Prospective analysis

the TRELONG study features a longitudinal follow- SIRT3 with mortality data, we decided to reanalyze variability considering mortality as a dependent and plotting survival curves according to genotype (Fig. 1). As mortality might be several other variables, we controlled for TRELONG disorders (diabetes, cardiovascular diseases, vasculopathies, and cancer), risk factors (cho- level, scholarity), age, and gender. We found between rs4980329 and survival, with allele (A) giving an advantage for longevity. HRs, considering the major allele homozy- genotype as reference, were 1.08 and 0.66 for (GA) (AA) genotypes, respectively. A second found for rs11555236, with the minor allele (T) an advantage. For this polymorphism, the using the major allele homozygous genotype were 1.18 and 0.58, respectively. No found for rs3825075. To investigate whether these had a different outcome in males or females, performed a gender-stratified analysis, correcting for the other variables influencing survival listed plots of the TRELONG population according to are shown in Fig. 2. We found a significant effect rs4980329 and rs11555236 minor alleles for p p (corrected values and HR: value, 0.02; HR,

Results

Cross-sectional analysis

We started evaluating the correlation of rs3825075, rs4980329, and rs11555236 with longevity by splitting the TRELONG population around 85 years of age, basing on the 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 p Weinberg equilibrium (HWE) ( =0.82, 0.71, and 0.93 for χ2 test assessing departure from HWE, respectively). Genotypic and allelic frequencies did not differ between

Table 1 Cross-sectional analysis of the genotypic and allelic frequencies of SIRT3 single nucleotide polymorphisms rs3825075, rs4980329, and rs11555236

SNP (no.) Genotype no. (%) Allele no. (%) ≤85y >85y χ2 statistics ≤85y >85y χ2 statistics rs3825075 (n=549) GG 136 (43.9) 117 (48.9) χ2=3.1; df=2; p=0.21 G 418 (67.4) 329 (68.8) χ2=0.24; df=1; p=0.62 GA 146 (47.1) 95 (37.4) A 202 (32.6) 149 (31.2) AA 28 (9.0) 27 (13.7) rs4980329 (n=542) GG 167 (54.7) 138 (58.2) χ2=2.0; df=2; p=0.37 G 458 (75.1) 360 (75.9) χ2=0.10; df=1; p=0.74 GA 124 (40.6) 84 (35.4) A 152 (24.9) 114 (24.1) AA 14 (4.7) 15 (6.4) rs11555236 (n=542) GG 169 (54.9) 137 (58.5) χ2=2.5; df=2; p=0.28 G 454 (73.7) 361 (77.1) χ2=1.7; df=1; p=0.19 GT 116 (37.7) 87 (37.2) T 162 (26.3) 107 (22.9) TT 23 (7.4) 10 (4.3)

no. number, 85y 85 years of age, df degree of freedom, p p value

Lor   183825075 rs4980329 rs11555236184980329 (G/A)183825075 (G/A) 1811555236 (GIT)18511744 (TIC) 1511246020 (C/T)| Le |9 er¢ vy  ¢ 1.0     's3825075-%ges5Bo2Se2c5a :7 p-value:0.93 acs -- GA— AA 0 500 1000 1500 2000 2500 3000(days since interview)  Survival function (%)04 06 08 1.0     0   184980329gzs32g2ap-value:0.03 .... gig- GIA— AA 500 1000 1500 2000 2500 3000(days since interview)1.002 04 06 1811555236p-value:0.040 500 1000 1500 2000 2500 3000(days since interview)

Fig. 1 Genotyping and survival curves of the TRELONG popu- lation. 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 value is corrected for possible confound- ing factors affecting longevity (age, gender, scholarity, cholesterol level, cardiovascular disease, vascular cerebropathies, diabetes, and cancer). c Survival plot of the whole TRELONG population strat- ified 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 report- ed 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

p and value, 0.03; HR, 0.27, respectively), while the association was no longer present in males (corrected p values, 0.96 and 0.52, respectively).

(Bellizzi et al. 2005), we decided to assess whether the rs11555236 genotype correlated with a 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 pro- tein relevant for mitochondrial metabolic homeostasis (Hallows et al. 2008). We analyzed SIRT3 protein level by Western blotting in PBMCs from the TRELONG n population, building up three groups ( =10 subjects

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

06 08 1.0Survival function (%)040.20.008 1.0Survival function (%)04 06020.0Survival function (%)04 06 08 1.00.20.0                  0          500  rs3825075Men08 Survival function (%)04 06    p-value:0.18 .... GIG % p-value:0.64  .... GIG-- GIA -- GIA— AIA a | — AIAo |1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000(days since interview) b (days since interview)rs4980329- ° .Men . Women  08 04 Survival function (%)06   p-value: 0.96 p-value: 0.02sere GIG 3S sere GIG-- GIA -- GIA— AIA — AIA2 |°o } a0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000(days since interview) (days since interview)drs11555236fe Men ~ Women#35BoSo85 x1 . a 5 .p-value:0.52  __ aie ‘i p-value:0.03 __ aic-- GIT © -- GIT| coos T/T ecoe VT| 2 |co. a a a a a, |0 500 1000 1500 2000 2500 3000 0 500 1000 1500 2000 2500 3000(days since interview) f (days since interview)