The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects’ genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e. g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e. g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen, e. g. for absinthin, the bitter principle of the beverage absinth. These findings illustrate the extent to which genetic influences on taste are complex, yet arise from both receptor activation patterns and linkage structure among receptor genes.
Bitter taste perception plays a fundamental role in dietary preferences and behaviours, by shaping aversions to foods and drinks. Indeed, averse responses to bitterness are instinctive and drive rejection and avoidance behaviours widely observed in animal models, but also in human infants [1–5]. They are hypothesized to originate with bitter perception’s role as a warning sensor against potentially harmful substances contained in the diet, such as toxins released by plants to deter herbivores, and they belong to diverse chemical classes including acetogenins, alkaloids, flavonoids, phenylpropanes, terpenoids, and thiol compounds [6–9]. These rejection behaviours mediated by bitter perception show evolutionary trends, with responses depending on the occurrence of bitter substances in animal typical diets [10]. Occasionally, bitter substances known to possess desirable pharmacological activities are also deliberately ingested (e. g. , [11,12]), nevertheless acceptation of bitter phytonutrient in food remains challenging (for review see [13]). Despite the importance of bitter taste in shaping nutritional behaviours and guarding against toxin ingestion, bitter responses in humans vary profoundly. The classic example of phenotypic diversity in humans is threshold sensitivity to phenylthiocarbamide (PTC), which differs by up to 10,000-fold among individuals. Such variation is due to a constellation of interacting factors including environmental effects, age, gender, experience and genetics (for reviews see [13–15]). Particularly strong effects have been found to arise from polymorphism in TAS2R genes, which encode a series of ~25 G protein-coupled receptors expressed in taste buds [16–20]. In the case of PTC perception, polymorphism in TAS2R38 accounts for more than 55% of observed phenotypic variance [17,21]. Moreover, genetic polymorphisms occurring at TAS2R loci are common, with numerous high-frequency alleles [22], suggesting the presence of functionally important receptor variants. Major changes in receptor activity due to such variants have been observed in a few other cases, i. e. , TAS2R9, TAS2R16, TAS2R43, and TAS2R31 [23–27]. In addition, gene association studies suggested functional polymorphic alleles at other TAS2R loci, e. g. TAS2R4 or TAS2R13 [28–30]. An essential aspect of interactions between TAS2Rs and bitter compounds relates to the overlapping agonist profiles, with most TAS2Rs responding to multiple agonists and many agonists stimulating multiple receptors [31]. This combinatorial activation pattern, together with the distribution of TAS2R genes among only four cytogenic locations, which can lead to false-positive genotype-phenotype associations arising from sites in linkage with the causal variants, have so far prevented the full elucidation of bitter perception’s molecular underpinnings [26,32–34]. To establish a genetic basis for the observed perceptual differences in the population, we used in this study an integrative approach, sequencing all known TAS2R loci in humans, inferring long-rang haplotypes, mapping their effects on perception of several chemically diverse compounds, and functionally characterizing all allelic variants associated with shifts in perception.
Genetic diversity was assessed by determining whole-gene genotypes of the 25 members of the TAS2R gene family and corresponding copy number variations (Figs 1 and 2, S1 Table). Across the 48 Caucasian subjects, a total of 93 coding SNPs (cSNPs), including 65 missense SNPs and 2 nonsense SNPs, were identified. Three indels, including 1 rare three-nucleotide in-frame deletion and 2 major deletions spanning TAS2R43 or TAS2R45 locus, were also detected. Genes harboured a mean of 4 cSNPs with a mean of ~1 synonymous and ~3 non-synonymous SNPs per gene. The mean number of haplotypes within genes was 3. 5 with a range of 1 to 6, which recombined to form a mean number of 5. 6 genotypes within genes with a range of 1 to 13. When only common cSNPs were considered (i. e. , with frequency ≥ 0. 05), a total of 67 SNPs, including 45 missense SNPs and 2 nonsense SNPs, and 2 indels were identified, all of which have been reported previously [22,26,36–40]. Sequenced genes harboured a mean of ~1 synonymous and ~2 non-synonymous common SNPs per gene. While some genes harboured no SNPs (e. g. , TAS2R10 and TAS2R39), others harboured many. Genes located in the proximal region of the cluster at 12p13 harboured also highest number of SNPs, with 8 at TAS2R31 and -42, and 9 at TAS2R20. Of these 45 common missense cSNPs, 27 corresponded to amino acid positions localised in TAS2R transmembrane (TM) domains, with 9 SNPs affecting amino acids in TM VI and 6 in TM V (Fig 3). The remaining SNPs were distributed in sequence areas coding for extracellular domains, intracellular domains, and COOH terminal region, with 8,5, and 5 SNPs, respectively. In addition, 2 nonsense cSNPs result in a premature stop codon in the reading frame and therefore to a putatively non-functional, truncated receptor variant. These common cSNPs recombined across genes to form a total of 59 coding haplotypes, with a mean of 2. 2 per gene and a range of 1 to 4. Again, genes localised to the proximal region of 12p13 were most diverse, with 4 haplotypes at TAS2R20, -31, and -50. As with common SNPs, most within-gene haplotypes have been reported previously [22,26,36–40]. Two common haplotypes were newly identified: one at TAS2R14 (frequency = 0. 34), and one at TAS2R42 (frequency = 0. 30). High levels of genetic diversity were observed although our population sample, only Caucasian subjects, was relatively homogeneous with respect to ethnic and geographic origin. Thus, chromosomal spatial relations, linkage and block structures among genes might be important contributors to TAS2R-mediated phenotypes. Indeed, the 25 members of the TAS2R gene family are restricted to just three cytogenetic locations 5p15,7q31-7q35, and 12p13 (Fig 1). Position 5p15 contains a single gene, TAS2R1,7q31-35 contains 9 genes distributed across a ~20. 5 Mb region, and 12p13 contains 15 genes distributed across a ~400 kb region. Further, most of the TAS2Rs at 7q31-7q35 (TAS2R3, -4, -5, -38, -39, -40, -60, and -41) reside in a 1. 8 Mb sub-region. Estimates of pairwise D′ and r2 revealed extensive linkage in both the 7q31-7q35 and 12p13 regions. Mean values of D′ and r2 between common cSNPs were 0. 66 and 0. 52 across 12p13, and associated with low p-values (Fig 4). Nearly identical trends were observed between TAS2R loci, with mean D′ and r2 values of 0. 66 and 0. 37. Similarly, across the 1. 8 Mb TAS2R3-38 cluster, mean values were 0. 76 and 0. 74 for common cSNPs and 0. 60 and 0. 63 between TAS2R loci. These trends indicate that TAS2Rs are tightly connected in long-range haplotypes spanning multiple genes. Haplotype block partitions and corresponding long-range haplotypes were then determined for the 25 TAS2R genes (Fig 5). Analyses inferring haplotype blocks identified six blocks distributed across the TAS2R clusters on chromosomes 7 and 12. These corresponded to blocks found in the region by previous studies [34,43,44]. Two were found on chromosome 7, encompassing the TAS2R3-5 and TAS2R39-60 regions, each of which harboured two long-range haplotypes with frequencies at or above 0. 05. Four blocks were found on chromosome 12, encompassing the TAS2R7-10, TAS2R13-14, TAS2R50-19, and TAS2R31-42 regions, which harboured 1,3, and 5 long-range haplotypes respectively. In addition, whereas blocks on chromosome 7 were flanked by recombination hot spots, we identified no hot spots in the TAS2R13-42 region on chromosome 12, which seems to indicate that these latter haplotype blocks are determined by linkage disequilibrium decay [45]. Genotype-phenotype association analyses were conducted to elucidate genetic liability for taste phenotypes; phenotype data consisting of individual detection and recognition thresholds for the bitter tastants, as well as concentrations for perceived weak, moderate, strong, and very strong intensities inferred from intensity ratings. Subsequent functional assays provided further insights into the identification of causal TAS2Rs.
Bitter taste perception has long been known to have major heritable components. Common dominant and recessive alleles shaping sensitivity to specific chemical compounds were identified as early as the 1930s (for review see [47]). It is now known that much of this heritability is due to coding variation in TAS2R genes altering receptor affinity, which shapes perception phenotypes. In the classic case, patterns of bitter sensitivity are driven by strong, single-gene effects [17,21,25,26]. However, such associations with only one TAS2R locus appear to be rare. Relationships between TAS2R variation and perception are likely most often complex due to high levels of genetic diversity, linkage between loci and overlap in receptor-agonist interactions, with some agonists stimulating multiple receptors and some receptors responding to multiple agonists [26,31]. This hinders elucidation of bitter perception’s molecular underpinnings and, consequently, their downstream effects on aversion and ingestive behaviours [34]. Therefore, until now only a limited number of TAS2R genes have been implicated in the modification of ingestive behavior (for a review see [48]). Our findings reveal the extent to which linkage constrains both, variation in TAS2R genes and patterns of overlap in receptor-agonist affinity, and the impact of these factors on bitter taste phenotypes. Human TAS2Rs are highly diverse, with per nucleotide heterozygosity significantly higher than genome-wide averages, elevated rates of non-synonymous substitution, and fixation indices (FST values) indicative of substantial population differentiation [22]. This suggests that combinatorial variation across loci could, in principle, be extremely high. However, patterns of linkage disequilibrium in our sample of the Caucasian population demonstrate that such diversity is limited. While a total of 93 SNPs were present, which could in principle recombine to form more than 7 quintillion (7. 71 x 1030) different combinations, most variation resided in just six blocks, each harbouring just 1 to 4 haplotypes. This finding has two implications. First, it suggests that while humans harbour 25 functional TAS2R loci, each of which encodes a receptor with alleles responsive to different ranges of agonists, phenotypic responses are likely correlated across compounds regardless of whether they are mediated by the same receptor. Second, linkage disequilibrium and block structures spanning loci are likely major sources of spurious genotype-phenotype associations. Because blocks often span several loci, yet few long-range haplotypes are present, true genotype-phenotype associations will most often be accompanied by false positives arising from sites in LD with the causal variants. This problem is compounded by the prevalence of non-synonymous variants in TAS2Rs, which stand out as potential functional receptor candidates, making them difficult to rule out as causal. These issues were evident in our association analyses, which were able to localize signals for amarogentin, grosheimin, and quassin at the block level but not with respect to specific SNPs (Figs 7,8 and S1–S3). Functional assays characterizing the kinetic properties of individual TAS2R variants in our sample were successful in resolving the positions of sites shaping phenotypes, revealing complex variation in response across loci and alleles (Figs 6–8; S2 Table). Within each haplotype block implicated in associations, we found at least one locus harboring functionally polymorphic alleles corresponding to receptor variants activated in the concentration range perceived as bitter by subjects, thus pinpointing the sites underlying variable perception of four tastants. However, the mechanisms underlying variation in sensitivity varied from locus to locus. At TAS2R30, the high- and low-sensitivity receptor variants, which differed drastically in response to amarogentin and quassin, were distinguished by a single amino acid position (L252F) in the third extracellular domain of the receptor, which is hypothesized to interact directly with agonists. High- and low-sensitivity TAS2R46 variants also differed at a single amino acid position (L228M); however, this site, located in the sixth transmembrane domain of the receptor, is highly conserved among TAS2Rs and thought to be essential to the basic functionality of the receptor, which likely explains the strong effects of TAS2R46-L228M on phenotype. TAS2R46 also harbored a common allele coding a premature stop codon (W250X), resulting in the production of a severely truncated, dysfunctional receptor [22]. At TAS2R43, the high- and low-sensitivity receptor variants differed at position W35S in the first intracellular domain and H212R in the fifth transmembrane domain; by considering the high degree of sequence similarity between TAS2R43 and TAS2R31, the sole presence of amino acid substitution W35S, corresponding to the deleterious substitution TAS2R31-R35W, likely explains the severe impaired functionality of the receptor [26]. Beyond these substitutions, TAS2R43 harboured a high-frequency whole-gene deletion allele completely lacking an open reading frame, and unable to produce protein at all [25,26]. In addition to identifying simple phenotypic associations with alleles at single loci, our analyses revealed associations arising from linkage disequilibrium across loci, demonstrating the complexity of relationships between TAS2R variation and phenotypic response. In the case of amarogentin and quassin, several loci (TAS2R30, -43, and -46) harboured both high- and low-sensitivity alleles, suggesting that loci could individually contribute up- or downward shifts in phenotype. However, only TAS2R30 harboured receptor variants responsive across the threshold ranges of subjects, indicating that it alone accounts for most variability in perception. This relationship is similar to long known patterns of bitter sensitivity driven by strong, single-gene effects. In the case of grosheimin, two loci (TAS2R43 and -46) harboured both high- and low-sensitivity alleles. However, these were maintained in the same linkage phase such that the sensitive allele of TAS2R43 was linked with the sensitive allele of TAS2R46 and the insensitive allele of TAS2R43 was linked with the insensitive allele of TAS2R46. Thus, while both loci harboured variation able to explain observed phenotypes, their contributions were strongly correlated. In the case of absinthin and cascarillin, again two loci (TAS2R30 and -46) harboured both high- and low-sensitivity alleles. However, linkage disequilibrium maintained these in opposite phase such that the sensitive allele of TAS2R30 was linked with the insensitive allele of TAS2R46, and the insensitive allele of TAS2R30 was linked with the sensitive allele of TAS2R46. Thus, most subjects carried at least one allele sensitive to absinthin and cascarillin, explaining both the low mean threshold response to these compounds in subjects and the weak statistical associations for individual loci. These findings, together with prior evidence of extensive overlap in sensitivity across loci and agonists, suggest that while strong associations between a single TAS2R locus and phenotype may occur, they are likely uncommon. Moreover, linkage between TAS2R loci can cause confounds resulting in both false positive and false negative results in association analyses. Thus, dissecting genetic effects on bitter taste sensitivity through association analysis alone is likely to be inaccurate in most situations. An essential aspect of TAS2R diversity in our sample of the Caucasian population, which has been broadly observed in population genetic studies, was that diversity is extremely high. In total we identified 93 SNPs, of which 67 SNPs were common, with frequencies above 5% (Fig 3). Moreover, every subject harboured a different allele combination at the SNP positions. Thus, inherited variation in taste responses was not a rarity as is the case for many phenotypes, such as diseases, but the norm. Further, our European sample, though ethnically homogeneous, captured most TAS2R variation found to date in worldwide populations [22,40]. For example, beyond the 93 SNPs found in our subjects, Kim et al. (2005) reported only 10 additional common SNPs (5 synonymous and 5 non-synonymous) across a panel of 55 Africans, Asians, Europeans, and Native Americans. Hence, differences at TAS2R loci between individuals from ethnically diverse populations are modest in comparison to differences among individuals from the same population, consistent with more general observations on large-scale data sets [49,50]. These patterns suggest that the association trends in our data are likely not restricted to Europeans, but relevant in most populations. Yet, even if receptor polymorphism and genomic structure dominantly shape bitter taste perception, further studies need to be performed to enable a complete comprehension of variation in bitter taste perception, taking into account that other relevant factors may also play a significant role. Indeed, besides receptor-agonist interactions, differences in taste receptor gene expression levels could also contribute to individual differences in bitter taste perception. The importance of polymorphisms in the putative promoter regions of taste genes further indicates overlapping genetic influences on receptor expression and functionality [51,52]. In addition, differences at peripheral level, e. g. in taste signaling cascade components [53], may also influence taste perception, as well as differences in signal transmission by afferent taste nerves and signal processing at central level. Hormones may also, at the level of the individual, impact bitter taste perception, e. g. hunger-satiety hormones as well as sex hormones (for review see [54,55]). Nonetheless, deciphering receptor activation patterns and linkage structure among TAS2R genes is an important prerequisite to establish a solid basis to assess bitter taste variations in the population. This may pave the way to evaluate the consequences of these variations in food rejection and ultimately help to improve public health.
This work was conducted in accordance to the Declaration of Helsinki on Biomedical Research Involving Human Subjects and approved by the Ethics Committee of the University of Potsdam (Germany) through decision 11 / 27. Session / 2009. All participants gave written informed consent. The subject panel was composed of 48 unrelated Caucasian subjects (39 women, 9 men; age range 21 to 59 years, mean age 30. 6 years), recruited at the German Institute of Human Nutrition Potsdam-Rehbruecke (Germany). All were pre-screened to avoid inclusion of individuals with health problems and overt taste pathologies; pregnant and breast-feeding women were also excluded. Each subject participated in the entire course of the study, which included one training session and nine experimental sessions. Visits consisted of DNA collection and psychophysical tests for genotyping and phenotypic analyses, respectively. General taste abilities were also assessed for the bitter, salty, sour, and sweet taste during the first session. Six structurally diverse bitter substances, found at low concentrations in various beverages, and known to exhibit pharmacological properties at high concentrations, were used to probe phenotypic and molecular responses (S1 Fig). These included absinthin (a dimeric sesquiterpene lactone), amarogentin (a secoiridoid glycoside), cascarillin (a diterpene lactone), grosheimin (a sesquiterpene lactone), quassin (a triterpene lactone), and quinine (a quinoline alkaloid). Absinthin, cascarillin, and grosheimin were isolated from crude vegetable material as detailed in previous studies [31,56]. Amarogentin and quassin were purchased from Chromadex Inc. (Irvine, CA, USA), quinine hydrochloride from Sigma-Aldrich Co. (St. Louis, MO, USA). Salicin, sodium chloride, citric acid, and sucrose (Sigma-Aldrich Co.) were used as reference tastants for assessing bitter, salty, sour, and sweet perception, respectively. Gene locations and coding sequences of the 25 TAS2R genes were obtained from genomic scaffolds 1103279188109,1103279188381,1103279188228, and 1103279188408 of the whole genome assembly released by the Venter Institute human reference genome as well as from all subjects in the present study [57] (Fig 1). Corresponding amino acid sequences were aligned according the modified version of the Feng-Doolittle progressive alignment algorithm (Align X, Vector NTI; Life Technologies, Carlsbad, CA, USA) [58]. Alignment was then manually adjusted on the basis of previous in silico and in vitro experiments, in order to conserve structural and functional key domains, e. g. , transmembrane domains and the conserved glycosylation site [41,42,59]. A neighbour-joining tree with bootstrap values was then constructed from aligned sequences using Clustal X [60]. Genomic DNA was obtained from saliva samples collected using Oragene DNA self-collection kits (Oragene DNA; DNA Genotek Inc. , Kanata, Canada), and purified using prepIT-L2P kits (prepIT-L2P; DNA Genotek Inc.). Complete nucleotide sequences of every TAS2R coding region were then obtained for all subjects. Locus-specific primers localized in the flanking regions of each gene, ~100 bp upstream of the start codon and ~100 bp downstream of the stop codon, were designed using the Primer-Blast tool or obtained from previous studies [22,26,40,61]. Corresponding DNA fragments of at least ~1 kbp were amplified by PCR using a high-throughput polymerase (Advantage 2 polymerase mix; Takara Bio Inc. , Otsu, Japan). Amplified DNA fragments were sequenced by capillary electrophoresis of both forward and reverse strands (Eurofins MWG Operon, Ebersberg, Germany). Reads were assembled and trimmed to remove low-quality sequence (Vector NTI; Life Technologies). All single-nucleotide polymorphisms (SNPs) were then identified and individual genotypes were determined. Departures from the Hardy-Weinberg equilibrium were tested to rule out genotyping problems. Previously reported major deletions at TAS2R43 and -45 loci, ~39k b and ~32 kb in length, respectively, were characterised by multiplex PCR reactions. These were performed using primer sets targeted within, outside, and spanning the deleted regions such that amplifications produced alternate products in deleted and non-deleted alleles (S4 Fig). Gel separation and sequencing of the resulting fragments (Advantage 2 polymerase; Takara Bio Inc.) revealed whether a subject carried zero, one or two copies of each gene. DNA sequence and copy-number data were jointly used to call genotypes. Haplotypes were then either directly ascertained for homozygous individuals or inferred using PHASE, which utilizes Bayesian algorithms to resolve haplotypes in heterozygous individuals [62,63]. In cases of uncertain phase, haplotypes were confirmed by comparison with previously published data or identified by cDNA cloning and sequence analysis [26]. Linkage disequilibrium measures were then obtained by calculating D’, r2 and corresponding p-values between multi-allelic loci for both SNPs and genes [64,65]. Haplotype block partitions were generated according to the four gamete rule with a 5% cut-off score, and manually adjusted for TAS2R19 and TAS2R42, which SNPs spanned several blocks [66,67]. Corresponding long-range haplotypes were then inferred [62,63]. Algorithms for visual representation were specifically implemented in Matlab, based on the population genetics and evolution toolbox (Matlab; The MathWorks Inc. , Natick, MA, USA) [68]. Experiments were conducted at the sensory analysis laboratory of the German Institute of Human Nutrition Potsdam-Rehbruecke, according to good practice guidelines [69,70]. Detection and recognition thresholds were assessed using a procedure adapted from the norm ISO 13301: 2002 of the International Organization for Standardization: a four-alternative ascending forced-choice procedure, followed by yes-no questions about the bitter taste quality of the quoted samples [71]. Perceived bitter taste intensities were rated on the general Labelled Magnitude Scale (gLMS; [72–74]). Concentration series consisted of geometric sequences of twelve steps, with a 1. 5 common ratio and the following concentration ranges: 2. 3x10-8–2. 0x10-6M, 3. 5x10-9–3. 0x10-7 M, 4. 6x10-7–4. 0x10-5 M, 5. 8x10-7–5. 0x10-5 M, 1. 2x10-8–1. 0x10-6 M, 4. 6x10-7–4. 0x10-5 M, for absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine, respectively. For each 4-AFC test, four coded samples containing 10 ml solution were presented simultaneously; one containing the bitter tastant diluted in mineral water (Evian; Danone, Paris, France) and three containing mineral water only. At each concentration, subjects were challenged to identify the different sample, specify whether the quoted sample tasted bitter, and rate the perceived bitter intensity. Tests were performed with nose clips and oral rinsing, with a 45 s pause between concentrations. Training sessions were first used to familiarize subjects with the experimental procedures, and secondly, to assess general taste abilities. Concentration series consisted of geometric sequences of six steps, with a 1. 5 common ratio and the following concentration ranges: 3. 5x10-5–2. 0x10-3M, 6. 9x10-4–4. 0x10-2 M, 2. 6x10-4–1. 5x10-2 M, 6. 9x10-4–4. 0x10-2 M, for salicin, sodium chloride, citric acid, and sucrose used as reference tastants for bitter, salty, sour, and sweet taste, respectively. Following training, subjects tested each tastant in triplicate over nine sessions. Latin square designs extended for first-order carry-over effects were used to counterbalance presentation order of the test compounds over the test sessions, across subjects and for each test compound, as well as presentation order of the samples at each concentration across both repetitions and subjects. Sensory sessions were monitored and data automatically collected (Fizz; Biosystèmes, Couternon, France). Detection and recognition probabilities of the bitter samples were analysed per repetition and subject. Relationships between probabilities (detection or recognition probabilities) and concentrations were fitted by a logistic regression model using the maximum-likelihood method. Threshold value and slope of the logistic curves were then obtained per repetition and subject, using a self-implemented toolbox (Matlab; The MathWorks Inc.). No repetition effect was observed. Where required, outliers were discarded according to the Peirce’s criterion [75,76]. Perceived intensities, expressed in percentage of the scale length, were analysed similarly. Concentrations were subsequently inferred for weak, moderate, strong, and very strong intensities, corresponding respectively to 6,17,34. 7, and 52. 5% of the scale length. General linear mixed model analyses were performed at SNP, gene, and LD block level (SAS Institute Inc. , Cary, NC, USA). Concentrations corresponding to detection thresholds, recognition thresholds, or perceived intensities were treated as dependent variables following a log-normal distribution. Genotype or haplotype with frequencies above 0. 05 were used as independent variables and subjects as random variables. For analyses at the SNP level, probability values were assessed at an uncorrected significance level of 0. 05, and at an experiment-wide significance threshold required to keep a significance level of 0. 05, thus correcting for multiple comparisons [77]. For analyses at gene-specific level and LD block level, a Bonferroni correction for multiple corrections was applied. Allelic responses to agonists were quantified using in vitro heterologous expression assays designed in previous studies of TAS2R receptor function, which successfully mimic responses in vivo with respect to both magnitude and concentration range [17,23,25,26,46]. DNA fragments containing TAS2R coding sequences were first amplified from genomic DNA by PCR using a proofreading polymerase (PfuUltra II Fusion HotStart DNA Polymerase; Agilent Technologies, Santa Clara, CA, USA) and cloned into a plasmid vector according to the manufacturer’s protocol (Zero Blunt TOPO PCR Cloning Kit; Life Technologies). A second PCR was performed using specific cloning primers to facilitate subcloning into the expression vector, which was then modified to add an N-terminal signal for cell surface localisation and a C-terminal epitope for immunocytochemical detection to the receptor sequence (Fast Link DNA Ligation Kit; Epicentre, Illumina Inc. , Madison, WI, USA) (pcDNA5/FRT Mammalian Expression Vector; Life Technologies). Empty vector was used as a negative control [23,31]. Functional assays were carried out in HEK 293T cells stably expressing the G protein chimera Gα16gust44 and transiently transfected with TAS2R alleles subcloned into the expression vector [78]. Calcium imaging was performed using an automated fluorometric imaging plate reader by exposing transfected cells to test compounds dissolved in assay buffer or to assay buffer alone (FLIPR Tetra; Molecular Devices, LLC, Sunnyvale, CA, USA). Changes in cytosolic calcium levels were monitored by measuring fluorescence intensity of a calcium-sensitive dye previously added (Fluo-4 AM; Life Technologies). Six replicates were carried out for each bitter stimulus, on separate experimental days, with each replicate consisting of concentration series of each bitter tastant. Prior to final analysis, fluorescence data from functional assays, expressed in relative fluorescence units (RFU), underwent three corrections. First, a correction calculated from baseline values was applied to compensate for well-to-well fluctuations. A second correction calculated from negative control values was applied to correct for receptor independent artefacts and signal drift. A third correction calculated from positive control values was applied to facilitate comparison of data obtained from different experimental days. Fluorescence ratios ( (F-F0) /F0), obtained by subtracting the background fluorescence from fluorescence peak height and then dividing the difference by the background fluorescence, were then used for data analysis. Variance analyses were performed, followed by Bonferroni multiple comparisons tests (SPSS 20; IBM Corporation, Armonk, NY, USA). Threshold response values were then defined as the first concentration eliciting a significant activation of the receptor, with empty vector acting as a negative control. Finally, dose-response curves were fitted to the Hill equation by nonlinear regression in order to determine half maximal effective concentrations (EC50) and maximal amplitude [79] (SigmaPlot; Systat Software Inc. , Chicago, IL, USA).
Human bitter taste is believed to protect us from the ingestion of poisonous substances, thereby shaping food rejections. Bitter perception differs, however, across individuals, due to genetic variations in the ~25 bitter taste receptor (TAS2R) genes. A famous example known since the 1930s is the inherited bitter taste sensitivity to phenylthiocarbamide, which is associated with genetic polymorphisms in a single TAS2R gene. Yet, such simple receptor-substance associations do not reflect the full complexity of bitter perception, since individual bitter substances frequently activate several TAS2Rs. Here, we provide an in-depth analysis of the genetic variability influencing human bitter taste. While each study subject carried a different set of genetic polymorphisms, we found that most variations reside in just six blocks, each harboring only one to five haplotypes. Thus, besides simple associations between taste and TAS2R gene polymorphisms, we revealed complex associations dependent on linkage between several high- and low-sensitivity alleles. Indeed, subjects carried either sensitive or insensitive alleles for receptors sensitive to grosheimin, a bitter compound in artichoke, or at least one sensitive allele for receptors specific for absinthin, the bitter principle of absinth. In short, simple associations and complex genomic linkage determine sensitivity to selected dietary bitter compounds.
Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception