The analyses were carried out on a group of participants carefully selected from the cohort of the PolSenior study. Information on age, sex, socio-demographic characteristics, medical history, health status, family history and lifestyle were obtained based on detail questionnaires in a standardized manner (Bledowski et al. 2011).
In the group of 1842 subjects with assessed telomere length (data not shown), 277 participants were treated for diabetes and among them 140 individuals were obese, according to WHO criteria. The number of obese participants without T2DM was 411. From this cohort, participants with inflammatory conditions, namely rheumatoid diseases, acute and chronic infections, history of cancer, stroke, congestive heart failure, dementia or chronic obstructive pulmonary disease (except hypertension) were excluded. The group of T2DM included only patients previously diagnosed and already pharmacologically treated for diabetes with insulin. The inclusion criterion was the coexistence of insulin resistance (HOMA-IR values above 2.5). Selected subjects from the group without T2DM were matched with age, sex and in equal proportion of men and women.
The final group, strictly fulfilling the presented above criteria, consisted of 140 participants, of which 70 were obese with T2DM and 70 were control obese without T2DM. For this group, the power of statistical tests, for calculated variance, was satisfied. Specifically, 28 of age 65–69 years included 15 with T2DM, 28 of age 70–74 years included 14 with T2DM, 28 of age 75–79 years included 13 with T2DM, 28 of age 80–84 years included 14 with T2DM and 28 of age 85–89 and 90–95 years included 14 with T2DM. Detailed characteristics of both groups are presented in Table 1.
Whole blood samples were obtained from all participants. Genomic DNA was extracted by salting-out method and stored at − 80 °C. The concentration and purity of the DNA were assessed by UV spectroscopy (Nanodrop, Thermo Fisher Scientific Inc., Wilmington, DE, USA).
The TL assay
In white blood cells, TL was measured using the real-time quantitative polymerase chain reaction (Q-PCR) as described previously (Gutmajster et al. 2013). Briefly, the amount of telomeric DNA (T) was divided by the amount of single-copy control gene DNA (S) which encodes acidic ribosomal phosphoprotein P0 (36B4, accession number NC_000012.12), producing a relative measurement of the telomere length (T/S ratio). Results were related to a control sample, used for standard curve generation. The quality of PCR products was assessed by the melting curve analysis. All samples were run in triplicates, and the control sample (Human Genomic DNA, ROCHE, Germany) was run in each experiment to ensure correct normalization among experiments.
DNA amplification was performed using the mix OptiTaq™ PCR Master Mix (Eurix®) as follows: 5 min at 94 °C, 35 cycles at 94 °C for 30 s, 56 °C for 30 s, 72 °C for 1 min 30 s, with a final step at 72 °C for 7 min. Primer sequences were as follows: forward primer for TERT (TERT_F): 5′ATTCGACCTCTCTCCGCTGG3′; reverse primer (TERT_R): 5′CTGGAAGGTGAAGGGGCAG3′. PCR product was treated with exonuclease/alkalic phosphatase mix (Eurix®).
The sequencing PCRs were performed with the BigDye v.3.1 (Fisher Scientific, USA) with internal primers and sequences thanks to courtesy of Prof. Grzybowska (Varadi et al. 2009). Products were cleaned with the BigDye Terminator X (Fisher Scientific, USA). DNA sequencing was performed using Sanger’s technique and analyzed with the ABIPrism3130xl instrument (Fisher Scientific, USA). Results were analyzed with the Blast software (NCBI, USA).
Serum total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, glucose, uric acid and C-reactive protein concentrations were assessed by an automated system (Modular PPE, Roche Diagnostics GmbH, Mannheim, Germany) in a single certified laboratory.
Serum insulin concentration was assessed by electrochemiluminescence method (ECLIA) using commercially available kits and the Cobas E411 analyzer (Roche Diagnostics GmbH, Mannheim, Germany). Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated with the standard formula: HOMA-IR = fasting serum insulin (μIU/ml) × fasting glucose (mg/dl)/405.
The plasma concentration of interleukin 6 (R&D Systems, Mineapolis, MN, USA) and adiponectin (B-Bridge International Inc., San Jose, CA, USA) was measured by the immunoenzymatic (ELISA) method.
Results from both the assays and tests and from the questionnaire-derived data on gender, age, body mass index (BMI), blood pressure and smoking status were analysed. Characteristics of the study population are presented as mean and standard deviation (SD) or median and lower-upper quartile (for non-normal distribution of the data). Categorical variables are reported as frequency and percentage. Pearson’s or Spearman’s correlation coefficients were calculated whenever necessary. Comparison between mean values in the two groups (patients and controls) according to RTL was evaluated using the Student’s t test or the Mann-Whitney U test when applicable. The association between TERT genotypes and variables of interest was initially assessed using multivariate logistic regression analysis, and then comparisons of groups were performed using chi-square test. The lowest analysed minor allele frequency (MAF) was accepted as 0.23, and the calculated power of the test was 0.81, which is the value statistically acceptable for the number of participants in this study (140). Univariate analysis of the association of RTL, anthropometric and biochemical findings between TERT genotypes of SNPs was conducted using Pearson correlation. For each SNP, Hardy-Weinberg equilibrium was assessed according to methods described elsewhere www.oege.org/software (Rodriguez et al. 2009). The association between TERT genotypes and T2DM risk was determined by calculating the odds ratio (OR) and 95% confidence interval (CI) using binary logistic regression. All the statistical analyses were performed using Statistica v.12 (StatSoft, DELL, USA), and the criterion for statistical significance was p < 0.05.