3. Results

The apo A-IV polymorphism was analyzed by geno- % %% ‘72% typing (Fig. 1a,a ,a ). The Thr Ser change, that 347 eliminates a HinfI site, was identified by the presence (allele(cid:27)) or absence (allele(cid:28)) of the restriction site in I] 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 ab- sence (allele 2) of this cutting site in the same amplifica- tion product. The VNTR alleles were identified by direct gel electrophoresis of the specific amplified frag- ment: the number of repeats was routinely calculated from the dimension of the fragment using a size marker and occasionally verified by sequencing. The locus showed three (allele 3) or four (allele 4) repeats in all the subjects. The cis:trans location of the three poly- morphic variants was directly assigned by this analysis with the exception of some double heterozygous indi- viduals. In the case of the heterozygotes for both the restriction sites the haplotype was assessed by the dou- ble Hinf(cid:29)Fnu digestion (Fig. 1b); in the few subjects carrying the Hinf–VNTR heterozygosity the haplotype was clarified by the digestion of the 472 bp fragment (Fig. 1c).

Apo(a) phenotyping

The isoforms were analyzed by a 4–15% gradient SDS-PAGE (Pharmacia’s PhastSystem). The samples were 1:4 diluted with the Lp(a) phenotyping buffer and run with the standard mix according to the manufactur- ers instructions (Immuno-Vienna). The separated proteins were western-blotted on nitrocellulose (Hoefer) for 1 h at 52°C and treated as first antibody with the polyclonal anti-human Lp(a) antibody (Immuno-Vi- enna) and as second antibody with a peroxidase-conju- gated anti-sheep IgG (SIGMA). The ECL-kit (Amersham, UK) was used for detection. The major isoproteins, classified according to their relative mobili- ties compared to apo B [36], were identified even in the

The frequencies of the polymorphisms at each locus were calculated. First we compared the Apulian adults with a normal German population [16]. The Hinf 347 and the Fnu polymorphisms were not significantly 360 different, the VNTR was dimorphic in the Apulian as well as in the German population, but had different

 a) i= M +]- +]+ b) M +1/+1 -1/4+2—bp bp bpbad PE em fm =<-180— <-183150-> catia!<-140'~ hat <osa') 1/2 Ww M 2/2 c) +4/-3 -4/-3 Mbp bp i.“aes430-> heli Mt189-> I i Js180-> ead (aalad ‘ /247-> so—_ 243-> ~a") 4/4 3/3 3/3 3/4 M 183-> Vemma bec |bp bp '221-> <-221217->

Fig. 1. Genotyping of apo A-IV by Amp-FLP. (a, a%) Identification of the Fnu4HI and HinfI polymorphisms at the codons 360 and 347 by 8% PAA gel (0.7(cid:29)10(cid:29)12 cm), 200 V, 30 min. (a%%) VNTR definition by 10% PAA gel (0.2(cid:29)20(cid:29)40 cm), 350 V, 18 h. (b) Cis:trans localization of the two loci 347 and 360 by 8% PAA gel (0.2(cid:29)20(cid:29)40 cm), 600 V, 3 h. (c) Cis:trans localization of the loci 347 and VNTR on the 472 bp fragment by 12% PAA, 350 V, 20 h. The gels were (24:1) PAA and the bands were visualized by silver staining.

distribution frequency of alleles 3 and 4 (0.49 vs. 0.61 and B 0.51 vs. 0.39, respectively; P 0.025). Then, the genotype distribution was compared in our study groups and the frequencies were tested for significance by the x2 test: they were found different at the Hinf locus, while they were similar at the Fnu and VNTR loci (Table 1).

the values but one being in the normal range. In particular, as far as the Lp(a) concentration was con- cerned, in centenarians it was more elevated (17.5 vs. 4.5; 12.0 vs. 5.2; 26.59 25.2 vs. 16.99 23.1 being the medians, geometric and arithmetic means, respectively), with the median value significantly higher than in B adults (P 0.002). A large part of centenarians (47 vs. \ 26% of adults) had an Lp(a) plasma level 20 mg:dl. Moreover, dividing the two groups by sex, the median values of Lp(a) were still significantly higher in cente- narians than in adults, both in males and females (M: B B 15.5 vs. 2.5, P 0.008; F: 19.0 vs. 5.8, P 0.04, respectively).

Table 2 summarizes the haplotypes found in the two study groups, their frequencies and the linkage disequi- librium D analysis for the pair of Hinf–VNTR loci. Plasma concentrations of lipids, apolipoproteins, and lipoproteins were analyzed in centenarians and adults (Table 3). We found significant differences in LDL-C, HDL-C, TG, B:L, and Lp(a) concentrations, despite all

Table 2 summarizes the haplotypes found in the two study groups, their frequencies and the linkage disequi- librium D analysis for the pair of Hinf–VNTR loci.

lipoproteins were analyzed in centenarians and adults (Table 3). We found significant differences in LDL-C, HDL-C, TG, B:L, and Lp(a) concentrations, despite all

A-IV genotype frequencies

Serum lipoprotein concentrations (mg:dl)

Centenarians Adults P TC 172938 186937 B0.02 LDL-C 103931 115933 B0.02 HDL-C 44914 54913 B0.0005 TG 121959 82942 B0.0005 Apo B 78917 80922 ns Apo A-I 133931 153927 B0.0005 B:L 0.7990.22 0.7090.08 B0.0005 Lp(a) 17.5 (5.0–45.5) 4.5 (1.0–27.8) B0.002

For the Lp(a), due to the non-Gaussian frequency distribution, the values are expressed as medians (in parentheses 25 and 75° percen- tiles).

Populations are in Hardy–Weinberg equilibrium.

heterozygotes and then to the ((cid:27)13) homozygotes some trends were observed, in particular the Lp(a) increasing, although the differences did no reach a statistical relevance (Table 6). The ((cid:27)13) homozygous centenarians having Lp(a) concentration \ 20 mg:dl were significantly more numerous than the adults with the same genotype (Fig. 2).