UCP2 and UCP3 genes cluster on chromosome 11q13 in a region linked to lower resting metabolic rate in humans [53], which is also syntenic to a region of mouse chromosome 7 linked to hyperinsulinemia and obesity [54]. Their chromosomal location and their association with fatty acid transport across mitochondrial inner membrane and b-oxidation, support the hypothesis that, in addition to functioning as regulators of oxidative stress, both UCP2 and UCP3 can modulate cellular energy metabolism [16,55]. While mounting evidence has documented the impor- tance of both UCP2 and UCP3 variability in the pathophysiology of different chronic metabolic diseases, such as diabetes and obesity [41,56], relatively little is known of their implication in human aging and longevity. In this study we found that the

Note: age-cut-offs to define the younger and older groups were 88 years for males and 91 years for females, as reported in Materials and Methods section. SD: standard deviation.

Table 3. Results of the Robust SNP association test for the most likely genetic model (additive, recessive and dominant) obtained in the analyzed sample.

Gene SNP MAF Z(ADD) Z(REC) Z(DOM) P(ADD) P(REC) P(DOM) P-value* UCP2 rs659366 A (0.295) 0.936 0.338 0.978 0.349 0.735 0.328 0.555 rs660339 T (0.341) 3.049 1.477 3.190 0.002 0.140 0.001 0.003 UCP3 rs15763 T (0.247) 2.163 2.468 1.454 0.031 0.014 0.146 0.029 rs1800849 T (0.099) 2.788 21.197 3.186 0.005 0.231 0.001 0.003 UCP4 rs9472817 G (0.498) 21.984 23.607 0.316 0.047 ,0.001 0.752 0.001 rs10498769 G (0.190) 20.085 20.660 0.130 0.933 0.509 0.897 0.761 UCP5 rs2235800 A(0.387) 1.554 2.120 0.849 0.120 0.034 0.396 0.058 rs5975178 C (0.474) 0.203 0.210 0.170 0.839 0.833 0.865 0.959

Z(ADD), Z(REC) and Z(DOM) are the z-statistics under the additive, recessive and dominant models respectively. P(ADD), P(REC) and P(DOM) are the p-values three genetic models. *p-value was adjusted for multiple testing of different genetic models obtained by the proposed analytic approach. MAF: Minor Allele Frequency. doi:10.1371/journal.pone.0029650.t003

Ala55Val (rs660339) of UCP2, and -55 C/T (rs1800849) and rs15763 of UCP3 were significantly associated with survival at very old age after Bonferroni correction.

[67–69], in the heart as a regulator of energy availability [70], in white adipose tissue and skeletal muscle as a regulator of fatty acid metabolism [71,72], and as a regulator of insulin dynamics in pancreatic b and a-cells [73,74]. In this scenario, variants of UCP2 may have positive and negative effects depending on the tissue and on the fine balance between energy production/consumption and mitochondrial ROS generation.

Ala55val SNP in exon 4 causes a conservative amino acid change that does not seem to cause a functional change in the protein. However, it has been reported that the Val/Val genotype causes a lower degree of uncoupling, an enhanced metabolic efficiency, and a lower fat oxidation than the Ala/Ala and Ala/Val genotypes [57]. In addition this genotype has been associated to higher exercise energy efficiency [58], higher risk in obesity, higher incidence of diabetes [59,60], and a higher acute insulin response to glucose [61]. Moreover, individuals with the Val/Val genotype had greater weight loss and a higher BMI [62]. Nevertheless, other studies have produced conflicting results [63–66]. Here we show that this polymorphism also contributes to extend lifespan. Given the wide distribution of UCP2, it is likely that systemic effects on global energy metabolism and redox state underlie the association with longevity. However, UCP2 also exhibits tissue-specific regulation suggesting tissue specific physiological effects. In this regard, evidence has been provided that in the brain, liver and other tissues UCP2 functions as regulator of oxidative stress

It is widely accepted that damage by free radicals to the molecules with which they come into contact are the underlying cause of aging, therefore the ability of UCP2 to attenuate steady- state levels of ROS strongly suggests an essential role in lifespan extension. Recently, 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

Table 4. Estimation of haplotype frequencies in the UCPs SNPs and association with longevity in the analyzed sample.

Gene SNPs Freqa Score P-valueb UCP2-UCP3 rs660339 rs659366 rs15763 C A C 0.029 22.876 0.003 C G C 0.596 21.747 0.086 C G T 0.028 0.317 0.754 C A T 0.004 0.877 0.400 T A C 0.077 0.953 0.347 T G C 0.050 0.971 0.339 T A T 0.184 1.390 0.165 T G T 0.030 1.985 0.046 UCP3 rs15763 rs1800849 C C 0.659 23.720 ,0.001 T C 0.243 2.029 0.044 C T 0.095 2.766 0.006

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Figure 1. Schematic representation of linkage disequilibrium (D’ coefficient) among the four SNPs of the UCP2-UCP3 gene cluster.

bMonte-Carlo p-value from 104 replications. Note: p-values of global score statistics based on 104 replications were 0.047 for UCP2-UCP3 haplotypes, 0.0028 for UCP3 haplotypes. doi:10.1371/journal.pone.0029650.t004

doi:10.1371/journal.pone.0029650.g001

positive effect of the rs659366T-rs15763T haplotype. The rs659366 (-866G/A) variant changes promoter activity, affects metabolic rate and oxidative stress, and has been associated with metabolic traits in several, although not all, studies [87].