Haplotypic analysis: Pairwise measures of linkage disequilibrium (LD) between the analyzed loci were calculated with Haploview 4.2 [50]. The amount of LD was quantified by Lewontin's coefficient (D). In order to model the effect of haplotypes on the probability to attain longevity, we used the haplo.stats package of R. It implements a haplotype-based association analysis within the generalized linear model (GLM) framework that allows for ambiguous haplotypes. The haplo.score function of this package has been used to obtain the GLM-based score statistics for testing global and individual haplotype effects on the probability to attain longevity [51]. Permutation-based p-values were used to evaluate the significance of the scores obtained (10,000 permutations). In this model, the effect of different haplotypes was assessed assuming a dominant model after adjusting for sex and BMI.

By using the MB-MDR approach, we did not find any significant interaction effect among the analyzed polymorphisms (data not shown). Subsequently, using the aforementioned GLM-based algorithm, we evaluated the effect of the UCP haplotypes on the probability to attain longevity. LD analysis showed that among the four SNPs in the UCP2-UCP3 gene region rs660339, rs659366 and rs15763 were in moderate LD (see Fig. 1), while rs1800849 was in LD with rs15763 (D = 0.83), but it was virtually unlinked with others (D < 0.30). Finally, we found weak LD between rs9472817 and rs10498769 in the UCP4 gene (D = 0.47), and between rs2235800 and rs5975178 in the UCP5 gene (D = 0.44). Based on these LD patterns, and using a D cutoff equal to 0.6, haplotypes were reconstructed at the UCP2-UCP3 and UCP3 loci.

Interaction analysis: In order to explore the interaction effects between analyzed polymorphisms on the probability of being part of the very old age group, the Model-Based Multifactor Dimensionality Reduction (MB-MDR) method was applied [52]. In this model, only all possible second-order interactions, after adjusting for main effects sex and BMI, were analyzed.

As shown in Table 4, the analysis of the UCP2-UCP3 haplotypes revealed that the CAC haplotype, consisting of the minor allele for rs660339, major allele for rs659366, and minor allele rs15763, was associated with a decreased probability of attaining longevity (P= 0.003), while the TGT haplotype (opposite combination of alleles) acted in a reverse fashion by increasing this probability (p =0.046). However, the strength of these associations was influenced by the allelic status at rs659366 of the relevant haplotype. Indeed, the CGC and TAT haplotypes, when compared to the above, differ only for rs659366 and did not show any effect. For the UCP3 haplotypes involving rs15763 and rs1800849, we found that carriers of the CC haplotype have a decreased probability of achieving longevity (P<0.001). Conversely, asfrom mitochondria extends lifespan in various organisms [61]. The Val/Val genotype has been associated with greater weight loss and higher BMI [62], although conflicting results have been reported in other studies [63-66]. Our study shows that the UCP2 polymorphism contributes to extending lifespan, likely due to its systemic effects on global energy metabolism and redox state. However, UCP2 also exhibits tissue-specific regulation, suggesting tissue-specific physiological effects. Evidence suggests that UCP2 functions as a regulator of oxidative stress in the brain, liver, and other tissues. The ability of UCP2 to attenuate steady-state levels of ROS strongly suggests an essential role in lifespan extension, as damage by free radicals is the underlying cause of aging. Andrews proposed that UCP2 promotes longevity by shifting a cell towards fatty acid fuel utilization, thus pointing to a major role of UCP2 in modulating metabolism [75]. This hypothesis is somewhat supported by Barbieri and colleagues' positive effect of the rs659366T-rs15763T haplotype [76]. 

Skeletal muscle, where UCP3 is mainly expressed, contributes considerably to the basal metabolic rate [80]. Carriers of the TT genotype (rs15763) and carriers of the -55 T allele (rs180084) were significantly over-represented among long-lived subjects. The -55 T allele has been associated with a significantly increased gene expression and has been shown to positively modulate the resting metabolic rate of skeletal muscle [81,82]. The over-expression of UCP3 in muscle cells has been shown to be associated with decreased production of ROS as well as with facilitating fatty acid oxidation [17,83]. The physiological consequence of an increased UCP3 activity in skeletal muscle might be to slow down the age-related decline of muscle performance as a result of decreased ROS production, an increased protection of mitochondria from lipid peroxidation, and better metabolic efficiency [28,83]. 

UCP4 and UCP5, together with UCP2, are known as neuronal UCPs due to their widespread distribution in the brain [6]. Andrews and co-workers observed that neuronal uncoupling activity leads to decreased ROS levels, a decreased Ca2+ voltage-dependent influx, and increased local temperature in the neuronal microenvironment [88]. Only the UCP4 rs9472817-GG genotype negatively impacts on the probability to attain longevity. UCP4 activity can induce an adaptive shift in energy metabolism from mitochondrial respiration to glycolysis that helps sustain neurons under conditions of metabolic and oxidative stress [89,90]. Therefore, by decreasing free radical production and stabilizing cellular calcium homeostasis, UCPs expressed in neurons may positively influence neuronal function (synaptic transmission and plasticity) and retard the cellular deterioration associated with aged-related neurological disorders.