genes play a much broader role in human health. Genes of TAS1R-TAS2R gene family express membrane taste receptors the neuroendocrine cells of several organs of the system [58]. These cells start the regulation of a variety of functions, including appetite, satiety, the proliferation of GI cells, secretory activity of the stomach, liver and intestine motility, and gall bladder contraction [59]. A correlation has been observed between polymorphic variants various taste receptors and homeostasis of glucose and insulin which in turn are strongly related with longevity [61]. allelic variants in the taste receptor family may be a link ageing and glucose homeostasis. Moreover candidate gene on various traits have indicated suggestive associations allelic variants in taste receptor genes and Body Mass Index [34,62,63], complex diseases such as cancer [64], consumption [65], smoking [66] and nicotine dependence although these associations did not emerge from association studies on BMI and diabetes, at the highly threshold that is typically used in such studies (p,1028). it has been shown that taste receptors are expressed in organs and tissues which are not directly implicated in food or digestion such as the lung, the airway smooth muscle, the and the testis. In the airways they are thought to enhance response to noxious stimuli, while in the testis they could involved in spermatogenesis. These findings point to the fact bitter sensing is just one of the functions performed by this of genes, which could have a central role in the homeostasis of organisms. Given the possible involvement of the taste genes in wide spectrum of aspects which are crucial to health and such as diet and nutrition or the defense of the organism aerial and ingested toxic compounds, and due to the fact that genetic variability within these genes is well documented to an impact on their function, we addressed the hypothesis that receptor genetic variants may have a role in longevity. particular, using a tagging approach, we investigated the association between the common genetic variation at the bitter taste gene clusters on chromosomes 5, 7 and 12 longevity in a population of 941 individuals ranging from 60 106 years of age from the South of Italy.

 Chrs ofc“rs _"redis inter gene“rs_" black is out of gene 15414672234233\Block 1 (1 kb)1 2 

chromosome 5 region. The value inside each diamond the linkage disequilibrium (r2) between each SNP. The values inside black arrows represent the distance between the SNPs. SNPs situated a coding region are written in red, while SNPs in non coding regions in black. The red arrows show the direction in which the gene transcribed. doi:10.1371/journal.pone.0045232.g001

grandparents were all born in Calabria, as ascertained from population registers.

Younger subjects were contacted through general physicians. Subjects older than 90 years were identified by screening of population registers in different municipalities distributed across the Calabria region. It is important to note that we can exclude the fact that older and younger people in Calabria are of two different ethnicities. Calabria is not subject to immigration from other parts of Italy and all the subjects in the study were of Caucasian origin excluding the possibility that the observed effect is due to ethnicity Eligible subjects were contacted and invited to participate in the study. Written informed consent was obtained from all partici- pants before the enrolment in the study. The health status was ascertained through a medical examination carried out by a geriatrician. Subjects with dementia and/or neurologic disorders were not included. At the time of the visit, peripheral venous blood samples were also obtained.

Materials and Methods

Ethical Statement

Samples were collected within the framework of several recruitment campaigns carried out for monitoring the quality of aging in Calabria (Southern Italy) 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.

Study Population

Subjects were recruited between 1994 and 2008 in Calabria and included 941 unrelated individuals, of which 348 were very elderly cases ($85 years, range 85–106 years, mean age 93.8264.44 years, median age 92) and 593 were non-elderly controls (,85 years, range 20–84 years, mean age 59.17619.45 years, median age 67 years). We chose 85 as a cut-off point because it has been shown that genetic factors contribute to the variation in human life span minimally before age 60 years and most profoundly from age 85 years onwards [13]. Study participants, their parents, and

Selection of Tagging SNPs

We sought to survey the entire set of common genetic variants in the selected bitter taste receptors situated at chromosome 5, 7 and 12. To this end, we followed a hybrid tagging-functional approach. We used the algorithm described by Carlson and coworkers [68] that was developed to select the maximally informative set of tag SNPs in a candidate gene/candidate region for an association study. The resulting SNPs captured all genetic

 Chr7    1370bp 18826637bp 266bp 182366bp 640bp 38191bp 220241 bp 191bpSOc a mo <_ aR <=209bp 181bp 876bp 15771bp 11664bp 101bp 1208195bp 1503bp 33679bpec Sf <—_ <> << 4H - <>          Lode aOLS J&2 2 2oo 2 en z Bo 3 =BF og a Si & So s 2ea RS 2 Si 8 3 on 3 882S Se 3 ot 5 San 3 288 38 8 en 5 ana N x 5as ge 3 SN g Ss = saBe 2B Z oP > 23 2 22—— 15135794938ge35ss    Block 1 (2 kb)1 2   

Figure 2. Figure 2 shows the selected polymorphisms in the chromosome 7 region. Symbols are as in Figure 1. doi:10.1371/journal.pone.0045232.g002