Allele and genotype frequencies were calculated using the genotype counting method, and Hardy-Weinberg Equilibrium (HWE) was assessed by comparing the genotype frequencies with the expected values using a contingency table 2 statistics. We tested genotype association under additive, dominant, and recessive models and employed Cochran-Armitage test of trend for the additive model. We used Pearson's 2 or Fisher's exact test (when expected is <5) to evaluate the dominant model (L*/L* + L*/S* vs. S*/S*), recessive model (L*/L* vs. L*/S* + S*/S*), and allelic association. We expressed the strength of association as odds ratios (O.R.s) with 95% confidence intervals.

For LTL measures, we assessed the normality of distribution by Kolmogorov-Smirnov test, while we analyzed differences between study groups using one-way ANOVA. We expressed data as mean ± S.D. Statistical analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL). We regarded two-tailed probability values of less than 0.05 as statistically significant. We did not perform Bonferroni's correction for multiple testing since we were assessing specific questions on a gene previously tested for association with human longevity, and we were not searching for associations without a priori hypotheses (Pernerger, 1998).

During the last decades, several studies have demonstrated a strong link between telomere length and human lifespan (Barrett and Richardson, 2011; Gomes et al., 2011). Telomere length shortens with age, leading 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 telomere maintenance genes (TERT and TERC) in human longevity.

In this study, we provide novel evidence for TERT in longevity by showing that the L* allele at VNTR MNS16A of the TERT gene influences negatively life expectancy. This result was further supported by the significant LTL attrition that we found along the three age classes, for L*/L* genotype carriers only. 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* genotype 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 suggest that the MNS16A*S allele is a sort of double-edgedIn complex traits, the association of VNTR in the INS gene with type-1 diabetes (Julier et al., 1991), the VNTRs located in the SLC6A3 and SLC6A4 genes with several psychiatric conditions (Ueno, 2003), and the VNTRs in SIRT3 and APOB genes with human longevity (Bellizzi et al., 2005; De Benedictis et al., 1998) has been previously demonstrated. Although we found LTL attrition along the three age classes (p = 0.0001), it remains significant only in L*/L* genotype carriers (p = 0.002) when the analysis was conducted according to MNS16A genotype. This result should be taken with caution due to limited sample size. Moreover, it should be noted that the difference between the average LTL among the two elderly groups (Age Class 2 and Age Class 3) (Table 2 and Fig. 2) seems insignificant, indeed appearing unchanged. This fact is supported by several studies that reported the leveling off of telomere length in advanced age groups (Ishikawa et al., 2012), the dependence of LTL on the health status of centenarians (Terry et al., 2008), and the lack of association of LTL with overall survival or lifespan in elderly persons (Njajou et al., 2009), respectively.

It has been reported that VNTR MNS16A affects the putative promoter region of the antisense transcript of TERT, giving rise to a potential allele-dependent regulation of telomerase expression (Wang et al., 2003). Longer alleles at MNS16A exhibit stronger promoter activity compared to shorter alleles (Wang et al., 2003). This leads to an increased expression of antisense TERT mRNA with a conceivable, at least partial, silencing of sense telomerase transcript (Wang et al., 2003). Accordingly, carriers of the MNS16A*S allele should be noted. It is noteworthy that some TERT polymorphisms correlate to telomere length but do not affect longevity or the parameters of healthy aging (Soerensen et al., 2012). This is probably due to the great environmental component affecting telomere length. Oxidative stress, depression, and uncertainty for the future have been found to significantly correlate with shorter telomeres (Cherkas et al., 2006; Epel et al., 2004; Lung et al., 2007; Yen and Lung, 2013). Thus, environmental factors play a significant role.

Table 2: Mean LTL expressed as T/S ratio among the MNS16A genotypes divided by age classes. Values are expressed as mean S.D. Values showing significant differences are shown in bold. p-Values were obtained by ANOVA.

Genotype | Age Class 1 (N = 24) | Age Class 2 (N = 24) | Age Class 3 (N = 24) | p-Value
---|---|---|---|---
L*/L* (N = 8 for each Age Class) | 1.455 0.274 | 1.019 0.198 | 1.003 0.257 | 0.002
S*/L* (N = 8 for each Age Class) | 1.285 0.331 | 1.035 0.262 | 1.073 0.280 | 0.207
S*/S* (N = 8 for each Age Class) |1.140 0.205 | 1.033 0.328 | 0.966 0.129 | 0.345

*p-Value obtained by comparing the mean T/S ratio along the three age classes.
**p-Value obtained by comparing the mean T/S ratio along the three MNS16A genotypes.