Methods

Population sample recruitment and ethics

The TRELONG study has been described in detail elsewhere [29]. In brief, it started in 2003 in the municipality of Treviso. Participants were systematically sampled from the list of resi- dents at the Registry Office of Treviso, based on an initial plan to include 100 participants per sex and 10-year age group; selection criteria included 125 women and 125 men aged 70–79 years and everyone over 100 years old. Of the 670 eligi- ble, 668 participated (99.7% response rate): 311 men and 357 women, aged 70 years and older (mean age 84 ± 8 years). An interviewer-administered questionnaire and a blood sample were collected at participants’ homes. Baseline characteristics of this study population and methodological details have been published previously, and form the basis for these analyses. The elderly sample was followed for seven years beginning in 2003; the dates of death were collected. Those who survived were assessed at the age they were on June 20th, 2009. The participants were evaluated using biologic, clinical and socio- economic measures, with blood samples and a structured interview.

Results

Effects of rs2229765 and rs1800795 on survival

The age and sex of the TRELONG study population are sum- marized in Table I. The sample included individuals from 70 years of age and older (six were ultracentenarians). The male- to-female ratio was 0.87. The survival curves were plotted by sex and stratifying for IGF1R rs2229765 genotype [Figure 1A and 1B]. No significant effect comes to light. For men, the odds ratio [and 95% confidence interval (CI)], corrected for age, and considering the A/A genotype as reference were: A/G 2.29 [0.54–9.59], p = 0.25; G/G 2.54 [0.57–11.27], p = 0.22. For women, the odds ratio [and 95% CI], corrected for age, and considering the A/A genotype as reference were: A/G 0.81 [0.38–1.73], p = 0.60; G/G 0.95 [0.43–2.10], p = 0.91. However, a multivariate regression analysis including several confound- ers (age, CVD, VC, total cholesterol, diabetes, cancer and smoking) (Table II) indicated that the rs2229765 G/G geno- type significantly increased the hazard ratio in comparison to the A/A genotype, suggesting a positive role of the latter on survival.

The study protocol was approved by the Ethics Committee of the National Institute on Research and Care of the Elderly (INRCA, Italy). Written informed consent was obtained from each participant or from a legally responsible person.

Blood sampling, rs2229765 and rs1800795 genotyping

Blood samples (about 30 mL) were collected by veni- puncture. One part was centrifuged to isolate leukocytes to be used for genomic DNA (gDNA) extraction, using a semi-automated nucleic acid extractor (AB6100, Applied Biosystems, CA, USA). Another portion was centrifuged at 2000 rpm for 10 min at 4°C (with sodium EDTA as the anticoagulant) to separate the plasma, which was stored at−80°C until needed. Consent for blood collection was obtained from 590 of the 668 participants and 587 plasma

For IL-6 rs1800795, we did not find any influence on sur- vival [Figure 1C–1D]. In fact, for men the odds ratio [and 95% CI], corrected for age, and considering the C/C genotype as reference were C/G 0.77 [0.29–2.03], p = 0.60; G/G 0.69 [0.25– 1.87], p = 0.47, while for women the odds ratio [and 95% CI], corrected for age, and considering the C/C genotype as refer- ence were C/G 2.3 [0.55–10.14], p = 0.24; G/G 2.2 [0.52–9.40], p = 0.28. A multivariate regression confirmed the lack of influ- ence on survival (data not shown).

The rs2229765 and rs1800795 genotypes were assessed as previously described [30,31].

Effects of circulating IGF-1 and IL-6 on survival

interval (CI)] for the single curves, corrected for age and con- sidering the 1st quartile as reference, were the following: 2nd quartile: 2.3 [0.95–5.71], p = 0.06; 3rd quartile: 3.5 [1.5–8.1], p = 0.003, 4th quartile: 3.0 [1.2–7.1], p = 0.012. For women, the odds ratio [and 95% CI] for the single curves, corrected for age and considering the 1st quartile as reference were as follows: 2nd quartile, 0.98 [0.4–2.1], p = 0.96; 3rd quartile 0.8 [0.3–2.0], p = 0.65; 4th quartile 1.1 [0.5–2.4], p = 0.80.

We have previously reported that circulating levels of IL-6 rise with age and correlate with disability and mortality [32]. We have now extended this observation, considering a possible sex-specific pattern [Figure 2A and 2B]. IL-6 had a more pro- nounced effect on survival in men than in women. In fact, in men IL-6 levels in the second quartile were already marginally significant in increasing mortality risk (p = 0.06) and became a clearly deleterious predictor of survival from the third quartile onwards. In men, the calculated odds ratio [and 95% confidence

Multivariate analysis, reported in Table II, showed that IL-6 levels in the fourth quartile were associated with a significant

Survival functionSurvival function     Men       T1500 2000Days since interviewMen2500          xa43— cic34 GIG     T T1500 2000Days since interview2500Survival function  Survival functionWomen       T1500 2000Days since interviewWomen2500      T1500 2000Days since interview2500  

Figure 1. Survival analysis according to sex and genotype. (A) Survival for males according to IGF-1R rs2229765 (G/A) polymorphic site. (B) Survival for females according to IGF-1R rs2229765 (G/A) genotype. (C) Survival for males grouped according to IL-6 rs1800795 (C/G) polymorphic site. (D) Survival for females stratified for IL-6 rs1800795 (C/G) polymorphism.