Nonsynonymous single nucleotide polymorphisms in human tas1r1, tas1r3, and mGluR1 and individual taste sensitivity to glutamate14

Mariam Raliou, Anna Wiencis, Anne-Marie Pillias, Aurore Planchais, Corinne Eloit, Yves Boucher, Didier Trotier, Jean-Pierre Montmayeur, and Annick Faurion
ABSTRACT Several studies indicate an essential role of the heterodimer Tas1R1Tas1R3 for monosodium L-glutamate (MSG) detection, although others suggest alternative receptors. Human subjects show different taste sensitivities to MSG, and some are unable to detect the presence of glutamate. Our objective was to study possible relations between phenotype (sensitivity to glutamate) and genotype (polymorphisms in candidate glutamate taste receptors tas1r1, tas1r3, mGluR4, and mGluR1) at the individual level. The sensitivity was measured with a battery of tests to distinguish the effect of sodium ions from the effect of glutamate ions in MSG. A total of 142 genetically unrelated white French subjects were categorized into 27 nontasters (specic ageusia), 21 hypotasters, and 94 tasters. Reverse transcriptase polymerase chain reaction and immunohistochemistry showed expression of tas1r1, tas1r3, and a-gustducin in fungiform papillae in all 12 subjects tested, including subjects who presented specic ageusia for glutamate. Amplication and sequencing of cDNA and genomic DNA allowed the identication of 10 nonsynonymous single nucleotide polymorphisms (nsSNPs) in tas1r1 (n = 3), tas1r3 (n = 3), and mGluR1 (n = 4). In our sample of subjects, the frequencies of 2 nsSNPs, C329T in tas1r1 and C2269T in tas1r3, were signicantly higher in nontasters than expected, whereas G1114A in tas1r1 was more frequent in tasters. These nsSNPs along with minor variants and other nsSNPs in mGluR1, including T2977C, account for only part of the interindividual variance, which indicates that other factors, possibly including additional receptors, contribute to glutamate sensitivity. Am J Clin Nutr 2009;90(suppl):789S99S.

respond to MSG by a decrease of intracellular cAMP (14) independently of the phospholipase C transduction pathway. The expression of several metabotropic glutamate receptors (mGluRs), including truncated forms, has been reported in rodent taste papillae: mGluR4, mGluR1, and mGluR2/3 (1520). NMDA (N-methyl D-aspartate) and non-NMDA ionotropic glutamate receptors (iGluRs) are also expressed in taste cells (2124), whereas MSG and glutamate receptor agonists NMDA and 2-amino-4-phosphonobutyrate induce complex membrane conductance changes in some taste cells (2527) as well as an increase or a decrease in intracellular calcium concentration on stimulation with MSG (28). Glutamate exhibits specic tastant properties as assessed with electrophysiologic and human psychophysical data (29, 30). Hamster chorda tympani responses to various agonists of brain glutamate receptors, including ionotropic a-amino-3-hydroxy-5methylisoxazole-4-propionic acidkainate iGluRs, suggest distinct receptor mechanisms (31). Glutamate elicits the umami taste (32) and enhances the palatability of various foods (33, 34). Ribonucleotides such as 5#-inosine monophosphate or 5#-guanosine-monophosphate (5#GMP), which reinforce the perceived intensity of MSG (35), also elicit an umami sensation but are discriminated from MSG by humans and by rats (36). The umami taste in humans is elicited by various agonists of iGluRs and mGluRs (31, 37). Gustatory neural responses in different strains of mice discriminating between compounds eliciting an umami taste suggest that these compounds involve distinct sensory pathways (38). Rats easily distinguish MSG from 5#GMP (36), MSG from 2-amino-4-phosphonobutyrate, and
1 From the NBS-NOPA, Institut National de la Recherche Agronomique, Jouy-en-Josas, France (MR, A-MP, AP, CE, DT, and AF); CESG-CNRS, pital Lariboisie ` re, Dijon, France (MR, AW, and J-PM); Service ORL, Ho Dentaire, UFR Odontologie, Paris, France Paris, France (CE); and Faculte (YB). 2 Presented at the 100th Anniversary Symposium of Umami Discovery: The Roles of Glutamate in Taste, Gastrointestinal Function, Metabolism, and Physiology, held in Tokyo, Japan, September 1013, 2008. 3 Supported by ACI INSERM-INRA, ANR (PNRA 1.5), COFAG-ECU, gion Ile de France, and the International Glutamate Technical Committee, Re a nongovernmental organization funded by industrial producers and users of glutamate in food. 4 Address correspondence to A Faurion, Neurobiologie Sensorielle, NBS t. 325, Domaine de Vilvert, Institut de la Recherche Agronomique NOPA, Ba (INRA), 78352 Jouy-en-Josas, France. E-mail: annick.faurion@jouy.inra.fr. First published online July 1, 2009; doi: 10.3945/ajcn.2009.27462P.

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INTRODUCTION

We have described subjects perceiving only the saltiness of monosodium L-glutamate (MSG) but not its specic taste nor its persistence (1), a specic ageusia independent of the ageusia to phenylthiocarbamide (2). The detection of glutamate in taste papillae is considered to involve Tas1R1 (taste receptor type 1, member 1) and Tas1R3 (taste receptor type 1, member 3) receptors (38) linked to phospholipase Cb2 and TRPM5 (transient receptor potential M5) activation and release of ATP to activate nerve bers (9). Knockout mice that lack a functional Tas1R3 receptor still exhibit behavioral and residual neural responses to MSG (1012). Furthermore, taste nerves still respond to MSG in mice with nonfunctional TRPM5 channels (13). Moreover, some taste cells

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MSG from NMDA (39). Altogether this body of literature suggests that responses to MSG in the taste system may involve other receptors than Tas1R1-Tas1R3 (11). Our aim was to examine a possible link between the glutamate taste sensitivity of subjects (phenotype) and their genotype. Taste sensitivity was measured with a battery of tests to distinguish the stimulatory effects of glutamate and the sodium cation in MSG. We looked for the expression of Tas1R1-Tas1R3 in taste buds and further explored for the presence of single nucleotide polymorphisms (SNPs) in tas1r1, tas1r3 (40, 41), as well as in mGluR1 and mGluR4 (41). We then established statistical relations between modications in the coding sequences of candidate receptors for umami and the observed phenotypes.
SUBJECTS AND METHODS

taste papillae. They were paid 10 euros/h for participating in further phenotype characterization by psychophysical testing in the laboratory. Available relatives (parents, grandparents, children, siblings, aunts, uncles, nephews, and nieces when available) of 11 tasters (n = 70; 28 men and 42 women) and of 17 nontaster or hypotaster subjects (n = 62; 28 men and 34 women) were selected to document the inheritance of both the phenotype and the genotype (total n = 274). Psychophysics All 142 subjects participated in tests addressing intensity, quality, and preference to document their individual capacity to discriminate between MSG and sodium chloride (study ap pital Saint Louis-Fernand Vidal, Paris; CCPPRB proved by Ho no. P0040805). Evaluation of isointense concentrations (MSG, GMP, and sucrose compared with 29 mmol NaCl/L) This test measures the stimulus concentration perceived as intense as the 29 mmol NaCl/L reference. Isointense concentrations (C-ISOs) were measured with the staircase Up & Down method (Up&D) of Dixon and Massey (42). Single tests were forced-choice paired comparisons; subjects answered the question: which is stronger? The concentration of stimulus varied according to subjects responses. One Up&D test represented an average of 78 paired presentations (15 min), and one session included 34 Up&D tests per stimulus. The experiment was computer assisted as described in Lugaz et al (1). Sixty-six subjects, including all putative nontasters and all hypotasters of the 142 independent subjects, participated in 6 sessions at intervals of several days. Questionnaire based on paired comparisons Four successive pairs, including 1) water compared with 29 mmol NaCl/L, 2) 29 mmol NaCl/L compared with 29 mmol MSG/L, 3) 29 mmol NaCl/L compared with 14.5 mmol GMP/L, and 4) 29 mmol MSG/L compared with 14.5 mol GMP/L, were presented, and the same series of 10 questions were asked for each pair: Do you perceive a difference of taste? Do you perceive a difference in the nature of the taste? Do you perceive a difference of intensity? Which tube has the stronger taste: A? or B? The hedonic value was assessed for each tube on a 210 to 10 scale. A description of the perception was required twice for each solution. A total of 252 subjects had this test. Some subjects not perceiving 29 mmol NaCl/L repeated this test with 43 mmol NaCl/L. Ranking test Three tubes that contained 29 mmol NaCl/L, 43 mmol NaCl/L, and 29 mmol MSG/L were ranked by 47 subjects according to perceived intensity. They also had to state whether the taste quality varied and whether they perceived persistence, the hallmark of MSG, in any solution. Electrogustometric threshold Electrogustometric test was used to detect and exclude subjects with general taste impairment. Electrogustometric threshold

Stimuli For tests at constant concentration, MSG (29 mmol/L; Ajinomoto Eurolysine, Paris, France) and disodium 5#GMP (14.5 mmol/L; Sigma-Aldrich, St Louis MO) were compared with isomolar sodium chloride (29 mmol/L; Merck Chimie SAS, Fontenay ss Bois, France) prepared in low mineral tap water. For some sodium-low sensitive subjects, test concentrations were increased to 43 mmol/L for MSG and sodium chloride and to 21.5 mmol/L for 5#GMP. For tests sent by mail with instructions, the substances were weighted in disposable tubes, and subjects were told to add a specied amount of water. For evaluation of isointensity concentrations, the reference for sodium chloride was 29 mmol/L; the maximal concentrations of solutions of MSG, 5#GMP, and sucrose were 60, 90, and 175 mmol/L, respectively. Subjects phenotype Screening A total of 3084 subjects were screened for their sensitivity to MSG in laboratories, Research Centers of Jouy and Dijon, Faculty of Odontology, Museums (La Villette, Palais de la couverte). The test was designed to preferentially select De subjects whose sensitivity to MSG was doubtful and included 2 paired presentations namely: water compared with 29 mmol NaCl/L and 29 mmol NaCl/L compared with 29 mmol MSG/L, each one associated with the question: Do you perceive a difference? If the subject could not perceive a difference between salt and water, the test was repeated with water and 43 mmol NaCl/L. If the subject clearly expressed a difference of perception between sodium chloride and MSG, he or she was supposed to be a glutamate taster. Otherwise, the subject was supposed to be a potential glutamate non or hypotaster and submitted to 1030 triangular tests. If the criterion of P = 5% (a risk) for discrimination was reached, the test was stopped, and the subject was categorized as glutamate taster. If not, the subject was categorized as a potential glutamate nontaster. Borderline subjects were categorized as potential hypotasters. All available potential non- and hypotasters as well as 94 tasters selected at random were included in the study. The nal study group consisted of 142 white, French, genetically unrelated subjects (55 men and 87 women). All were submitted to further psychophysics evaluation and provided oral cells on swabs; 20 provided fungiform
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was measured at 9 loci in 51 subjects; medication, smoker status, and the number of dental deafferentations were noted (4345). The family subjects received the questionnaire or the ranking tests or both by mail. If they did not obviously perceive glutamate different from isomolar sodium chloride, they were submitted to the discrimination triangular test, and eventually to the C-ISO evaluation test (the latter 2 in the laboratory). In case no quantitative measurement was possible on these relatives, putative nontasters were named hypotasters.

RESULTS

Phenotype On the basis of the complete set of test results, 142 genetically independent subjects were classied as 94 tasters, 27 nontasters, and 21 hypotasters (Table 1). It should be emphasized that these gures do not represent the natural frequency in the population (1): the proportion of nontasters was articially overrepresented for the sake of the phenotype-genotype correlation study. Tasters clearly perceived a peculiar taste for MSG and its persistence in the mouth; tasters type T (n = 70) disliked the taste of MSG and tasters type TL (n = 24) liked it. Nontasters (NT; n = 27) did not perceive glutamate: type NT subjects (n = 9) perceived sodium in MSG and in isomolar sodium chloride equally well, whereas type NTI subjects (n = 14) needed stronger MSG solutions to match the intensity of 29 mmol NaCl/L and evaluated sodium chloride stronger than MSG in isomolar solutions; their sensitivity to sodium was decreased (inhibited) in the presence of the glutamate ion. In type NTR (n = 2) conversely, the saltiness of MSG (the only taste perceived) was higher (reinforced) in MSG than in isomolar sodium chloride, and the isointense MSG concentration ,29 mmol NaCl/L, demonstrating an enhanced sensitivity to sodium by glutamate. Both NTI and NTR subjects may be missed at screening because they discriminate MSG from sodium chloride on an intensity criterion, as opposed to NT subjects. It was remarkable that in this group of 142 subjects, 2 of them acquired the sensitivity to glutamate on repeated exposure during the 6 sessions of isointense concentration evaluation to which every nontaster and hypotaster was submitted together with some tasters (Figure 1). This shows the pressing necessity of familiarization and repeated tests before categorizing subjects. Potential nontasters and hypotasters and some tasters measured their concentration of MSG and of sucrose isointense to sodium chloride during 6 sessions covering a minimum of 3 wk which ensures reproducibility. One subject who was measured at a 6-y interval, for example, gave the same isointense concentration [mean 6 SD: 56.1 6 7.8 (n = 26) and 53.6 6 12.1 (n = 5)], and similarly for several subjects who were repeatedly measured during 19 sessions and retested semiquantitatively after several years. Finally, hypotasters (n = 21) could discriminate MSG from sodium chloride at the limit of statistical criterion. Two types were distinguished: type H subjects (n = 11) perceived MSG as strong as or slightly stronger than isomolar sodium chloride; type HI subjects (n = 10) perceived glutamate less strong than isomolar sodium chloride. Genotype Expression of tas1r1, tas1r3, and gustducin Reverse transcriptasePCR on cDNA from taste papillae tissue showed the expression of the mRNA coding the candidate receptors Tas1R1, Tas1R3, and the G protein a-gustducin in tasters, hypotasters, and nontasters (Figure 2). Immunohistochemistry also showed that Tas1R1, Tas1R3, and a-gustducin were present in all subjects (41). Single nucleotide polymorphisms The 142 genetically independent subjects showed 20 SNPs, among which 10 nonsynonymous SNPs (nsSNPs) change the

Subject genotype mRNA Twenty subjects gave 4 fungiform papillae (study approved by pital Saint Louis-Fernand Vidal, Paris; CCPPRB no. Ho P0040805). The inclusion criteria were nonsmokers or subjects smoking ,6 cigarettes/d, no medication besides contraceptive pills or patches, ,7 dental deafferentations, and not pregnant. A small part of the anterior tongue was anesthetized by lidocaine (5%) and disinfected by chlorhexidine digluconate (0.2%). Fungiform papillae were gently pulled up with ne Brussels, and the upper part carefully sectioned with ophthalmological scissors by the study surgeons under sterile conditions. Papillae were instantaneously frozen (280C) so as to extract total mRNA. For histologic analysis, papillae were xed (4% paraformaldehyde in phosphate-buffered saline). Genomic DNA All 142 subjects collected their own oral swabs before or long after eating, gum chewing, or teeth brushing; the samples were kept at 20C for extraction of genomic DNA (BuccalAmp DNA Extraction, tebu-bio; Epicentre Biotechnologies, Madison, WI). Polymorphisms were identied by amplication and sequencing of the entire coding regions of tas1r1, tas1r3, mGluR1, and mGluR4 receptor genes from the genomic DNA of 20 subjects. Then, the presence of each SNP was further analyzed in the genomic DNA of 142 subjects. Polymerase chain reactions (PCRs) were performed with the Turbo Hotstart Pfu DNA proofreading polymerase (Stratagene, La Jolla, CA). Oral swabs of relatives were treated similarly.

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Statistics Factor analysis of correspondences (SPAD, version 5.5, 2002; Decisia, Pantin, France) was used to analyze the multidimensional space built from the individual phenotype and genotype characteristics (columns) of all subjects (lines). The analysis gives the dimension and structure of the subjects sensitivity space and the proximity relations between SNPs and subjects. Chi-square statistics (XLSTAT, version 2007; Addinsoft, Paris, France) evaluated the signicance of the prevalence of polymorphisms in each group of subjects compared with expectation if these polymorphisms were distributed independently from the observed phenotype. The table of contingency tested the independence of 2 characteristics: having or not having a given phenotype trait, and having or not having a given nucleotide polymorphism.

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TABLE 1 Categorization of 142 genetically unrelated subjects according to their sensitivity characteristics for monosodium L-glutamate (MSG)

Subject status ++ Unpleasant Signicant , Na29 , Na43 , MSG29

Perceived taste for 29 mmol MSG/L Persistence Intensity ranking1

Hedonic evaluation of MSG

Discrimination test (triangles), NaCl-MSG

MSG isointense concentration (vs 29 mmol NaCl/L)

Comments

Tasters disliking MSG (n = 70) ++ 0 Depending on judgment on salt Depending on judgment on salt Signicant . Nonsignicant = Pleasant Signicant , Na29 , Na43 , MSG29 Na29 = MSG29 , Na43

Strong taste, qualitatively different from NaCl

Tasters liking MSG (n = 24) Nontasters (n = 9)

Strong taste, qualitatively different from NaCl Salty

Nontasters who perceive sodium less intense in MSG than in isomolar NaCl (n = 14)

Water or slightly salty

MSG29 , Na29 , Na43

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Nontasters who perceive sodium stronger in MSG than in isomolar NaCl (n = 2)

Salty

Depending on judgment on salt

Signicant

Na29 , MSG29  Na43

Subjects becoming tasters with exposure (n = 2)

Nothing or salty; but, after several tests, a specic taste appears and becomes obvious

0 / ++

Nonsignicant, then signicant

 then ,

MSG29  Na29 , Na43 at screening then like tasters

Hypotasters (n = 11) Slightly Not very unpleasant

Slight taste, different or maybe different from salt Slightly Not very unpleasant

Just signicant or signicant

Na29  MSG29 , Na43

Hypotasters who perceive sodium less intense in MSG than in isomolar NaCl (n = 10)

Slight taste, different from salt

Signicant

MSG29 , Na29 , Na43

Perceive the glutamate anion stronger than the sodium cation Perceive glutamate stronger and like it Do not perceive the glutamate anion, only the sodium cation Do not perceive the glutamate anion; moreover, the perception of the sodium cation is reduced in presence of glutamate anion Do not perceive the glutamate anion, but the perception of the sodium cation is reinforced in presence of glutamate anion Nontasters becoming tasters by repeated exposure to MSG (at least four to ve1-h repeated quantitative testing sessions) Perceive the glutamate anion stronger or slightly stronger than the sodium cation Perceive the taste of glutamate anion, but the intensity perceived for MSG is reduced compared with the perception elicited by isomolar NaCl

Na29, 29 mmol NaCL/L; Na43, 43 mmol NaCl/L; MSG29, 29 mmol MSG/L.

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FIGURE 1. Individual mean (6SD) data showing examples of quantitative evaluation of suprathreshold sensitivity [monosodium Lglutamate (MSG) concentration isointense to 29 mmol NaCl/L] with repeated exposure to the stimulus. Subjects were tested during six 1-h sessions comparing variable concentrations of MSG to 29 mmol NaCl/L; the horizontal line shows the reference of 29 mmol NaCl/L. The taster subject T rst evaluates 9 mmol MSG/L, which elicits a sensation isointense to 29 mmol NaCl/L, then, from the third session on, the isointense concentration (C-ISO) is stable at 4.8 6 1.2 mmol MSG/L. NT represents the nontaster subject who had difculty in evaluating the concentration of MSG isointense to 29 mmol NaCl/L (reference) as 60 mmol/L, the maximum available concentration is not sufcient, and C-ISO often nears 60 mmol/L with a ceiling effect; this subject (nontaster inhibited) shows inhibition of his perception of sodium in the presence of the glutamate anion, although he does not taste glutamate in MSG. The third subject (subject shifts to T) could not match, at rst, the intensity perceived for 29 mmol NaCl/L, even with 60 mmol MSG/L, but, by the fth session, he had reached the level of sensitivity of tasters (below the 29 mmol/L line). Subjects were categorized as nontasters or hypotasters only after repeated quantitative evaluation of MSGsodium chloride C-ISO (repeated exposure).

homozygous. Seven subjects were heterozygous for G1520A (R507Q, in the protein ECD). tas1r3: Fourteen subjects were mainly heterozygous (n = 13) for mutation G13A (A5T in the protein ECD). Seven subjects presented the mainly heterozygous (n = 6) mutation C2269T (R757C, at the border between the transmembrane domain and the internal C-terminal of the protein). Two subjects presented the mutation T2246C (F749S, in the protein transmembrane domain). mGluR1: One hundred ten subjects presented the mutation T2977C in the exon 8 (P993S rs6923492), among them 39 were homozygous. Other mutations corresponded to A1670C (Q557P) in the exon 6 (1 subject), G2651A (G884E rs362936) in the exon 7 (5 subjects), and C3206T (P1069L) in exon 8 (3 subjects). All these nsSNPs are located in the C-terminal tail of the protein. No nsSNPs, but only 3 silent SNPs, were identied in mGluR4. Allelic frequencies of the nsSNPs are very low (12%) except for G1114A and T2977C (41)
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Phenotype-genotype relations: occurrence of nsSNPs in the different groups of subjects tas1r1 The mutation C329T, found in 10 subjects, was more frequent in nontasters (5 of 27) than expected if this polymorphism was distributed independently from the observed phenotype (P = 0.007, chi-square test) and similarly in hypotasters (2 of 21; P = 0.01). It was less frequent (3 of 94) than expected in tasters (P = 0.005; Table 3, top). This mutation was also associated with the inhibition of salt taste in the presence of the glutamate anion (P = 0.04). Conversely, the mutation G1114A found in 44 subjects (40 heterozygous and 4 homozygous subjects) was more frequent than expected in tasters (P = 0.008). The mutation G1520A (Table 2), found in 7 subjects, only tended to be more frequent in nontasters (P , 0.1; not shown in Table 3). tas1r3 The mutation C2269T, found in 7 subjects, was less frequent than expected in tasters (P = 0.04) and tended to be more frequent in nontasters (P = 0.08). The mutation G13A, found in 14 subjects, was associated with nontasters and hypotasters without inhibition of sodium taste by glutamate (P = 0.04). mGlur1 The mutation T2977C was found in 71 heterozygous subjects and 39 homozygous subjects. As for C329T in tas1r1 and C2269T in tas1r3, the mutation T2977C in mGluR1 tended to be associated with the nontaster phenotype: the heterozygous mutation tended to be more frequent than expected in nontasters and hypotasters without inhibition of salt taste in the presence of glutamate (P = 0.08), and the homozygous mutation tended to be less frequent than expected in tasters (P = 0.10). Other nsSNPs were too rare to be considered individually for statistical evaluation but were considered all together in the group of minor variants. Relation between the occurrence of nsSNPs and assessments obtained in the questionnaire Twenty-six nontasters, 19 hypotasters, and 79 tasters lled in the questionnaire, totalling 124 genetically unrelated subjects

amino acid in the protein (Table 2). For the statistical evaluation of the genotype-phenotype relation, only nsSNPs were considered in this study. tas1r1: Ten subjects presented the heterozygous mutation C329T [A110V in the extracellular domain (ECD) of the protein]. Forty-four subjects presented the mutation G1114A (A372T in the protein ECD), among them 4 were

FIGURE 2. Nested reverse transcriptasepolymerase chain reaction (RTPCR) was performed on total RNA extracted from 2 fungiform papillae per subject. Fungiform papillae of tasters (T), one hypotaster (H) and nontasters (NTs) expressed mRNA coding for a-gustducin, Tas1R1, and Tas1R3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a positive control of RT-PCR, and a-gustducin was used as a positive control for the presence of taste buds in our samples. Human tongue RNA (Ton) was used as a positive control of the presence of a-gustducin. Filiform papillae (Filif), without taste buds, were used as a negative control. Testis RNA (Testis) was used as a control for tas1r1 and tas1r3 primers. The (+) and (2) indicate whether the reverse transcriptase was present (+) or omitted (2) in the reverse transcription reaction; Con2 indicates negative control for RT-PCR; M, molecular weight marker.

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TABLE 2 Nonsynonymous single nucleotide polymorphisms (nsSNPs) in candidate taste receptors1 tas1r1 C329T Heterozygous subjects Homozygous subjects3 Nontasters and hypotasters (n = 48) Tasters (n = 94)
1 2

tas1r3 G1520A 7 3 4 G13A T15A 13 1 7 7 T2246C 2 C2269T 6 1 4 3 A1670C 1

mGluR1 G2651A 5 T2977C 71 24 47 39 17 22 C3206T 3

G1114A 40 4 8 36

10 7 3

Values are numbers of nsSNPs observed in 3 candidate taste receptor genes in this studys 142 white French subjects that led to modication of an amino acid in the receptor protein. 2 One allele was mutated. 3 Both alleles were mutated.

(Table 3, bottom). Subjects with the mutation C329T in tas1r1 (n = 9) were more often unable to propose words to describe the taste of MSG than expected if the perception were independent of the genotype (P = 0.01). Subjects with the mutation G1114A in the same gene (n = 37) could discriminate MSG from sodium chloride more often than expected, from the nature of the taste (P = 0.05); they could discriminate MSG from GMP, from the perceived intensity (P = 0.01). Subjects with the mutation G1520A in tas1r1 (n = 6) tended to be unable to perceive the difference between water and 29 mmol NaCl/L at rst testing (P = 0.06) and could not describe MSG (P = 0.03; not shown in Table 3). Subjects with the mutation C2269T in tas1r3 (n = 5) did not perceive a difference (P = 0.0001), neither in terms of a qualitative difference of taste nor of intensity,- between so-

dium chloride and MSG as often as would be expected if the perception were independent of the SNP. They also did not perceive the difference between sodium chloride and 5#GMP (P = 0.009) and were not able to propose words for the taste of umami solutions (P = 0.04). Subjects with the mutations G13A in tas1r3 (n = 14) did not reach any signicant statistical criterion for any tentative relation in these data. Subjects with one allele mutated in T2977C in mGluR1 (59 heterozygous of 124 subjects, not shown in Table 3) tended not to discriminate MSG from sodium chloride (P = 0.07) and did not describe a taste for MSG as often as expected (P = 0.04). Adding both the homozygous and heterozygous together led to a group of 96 of 124 subjects who did not discriminate, as often as expected, MSG from sodium chloride (P = 0.01) or GMP from

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TABLE 3 Statistical evaluation of phenotype-genotype relations1 tas1r1 C329T Cov Categorization (from all tests) NT (n = 27) H (n = 21) T (n = 94) NT and H with sodium taste inhibited in presence of glutamate (n = 25) NT and H without inhibition to sodium (n = 23) TL (n = 24) Questionnaire3 Discriminate water NaCl Discriminate NaCl MSG Discriminate NaCl GMP Discriminate intensity of MSG GMP Identify water Identify salt Describe MSG
2

tas1r3 G1114A G13A Cov 0.04 Cov 0.08 0.08 C2269T Anti-cov 0.04 0.0001 0.009 0.02 0.04 Cov 0.08

mGluR1 T2977C Anti-cov 0.10 0.01 0.03 0.06

Anti-cov 0.005 0.01 0.01

Cov 0.008 0.05 0.01

Anti-cov 0.008 0.03 0.003

0.007 0.01 0.04 0.03

1 Cov, covariance between the genotype and the phenotype tested; Anti-cov; anticovariance between genotype and phenotype; NT, nontasters; H, hypotasters; T, tasters; MSG, monosodium L-glutamate; TL, tasters who like MSG; GMP, guanosine-monophosphate. 2 Subjects (n = 142) were grouped according to all sensory tests. P values (chi-square test) that resulted from a test of independence between the presence of nonsynonymous single nucleotide polymorphisms (nsSNPs; columns of the contingency table) and the phenotype characteristics of subjects (lines of the contingency table) are shown. n = 10 for C329T; n = 44 for G1114A; n = 14 for G13A; n = 7 for C2269T; n = 110 for T2977C. 3 P values (chi-square test) that resulted from the test of independence between the presence of nsSNPs and some of the questions of the questionnaire on MSG-NaCl discrimination and MSG identication ability of 124 subjects are shown. n = 9 for C329T; n = 37 for G11114A; n = 14 for G13A; n = 5 for C2269T; n = 96 for T2977C.

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sodium chloride (P = 0.03). The high prevalence of the mutation in all subjects is responsible for low levels of signicance of the differences between nontasters and tasters; however, this gene seems to contribute to signaling MSG. Relation between C-ISOs and nsSNPs The concentration of MSG found to be isointense to the sodium chloride reference varied across subjects, from 1.8 mmol/L to values above mother solutions (60 mmol/L for MSG and 90 mmol/L for 5#GMP) hence, .29 mmol NaCl/L. Results were very consistent at least in the last 2 d of a 6-d protocol, the learning phase being visible in the rst 24 d. These data showed that paired GMP and MSG C-ISOs were correlated at r = 0.75 (Pearson r; df = 19), whereas the correlation between MSG and sucrose or GMP and sucrose were very low (r = 0.36; Pearson r; df = 42; r = 0.23; Pearson r; df = 19), showing that mechanisms signaling sucrose are clearly discriminated from mechanisms discriminating either MSG or GMP. Chi-square calculations on 53 subjects showed a trend to associate C329T with high C-ISO (meaning low sensitivity: P , 0.10), a trend for G1114A not to be associated with elevated CISO (P , 0.10), and a trend for the mutation G1520A to be associated with elevated C-ISO (P = 0.06). These data are reected in factor analysis of correspondences (Figure 3). Inheritance of phenotype and genotype: a family study The group of 62 relatives of nontasters or hypotasters as dened in Subjects and Methods contained 11 nontasters or hypotasters, whereas the group of 70 relatives of tasters did not contain any nontaster or hypotaster. This statistically signicant

difference (P , 0.001, chi-square test) points at glutamate ageusia being an inherited trait. The proportion of mutations compared with wild alleles was compared across the 4 groups (17 nontasters and hypotasters, their 62 relatives; 11 tasters and their 70 relatives) for nsSNPs C329T in tas1r1, C2269T in tas1r3, and T2977C in mGluR1. The 17 nontasters or hypotasters presented 20% of mutated alleles, their 62 relatives presented 11.4%, the 11 tasters presented 10% and their 70 relatives presented 5.2%. These gures are signicantly different from a random distribution (P , 0.001, chi-square test; ddl = 3). The difference of the prevalence of nsSNPs in nontasters and hypotasters compared with the group of their relatives was signicant (P = 0.04, chi-square test; ddl = 1); this group, genetically related to nontasters and hypotasters, showed a lower prevalence of nsSNPs. The difference of proportion of polymorphisms between tasters and their relatives was not signicant. The group of tasters relatives and the group of nontasters or hypotasters relatives, on the contrary, had signicantly different prevalence of nsSNPs (P = 0.005). Hence, the signicant difference of the prevalence of nsSNPs shown in tasters compared with nontasters and hypotasters in genetically unrelated subjects is also found between relatives of nontasters or hypotasters and relatives of tasters. Looking at minor variants specically, without nsSNPs C329T and G1114A in tas1r1, C2269T in tas1r3, and T2977C in mGluR1, showed signicantly more of these minor nsSNPs in the families of nontasters or hypotasters (3.9%) than in the families of tasters (1.4%; P = 0.02). The same calculation across 4 groups taking in account the 9 nsSNPs in tas1r1, tas1r3, and mGluR1, including C329T, C2269T, T2977C, and minor variants, comes up to 5.2%, 4.6%, 3.7%, and 2.5%, respectively

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FIGURE 3. Factor analysis of correspondences. Factor analysis of data (SPAD, version 5.5; Decisia, Pantin, France) included both subjects phenotypes classied into categories and genotypes noted as occurrence or no occurrence of each nonsynonymous single nucleotide polymorphism (nsSNP) in each subject (n = 142). T, tasters; TL, tasters liking monosodium glutamate (MSG); NT, nontasters; NTI, nontasters who perceive sodium less strong in the presence of the glutamate anion (so-called inhibited); H, hypotasters; HI, hypotasters with sodium taste inhibited in presence of glutamate. This gure shows particularly well the discrimination of tasters compared with nontasters and hypotasters on the rst axis and nontasters compared with nontasters whose sensitivity for sodium is inhibited in the presence of glutamate on the third axis. The nsSNPs C329T in tas1r1 and C2269T in tas1r3 are associated with nontasters and hypotasters, whereas the nsSNP G1114A in tas1r1 is associated with tasters. Other nsSNPs, including T2977C in mGluR1 in homozygous subjects (ho), are tentatively associated with nontasters and hypotasters.

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(P = 0.009, chi-square test; ddl = 3). Again, the prevalence of nsSNPs in the relatives of nontasters and hypotasters was signicantly different from the prevalence of nsSNPs in the relatives of tasters (P = 0.003, chi-square test; ddl = 1). Hence, the phenotype seems to be transmitted to relatives together with the prevalence of nsSNPs studied here. The observation of mutations in 28 families showed that nsSNPs are transmitted from generation to generation.

DISCUSSION

MSG concentrations that were found to be isointense to 29 mmol NaCl/L varied among subjects continuously from 1 to .60 mmol/L. This study could categorize subjects into 3 classes. Tasters, who easily discriminated between MSG and isomolar sodium chloride, perceived a typical persistence and a typical taste for MSG; 2 subclasses were observed according to hedonic valence, most subjects considering it as unpleasant, fewer liking it. Nontasters were not able to perceive either the taste or the persistence of glutamate itself and described only a salty sensation. Three subclasses of nontasters could be distinguished according to the intensity of the salty perception elicited by MSG compared with isomolar sodium chloride. Apparently, in some nontasters, the presence of glutamate interfered with the detection of sodium ions either by decreasing it (56% of NT) or by increasing it (8%). It should be emphasized that nontasters with sodium taste modied by glutamate may easily be confused with tasters, eg, inhibited nontasters or NTI, who discriminate MSG from sodium chloride because they perceive MSG less intensely than an isomolar solution of sodium chloride. Clearly, various complementary tests are needed to properly establish individual phenotypes; in this study, the evaluation of the concentration of MSG eliciting the same intensity as the reference was a key tool to identify NTI and discriminate them from tasters. In addition, in some cases, tasters perceiving isointensity at isomolarity can be identied because their hedonic valence for MSG is reproducibly highly different from their hedonic valence for sodium chloride. Finally, subjects classied as hypotasters were subjects who were at the statistical limit for discrimination and who were tremendously less sensitive to glutamate than tasters, even after the six 1-h repeated familiarization testing sessions ensuring sufcient repeated exposure to the stimulus (46). Moreover, these subjects were doubtful about the nature of their perception and some referred only to textural semantics (viscous, thick, fat, or a sensation). Two subclasses of hypotasters could be distinguished; as for nontasters, 50% of hypotasters showed an inhibition of the sodium sensitivity in the presence of glutamate. In tasters, the sensitivity to the stimulus increased on repeated exposure and C-ISOs were modied in a factor by 23 as already shown (46). This is reminiscent of data obtained when recording from hamster chorda tympani (47). But most remarkably, 2 subjects, apparently nontasters after discrimination tests, became tasters through repeated exposure to MSG in quantitative evaluations (the six 1-h testing sessions). In these cases, the decrease of the MSG concentration isointense to the reference after exposure was about 20-fold. For this reason, in this study, all nontasters and hypotasters together with some control tasters were submitted to the C-ISO evaluation test.

Furthermore, most subjects were retested 1 y (and 8 y) after to conrm their phenotype, before classication. Only C329T, G1520A, and G1114A in tas1r1; G13A and C2269T in tas1r3; and T2977C in mGluR1 were submitted to individual statistical analysis, the other nsSNPs being too rare to be treated individually. C329T in tas1r1, C2269T in tas1r3, and T2977C in mGluR1, taken individually, were signicantly associated with the nontaster trait. Conversely, G1114A in tas1r1 was associated with tasters. G1520A in tas1r1 and G13A in tas1r3 showed only a trend in their association with the NT phenotype. Taste spaces obtained from factor analysis of correspondences on subjects and phenotype categories (Figure 3) or on single responses in the questionnaire or on C-ISOs (not shown) all exemplied the association of C329T and C2269T to nontasters or hypotasters and G1114A to tasters. C329T and G1114A are both localized on the external domain of Tas1R1, which suggests that they may modify the binding of glutamate. Interestingly, all subjects with both C329T and G1114A were tasters, which suggests that G1114A might be compensating the possible decit because of C329T. Interestingly, G1114A exhibits a much higher allelic frequency (20%) than does C329T or C2269T (11.5%) (41). C2269T is localized at the border between the transmembrane domain and the C terminal tail and therefore could affect signal transduction. This study shows that a part of the interindividual variability of sensitivity to MSG and 5#GMP is likely controlled by nsSNPs in tas1r1 and tas1r3, thus emphasizing the role of Tas1R1-Tas1R3 in MSG detection. This work adds to a long list of genetic polymorphisms affecting taste receptor function (4851). The fact that not all nontasters exhibited either C329T or C2269T, which, conversely were possibly found in some taster subjects, suggests that none of these nsSNPs is either necessary or sufcient to explain the taster or nontaster phenotypes. Extending the analysis to mGluR4 coding for another candidate glutamate taste receptor did not show nonsynonymous polymorphisms. In contrast, 4 nsSNPS were found in mGluR1, including T2977C, which was frequent enough (50% heterozygous and 28% homozygous subjects) to allow statistical evaluation to show a higher prevalence than expected in subjects who did not discriminate either MSG from sodium chloride or 5#GMP from sodium chloride. Although P values were moderately low, several criteria in the data converged to correlate the occurrence of this T2977C nsSNP and the non taster or hypotaster phenotype. Factor analyses indicated a low weight, hence a discrete contribution of T2977C to the discrimination of tasters from nontasters. The prevalence of C329T or C2269T is signicantly different between nontasters or hypotasters (n = 11 of 48) and tasters (n = 6 of 94; P = 0.005, chi-square test; df = 1), but 37 of the former group do not display these nsSNPs. Including minor variants, 18 of 48 nontasters or hypotasters presented 1 or 2 of the 5 nsSNPs found in tas1r1 and tas1r3 (except G1114A which is associated with tasters) compared with 17 of the 94 taster subjects. The difference is statistically signicant despite that 30 of 48 nontasters or hypotasters did not show any nsSNPs in tas1r1 and tas1r3. When considering 4 nsSNPs found in mGluR1 together with 5 nsSNPs in tas1r1 and tas1r3 (excluding G1114A), 98% of nontasters and hypotasters compared with 75% of tasters have 13 of the 10 possible nsSNPs found in these 3 genes (P , 0.001, chi-square test; df = 1). Moreover, comparison of the

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genotype of family members disclosed the apparent inheritance of the phenotype together with a signicantly higher prevalence of C329T, C2269T, and T2977C in the relatives of nontasters and hypotasters compared with the relatives of tasters. Focusing on all possible combinations of the 10 nsSNPs found in the 48 nontasters and hypotasters, we observe 20 combinations. Looking at nontasters specically and excluding G1114A, 5 of the 13 different combinations observed in the 27 nontasters were also represented in tasters. This constitutes a further argument adding to our nding that the interindividual variance is not entirely explained by the 10 nsSNPs we found in tas1r1, tas1r3, and mGluR1. Taken together these results indicate that nsSNPs found in tas1r1, tas1r3, and mGluR1 account for part of the interindividual variance to MSG. Still, all combinations of nsSNPs in the 3 genes are not sufcient to explain all the phenotypic data. Regulation factors might justify the increase of sensitivity during repeated exposure to the stimulus but could not explain specic ageusia to glutamate because Tas1R1 and Tas1R3 receptors were present in nontasters. Hence, the present results strongly suggest that other factors than Tas1R1-Tas1R3 need to be taken into account to fully explain the sensitivity to MSG, as suggested by other studies (1013, 52, 53). Indeed, there are strong arguments in favor of the involvement of several receptors for umami taste (10, 11, 29, 39). MSG and 5#GMP are discriminated, several compounds, including ionotropic nonNMDA agonists (31, 37), mGluR type I (18, 19), mGluR type III (1517), and mGluR type II (20) agonists, taste umami (37). Moreover, some antagonists of these receptors, namely methylserine-O-phosphate for mGluR type III, 6-cyano-7nitroquinoxaline-2,3-dione for ionotropic receptors can inhibit chorda tympani responses in the rodent (Vandenbeuch and Faurion, unpublished data, 2009).
CONCLUSIONS

phenotypes and tastes elicited by glutamate. Therefore, the possibility remains that, besides nsSNPs in these 3 genes, other independently coded receptor mechanisms functionally cooperate to perceive glutamate. (Other articles in this supplement to the Journal include references 5482.)
The PCR conditions, primer sequences, and sequences containing the polymorphisms have been deposited in GenBank under accession numbers BV725433BV725462. We thank Patrick MacLeod (Prof E.P.H.E. Paris) and Victoria Barham (University of Ottawa) for reading the manuscript; Jean Claude Pernollet rie Be zirard and and Lo c Briand for comments throughout the study; Vale atrice TrotSylvie Grall for technical assistance; and Camille Legrand and Be ier for on-site psychophysical testing. The authors responsibilities were as followsMR, AW, and J-PM: carried out immunohistochemistry, genotyping, and RT-PCR; A-MP and AP: collected psychophysical data and designed and constructed the mobile prototypes; CE and YB: collected taste papillae; DT and AF: wrote the manuscript; and AF: designed the study. The presenting authors (AF) expenses associated with participation in the symposium were paid by the conference sponsor, the International Glutamate Technical Committee, a nongovernmental organization funded by industrial producers and users of glutamate in food. None of the authors had a conict of interest.

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