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Blackburn, 2012; Epel et al., 2004;Willeit et al., 2011). Moreover, sever- al SNPs in TERT and TERC genes were associated to LTL (Codd et al., 2010; Levy et al., 2010; Melin et al., 2012; Shen et al., 2011). The collec- tive evidence clearly suggests the telomere maintenance genes (TERT and TERC) as candidate genes for human longevity.

individuals b66 years old [b73 years old], the second class of individ- uals 66–88 years old [73–91 years old], and the third class of individ- uals >88 years old [>91 years old]. These gender-specific age classes were defined according to demographic information and accounting for the different survival rates of men and women in the Italian popula- tion (Passarino et al., 2006). Briefly, the age classes ranging from 18 to 106 years were obtained on the basis of a synthetic survival curve constructed using historic mortality data taken from the Italian popula- tion from 1890 onward (computed using death counts per calendar year and per year of birth as reported for the Italian population in the Human Mortality Database, available online at www.mortality.org) (Passarino et al., 2006). The study protocol was approved by the Joint Ethical Committee (JEC) University of Camerino-Azienda ASUR Marche ZT-10Camerino,inaccordancewiththeDeclarationof Helsinkiinitsre- vised edition and with international and local regulatory requirements.

and Danish populations, with conflicting results. First, Atzmon et al. (2010), using a cohort of Ashkenazi Jewish centenarians, their off- spring, and offspring-matched controls, found an overrepresentation of synonymous and intronic mutations in TERT and TERC genes among centenarians compared to controls. Moreover, they reported a signifi- cant association of a common TERT haplotype (defined by rs2853669, rs2736098, rs33954691, and rs2853691 SNPs) with both exceptional longevity and longer telomere length. In the second study, Soerensen et al. (2012) investigated two TERC (rs3772190 and rs12696304) and four TERT (the same defining the haplotype associated with lon- gevity by Atzmon et al. (2010)) SNPs in middle-aged, old, and oldest- old Danes (58–100 years) and their association with LTL and longevity. TheyfailedtoreplicatetheassociationofTERTSNPsreportedbyAtzmon et al. (2010), but they found the association of TERC SNPs with both LTL and longevity. They also investigated 11 tag-SNPs, covering the com- mon variation in TERT in Caucasians, on 1089 members of the “1905 cohort” and 736 middle-aged controls, finding no significant associa- tions (Soerensen et al., 2012).

2.2. TERT MNS16A genotyping

Genomic DNA extraction was carried out from peripheral blood through standardized salting-out method and DNA was stored at −20 °C until gene analysis.

TERT VNTR MNS16A was genotyped according to the allele-specific PCR method as previously reported (Wang et al., 2003). PCR was performed using PCR Master Mix (2×) (Fermentas), with a primer set, 5′-AGGATTCTGATCTCTGAAGGGTG-3′ and 5′-TCTGCCTGAGGAAGGAC GTATG-3′ (Sigma-Aldrich, MO, USA). The amplification procedure consisted of an initial denaturing step for 5 min at 95 °C followed by 35 cycles for 30 s at 95 °C, 45 s at 60 °C, and 1 min at 72 °C, as well as a final extension step for 10 min at 72 °C. Each sample was genotyped in duplicate. The PCR products were visualized on a 2% agarose gel containing 0.25 mg/ml of ethidium bromide and genotypes were as- signed as previously reported (Wang et al., 2003): the 243 bp and 272 bp bands were classified as the short (S*) allele, and the 333 bp and 302 bp bands were classified as the long (L*) allele, thus defining the MNS16A genotypes as L*/L*, S*/L* and S*/S*.

In the present study, we attempt to add knowledge on the role of the TERT gene in longevity by analyzing the functional variable num- ber of tandem repeat (VNTR) MNS16A (Wang et al., 2003). The VNTR MNS16A is located down-stream of exon 16 of the TERT gene and up-stream the putative promoter region of an antisense TERT tran- script (Hofer et al., 2011; Wang et al., 2003). MNS16A was found to have two repeat elements forming a 23 bp core sequence or a 26 bp core sequence with a CAT insertion representing a transcription factor bindingsiteforGATA-1.Todate,sixdifferentVNTRalleleshavebeende- scribed.ThetwomostcommonallelesaretheVNTR-302andVNTR-243, followed by rare VNTR-333 and VNTR-272. Recently, two very rare alleles, namely VNTR-364 and VNTR-213, were found in just one sub- ject from Austria (Hofer et al., 2011) and one subject from Taiwan (Chang et al., 2011), respectively. Promoter activity was demonstrated to depend on the number of tandem repeats implicating functionality of MNS16A genotype (Wang et al., 2003). It was proposed that this minisatellite might act as a repressor for the promoter of the TERT anti- sense transcript (Wang et al., 2003).

2.3. LTL measurement