Statistical analyses

The age-related trajectories of the frequencies of both allele (A)−110 and genotypes carrying the allele (A)−110 were analysed by the least squares method. The MATLAB function polyfit was used to fit a straight line y = β + β x to the relevant frequency 0 1 data observed in five age classes (18–29 years; 30– 49 years; 50–69 years; 70–89 years; 90–109 years), thus obtaining estimates b and b . The mean of 0 1 the ages of individuals belonging to each age class was taken as the ‘x’ value for that class. Ten thou- sand replicates of b and b were then generated by 0 1 the bootstrap method (MATLAB Statistics toolbox) from which the empirical distribution of β and β 0 1 was obtained, permitting statements to be made about the age-related frequency trajectory. It was assumed that the age-related frequency trajectory decreases (increases) if the estimated value b of the slope coef- 1 ficient is negative (positive) and the empirical 95% confidence interval (c.i.) of β does not include the 1 zero value.

DNA analyses

Total DNA was extracted from blood buffy-coats following standard procedures. DNA fragments of 288bp (from 5nt to 292nt of the gene sequence reported in Gene Bank accession no. M59828) were amplified by PCR. Amplification was performed on 150ng of total DNA in 50 µl of reaction mixture containing 200 µM each of dNTP, 0.4 µM of both 5! phat3’) forward (5 -ATG GAG ACC AAC ACC CTT-3 ) Ad5). 3’) and reverse (5 -TCC TCA GGC TAG CCG TTA-3 ) primer, 1.5 Units of Taq DNA polymerase and 1.5 mM MgCl , 1X polymerase buffer under the following 2

The odds ratio (O.R.) relevant to allele (A)−110 was computed by taking as ‘cases’ the carriers of allele (A)−110 in the 90–109 years age class and as ‘controls’ those in 18–29 plus 30–49 years age class. The boot- strap method was used to estimate the 95% c.i. of O.R.

However, two points are crucial to the reliability of such studies: i) the homogeneity of the population, in order to avoid confounding factors due to population stratification; ii) the selection of the candidate gene, which should be founded on strong biological hints (Tabor et al. 2002). The data presented here show that the (A/C)−110 variability in the promoter region of the HSP70-1 gene is associated, in women, with the probability of survival to oldest age (O.R. = 0.403, with 95% c.i. [0.163, 0.910]). This positive associa- tion seems to be reliable and not due to population stratification, taking into account data regarding the structure and the demographic history of the Calabrian population (Rose et al. 1996; Danubio et al. 1999; Baudi 2001). What is more, the biological role of Hsp70 proteins in stress response and the link between stress response and aging ascertained in model orga- nisms (Minois 2000) reinforce the choice of HSP70-1 gene as a candidate gene in human gene/longevity association studies.

Results

In order to verify whether the genotypic/allelic pool changed as the population aged and selection for health or survival occurred, both female and male samples were subdivided into five age classes (18– 29 years, 30–49 years, 50–69 years, 70–89 years, 90–109 years) and the (A/C)−110 genotypic/allelic frequencies were estimated in each age class. Data are reported in Table 1. In each age class, and in both the sexes, the observed genotypes were in agreement with those expected at Hardy-Weinberg equilibrium (P > 0.05).

Linear regression analysis was then applied to allele frequency data. In females (Figure 1) a decreasing trend of the frequency of allele (A)−110 was observed with increasing age. The estimated value of the slope coefficient of the regression line, b = 1 –0.0018, was significantly lower than zero because the 95% empirical confidence interval (c.i.) of β 1 was [–0.0030, –0.0005]. Moreover, the frequency of the allele (A)−110 carriers (AA plus AC genotypes) showed a significant decrease with increasing age (b 1 = –0.0013, 95% c.i. of β 1 [–0.0027, –0.00013]). The age-related decrease of allele (A)−110 indicated that this allele is unfavorable to female longevity. To check this indication we estimated the O.R. relevant to allele (A)−110 by assuming as ‘cases’ the carriers of allele (A)−110 in the 90–109 years age class and as ‘controls’ those in the 18–29 plus 30–49 years age class (Table 1a). It should be noted that both allelic and genotypic frequency distributions were different between ‘cases’ and ‘controls’ (χ 12 = 7.76, P = 0.005 for alleles; χ 2 = 8.07, P = 0.018 for genotypes). The 2 O.R. of 0.403 (95% c.i. [0.163, 0.910]) confirmed that the (A)−110 allele is unfavorable for longevity in women.