Genomic DNA was extracted from peripheral blood samples by the GFXe Genomic Blood DNA Purification Kit (Amersham Biosciences). APOE genotypes were determined as detailed elsewhere (Panza et al., 1999). The IL-6 promoter genotyping was performed on Light Cycler system (Roche, Mannheim Germany) by melting curve analysis using specifically designed hybridization probes with fluorescent dyes: Fluoresceine Isothiocyanate (FITC) 0 labeled anchor (5 -CGACCTAAGCTGCACTTTTCCCCC- 0 TAX-3 ) and Light Cycler (LC) Red 705 labeled sensor 0 0 (5 -TGTGTCTTGCCATGCTAAAGGACGp-3 ; TIBMOL BIOL) probes. The primers used in the amplification 0 were: forward primer, 5 - AAATTCTAGCCTGT- 0 0 TAATCTGGTC-3 , reverse primer, 5 - ATCCCACATTT- 0 GATAAATCTTTGT-3 (Moos et al., 2000). The following 20 ml reaction mix was used for PCR amplification and subsequent detection of fluorescence from hybridization probes: 100 ng of genomic DNA, 50 pmol each primer, 3 pmol each probe, 3.5 mM MgCl , 1 £ DNA master 2 hybridization probes (Roche Diagnostics). A PCR ‘touch- down’ was performed to reduce mispriming and increase efficiency. The amplification program consisted of initial denaturation of 2 min at 95 8C, then 45 cycles with three temperature segments. The first segment was 94 8C for 10 s at 20 8C/s of temperature transition rate for denaturation; a second segment for both primer and probe annealing was of 15 s at 62 8C for five cycles followed by a 0.4 8C decrease of the annealing temperature every second cycle up to 55 8C; the temperature transition rate was 20 8C/s. A third temperature segment for primer extension was 72 8C for 10 s at 20 8C/s of temperature transition rate. After amplification, the melting curve analysis program was performed with three different temperature segments, as well: the temperature was initially raised to 94 8C for 30 s for denaturation, then lowered to 40 8C at 20 8C/s, and held at 40 8C for 1 min to permit probe annealing, and finally raised to 90 8C at 0.15 8C/s of temperature transition rate while collecting fluorescence data continuously to obtain the melting curve analysis profile temperature. A final cooling at 40 8C was carried out. The melting temperatures were 61 8C for G allele and 69 8C for C allele (Table 1).

Apolipoprotein E (APOE) is a major constituents of very low density lipoproteins, and a polymorphism in APOE has been extensively examined in populations worldwide for its association with an increased risk of CAD and AD (Davignon et al., 1988; Saunders et al., 1993). Recent findings of a higher prevalence of the APOE 1 2 allele and a lower prevalence of the APOE 1 4 allele in centenarians than in younger controls suggested a role of the APOE polymorphism in longevity (Schachter et al., 1994; Louhija et al., 1994; Panza et al., 1999). The first aim of the present study was to investigate whether there was evidence in Southern Italy to support findings of an association between the IL-6 2174 G/C promoter polymorphism and the extreme longevity. Secondly, we tested the possible interaction of APOE alleles with the IL-6 2174 G/C promoter polymorphism in view of our recent findings for reduced APOE 1 4 allele in centenarians (Panza et al., 1999).

2. Material and methods

2.1. Subjects selection

Italy) have been studied. These included 81 centenarians and 122 middle-aged, unrelated subjects or volunteers who presented themselves to the Department of Geriatrics, Bari University Hospital, Italy, between March 1995 and December 1998. The population of 81 centenarians was composed of 62 women and 19 men with a mean age of ^ 100 2 years. Centenarians were recruited from all over the Apulia region (Southern Italy) (regional distribution: 40% Bari region, 18% central, 4% northern, 5% eastern, 15% western, 18% southern), at their home, through the Central Population Register of Apulia. They were all Caucasians residing in Southern Italy. The criteria for inclusion were to be at least 99 years old on the day of blood collection and no known diagnosis of dementia or other chronic neurological diseases or psychiatric syndromes with cognitive impairment (Solfrizzi et al., 1999), cerebrovascu- lar disease, nephropathy or end-stage renal disease, and no

C. Capurso et al. / Experimental Gerontology 39 (2004) 1109–1114

population were in Hardy-Weinberg equilibrium (centenar- ians: Pearson x2 ¼ 0: 41; df ¼ 1; p ¼ 0: 52; middle-aged subjects: x2 ¼ 0: 54; df ¼ 1; p ¼ 0: 36). No statistically significant differences were found in the IL-6 2174 G/C promoter genotype between centenarians and middle-aged subjects in our population (Table 3). Similarly, the frequency of the IL-6*C allele (or vice versa IL-6*G allele) was comparable between centenarians and middle-aged control subjects (Table 3). Finally, we did not find any 3-way statistically significant interaction among IL-6 2174 G/C promoter genotype, APOE alleles, and longevity (odds ratio: 1.49 CI 95% 0.64–3.84, Exact p-value ,0.45) (Table 4).

Table 1

Interleukin-6 (IL-6) promoter 2174 G/C genotyping: amplification and melting curve analysis conditions

Amplification: cycles 45 Segment 1: Segment 2: Segment 3: denaturation annealing extension Target temperature (8C) 94 62 72 Incubation time (s) 10 15 20 Temperature transition 20 20 20 rate (8C/s) 2nd Target temperature (8C) 0 55 0 Step size (8C) 0 0.4 0 Step delay (cycles) 0 5 0 Melting curve Segment 1: Segment 2: Segment 3: analysis: cycle 1 denaturation probe annealing probe detachment Target temperature (8C) 94 40 90 Incubation time (s) 30 60 0 Temperature transition 20 20 0.15 rate (8C/s)

4. Discussion