Control Long-lived Odds ratio (95% CI) p crude p Adjusteda IGF1R GG 35.0 (171) 25.1(46) 1 1 GA 46.3 (226) 45.9 (84) 1.382 (0.91–2.08) 0.123 1.66 (1.01–2.73) .043 AA 18.6 (91) 29.0 (53) 2.165 (1.35–3.46) 0.001 2.18 (1.24–3.84) .006 χ2=10.6; p=0.005 Allele frequency g 58.2 (568) 48.1(176) 1 1 a 41.8 (408) 51.9 (190) 1.50(1.18–1.91) 0.001 1.58 (1.03–2.43) .034 Fisher’s test: p=0.001 IRS2 Gly/Gly 46.3 (209) 35.2 (51) 1 1 Gly/Asp 41.9 (189) 48.3 (70) 1.518 (1.03–2.28) 0.034 1.61 (1.01–2.60) .048 Asp/Asp 11.8 (53) 16.6 (24) 1.856 (1.07–3.28) 0.042 1.84 (0.93–3.64) .079 χ2=6.11; p=0.04 Allele frequency g 67.3(607) 59.3(172) 1 1 a 32.7(295) 40.7 (118) 1.41(1.07–1.85) 0.013 1.80(1.01–3.22) .044 Fisher’s test: p = 0.01 UCP2-55 Ala/Ala 49.3(232) 43.1 (50) 1 1 Ala/Val 42.0 (198) 45.7 (53) 0.84(0.42–1.68) 0.632 1.07 (0.48–2.39) .324 Val/Val 8.7 (41) 11.2 (13) 0.68 (0.33–1.36) 0.276 0.73 (0.33–1.63) .452 χ2=1.65; p=0.43 Allele frequency t 70.3(662) 65.9 (153) 1 1 c 29.7 (280) 34.1(79) 1.22 (0.90–1.65) 0.200 1.43(0.89–2.32) .130 Fisher’s test: p = 0.03

found. In particular, Asp/Asp-IRS2 and Val/Val-UCP2 genotypes had the highest RMR and RQ. Accordingly, the effect of Gly1057Asp-IRS2 and UCP2-Ala55Val polymorphisms on energy expenditure was assessed by means of a general linear model (GLM) ANOVA, including age, sex, IR (HOMA), and BMI, as cova- riates. The analysis revealed higher Rq (0.82±0.07 vs. 0.80±0.06 and 0.78±0.06, F=15.47, p=0.0001) and RMR (1,420±135 vs. 1,377±173 and 1,324±220, F= 9.27, p=0.001) in subjects carrying Asp/Asp genotype in comparison to Gly/Asp and Gly/Gly genotype, as well as in Val/Val individuals in comparison to Ala/Ala and Ala/Val individuals: Rq (0.83±0.05 vs. 0.79±0.06 and 0.80±0.06, F=4.129, p=0.01) and RMR (1,431± 139 vs. 1,340±197 and 1,334±208, F=3.881, p=0.03)

Test for the association between UCP2-Ala55Val polymorphism and longevity was not statistically significant.

Genotype and phenotype correlation

The impact of genetic variability at IGF1R (G to A transition at nucleotide 3174), IRS2 (Gly1057Asp), UCP2 (Ala55Val) loci on anthropometric, metabolic and energy expenditure parameters, was tested in univariate analysis (Table 3). No significant differ- ences were observed among the genotypes regarding BMI and beta cell function. Subject with IGF1R-AA genotype had lower plasma glucose levels and insulin resistance degree compared to GG and GA carriers. Subjects with IRS-2 Asp/Asp genotype had lower plasma insulin levels compared to Gly/Gly and Gly/ Asp. Different energy expenditure parameters among Gly/Aps IRS2 and Ala/Val UCP2 genotypes were

Haplotype analysis

Considering the possibility of interchromosomal interaction, allele combination analysis of IGF-1, IRS2, UCP variants have been performed. Allele combination frequencies in long-lived and control

Table 3 Anthropometric and metabolic variables among different IGF1R, IRS2, UCP2 genotypes

SNP Variable Homozygous for Heterozygous Homozygous for p the common allele the rare allele IGF1R G→A BMI 25±3 24±3 24±3 0.297 Insulin 11.0±4.7 10.9±4.3 10.5±4.2 0.544 Glucose 94.9±32.5 90.38±22.1 87.6±23.4 0.027 HOMA-IR 2.64±1.53 2.46±1.21 2.27±1.03 0.031 HOMA-B cell 40.7±22.6 41.3±19.5 41.5±20.4 0.922 RMR 1352±231 1658±144 1795±224 0.685 RQ 0.80±0.06 0.79±0.07 0.80±0.05 0.623 IRS-2 Gly→Asp BMI 24±3 24±3 25±3 0.574 Insulin 11.4±3.7 10.7±4.1 10.0±4.9 0.022 Glucose 91.4±25.23 91.7±26.2 87.6±17.4 0.426 HOMA-IR 2.58±1.15 2.44±1.20 2.23±1.31 0.067 HOMA-B cell 43.0±17.8 40.2±18.8 38.4±20.3 0.097 RMR 1324±213 1381±175 1421±133 0.003 RQ 0.78±0.05 0.80±0.06 0.82±0.07 0.000 UCP2 Ala→Val BMI 24±2 25±3 25±3 0.891 Insulin 10.9±3.6 11.2±4.7 10.0±3.6 0.148 Glucose 92.8±28.8 91.8±26.1 91.3±26.8 0.894 HOMA-IR 2.53±1.21 2.57±1.31 2.33±1.31 0.449 HOMA-B cell 40.8±16.5 42.5±22.2 37.3±14.9 0.172 RMR 1334±204 1347±194 1430±137 0.026 RQ 0.79±0.06 0.79±0.07 0.83±0.05 0.004

– analyses (CVD, HR 0.88; 95% CI, 0.75 1.32; p=0.325; – cancer mortality, HR 0.96; 95% CI, 0.36 2.56; p= – 0.938; other causes, 0.75; 95% CI, 0.26 2.16; p=.601) across different allele combination. By contrast, after adjustment for sex, age, BMI, and HOMA, there were less all-cause deaths in the individual carrying the AAV allele combination compared to reference (HR – 0.72; 95% CI, 0.63 0.91; p=0.03).

subjects are shown in Table 4. When the most common allele combination consisting of the three major alleles, G-Gly-Ala, is considered as reference, as expected, the all-minor-allele combination A-Asp- Val (AAV) was associated with an increased proba- bility to reach extreme old age (OD=3.185 95% CI, – 1.63 6.19; p<0.0006). The effect of each allele according to the different background has been reported in Table 4. Further allele combination phenotype analyses revealed a significant association between A-Asp-Val allele combination and HOMA-IR and RQ and RMR parameters after adjustment for age and sex and BMI and WHR. This allele combination carriers − − had HOMA-IR (Diff= 0.532 95% CI, 0.886 to − 0.17; p=0.003) 7.6% lower and RQ (Diff=0.0363 95% CI, 0.014 to 0.05; p=0.001) and RMR (Diff= − – 101.80693 95% CI, 5.26 204.278; p=0.038), respec- tively, 8.9% and 12%, higher compared to the reference allele combination.

Discussion

The major finding of this study is that AAV allele combination is associated with a decreased all-cause mortality risk and with a higher probability to reach the extreme of old age. The analysis also revealed lower HOMA-IR, higher RQ and RMR for AAV allele combination carriers.