DNA was extracted from 500 l of peripheral blood in 0.1M EDTA pH 7.5 [34]. PCR amplifications were performed using 500 ng of DNA, 12.5 pmol of primers, and 200 M dNTPs. The gene region containing the two polymorphic sites at codons 347 and 360 was analyzed for the amplified fragment length polymorphism (Amp-FLP). The fragment of 244 bp was amplified using the primers A1 and A2. The product was digested by HinfI or by Fnu4HI restriction enzyme and each polymorphic pattern was analyzed on a suitable % selective gel. The 3 untranslated portion of the gene containing the VNTR was separately amplified with primers A3: nt.2414 5 GATGCTGGCCCCTTTGG- AGAG and primer A4: nt.2634 5GCTGCAAGGA- GGATTCATCCG (the positions are according to [10]). PCR conditions were 1 at 94C, 1 at 62C, and 1 at 72C, for 30 cycles. The product was directly analyzed by selective gel electrophoresis. Samples with different migrations were sequenced (USB Sequenase 2.0 version) to control the actual dimension of the repeat. Whenever required, the cis:trans location of the three polymorphisms was determined by the double HinfxFnu digestion of the 244 bp fragment or by the Hinf digestion of the 472 bp fragment amplified using the primers A1 A4 (same conditions as for the primers A3A4).

The apo A-IV polymorphism was analyzed by genotyping. The Thr Ser change that eliminates a HinfI site was identified by the presence (allele') or absence (allele'') of the restriction site in the 244 bp amplified fragment. The Gln H His 360 change that results in the loss of a Fnu4HI site was separately identified by the presence (allele 1) or absence (allele 2) of this cutting site in the same amplification product. The VNTR alleles were identified by direct gel electrophoresis of the specific amplified fragment.

Isoforms of apo(a) were analyzed using a gradient SDS-PAGE and western blotting on nitrocellulose.

The genotype distribution was compared in our study groups, and the frequencies were tested for significance by the x2 test. The haplotypes found in the two study groups, their frequencies, and the linkage disequilibrium D analysis for the pair of HinfVNTR loci were summarized in Table 2.

Plasma concentrations of lipids, apolipoproteins, and lipoproteins were analyzed in centenarians and adults. We found significant differences in LDL-C, HDL-C, TG, B:L, and Lp(a) concentrations between the two groups. The VNTR itself did not appear to be significantly associated with any of the lipoprotein plasma concentrations in either group. We searched for a possible association between the three apo A-IV polymorphisms considered together in the haplotype phase and lipoprotein concentrations. In centenarians, going from the ((cid:27)14) homozygotes to the ((cid:27)13:(cid:27)14) heterozygotes and then to the ((cid:27)13) homozygotes some trends were observed, in particular, Lpprocedure used allowed for the identification of isoproteins into major categories, which was considered satisfactory for a preliminary investigation (Fig. 3). No subjects with the F type were found. The S2 and S3 isoforms were the most frequent and prevalent in subjects with Lp(a) levels below 25 mg/dl, while the larger S4 isoform was most represented in subjects with Lp(a) levels below this value. S2 and S3 characterized respectively the 50 Apo A-IV haplotypes ((cid:27)13), ((cid:27)14), ((cid:28)14), ((cid:28)13), ((cid:27)23), ((cid:27)24), ((cid:28)23), and ((cid:28)24). Centenarians had allele No. 61 (54%), 34 (30%), 10 (9%), 2 (2%), 3 (3%), 2 (2%), 0, and 0 for the respective haplotypes. Adults had allele No. 67 (39%), 67 (39%), 19 (11%), 10 (6%), 6 (3%), 3 (2%), 0, and 0 for the respective haplotypes. Linkage disequilibrium values (D) for pairs of polymorphic sites at Hinf and VNTR loci were analyzed, revealing significantly different D values in centenarians compared to adults. The results 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 favor longevity.

In Table 6, serum lipoprotein concentrations with respect to genotype in centenarians are shown. The major findings concern the LDL-C and Lp(a) concentrations. The mean concentration of LDL-C in centenarians is low, very close to that considered as the optimal cut-off level in CAD secondary prevention, 100 mg/dl. This probably makes it one of the major factors which confer protection against this disease in such individuals, thus representing a possible selective feature for longevity. The Lp(a) concentration is unexpectedly high in centenarians, revealing to be mostly unaffected by other factors such as drugs, smoking, alcohol, etc. The increased value, in the absence of chronic or acute-phase diseases, can derive from a general remolding of the plasma parameters with aging or is genetically determined by apo(a). Thus, the data on Lp(a) in centenarians draw an unusual pattern since a so-called atherogenic marker does not preclude longevity.