2.3. Statistical analysis

The statistical analysis was performed by the Pearson x2 test to undertake genotype and allele comparisons as well as test for agreement of data with Hardy–Weinberg principles. Allele frequencies were determined by allele counting. To express variances of the allele frequencies, we used 95% confidence intervals, the upper and lower values of which were calculated according to the Wilson’s formulae. A Pearson x2 test was performed to evaluate differences between ACE1 and APOE allele frequencies in centenarians and age-matched controls in Southern Italy. The results of statistical inference were adjusted according to Bonferroni inequality. To evaluate whether the association between ACE1 alleles and longevity were homogeneous in all APOE strata we used a permutation-based exact logistic model by LogXact procedure implemented in the SAS system (Proc-LogXact 5). In these analyses we used the x2 value corresponding to 0.05/3 ¼ 1.7% for each of the individual comparisons, adjusted for Bonferroni inequality. The Cochran–Armitage trend test was carried out to evaluate the geographical gradient among ACE1 allele and genotype frequencies in centenarians and younger controls of three European countries (Italy, France, and Denmark), from published studies (Bladbjerg et al., 1999; Blanche` et al., : : 2001). The threshold of significance was set at p , 0 05 The x2 or z tests were calculated by asymptotic and exact p- values. We reported in most cases the asymptotic p-values except in that situations, essentially due to a small sample size, in which asymptotic and exact p-values give a different conclusion in rejecting the null hypothesis at the 0.05 significant level. In these two last situations we preferred to use exact p-values. The data were analysed by SAS FREQ procedure (version 8.2). Exact tests were evaluated by Proc- StatXact (version 4.0.1).

2. Materials and methods

2.1. Subject selection

studied. These included 82 centenarians and 252 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 82 centenarians was composed of 62 women (75.6%) and 20 men (24.4%) 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. Furthermore, they had to have no known diagnosis of dementia or other chronic neurological diseases or psychiatric syndromes with cognitive impairment (Solfrizzi et al., 1999), cerebrovascular disease, nephro- pathy or end-stage renal disease, and no severe functional limitations. The group of middle-aged healthy adults ^ included 252 healthy subjects (mean age: 50.5 18.5SD; range: 19–70 years). Finally, these two groups had the same geographical origin and were sex-matched, reflecting the sex differential in longevity in Italy (Capurso et al., 1997). Informed consent was obtained from all subjects or their relatives; the study project was approved by the Local Ethics Committee.

3. Results

Table 1 shows ACE1 allele and genotype frequencies in centenarians and controls in Southern Italy. The frequencies of the different ACE1 genotypes in our centenarian and control group were in Hardy–Weinberg equilibrium (respectively: x2 ¼ 0: 31; p ¼ 0: 58; x2 ¼ 0: 008; p ¼ 0: 93). No statistically significant differences were found in ACE1 genotype frequencies between centenarians and controls in this Southern Italian population (ACE1*I/*I versus ACE1*I/*D and ACE1*D/*D: Pearson x2 : 0.09

F. Panza et al. / Experimental Gerontology 38 (2003) 1015–1020

Table 1

Angiotensin I converting enzyme 1 (ACE1) genotype and allele distributions in European centenarians and middle-aged subjects

Mean age at collection Genotypes n frequency (95% CI) Alleles n frequency (95% CI) (SD) (years) ACE1*I/*I ACE1*I/*D ACE1*D/*D ACE1*I ACE1*D Italian centenarians 100 (2) 10 34 38 54 110 ðn ¼ 82Þ 0.12 (0.21–0.07) 0.42 (0.52–0.31) 0.46 (0.57–0.36) 0.33 (0.40–0.26) 0.67 (0.74–0.60) French centenarians 103.1 103 261 196 467 653 (Blanche` et al., 2001) ðn ¼ 560Þ 0.18 (0.22–0.15) 0.47 (0.51–0.43) 0.35 (0.39–0.31) 0.42 (0.45–0.39) 0.58 (0.61–0.55) Danish centenarians .99 41 95 48 177 191 (Bladbjerg et al., 1999) ðn ¼ 184Þ 0.22 (0.28–0.16) 0.51 (0.59–0.45) 0.27 (0.33–0.20) 0.48 (0.53–0.43) 0.52 (0.57–0.47) Italian middle aged 50.5 (18.5) 34 118 100 186 318 subjects ðn ¼ 252Þ 0.13 (0.18–0.10) 0.47 (0.53–0.41) 0.40 (0.46–0.34) 0.37 (0.41–0.33) 0.63 (0.67–0.59) French middle aged 51.2 101 288 171 490 630 subjects (Blanche` et al., 2001) ðn ¼ 560Þ 0.18 (0.21–0.15) 0.51 (0.56–0.47) 0.31 (0.35–0.27) 0.44 (0.47–0.41) 0.56 (0.59–0.53) Danish middle aged subjects 42 46 102 51 194 204 (Bladbjerg et al., 1999) ðn ¼ 199Þ

0.23 (0.29–0.17) 0.51 (0.58–0.44) 0.26 (0.32–0.20) 0.49 (0.54–0.44) 0.51 (0.56–0.46)

I, ACE1*I; D, ACE1*D; II, ACE1*I/*I; DD, ACE1*D/*D; ID, ACE1*I/*D; n; number of individuals genotyped; CI, confidence interval.

Bonferroni-p . 0: 05; df ¼ 1; ACE1*I/*D versus ACE1*I/ *I and ACE1*D/*D: Pearson x2 : 0.72 Bonferroni-p . 0: 05; df ¼ 1; ACE1*D/*D versus ACE1*I/*I and ACE1*I/ *D: Pearson x2 : 1.13 Bonferroni-p . 0: 05; df ¼ 1). Similarly, the frequency of the ACE1*D allele (or vice versa ACE1*I allele) was comparable between centenarians and middle-aged control subjects (Pearson x2 : 0.85 Bonferroni-p . 0: 05; df ¼ 1). We also tested the ACE1