Significant association was found between rs4980329 and whole population survival, with a gender effect significant in women only. Importantly, we also observed a parallelism between the results of rs4980329 and rs11555236 as expected due to the predicted location of these SNPs in a linkage block according to the available HapMap (www.hapmap.org) (see Fig. 1). Our experiment allows for the consideration of these SNPs as an alternative to be genotyped in association with longevity. The fact that these polymorphisms were associated with survival in the whole population but only in females under gender stratification might tune down their effect on survival, but we and others have already seen several cases of analogous outcomes after stratification (Albani et al. 2009; Napolioni et al. 2011; Anselmi et al. 2009; Cederholm et al.). SIRT3 is linked to increased survival at oldest ages (Tazearslan et al. 2012). This gene deserves attention both for its role in mitochondrial homeostasis, a fundamental aspect of cell physiology, and for the available data associating it with survival in animal models or in the elderly (Benigni et al. 2009; Halaschek-Wiener et al. 2009; Rose et al. 2003; Bellizzi et al. 2005). We analyzed an informative set of SNPs of the SIRT3 gene in a prospective study (TRELONG) allowing us to have access to survival data over a 7-year follow-up. This work was carried out on a unique group of subjects: a community-based population with the highest longevity rates in Italy (Gallucci et al. 2007). Therefore, our analysis closely reflects the correlation between SIRT3 and longevity in the Italian population in the real world. As in a previous genetic study performed in our laboratory where we had already investigated a panel of SNPs (Polito et al. 2013), we decided to select from this panel those SNPs matching or tagging a list of genetic variants whose presence was highlighted in a population of Caucasian elderly, suggesting their involvement in a prosurvival background (Halaschek-Wiener et al. 2009). Thanks to the existence of linkage disequilibrium blocks within SIRT3, we were able to choose a couple of SNPs from Polito et al. (2012) (rs3825075 and rs4980329) that were linked to the SNPs rs511744 and rs11246020 reported by Halaschek-Wiener et al. (2009; Fig. 1). The third SNP, rs11555236, was linked to longevity in an Italian study and was supposed to be in linkage disequilibrium with a putative functional enhancer (a variable number of tandem repeat (VNTR) element) located within intron 5 (Bellizzi et al. 2005). The cross-sectional association study was performed by splitting the TRELONG population around 85 years, a critical point discriminating long-living individuals that has allowed us to find other informative genetic variability in the same population (Albani et al. 2009). However, no association was found with this experimental design. To reanalyze data taking into account the real survival curve of each subject, a prospective analysis was conducted by inputting mortality data of the TRELONG study over a 7-year follow-up. The main result was the association between rs11555236 and survival. In this study, we have not assessed the putative VNTR enhancer element of the SIRT3 gene described by Bellizzi et al. (2005). Still, taking advantage of its reported linkage disequilibrium with rs11555236, we decided to investigate further the functional action of the VNTR element in a group of TRELONG subjects bearing the rs11555236 variants. We hypothesized that some sequences of the cis SIRT3 VNTR element acted as an enhancer on the gene, leading to an increase in SIRT3 protein levels. We verified this by measuring SIRT3 protein expression in PBMCs, as this was the only available tissue specimen from the TRELONG study. Our observation that the (T) allele, which correlated with longevity, was also associated with an increased level of SIRT3 protein is intriguing, as it might suggest that increased SIRT3 levels are beneficial, as expected from the current knowledge on this protein's functions, dealing with response to oxidative stress and control of the mitochondrial acetylome (Bause and Haigis 2012; Fritz et al. 2012). This study points to an involvement of SIRT3 variability in human longevity, with a particular emphasis on the role of the rs11555236 polymorphic site as aS, De Rango F, Greco V, Maggiolini M, Feraco E, Mari V, Franceschi C, Passarino G (2014) A novel NTR enhancer within the SIRT1 gene, a human homologue of SIR2, is associated with survival at oldest ages. Genomics 104(1):15-21.

Benigni A, Corna D, Zoja C, Sonzogni A, Latini R, Salio M, Conti S, Rottoli D, Longaretti L, Cassis P, Morigi M, Coffman TM, Remuzzi G (2009) Disruption of the Ang II type 1 receptor promotes longevity in mice. J Clin Invest 119(3):524-530.

Cederholm T, Persson M, Andersson P, Stenvinkel P, Nordfors L, Madden J, Vedin I, Wretlind B, Grimble RF, Palmblad J (2007) Polymorphisms in cytokine genes influence long-term survival in patients with rheumatoid arthritis treated with TNF antagonists. J Immunol 179(5):3232-3239.

Yu W, Dittenhafer-Reed KE, Denu JM (2012) SIRT3 protein deacetylates isocitrate dehydrogenase 2 (IDH2) and regulates mitochondrial redox status. J Biol Chem 287:14078-14086.