Survival functionSurvival function 1.008                                                       3 : B24{ 2} =i 3i zi £3 of ° 34“ ad— First Ibs quart — First IL6 quartile2nd IL6- quartile ~ 2nd IL6 quartile° 3rd IL quartile o 3rd IL8 quartileS 7) --+ 4th Le quartile 37 4th IL6 quartilet T T T , T1500 2000 2500 1500 2000 2500Days since interview B Days since interview27 sone gei 2i 3iL. 32x | ata J a3 $First IGF1 quartile —— First |GF1 quartile2nd IGF1 quartile <== 2nd IGF1 quartile° ‘3rd IGF1 quartile o ‘3rd IGF1 quartileSo 4th 1GFt quartile S 7) <o+ 4th 1GF1 quartileT T Yi T Y T1500 2000 2500 1500 2000 2500Days since interviewDays since interview  

Figure 2. Survival curves on the basis of sex and circulating levels of IL-6 or IGF-1. (A) Survival in men stratified by IL-6 level in plasma (in quartiles). (B) Similar analysis in females. (C) Survival analysis according to sex and circulating levels of IGF-1 (in quartiles) in men only. No significant difference came to light considering the 1st quartile as reference and correcting for age. (D) The same analysis as in (C), for women. The individual functions showed no significant differences.

increase in mortality, although the association was attenuated in women.

Table I. Distribution by age of the elderly subjects in the TRELONG study. For a full study description see Gallucci et al. [29].

Age (years) (no. of individuals) Sex (male:female) 70–74 (129) 61:68 75–79 (132) 72:60 80–84 (126) 64:62 85–89 (73) 29:44 90–94 (168) 74:94 95–99 (34) 10:24 100–106 (6) 1:5 Whole study (668) 311:357

We have already shown an age-related decrease in IGF-1 levels that was more significant in males [30]. However, the survival curves of participants were similar when plotted according to IGF-1 level and sex [Figure 2C and 2D]. These data were confirmed in the multivariate regression analysis (data not shown).

We also checked a possible combined effect of IL-6 and IGF-1 on survival. First, we divided our population accord- ing to combinations of IL-6 and IGF-1 (low or high level,

to levels of IL-6 and IGF-1 [Figure 3A]. The statistical analy- sis demonstrated a global effect (log-rank test p < 0.001), even if the individual curves had no significant difference (data not shown). However, the group with reduced performance in survival was the opposite of the reference (high IL-6 and low IGF-1). We also tried to analyze survival according to a combination of IL-6 and IGF-1 level and sex [Figure 3C and 3D]. The situation was similar to the entire population, with a global significant effect (log-rank test p < 0.001 both for males and females); for men and women, the group with the appar- ent reduced longevity had a high level of IL-6 and a reduced level of IGF-1, even if the statistic analysis for the single curve did not evidence any significance.

polymorphic site (a) and of circulating IL-6 (b). CVD: cardiovascular disease (including myocardial infarction, ischemic cardiopathy, periph- eral vasculopathy and chronic heart failure); VC: vascular cerebral disease (including stroke and cerebral vasculopathies). C: regression coefficient; HR:

MEN C HR St. er t-Value Age 0.1 1.11 0.02 4.4 <0.01 CVD 1.14 3.14 0.37 3.06 <0.01 VC −0.01 0.99 0 -1.99 0.05 Cholesterol (total) 0.01 1.01 0.39 0.02 0.99 Diabetes 0.85 2.33 0.34 2.46 0.01 Cancer 0.01 1.01 0.32 0.04 0.97 Smoking 1.35 3.88 1.03 1.32 0.19 IGF1, A/G (ref: A/A) 1.42 4.14 1.04 1.37 0.17 IGF1, G/G (ref: A/A) 0.1 1.11 0.02 4.4 <0.01 WOMEN Age 0.11 1.12 0.02 4.58 <0.01 CVD 0.28 1.32 0.38 0.72 0.47 VC 0.08 1.08 0.42 0.19 0.85 Cholesterol (total) −0.01 0.99 0 −1.99 0.05 Diabetes −0.35 0.71 0.39 −0.88 0.38 Cancer 0.04 1.04 0.45 0.09 0.93 Smoking 0.06 1.07 0.38 0.17 0.87 IGF1, A/G (ref: A/A) 0.41 1.51 0.38 1.07 0.29 IGF1, G/G (ref: A/A) −0.36 0.7 0.45 −0.79 0.43 b. circulating IL-6 MEN C HR St. er t-Value p-Value Age 0.09 1.1 0.02 4.05 <0.01 CVD 0.35 1.42 0.33 1.06 0.29 VC 0.67 1.95 0.41 1.64 0.1 Cholesterol (total) −0.01 0.99 0 −1.49 0.14 Diabetes −0.18 0.83 0.38 −0.49 0.63 Cancer 0.64 1.9 0.36 1.8 0.07 Smoking −0.01 0.99 0.32 −0.03 0.98 IL-6, 2nd quartile (vs. 1st) 0.51 1.67 0.47 1.09 0.28 IL-6, 3rd quartile (vs.1st) 1.02 2.77 0.44 2.31 0.02 IL-6, 4th quartile (vs. 1st) 0.91 2.49 0.46 1.98 0.05 WOMEN Age 0.13 1.14 0.03 4.84 <0.01 CVD 0.05 1.05 0.41 0.12 0.9 VC 0.06 1.06 0.42 0.14 0.89 Cholesterol (total) 0 1 0 −0.92 0.36 Diabetes −0.49 0.61 0.41 −1.19 0.24 Cancer −0.16 0.85 0.54 −0.30 0.76 Smoking 0.25 1.28 0.39 0.64 0.52 IL-6, 2nd quartile (vs. 1st) −0.02 0.98 0.41 −0.06 0.96 IL-6, 3rd quartile (vs. 1st) −0.20 0.82 0.49 −0.40 0.69 IL-6, 4th quartile (vs. 1st) 0.13 1.14 0.03 4.84 <0.01

Discussion

According to the Italian National Institute of Statistics (ISTAT), the province of Treviso has one of the longest life spans in Italy, particularly for women (http://demo.istat.it/) [33]. Therefore, the TRELONG study is uniquely positioned to investigate genetic and environmental factors in longevity. We first considered the genetic variability due to rs2229765 poly- morphism of the IGF-1R gene. The survival curves suggested the homozygous A-allele genotype was a positive predictor of survival in men, in comparison to the genotype G/G; this was confirmed by multivariate regression analysis. We previously reported that in a cross-sectional analysis in the TRELONG population, there was a significantly (p = 0.04) higher A-allele frequency in males over 85 [30]. Another study in Italy by Bonafé et al. reported an increase of the same rs2229765 A-allele in men and women over 85 years of age [19]. To the best of our knowledge, our prospective study that supports the pro-surviving effect of the A/A homozygous genotype, at least in men, is the only one available in the literature.