normalized mean densitometric values for the three groups were as follows: (GG)=0.71±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).

Fig.

Longitudinal survival analysis in the TRELONG popu- lation after gender stratification. Survival curves were plotted according to genotype and gender to search for a gender-specific effect of the investigated SIRT

SNPs. a–b rs

survival curve in males and females, respectively. c–d rs

survival curve in males and females, respectively. e–f rs

sur- vival curve in males and females, respectively. The reported p values are corrected for potential confounders (age, cholesterol level, scholarity, cardiovascular disease, vascular cerebropathies, diabetes, and cancer)

Discussion

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 signif- icantly enhanced in comparison to the (GG) carriers, using α -tubulin as normalization internal control. The

Longevity is a complex phenotype arising from multiple variables, and genetic background plays a pivotal role not only for a deterministic control on hereditary disor- ders, but also in modulating the individual response to environmental hits and drug therapies (Eaton et al. 2012; Murabito et al. 2012; Chung et al. 2010; Seripa et al. 2010). Genes with involvement in basic physiologic

 1s11555236 (G/G)440 440 236 236 421 421 575 575 111555236 (G/T)375 375 300 300 524 524 581 5812S oe oe oe Se es  111555236 (T/T)308 308 544 544 519 519 540 540   SIRT3/ a-tubulin ratioGG GT TTd (SIRT3 rs11555236)  SIRT3(30 KDa)a-tubulin(50 KDa)+4 = siRT3(30 KDa) a-tubuiin(60 KDa)SIRT3(30 KDa)4 c-tubulin(50 KDa)+

ting in peripheral blood mononucleate cells. To assess a possible functional correla- tion of rs11555236 variants with SIRT3 expression, we selected a group of subjects from the TRELONG study, balanced for gender, and evaluated SIRT3 protein im- munoreactivity. Each subject, identified in the figure by the number above the corre- sponding gel lane, was assessed in duplicate. a–c Representative Western blot- ting where subjects were di- vided according to rs11555236 genotype (GG, GT, or TT). A total of ten subjects for each genotype were analyzed. Alpha-tubulin immunoreactivity is also shown as loading control for normalization. Variation among subjects of α-tubulin reactivity was due to different cellularity of the correspond- ing PBMC fraction. d Box plot showing the densitomet- ric analysis of the Western blotting data. Optical density values of SIRT3 band were quantified and normalized to α-tubulin. Values were com-

study, where the association was significant in only, we found a detectable effect in the whole and by splitting according to gender the asso- kept significant in women only. Importantly, we also association between rs4980329 and whole pop- survival, with a gender effect significant for From the genetic point of view, the parallelism rs4980329 and rs11555236 results was expected to the predicted location of these SNPs in a linkage block according to the available HapMap (www.hapmap.org) (see Fig. 1). Our experimental allows to consider these SNPs as alternative to be genotyped in association to longevity. fact that these polymorphisms were associated to in the whole population but under gender in females only might tune down their to survival, but we and others have already several cases of analogous outcome after stratification (Albani et al. 2009; Napolioni al. 2011; Anselmi et al. 2009; Cederholm et al. SIRT3 This might suggest that genetic elements a gender-selective role in modulating longevity, though our results are not deciding to this re- as we found association only after correction several other variables affecting longevity, while uncorrected interaction (gender × polymorphism) not significant (data not shown).