Gene Function symbol dbSNP ID Physical location annotation * UCP2 rs660339 C/T exon 4 Ala55Val rs659366 G/A 59-proximal region 2866 G/A UCP3 rs15763 C/T 39UTR rs1800849 C/T 59-proximal region 255 C/T UCP4 rs9472817 C/G intron 8 rs10498769 C/G 59UTR UCP5 rs2235800 A/T intron 7 rs5975178 C/T 59-proximal region

Given the tissue-specific expression of UCP genes, it seems likely that they may affect the senescence of different tissues with important effects on the overall aging process and consequently on lifespan. Thus, the goal of the present study was to assess both the primary effects of single loci and gene-gene interactions to test the hypothesis that other UCP genes may contribute to survival at very old age either independently and/or through complex

*Provided only for coding SNP (amino acid residues for two alleles) and for SNPs in the 59 flanking region (the nucleotide positions relative to the transcription start sites in the promoter regions are indicated). doi:10.1371/journal.pone.0029650.t001

Genotyping

DNA was prepared from peripheral blood lymphocytes using standard techniques. Genotyping of the eleven polymorphic sites was carried out using a TaqMan Real Time PCR (SNP Genotyping kit, Applied Biosystems). In all assays the fluorescent FAM dye was used to label the wild-type allele, while the mutant allele was labeled by fluorescent VIC dye. PCR reactions were carried out in a total volume of 5 ml containing 20 ng of genomic DNA, 2.5 ml of TaqMan Universal Master mix (concentration of 26), 0.25 ml of Custom TaqMan SNP Genotyping Assay (concentration of 206) containing both primers and probes. The amplification protocol (60uC for 30 seconds, 95uC for 10 minutes followed by 40 cycles at 95uC for 15 seconds and 60uC for 1 minute) was performed by using a StepOne thermal cycler (Applied Biosystems). Random regenotyping was conducted to confirm the results.

Methods

Ethical statement

Samples were collected within the framework of several recruitment campaigns carried out for monitoring the quality of aging in Calabria from 2002.The recruitment campaigns and subsequent analyses received the approval of the Ethical committee of the University of Calabria. All subjects provided written informed consent for studies on aging carried out by our research group. White blood cells (WBC) from blood buffy coats were used as a source of DNA.

The Sample

105 years; mean ages 82.74 (611.66) and 85.23 (610.84) years, respectively) participated in this study. All subjects were born in Calabria (southern Italy) and their ancestry in the region was ascertained up to the grandparents’ generation. Younger subjects were contacted through family physicians. Subjects older than 90 years were identified through the population registers and then were contacted by specialized personnel and invited to join the study. Each subject underwent a medical visit carried out by a geriatrician who also conducted an interview including the administration of a structured questionnaire, validated at Euro- pean level. The questionnaire collected socio-demographic information, evaluated physical and cognitive status, and self- reported health status. Subjects with dementia and/or neurologic disorders were not included.

Genetic and statistical analyses

For each polymorphism of the UCP genes, allele frequencies were estimated by counting genes from the observed genotypes. The Hardy-Weinberg equilibrium (HWE) was tested using the exact test proposed by [45]. Standard errors for alleles were computed according to the hypothesis of the multinomial distribution.

Single-locus analysis

genotype-phenotype associations in case-control studies. One of these is the MAX3 test which assumes the maximum of three different Cochran-Armitage trend tests (CATTs) evaluated with respect to three different genetic models of inheritance: dominant, additive or recessive [46,47]. The main drawback of such tests is represented by the fact that they could be seriously affected by confounding factors [48]. In this context, So and Sham recently proposed a robust association test allowing for quantitative or binary traits as well as covariates [49]. This test was based on the score test and has been implemented in the R package RobustSNP. In the present study the robust association test proposed by So and Sham was applied to estimate the association between the variability in the UCP genes and the probability of reaching advanced ages. In each test we included sex and Body Mass Index (BMI) as covariates.

The analyses were carried out by dividing the sample into two specific age classes obtained according to the survival function of the Italian population from 1890 onward [44]. The two ‘‘thresholds of longevity’’ used to define these age classes were 88 years for men and 91 years for women.

SNPs selection

Polymorphisms within the UCP genes were selected from literature data, and using information from online databases such as NCBI dbSNP and HapMap. SNP selection was based on allele frequency, position, and functional effects. For each gene the selected SNPs are reported in Table 1.

From the z-statistics of these models we observed that carriers of the T allele at rs660339 and at rs1800849 (dominant models) significantly influenced the probability to be part of the oldest group (positive scores). For the recessive models, homozygous subjects for the T allele at rs15763 variation also increased the probability to be part of the oldest group, while this probability was significantly decreased (negative score) for homozygous subjects with the T allele at rs9472817.

Haplotypic analysis

Pairwise measures of linkage disequilibrium (LD) between the analyzed loci were calculated with the Haploview 4.2 [50]. The amount of LD was quantified by Lewontin’s coefficient (D’).