To investigate whether these associations had a different outcome in males or females, we performed a gender-stratified analysis, correcting for the other variables influencing survival. Plots of the TRELONG population according to genotype are shown in Fig. 2. We found a significant effect of rs4980329 and rs11555236 minor alleles for females (corrected p-values and HR: value, 0.02; HR, 0.27, respectively), while the association was no longer present in males (corrected p-values, 0.96 and 0.52, respectively).

We started evaluating the correlation of rs3825075, rs4980329, and rs11555236 with longevity by splitting the TRELONG population around 85 years of age, based on previous evidence from the same study (Albani et al. 2009, 2011). Results of this analysis are reported in Table 1. Each SNP was in Hardy-Weinberg equilibrium (HWE) (p=0.82, 0.71, and 0.93 for 2 tests assessing departure from HWE, respectively). Genotypic and allelic frequencies did not differ between age groups.

As it was hypothesized that rs11555236 might be a genetic marker of longevity in linkage disequilibrium with a putative functional enhancer within intron 5 of SIRT3 (Bellizzi et al. 2005), we decided to assess whether the rs11555236 genotype correlated with SIRT3 modulation of expression level. We focused on the SIRT3 isoform having a molecular weight of around 30 kDa that is considered the fully active protein relevant for mitochondrial metabolic homeostasis (Hallows et al. 2008). We analyzed SIRT3 protein level by Western blotting in PBMCs from the TRELONG population, building up three groups (=10 subjects each) balanced for age and gender, stratified according to rs11555236 genotype (GG, GT, or TT). Figure 3 shows SIRT3 protein immunoreactivity and the densitometric quantification. In the group homozygous for the rs11555236 minor allele (T), SIRT3 reactivity was significantly enhanced in comparison to the (GG) carriers, using α-tubulin as a normalization internal control. The normalized mean densitometric values for the three groups were as follows: (GG)=0.710±0.08; (GT)=0.80±0.4; and (TT)=1.11±0.3. These values fitted an additive model of allelic effect (linear correlation coefficient r2=0.892).

In the longitudinal survival analysis of the TRELONG population after gender stratification (Fig. 4), survival curves were plotted according to genotype and gender to search for a gender-specific effect of the investigated SIRT3 SNPs. The reported p-values are corrected for potential confounders (age, cholesterol level, scholarship, cardiovascular disease, vascular cerebropathies, diabetes, and cancer).