Modulation of human longevity by SIRT3 single nucleotide polymorphisms in the prospective study "Treviso Longeva (TRELONG)"

Abstract

Human sirtuins are seven proteins with deacetylase activity that are emerging as key modulators of basic physiological functions. Some evidence links SIRT3 to longevity in mammals. This study aimed to investigate whether variants within SIRT3 gene were associated to human longevity. We analyzed 549 genomic DNA collected during the prospective study "Treviso Longeva," including elderly over 70 years of age from the municipality of Treviso, a small city in the northeast of Italy. We genotyped SIRT3 rs3825075, rs4980329, and rs11555236 single nucleotide polymorphisms (SNPs) by real-time polymerase chain reaction allelic discrimination assay. A cross-sectional analysis performed by comparing people over and under 85 years of age did not evidence association among the SIRT3 SNPs and longevity. However, when we performed a longitudinal analysis considering mortality as a dependent variable, we observed an association of SIRT3 rs11555236 and rs4980329 with longevity in the whole population (p values corrected for potential confounders = 0.04 and 0.03, respectively). After stratification according to gender, the same SNPs were associated to female longevity only (p values corrected for potential confounders = 0.03 and 0.02, respectively). Finally, as rs11555236 was reported to be in linkage disequilibrium with a putative functional enhancer within the SIRT3 gene, we assessed whether rs11555236 genotypes correlated with a different level of SIRT3 protein in peripheral blood mononuclear cells. We found an increased level of SIRT3 in subjects homozygous for the (T) allele. We suggest that SIRT3 genetic variability might be relevant for the modulation of human longevity in the Italian population.

Fig. 1

Genotyping and survival curves of the TRELONG population. a Graphical representation of the SIRT3 gene portion encompassing the single nucleotide polymorphisms genotyped in the TRELONG population. The genotypic frequency is graphically indicated for rs3825075, rs4980329, and rs11555236, while just the position is shown for the tagged SNPs rs511744 and rs11246020. Below the gene structure, where exons are in yellow, a linkage disequilibrium graphical representation is reported; red diamonds are suggestive of high LD (D' >80 %). b Survival plot of the whole TRELONG population stratified according to rs3825075 genotype. The reported p value is corrected for possible confounding factors affecting longevity (age, gender, scholarity, cholesterol level, cardiovascular disease, vascular cerebropathies, diabetes, and cancer). c Survival plot of the whole TRELONG population stratified according to rs4980329 genotype. The p value was corrected as above. d Survival plot of the whole TRELONG population stratified according to rs11555236 genotype. This SNP was reported to be in linkage disequilibrium to a putative enhancer variant within SIRT3 intron 5 (Bellizzi et al. 2005). The p value was corrected for the same variables as previously detailed

Fig. 2

Longitudinal survival analysis in the TRELONG population after gender stratification. Survival curves were plotted according to genotype and gender to search for a gender-specific effect of the investigated SIRT3 SNPs. a–b rs3825075 survival curve in males and females, respectively. c–d rs4980329 survival curve in males and females, respectively. e–f rs11555236 survival 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)

Fig. 3

SIRT3 Western blotting in peripheral blood mononucleate cells. To assess a possible functional correlation of rs11555236 variants with SIRT3 expression, we selected a group of subjects from the TRELONG study, balanced for gender, and evaluated SIRT3 protein immunoreactivity. Each subject, identified in the figure by the number above the corresponding gel lane, was assessed in duplicate. a–c Representative Western blotting where subjects were divided 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 corresponding PBMC fraction. d Box plot showing the densitometric analysis of the Western blotting data. Optical density values of SIRT3 band were quantified and normalized to α-tubulin. Values were compared by nonparametric Mann–Whitney U test