Table 1. SNPs in chromosome 7 cluster associated with longevity.

ID_Gene SNP $85 yrsa ,85 yrsa OR (95%CI)b P P value trend T2R16 rs6466849 G/G 233 316 1 0.043 A/G 86 172 0.69 (0.50–0.94) 0.018 A/A 14 22 0.89 (0.44–1.77) 0.730 (A/G+A/A) 0.71 (0.53–0.95) 0.023 T2R16 rs860170 A/A 139 253 1 0.042 A/G 153 224 1.25 (0.93–1.67) 0.135 G/G 39 46 1.51 (0.94–2.43) 0.090 (A/G+G/G) 1.29 (0.98–1.71) 0.069 T2R16 rs978739 A/A 185 245 1 0.004 A/G 125 284 0.59 (0.45–0.79) 3.26*1024 G/G 30 54 0.76 (0.46–1.23) 0.262 (A/G+G/G) 0.62 (0.47–0.81) 0.001 T2R4 rs2233998 C/C 118 159 1 0.057 C/T 146 282 0.71 (0.52–0.97) 0.029 T/T 78 146 0.74 (0.51–1.06) 0.102 (C/T+T/T) 0.72 (0.54–0.96) 0.024 T2R5 rs2227264 T/T 114 158 1 0.047 G/T 148 287 0.72 (0.53–0.99) 0.044 G/G 74 146 0.72 (0.50–1.04) 0.081 (G/T+G/G) 0.72 (0.54–0.97) 0.029

aNumbers may not add up to 100% of subjects due to genotyping failure. Data points that were still not filled after this procedure were left blank. bOR: odds ratio; CI: confidence interval. doi:10.1371/journal.pone.0045232.t001

p,0.05. Three were observed in the TAS2R16 gene (rs6466849, rs860170 and rs978739), one in the TAS2R4 gene (rs2233998), and one in the TAS2R5 gene (rs2227264) (shown in Table 1). All the variant alleles were associated with a decreased probability to attain longevity (ORs,1).

To take into account multiple testing, we calculated for each region the number of effective independent variables (M ). The M values eff eff for the regions on chromosome 5, chromosome 7 and chromosome 12 were 2, 11 and 8, respectively. The experiment-wise significance threshold was therefore set at 0.05/(2+11+8)=0.0024. After correction for multiple testing, only the carriers of the G allele of rs978739 variation showed a statistically significant association.

Haplotype analysis was performed for each LD block in the study. None of the haplotypes showed a study-wise statistically significant association with longevity. However, the haplotype (rs1357949– rs6466849–rs860170–rs978739: T_A_A_G) of the TAS2R16 gene showed a suggestive association with longevity (OR=0.74; 95% CI 0.56–0.98; p=0.033). Moreover the haplotype rs2588350 (TAS2R7)- rs619381(TAS2R9)-rs3741845(TAS2R9): G_G_T showed an associ- ation with increased longevity at the conventional 0.05 P-value (OR=1.35; 95% CI 1.02–1.78; p=0.037). Tables S3, S4, S5, S6, S7, S8, S9, S10, and S11 show the distribution of all the haplotypes and their associations with longevity. We also analyzed all the possible pair- wise interactions between SNPs using the MDR method. No interactions between SNPs emerged with a study-wise significant p- value.

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Figure 4. Bar chart of age by genotypes showing the association between the polymorphism of TAS2R16rs 978739 and longevity. The A/A genotype frequency is about 40.5% in the young and adult classes (20–50 and 51–70 years). Thereafter the A/A frequency increases through older classes reaching the 55.5% in the oldest (x2=17,08, p,0.029).

Discussion

In the present study, we have investigated possible associations between longevity and 41 SNPs in 20 candidate genes involved in

harmful compounds are recognized by the receptor throughout the life span and that this could, in the long term, modify the chances to reach very old ages. However there is also another possible, even though highly speculative, explanation of the involvement of TAS2R16 genetic variability in healthy aging. Numerous recent reports investigated non-gustatory actions of taste receptors. They have been shown to be expressed in a plethora of tissues such as the respiratory system where they affect respiratory functions in response to noxious stimuli [87], and the gastrointestinal tract where they are suspected to regulate the activation of metabolic and digestive functions [87]. Recently it has been shown that taste receptors are expressed also in the testis in mouse, where they can be involved in spermatogenesis [88]. The emerging picture is therefore that taste receptors could behave as pleiotropic genes, whose products are used by various cells, or have signaling function on various targets not linked one to the other. Probably the bitter sensing is just one of the functions performed by this cluster of genes, which could have a central role in the homeostasis of the organisms. Therefore their genetic variations can affect profoundly various traits, including longevity, in a way that we are just beginning to understand [89].

bitter taste perception. Our intensive SNP tagging approach along with the analyses of haplotypes provides a close to exhaustive analysis of the possible associations of longevity with the known common polymorphic variants at these bitter taste receptor loci.

We found that five polymorphisms were associated with longevity: three in the TAS2R16 gene (rs6466849, rs860170 and rs978739), one in the TAS2R4 gene (rs2233998) and two in the TAS2R5 (rs2227264). rs6466849 and rs978739 share a modest degree of LD (r2=0.52) indicating that they might not be statistically independent findings, while rs2233998 and rs2227264 are in complete LD (r2=0.99) and therefore represent the same association. Two haplotypes, (rs1357949–rs6466849–rs860170– rs978739: T_A_A_G) of the TAS2R16 gene and rs2588350 (TAS2R7)-rs619381(TAS2R9)-rs3741845(TAS2R9): G_G_T showed a suggestive association with longevity. After correction for multiple testing only one polymorphism, rs978739 showed a statistically significant association with longevity (p=0.001). In particular, the frequency of homozygotes A/A increases gradually from 35% in the subjects aged 20 to 70 up to 55% in centenarians. A distribution of the genotypes through various age strata is given in Figure 4.