2.4. Statistical analysis

Allele and genotype frequencies were calculated by genotype counting method. Hardy–Weinberg Equilibrium (HWE) was assessed by comparing the genotype frequencies with the expected values using a contingency table χ2 statistics. Genotype association was tested under additive, dominant and recessive models. Cochran–Armitage test of trend was employed for the additive model. Pearson's χ2 or Fisher's exact test (when expected is b5) was used to evaluate dominant model (L*/L* + L*/S*vs.S*/S*),recessivemodel(L*/L*vs.L*/S* + S*/S*),andal- lelic association. The strength of association was expressed as odds ratios (O.R.s) with 95% confidence intervals. For LTL measures, the normality of distribution was assessed by Kolmogorov–Smirnov test, while the differ- ences between study groups were analyzed using one-way ANOVA. Data are expressed as mean ± S.D. Statistical analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL). Two-tailed probability values of less than 0.05 were regarded as statistically significant. Bonferroni's correc- tion for multiple testing was not performed, since we are assessing spe- cific questions on a gene previously tested for association with human longevity, and we are not searching for associations without a priori hy- potheses (Pernerger, 1998).

4. Discussion

Duringthelastdecadeseveralstudieshavedemonstratedastronglink between telomere length and human lifespan (Barrett and Richardson, 2011;Gomesetal.,2011).Telomerelengthshortenswithage.Progressive shortening of telomeres leads to senescence, apoptosis, or oncogenic transformation of somatic cells, affecting the health and lifespan of an individual. Shorter telomeres have also been associated with increased incidence of diseases and poor survival (Shammas, 2011). Thus, recent studies attempted to uncover the role of the telomere maintenance genes (TERT and TERC) in human longevity (Atzmon et al., 2010;

3. Results

3.1. MNS16A genotype and allele distributions

Thealleleand genotypefrequency distributionsof theMNS16A were compared among the age classes, males and females together because no significant differences were noticed between the sexes (data not shown). No significant deviation from HWE expectation was detected among age classes (Supplementary Table 1). The two very rare alleles

Here, we provide novel evidence for TERT in longevity; in the Cen- tral Italy population we studied, that the L*allele at VNTR MNS16A of the TERT gene influences negatively life expectancy. This result was

Table 1

TERT MNS16A genotype and allele distributions in age classes studied. O.R. values showing significant differences are shown in bold.

Genotype (N, %) Allele (N, %) Comparison 1 Comparison 2 (Age Class 2 vs. Age Class 1) (Age Class 3 vs. Age Class 2) Group L*/L* S*/L* S*/S* L* S* Additive (L*/L* vs. L*/S* vs. S*/S*) Additive (L*/L* vs. L*/S* vs. S*/S*) Common O.R. 1.099 p = 0.415 Common O.R. 0.749 p = 0.019 Age Class 1 (N = 333, 125 (37.5%) 156 (46.9%) 52 (15.6%) 406 (61.0%) 260 (39.0%) Dominant (L*/L* + L*/S* vs. S*/S*) Dominant (L*/L* + L*/S* vs. S*/S*) females 189, males 144) O.R. 1.240 (0.841–1.828) p = 0.277 O.R. 0.579 (0.378–0.886) p = 0.011 Age Class 2 (N = 539, 209 (38.8%) 260 (48.2%) 70 (13.0%) 678 (62.9%) 400 (37.1%) Recessive (L*/L* vs. L*/S* + S*/S*) Recessive (L*/L* vs. L*/S* + S*/S*) females 280, males 259) O.R. 1.054 (0.795–1.397) p = 0.718 O.R. 0.778 (0.553–1.095) p = 0.149 Age Class 3 (N = 200, 66 (33.0%) 93 (46.5%) 41 (20.5%) 225 (56.3%) 175 (43.7%) Allele (L* vs. S*) O.R. 1.085 Allele (L* vs. S*) O.R. 0.759 females 100, males 100) (0.890–1.324) p = 0.419 (0.601–0.958) p = 0.020 Comparison along the χ2 = 6.91 d.f. 4 p = 0.141 χ2 = 5.42 d.f. 2 p = three Age Classes 0.067

Women, Age Class 1: b73 years old; Class 2: 73–91 years old; Class 3: >91 years old Men, Age Class 1: b66 years old; Class 2: 66–88 years old; Class 3: >88 years old subjects.

display higher telomerase activity than the L*/L* genotype carriers (Wang et al., 2003). This evidence clearly supports the results we obtained herein, where L*/L* subjects are less likely to attain longer life-span probably because their telomerase may be less effective in preserving telomere length. Even if this proof of principle remains to be tested, a further clue corroborating our hypothesis comes from our results on LTL attrition along the three age classes, that resulted significant only for L*/L* genotype carriers.

Mean T/S1.5o0.50.0 

Several association studies of MNS16A in cancer can further help us to address its role in human longevity. A survival analysis on 299 non- Hispanic white patients with glioblastoma multiforme demonstrated that L* carriers have a worse survival duration compared to S*/S* geno- type carriers (Wang et al., 2006). L*/L* genotype carriers were also found to display a worse overall survival in surgically resected non- small cell lung cancer (Jin et al., 2011). On the other side, the S* allele seems to confer an increased risk for glioma (Carpentier et al., 2007), breast cancer (Wang et al., 2008), colorectal cancer (Hofer et al., 2011) and lung cancer (Jin et al., 2011). Thus, these findings may sug- gest that the MNS16A*S allele is a sort of “double-edged sword” with conflicting roles of increasing the risk of cancer development but, at the same time, improving survival in some cancer types. This intricate tanglecanbeunraveledbyassumingthatthestrongertelomeraseactiv- ity of S* carriers can not only promote cancer cell immortalization and tumor development, but also expands normal organismal and cellular life-span.

Fig. 2. Mean LTL expressed as T/S ratio among the three age classes studied. Values are expressed as mean ± S.D. Twelve females and twelve males were studied for each age class. Age Class 1, mean age = 39.7 ± 3.7 years; Age Class 2, mean age = 80.3 ± 1.0 years; Age Class 3, mean age = 97.9 ± 2.5 years.