’ Analysis of variance (ANOVA) with Scheffe s test was used for comparing plasma insulin levels, BMI,

WHR, HOMA-IR, HOMA-B cell, energy exependi- ture, among the genotype groups. Logistic regression analysis was used to test the association of gene variants with longevity independently of multiple covariates. Odds ratio and 95% confidence intervals (CI) were also calculated. Associations between genotypes and metabolic profile were analyzed using the general linear model.

In the whole population, HOMA-B cell displayed − an age-related decrease (r= 0.174; p<0.05), whereas HOMA-IR increases with advancing age up to 80 years (r=0.303; p<0.001), while later it declines − (r= 0.235; p=0.001).

Gender ratio confirmed a prevalence of females in the long-lived group. No difference in gender distri- bution was found in control subjects.

All-cause and cause-specific mortality risks with 95% CI were calculated with the Cox proportional hazard model, using left censoring to correct for the delayed entry into the risk set according to age.

As expected and in agreement with that previously reported in recent studies on Italian centenarians, long- lived subjects had a significantly lower BMI, WHR, lower fasting plasma cholesterol, and higher HDL cholesterol. Furthermore, long-lived subjects displayed a preserved insulin action despite a decreased HOMA-B cell compared to controls (Table 1).

Linkage disequilibrium and allele combination analyses were performed by use of the Thesias program based on the stochastic-EM algorithm (Tregouet et al. 2004). The Thesias program allows estimation of both haplotype frequencies and covariable-adjusted haplotype effects by comparison with a reference haplotype taken as the most frequent haplotype in the current analyses. A global test of association between haplotypes and any studied phenotype was performed by means of a chi-square − test with m 1 df in the case of m haplotypes.

eters were also evaluated. Rq displayed an age-related − decrease (r= 0.17; p<0.02) and was negatively corre- − lated with plasma glucose levels (r= 0.242; p<0.001). Similarly, RMR decreases with advancing age up to − 85 years (r= 0.407; p<0.001), without significant − changes in long-lived subjects (r= 0.086; p=.482) − while a negative association with WHR (r= 0.192; − p<0.05), plasma glucose levels (r= 0.214; p=0.001) − and HOMA-IR (r= 0.168; p=0.001) was found.

Statistical analyses were performed using SPSS software package. All metabolic parameters are presented as means±standard deviation (SD).

Due to the well-known effect of age on energy expenditure parameters, to better highlight the diverse role of long-lived vs. control people, the whole study population was subdivided into three age groups. Long-lived subjects (n=82; mean age, 96±3 years) had fasting Rq significantly higher (0.79±0.05 vs.

Results

Clinical characteristics of study groups are reported in Table 1. All subjects were old, not obese or malnourished, in sufficient metabolic control with a greater proportion of female (Table 1).

All (n=722) Control (n=514) p Long-lived Age (yrs) 62±25 49±16 0.001 96±4 Sex (male/female) 321/401 270/244 − 51/157* BMI (kg/m2) 24.7±3.3 25.3±3.0 0.001 23.1±3.5 WHR 0.83±0.01 0.85±0.01 00.01 0.81±0.007 Glucose (mmol/dL) 5.0±1.4 5.2±1.5 0.002 4.8±1.2 Insulin (mU/mL) 10.7±4.4 11.6±4.1 0.0001 8.6±4.4 Triglycerides (mg/dl) 109±48 110±54 0.540 107±38 Total cholesterol(mg/dl) 197±48 207±51 0.020 193±39 HDL cholesterol(mg/dl) 54.3±11.7 48.5±7.2 0.001 54.1±12.1 HOMA-IR 2.46±1.29 2.70±1.28 0.0001 1.85±1.13 HOMA-B 40.4±20 43.3±19 0.0001 33.3±21

0.75±0.05; p<0.001) than aged subjects (n=65; mean age, 70±4 years) but lower (0.79±0.05 vs. 0.80± 0.07; p<0.05) than adult subjects (n=113; mean age, 40±12 years). In addition, long-lived subjects had RMR greater than aged subjects (1,394±181 vs. 1,184±204; p<0.001) but not different from ones found in adults (1,394±181 vs. 1,440±157; p=NS)

A strong linkage disequilibrium among the three ′ UCP2 gene polymorphisms studied was observed (D values were between 0.97 and 0.99, whereas r2 varied from 0.75 to 0.88). Since these three variants would be predicted to provide identical/nearly identical − − genotypic information, the 866 G/A and 8 ins/del variant were excluded from further analyses.

The genotype frequency distributions of all var- iants studied in control and long-lived people are reported in Table 2. As previously demonstrated, carriers of the Asp1057Asp-IRS2 genotype and carriers the A/A genotype of IGF1 R gene were found to be more represented among long-lived in comparison to young people (Table 2). Both IRS2 Gly/Asp and IGF1R G/A polymorphism were signif- icantly associated with an increased probability to

Genetic analysis

In all study groups, the genotype frequencies of all – gene variants studied respected the Hardy Weinberg equilibrium (p>0.05 for all the SNP investigated). The genotypic frequencies observed for all gene variants studied were almost similar to those reported in previous studies among Caucasians.