2. Methods

2.1. Subjects

gene, localized at the 11q23-qter locus has a well known sequence and genomic organization [9,10]. The protein has also been sequenced and its structure has been predicted [11,12]. A possible role of apo A-IV has been suggested in the assembly and metabolism of the HDL, in reverse cholesterol transport and activation of lecithin-cholesterol acyltransferase (LCAT) and lipo- protein lipase (LPL)[13]. Several common and rare isoforms have been detected in a broad range of popu- lations with different ethnic backgrounds and the corre- sponding allelic variations have been analyzed [12,14–16]. The effect of the different isoproteins on plasma lipid and apolipoprotein concentrations is still controversial [6,14,17–20]. We studied the two common restriction site polymorphisms at codons 347 (Hinf ) 347 % and 360 (Fnu ), and the (CTGT) VNTR in the 3 360 untranslated region [16], examining their respective fre- quencies in centenarians and adults, in search of a longevity-associated allele. We also investigated whether the lipid, apolipoprotein and lipoprotein plasma concentrations were peculiar in centenarians and explored the existence of some association with the apo A-IV polymorphism that could be related to the longevity unaffected by atherogenic disorders. Sec- ondly, we focused on Lp(a), the LDL-like particle characterized by the unique apo(a) protein [21,22]. The human apo(a) gene has been localized at the 6q2.6–2.7 locus and the cDNA cloned and sequenced [23]. The apo(a) gene has a two order heterogeneity: a size poly-

A total of 71 centenarians (18 men and 53 women, 100–107 years of age, mean(cid:30)102.3 years) were re- cruited using the Central Population Register of Apu- lia. They were self-sufficient and lived at home. Admission criteria excluded acute-phase reactions or chronic disorders such as diabetes, malignancy, nephropathy or end-stage renal disease, cardiac failure, myocardial infarction and dementia. They were not on medications known to affect lipids. All these subjects were analyzed for the standard lipoprotein plasma parameters; the blood sample of 58 of them was also available for DNA analysis. A total of 100 unrelated adults (21 men and 79 women, 19–59 years of age, mean(cid:30)35.7 years) were analyzed as a control popula- tion. Such a population consisted of healthy people, free of the above mentioned diseases and related drug intake, with moderate to no smoking and alcohol as- suming behaviours. Moreover, the two groups had homogeneous geographic origin and were sex matched, reflecting the sex differential in longevity in Italy, ac- cording to the last estimation of 1993 [30]. All subjects gave informed consent to the study.

Blood samples were taken after a 13 h overnight fast and serum was used for quantitative analyses, essen- tially as already described [31]. Total cholesterol (TC), triglycerides (TG) and HDL-cholesterol (HDL-C) were

plasma having a low Lp(a) concentration (1 mg:dl). All the serum samples were frozen at (cid:28)80°C within 10 h of collection and thawed only once for the phenotyping within 1 month [33].

determined by standard enzymatic-colorimetric meth- ods (Boehringer, Mannheim). LDL-cholesterol (LDL- C) was calculated with the Friedewald formula [32]. Serum apolipoprotein B and A-I (apo B, apo A-I) levels were measured by nephelometry. Lp(a) plasma concen- trations were determined in duplicate by ELISA using the Immunozym Lp(a) Kit (Immuno-Vienna). All the samples were stored at (cid:28)80°C for not more than 1 month and thawed only once, at the time of quantita- tion, to avoid a significant effect of the storage on the results [33].

2.5. Statistical methods

Allele frequencies in centenarians and controls were estimated by gene counting and compared using the x2 test. The linkage disequilibrium D for the pairs of loci was estimated by the methods of Nei [37]. Lipoprotein concentrations in the two groups were compared by the unpaired Student’s t test and U Mann–Whitney test (the latter employed for Lp(a), due to its non-Gaussian distribution). The association of the different apo A-IV polymorphisms with plasma lipoprotein levels was ana- lyzed by means of the unpaired Student’s t test, the U Mann–Whitney test when comparing two groups, and using the oneway ANOVA and Kruskal–Wallis oneway ANOVA for comparison of three groups. The statistical analysis was performed using the SPSS package.

m DNA was extracted from 500 l of peripheral blood m in 0.1M EDTA pH 7.5 [34]. A total of 50 l PCR amplifications were performed using 500 ng of DNA, m 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 and the conditions already described [35]. 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 5’GCTGCAAGGA- GGATTCATCCG (the positions are according to [10]). % % % PCR conditions were: 1 at 94°C, 1 at 62°C, 1 at 72°C, for 30 cycles. The product was directly analyzed by selective gel electrophoresis. Samples with different mi- grations were sequenced (USB Sequenase 2.0 version) to control the actual dimension of the repeat. Whenever required, the cis:trans location of the three polymor- phisms was determined by the double HinfxFnu diges- tion 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 A3–A4).

3. Results