2.3. LTL measurement

A total of 72 samples were selected from our population to check a possible genotype-dependent telomere length variability along the three age classes studied. Eight samples (4 males and 4 females) were chosen for each genotype (L*/L*, S*/L*, S*/S*) from each age class. The total amount of telomeric DNA was measured by monochrome multi- plex Real-Time Quantitative PCR, by measuring for each sample the relative amount of telomeric DNA (T) as compared to the amount of a single copy gene DNA (S) in the same sample (T/S ratio) (Cawthon, 2009). This assay allows the measurement of both telomere and single copy gene (albumin) signals in the same well by using 2 specifically designed couples of primers: telg (5′-ACACTAAGGTTTGGGTTTGGGTTT GGGTTTGGGTTAGTGT-3′), telc (5′-TGTTAGGTATCCCTATCCCTATCCCTA TCCCTATCCCTAACA-3′), albugcr2 (5′-CGGCGGCGGGCGGCGCGGGCTG GGCGGCCATGCTTTTCAGCTCTGCAAGTC-3′), and albdgcr2 (5′-GCCCGG CCCGCCGCGCCCGTCCCGCCGAGCATTAAGCTCTTTGGCAACGTAGGTTTC- 3′). PCR reactions were conducted on a BioRad MyiQ single color ther- mal cycler using iQ5 2.0 Standard Edition Optical System Software to collect raw data. A total 25 μl of PCR reaction mix containing 20 ng ge- nomic DNA, SYBR Green JumpStart Taq ReadyMix 2× (Sigma), 1 M be- taine, 1 mM DTT, 100 nM telg, 900 nM telc, 700 nM albugcr2, and 500 nM albdgcr2 was prepared for each sample. Each sample as well as calibrator sample and standard curve points were run in triplicate. During the thermal cycle (95 °C for 15 min; 34 cycles at 98 °C for 2 s, 48 °C for 60 s, 74 °C for 15 s with signal acquisition, 84 °C for 20 s, 85 °Cfor15 swithsignalacquisition;meltingcurvestep)telomeream- plification signal is collected at 74 °C in the early cycles when albumin signal is under baseline, while acquisition at 85 °C provided the albu- min Ct values. To check for PCR reaction specificity, a melting analysis

Herein, we tested the association of VNTR MNS16A of TERT with human longevity by performing a cross-sectional study on a popula- tion from Central Italy divided into three gender-specific age classes defined according to demographic information and accounting for the different survivals between sexes. We also determined LTL in a se- lected group of subjects (age-, sex- and genotype- matched) to assess the association of VNTR MNS16A with telomere length.

2. Material and methods

2.1. Demographics

Peripheral blood was obtained from 1072 (569 females and 503 males) unrelated individuals, 18–106 years old, recruited from the same geographical area of Central Italy (Marche region) on the eastern side of the Apennines Mountains. The whole population studied is com- posed of Caucasian individuals. The same donors provided information concerning their health condition so as to ascertain that no pathological condition existed (e.g. cancer, diabetes, heart diseases, hypertension, obesity, and chronic inflammatory diseases). Subject's age was self- reported and was corroborated with official documentation. Almost all subjects were recruited in blood donor centers and through fam- ily physicians. The population under study was divided into three sex-specific age classes: for men [women], the first class consists of

 

VNTR-364 and VNTR-213 were not present in our population. In Fig. 1 we report the electrophoretic patterns of the nine genotypes found in the present study. In accordance with a previous report (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. The MNS16A genotypes were therefore defined as L*/L*, S*/L* and S*/S* and their distribution along the three age classes is reported in Table 1. While both genotype and allele distributions did not change significantly along the three age classes (Table 1; genotype:χ 2 = 6.91 d.f. 4 p = 0.141; allele: χ2 = 5.42 d.f. 2 p = 0.067), significant associa- tions of TERT VNTR MNS16A were found in Comparison 2 (Age Class 3 vs. Age Class 2) under both additive (odds ratio [O.R.] 0.749; p = 0.019) and dominant (O.R. 0.579; p = 0.011) models (Table 1). The MNS16A*L allele was significantly underrepresented in Age Class 3 (O.R. 0.759; p = 0.020) compared to Age Class 2 (Table 1).

Fig. 1. Representative electrophoresis gel displaying the nine MNS16A genotypes found in our population. Lanes 1 and 11: 100 bp ladders (Fermentas, 100 bp plus DNA ladder 100–3000 bp), lane 2: 243/243 (S*/S*); lane 3: 302/302 (L*/L*); lane 4: 333/333 (L*/L*); lane 5: 243/272 (S*/S*); lane 6: 272/302 (S*/L*); lane 7: 302/333 (L*/L*); lane 8: 243/302 (S*/L*); lane 9: 272/333 (S*/L*); lane 10: 243/333 (S*/L*).

stepwasincluded attheend of eachPCRrun:nonon-specificPCRprod- uct was detected. In our experimental conditions the standard curves' correlation coefficients were all R2 > 0.99.

3.2. Average LTL and MNS16A

Age-associated telomere shortening is a well documented feature of leukocyte cells from human blood (Lindsey et al., 1991; Slagboom et al., 1994). LTL was measured on 72 samples (age-, sex- and genotype-matched), selected from the whole population studied and expressed as T/S ratio, to detect the effect of MNS16A genotype on LTL. The mean value of T/S ratio was 1.111 ± 0.290, ranging from 0.683 to 1.833, and it followed a normal distribution in the study population (Kolmogorov–Smirnov test p = 0.153). No significant dif- ference was noticed between the sexes (females, 1.099 ± 0.283; males 1.124 ± 0.301; p = 0.721). As expected, LTL showed a signifi- cant age-dependent cross sectional decrease (Age Class 1, mean T/S = 1.315 ± 0.298; Age Class 2, mean T/S = 1.029 ± 0.256; Age Class 3, mean T/S = 1.011 ± 0.223; p = 0.0001) (Fig. 2). LTL did not differ among the three MNS16A genotype groups (Table 2). However, the LTL attrition along the three age classes was significant only for MNS16A L*/L* genotype carriers (p = 0.002) (Table 2).

2.4. Statistical analysis