) concentration than heterozygotes. The VNTR itself did not appear to be significantly associated to any of the lipoprotein plasma concentra- tions in either groups. Furthermore, we searched for a possible association between the three apo A-IV poly- morphisms considered together in the haplotype phase, and lipoprotein concentrations. Indeed, in centenarians, going from the ((cid:27)14) homozygotes to the ((cid:27)13:(cid:27)14)

Finally, we looked at the apo(a) isoforms in cente- narians. The procedure used allowed the identification of the isoproteins into the major categories, which was considered satisfactory enough for a preliminary inves- tigation (Fig. 3). No subjects with the F type were found. The S2 and S3 isoforms were the most frequent ones, and were prevalent in the subjects with Lp(a) level \ 25 mg:dl; the larger S4 isoform instead was the most represented in the subjects with the Lp(a) level below this value. S2 and S3 characterized respectively the 50

Apo A-IV haplotypes

a ((cid:27)13) ((cid:27)14) ((cid:28)14) ((cid:28)13) ((cid:27)23) ((cid:27)24) ((cid:28)23) ((cid:28)24) Centenarians Allele No. 61 34 10 2 3 2 0 0 Frequency 0.54 0.30 0.09 0.02 0.03 0.02 0.00 0.00 Adults Allele No. 67 67 19 10 6 3 0 0 Frequency 0.39 0.39 0.11 0.06 0.03 0.02 0.00 0.00 b ((cid:27)3) ((cid:28)3) ((cid:27)4) ((cid:28)4) D D:D x2 P max Centenarians Observed 0.571 0.017 0.321 0.089 0.045 71% 9.88 B0.005 Expected 0.526 0.063 0.366 0.043 Adults Observed 0.424 0.058 0.406 0.110 0.023 28% 2.64 ns Expected 0.401 0.081 0.430 0.087

(a) Haplotypic frequencies in 56 centenarians and 86 adults; (b), linkage disequilibrium values (D) for pair of polymorphic sites at Hinf 347 VNTR loci. Statistical significance of the difference found in the two populations evaluated according to Nei [37].

(a) Haplotypic frequencies in 56 centenarians and 86 adults; (b), linkage disequilibrium values (D) for pair of polymorphic sites at Hinf and 347

stludAsnairanetneC unFfniHunFfniH sn87188150.0 sn80171150.0 50.0 5000.0 50.0 B272850.0 sn5.53.4sn5.43.4sn0.120.9120.0





Serum lipoprotein concentrations (mg:dl) with respect to VNTR polymorphism

Centenarians Adults Genotype (3:3) (3:4) (4:4) P (3:3) (3:4) (4:4) P No. 23 21 14 27 44 29 TC 171 171 169 ns 179 188 188 ns LDL-C 96 102 106 ns 111 116 119 ns HDL-C 46 46 40 ns 53 57 51 ns TG 142 116 113 ns 76 80 91 ns Apo B 77 75 81 ns 76 81 82 ns Apo A-I 141 136 129 ns 149 158 150 ns Lp(a)* 36.5 16.0 10.0 ns 7.3 3.8 4.0 ns

* Lp(a) values expressed as medians.

and 42% of the ((cid:27)13) homozygotes with high Lp(a) (not shown).

the genetic background, which is often observed for other genes. Considering our study groups, the haplo- type analysis reveals some differences: in the adults the ((cid:27)13) and ((cid:27)14) alleles are present in the same amount, in centenarians ((cid:27)13) is the most frequent allele. By the analysis of the linkage disequilibrium for pairs of loci, Hinf and VNTR show a D value signifi- cantly different from 0 in centenarians, whereas in adults the values does not reach statistical significance. The association of ((cid:27)3) and (cid:28)4) is greater in cente- narians than in adults (D:D 71 vs. 28%). The results max for the Fnu–VNTR and Hinf–Fnu pairs are not sig- nificant (not shown). These data, on the whole, suggest that some apo A-IV polymorphic variants, namely Hinf((cid:27)) and VNTR(3), are enriched with aging, and the ((cid:27):(cid:27)) and ((cid:27)13) alleles are somehow selected in long-lived people, possibly because they are of long- term benefit and favour longevity.

4. Discussion

We have studied the apo A-IV polymorphism in Apulian centenarians searching for longevity:associated allele frequencies by comparing them with adults of the same region.

347 genotype distribution with the prevalence of the ((cid:27):(cid:27) ) homozygosity in centenarians. No significant differ- ences are observed at the Fnu locus. The VNTR, that 360 proved to be diallelic in every cases, appears not to be so informative as other repeat markers. However, it is interesting to notice that the (3:3) genotype is predomi- nant in centenarians with respect to adults, although with no statistical significance. The analysis of the three polymorphisms in phase reveals six of the eight possible haplotypes, the most represented four corresponding to the only ones found in the German population [16], but with a quite different frequency. The ((cid:27)23) and ((cid:27)24) haplotypes, absent in Germans, also exist in Apulians. These data probably reflect the different VNTR allele distribution in the two populations due to a diversity in

directly involved in many age-related disorders, the question arises as to whether centenarians are peculiar individuals in this respect. Indeed, our results validate the idea that centenarians are exceptionally healthy people with respect to several biological features, in particular those concerning the lipoprotein metabolism.

can be explained by the low energy consuming style, and the almost absent physical activity of subjects. The same can be inferred for the decrease HDL-C concentration. However both these values in the range of normality.

Table 6

Serum lipoprotein concentrations (mg:dl) with respect to genotype in centenarians

Genotype ((cid:27)13) ((cid:27)13:(cid:27)14) ((cid:27)14) P No. 20 15 7 TC 179 169 171 ns LDL-C 112 100 99 ns HDL-C 43 46 47 ns TG 117 116 122 ns Apo B 81 73 77 ns Apo A-I 135 137 142 ns Lp(a)* 37.5 16.0 18.3 ns