region of the gene. However, the combined effect of these variants with other genes involved in energy metabolism and longevity has not been investigated in depth. In this study, we analyzed the interchromosomal interactions among IGF1R, IRS2, and UCP2 genes to determine their impact on metabolic profile, energy expenditure parameters, and mortality rate in a group of 722 Italian subjects. Our results showed that the AAV allele combination of these genes was associated with a decreased risk of all-cause mortality and an increased probability of reaching extreme old age. Additionally, individuals with this allele combination exhibited lower HOMA-IR, higher respiratory quotient, and resting metabolic rate. These findings suggest that the combined effect of IGF1R, IRS2, and UCP2 genes on energy metabolism and age-related metabolic remodeling capacity is responsible for the observed effects on longevity. Overall, our study highlights the importance of investigating gene-gene interactions in understanding the genetic basis of human longevity.were calculated by using the homeostasis model assessment (HOMA) method. Statistical analysis was performed using SPSS software version 20.0 (IBM Corp., Armonk, NY). The chi-square test was used to compare genotype distribution and allele frequency between groups. Continuous variables were expressed as means ± standard deviation (SD) and were compared using Student's t-test or ANOVA, as appropriate. A p-value <0.05 was considered statistically significant. Logistic regression analysis was used to evaluate the association between genotype and longevity, adjusting for potential confounding factors such as age, sex, BMI, and smoking status.(HOMA-B cell) were calculated according to the homeostasis model assessment (HOMA) (31-32): insulin resistance (IR) = FIG/22.5 and B cell function: 20FI/G 3.5, where FI = fasting insulin (mU/ml) and G = fasting glucose (mmol/l; Matthews et al. 1985; Bonora et al. 2000). To approximate normal distributions, plasma insulin, HOMA-IR, and HOMA-B cell were logarithmically transformed and used in all calculations and back-transformed for result presentations. 

Genetic analysis: Genomic DNA was obtained from blood lymphocytes collected into EDTA-containing tubes using a commercial DNA extraction kit (Illustra, GE Healthcare UK Limited, Buckinghamshire, HP7 9 NA, UK). For each single-nucleotide polymorphism (SNP), the genotypic and allelic frequencies were calculated. The Chi-square test was used to compare the expected genotypic frequencies with the actual frequencies observed based on the Hardy Weinberg equilibrium. The difference in genotype frequency between cases and controls was analyzed by standard contingency Chi-square test. In all subjects, the following UCP2 gene polymorphisms on Chromosome 11 were evaluated using PCR and restriction analysis: Ala55Val in exon 4 (rs660339), 866 G/A in the 5' region (rs659366), 45 ins/del in position 173247 of the AC019121. In particular, Ala55val gene polymorphism was evaluated using Analysis of variance (ANOVA) with Scheffe's test used for comparing plasma insulin levels, BMI, WHR, HOMA-IR, HOMA-B cell, energy expenditure 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 increased with advancing age up to 80 years (r=0.303; p<0.001), while later it declined (r=0.235; p=0.001). Gender ratio confirmed a prevalence of females in the long-lived group. No difference in gender distribution 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 preserved insulin action despite a decreased HOMA-B cell compared to controls (Table 1). Linkage disequilibrium and allele combination analyses were performed using 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 degrees of freedom in the case of m haplotypes.

Parameters were also evaluated. Rq displayed an age-related decrease (r=0.17; p<0.02) and was negatively correlated with plasma glucose levels (r=0.242; p<0.001). Similarly, RMR decreased with advancing age up to 85 years (r=0.407; p<0.001), without significant changes in long-lived subjects (r=0.086; p=0.482) 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.