e-mail: lcaberlotto@ulss.tv.it

e-mail: azanardo@ulss.tv.it

FORGEI, Interdisciplinary Geriatric Research Foundation, Viale Trento Trieste 19, 31100 Treviso, Italy

2009). Rs11555236 was already linked to longevity in the Italian population (Bellizzi et al. 2005; Rose et al. 2003). To assess rs3825075, rs4980329, and rs11555236 genotypes, a gDNA aliquot (about 20 ng) was used in an allelic discrimination assay using a real-time PCR appa- ratus and TaqMan technology according to the manufac- turer's instructions (Applied Biosystems, Foster City, CA). The successful genotyping rate was around 92 %. Subjects' genotypes were independently confirmed in a random sample representing 10 % of the population, with 100 % replication rate. Subjects who were not genotyped were equally distributed in the TRELONG population (subjects belonged to almost all the age brackets) and had no peculiar demographic or survival features in compar- ison to the genotyped population.

Materials and methods

Population

A thorough description of the TRELONG study has already been reported (Gallucci et al. 2007). In short, after the selection of the 13,861 Treviso inhabitants over 70 years of age (17 % of total inhabitants) from the residents listed in the Registry Office of Treviso, partic- ipants were systematically sampled, planning to include 100 participants according to gender and 10-year-age – group, 125 women and 125 men aged 70 79 years, and all people over 100 years. A total of 668 people were selected: 311 men and 357 women (mean age 84.0 – ±8.0 years, range 70.0 105.5 years). A 7-year follow-up was then performed. Included subjects underwent exam- ination from the biologic, clinical, and socioeconomic point of view. A blood sample was collected and each participant was administered with a structured interview. The study protocol, blood collection procedure, and the questionnaire to be administered at home were submitted to and approved by the ethical committee of the National Institute on Research and Care of the Elderly (INRCA, Italy). The protocol included a written informed consent for clinical and genetic studies.

Protein extraction and Western blotting

sample buffer, subjected to SDS-PAGE electrophore- sis, and transferred to a nitrocellulose membrane (BioRad Laboratories, Hercules, CA, USA). The mem- brane was incubated overnight with a primary antibody diluted in 5 % nonfat dried milk in PBS + 0.1 % Tween- 20 (PBS-T). After washing, the membrane was incubat- ed for 1 h with a secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnologies, Santa Cruz, CA) diluted 1:5,000 in 5 % nonfat dried milk in PBS-T. The membrane was finally subjected to chemiluminescence detection using an enhanced re- agent (electrochemiluminescence (ECL) Millipore, Billerica, CA, USA). To quantitatively assess SIRT3 immunoreactivity, the ECL signal was measured by a densitometric method setup by a free image analyzer program (ImageJ, http://rsbweb.nih.gov/ij/), using an α internal normalization control ( -tubulin).

Fasting peripheral blood samples (30 mL) were col- lected by venipuncture; one aliquot was used to sepa- rate peripheral blood mononuclear cells (PBMCs) by a standard Ficoll centrifugation procedure. PBMC pel- lets were washed with ice-cold phosphate-buffered saline (PBS), divided into aliquots and stored at − 80 °C for further analysis. From the enrolled popula- tion of 668 subjects, 590 gave their consent for blood collection and PBMC preparation.

Antibodies

Genomic DNAwas extracted from PBMC pellet using a vacuum-based semiautomated nucleic acid extractor (AB6100, Applied Biosystems, Foster City, CA, USA), checked for concentration by a UV spectrophotometer (Eppendorf, Hamburg, Germany), and stored at 4 °C.

The following primary antibodies were used to run α immunoblotting: anti- -tubulin monoclonal antibody (dilution 1:5,000) (Abcam, Cambridge, UK) and anti- SIRT3 polyclonal antibody (dilution 1:1,000) (Abcam, Cambridge, UK).

SIRT3 We decided to assess rs3825075, rs4980329, and rs11555236 as the first two ones (which we geno- typed in a previous study (Polito et al. 2013)) captured the variation of rs511744 and rs11246020, respectively, that were reported to be overrepresented in long-living elderly of Caucasian ancestry (Halaschek-Wiener et al.

Genotypic and allelic frequency distributions and depar- – ture from Hardy Weinberg equilibrium were assessed

the considered groups, even after gender stratification (data not shown).

– Survival curves were estimated by the Kaplan Meier method. Hazard ratios (HRs) were calculated using Cox proportional hazard model. The propor- tional hazard has been tested using Schoenfeld's residual test, and it has never been rejected, thus confirming the suitability of the model. Multivariate re- gression analysis was performed considering mortality as dependent variable. These statistic analyses were com- “ ” “ ” puted using the package survival of the R software. After correction for possible confounders (diabetes, car- diovascular diseases, cerebral vasculopathies, cancer, cholesterol level, scholarity, age, and gender), results p were considered significant at <0.05, using two-tailed tests of significance.

Prospective analysis