Table 3

Maximum likelihood estimations of the parameters of the fitted binomial logistic regression models in the analyzed sample.

Parameter β SE p-Value 95% CI Haplotype A-C Age 0.009 0.009 0.288 −0.008–0.026 [Sex=males] 0.128 0.285 0.654 −0.431–0.687 Constant 2.035 0.675 0.003 – Haplotype G-A Age −0.014 0.005 0.010 −0.024 to −0.003 [Sex=males] 0.257 0.173 0.137 −0.082–0.596 Constant −0.604 0.414 0.144 – Haplotype G-C Age −0.001 0.005 0.752 −0.010–0.007 [Sex=males] −0.163 0.150 0.279 −0.457–0.132 Constant −0.813 0.364 0.025 –

Fig. 1. The observed and the estimated frequency for haplotype G-A carriers.

Table 4

         3.g_e3ie |1H °tho     Lovo s90 090 sso oso sro oro(9-W/9-v) Aouenbey edhjo\diq7060502°+Age Fitted (Mal© Observed (FObserved (Males)    |-- Fitted (Females)  Moco szo ozo sto oo(v-9/0-V) Aousnbay adAjojdiq 10'080 90 100 110Age70

Maximum likelihood estimations of the parameters of the fitted binomial logistic regression models in the analyzed sample.

Parameter β SE p-Value 95% CI Diplotype A-C/A-C Age 0.009 0.004 0.032 0.001–0.017 [Sex=males] −0.045 0.135 0.738 −0.309–0.219 Constant 0.346 0.328 0.291 – Diplotype A-C/G-A Age −0.014 0.006 0.014 −0.026 to −0.003 [Sex=males] 0.376 0.190 0.048 0.003–0.748 Constant −0.903 0.452 0.045 – Diplotype A-C/G-C Age 0.001 0.005 0.892 −0.009–0.010 [Sex=males] −0.166 0.161 0.300 −0.481–0.148 Constant −1.227 0.389 0.002 – Diplotype G-A/G-A Age −0.010 0.019 0.614 −0.048–0.028 [Sex=males] −0.343 0.650 0.599 −1.617–0.932 Constant −3.615 1.505 0.016 – Diplotype G-A/G-C Age −0.007 0.013 0.585 −0.032–0.018 [Sex=males] −0.194 0.427 0.649 −1.031–0.643 Constant −3.035 1.009 0.003 – Diplotype G-C/G-C Age −0.010 0.014 0.451 −0.038–0.017 [Sex=males] 0.067 0.453 0.882 −0.821–0.956 Constant −3.051 1.076 0.005 –

activity. Each transfection experiment was carried out three times in duplicate.

Statistical analyses were performed by using R statistical software for windows (www.cran.com). One-way analysis of variance (ANOVA) and Student's t-test with a significance level of 0.05, were adopted to check the significance of the difference between the fold induction values of the constructs containing the different haplotypes at basal conditions and after hormone stimulation.

Fig. 2. The observed and the estimated frequency for diplotype A-C/A-C (A) and A (B) carriers.

harmful haplotype. The slope parameters for both A-C and G-C haplotypes were not statistically different from 0, suggesting that their frequencies were independent of age. Fig. 1 shows the frequency patterns for the G-A haplotype carriers. As expected, both the observed and the fitted frequencies indicated that there was a constant decline of this haplotype with increasing age. In order to check if the detrimental effect of the G-A haplotype observed in both sexes involved a particular diplotype, we used the same model to analyze the frequency of the carriers of each of the six reconstructed diplotypes as a function of age. Table 4 reports the parameter estimates for all the reconstructed diplotypes. Diplotype A-C/A-C showed a significant positive slope parameter (β=0.009; p=0.032), indicating that the frequency of subjects with this diplotype increases with age. The diplotype A-C/G-A also showed a significant negative slope parameter (β=−0.014; p=0.014), which indicates that the frequency of subjects with this diplotype decreases with age (Fig. 2). As for the haplotypes, if one considers an age interval of 15 years the corresponding ORs from the relevant fitted models were 1.144 and 0.811 for the diplotype A-C/A-C and A-C/G-A, respectively. The diplotype A-C/G-C did not show any significant variation with age. The other diplotypes failed to reach the level of significance probably due to their low frequencies. On the whole, these results indicate that the A-C haplotype has a recessive and beneficial effect, while the G-A haplotype has a dominant and deleterious effect on human aging and longevity.

3. Results

3.1. Haplotype/diplotype analyses

on a sample of 910 subjects categorized by age and sex as reported in Passarino et al. (2006). In each group, there was no evidence of departure from Hardy–Weinberg equilibrium (pN0.05). The two SNPs, which are very close to each other, resulted to be in linkage disequilibrium (D′=0.99, r2=0.58). Frequency distributions of two- marker haplotypes and diplotypes are summarized in Tables 1 and 2, respectively. We modeled the frequency of the carriers of each of the reconstructed haplotype/diplotype as a function of age by using the binary logistic regression model. Table 3 reports the maximum likelihood estimations of the parameters of the fitted haplotype-based model in males and females. Of the three reconstructed haplotypes, only the G-A haplotype showed a significant variation with age in both sexes. In fact, the slope parameter was significantly lower than 0 (β=−0.014) with a corresponding confidence interval ranging from −0.024 to −0.003. If one considers an age interval of 15 years the corresponding odds ratio (OR) from the fitted model is:

3.2. Functional analyses of haplotypes

To ascertain whether the observed associations reflected a different functional relevance of the different haplotypes, we first

This means that for haplotype G-A carriers a remarkable reduction (around 20%) occurs in an age interval of 15 years indicating that it is a

Fig. 3. Cloned sequence of the UCP1 gene containing the A-3828G (R) and the C-3737A (M) polymorphisms. Retinoic Acid-Response Element (RARE), Progesterone-Response Element (PRE), Activating Transcription Factor/cAMP Response Element binding protein (ATF/CREB), and Estrogen-Response Element (ERE) are underlined.

submitted a sequence of 463 bp (from −3967 to −3504) the two polymorphic sites to the MatInspector online software. shown in Fig. 3, a putative retinoic acid response element