Genomic, genetic and functional dissection of bitter taste responses to artificial sweeteners.

Bitter taste perception is initiated by TAS2R receptors, which respond to agonists by triggering depolarization of taste bud cells. Mutations in TAS2Rs are known to affect taste phenotypes by altering receptor function. Evidence that TAS2Rs overlap in ligand specificity suggests that they may also contribute joint effects. To explore this aspect of gustation, we examined bitter perception of saccharin and acesulfame K, widely used artificial sweeteners with aversive aftertastes. Both substances are agonists of TAS2R31 and -43, which belong to a five-member subfamily (TAS2R30-46) responsive to a diverse constellation of compounds. We analyzed sequence variation and linkage structure in the ∼140 kb genomic region encoding TAS2R30-46, taste responses to the two sweeteners in subjects, and functional characteristics of receptor alleles. Whole-gene sequences from TAS2R30-46 in 60 Caucasian subjects revealed extensive diversity including 34 missense mutations, two nonsense mutations and high-frequency copy-number variants. Thirty markers, including non-synonymous variants in all five genes, were associated (P< 0.001) with responses to saccharin and acesulfame K. However, linkage disequilibrium (LD) in the region was high (D', r(2) > 0.95). Haplotype analyses revealed that most associations were spurious, arising from LD with variants in TAS2R31. In vitro assays confirmed the functional importance of four TAS2R31 mutations, which had independent effects on receptor response. The existence of high LD spanning functionally distinct TAS2R loci predicts that bitter taste responses to many compounds will be strongly correlated even when they are mediated by different genes. Integrative approaches combining phenotypic, genetic and functional analysis will be essential in dissecting these complex relationships.

Genetic, functional, and phenotypic diversity in TAS2R38-mediated bitter taste perception.

Mutational polymorphism in the TAS2R38 bitter taste receptor is a key determinant of threshold taste detection of isolated compounds, such as phenylthiocarbamide (PTC) and propylthiouracil (PROP), as well as complex orosensation-mediated traits such as diet choice and smoking habits. These relationships are accounted for, in part, by 2 common alleles differing in functionality, TAS2R38-PAV and TAS2R38-AVI. However, TAS2R38 harbors extensive additional polymorphism whose functional significance remains unknown. To examine this variation, we ascertained genetic diversity in 56 Caucasian subjects via whole-gene sequencing, analyzed allele-specific responses to 5 TAS2R38 agonists (PTC, PROP, goitrin, methimazole, and sinigrin) using in vitro assays, and assessed genotypic associations with threshold detection phenotypes. Sequencing identified 3 single-nucleotide substitutions encoding 3 amino acid changes (C145G/P49A, C785T/A262V, and A886G/I296V), which combined to form 6 haplotypes in our sample. In vitro assays revealed a continuous range of response across alleles, and associations with threshold were significant for all single nucleotide polymorphisms (P < 0.002) and PAV/AVI haplotypes (P < 0.001). Haplotypes other than PAV and AVI did not exhibit phenotypic associations in our sample, possibly as a result of their low frequencies. However, prior studies have indicated that these alleles are common in some global regions, suggesting that alleles rare in our sample may be phenotypically relevant in other populations.

Association of a bitter taste receptor mutation with Balkan Endemic Nephropathy (BEN).

BACKGROUND: Balkan Endemic Nephropathy (BEN) is late-onset kidney disease thought to arise from chronic exposure to aristolochic acid, a phytotoxin that contaminates wheat supplies in rural areas of Eastern Europe. It has recently been demonstrated that humans are capable of perceiving aristolochic acid at concentrations below 40 nM as the result of high-affinity interactions with the TAS2R43 bitter taste receptor. Further, TAS2R43 harbors high-frequency loss-of-function mutations resulting in 50-fold variability in perception. This suggests that genetic variation in TAS2R43 might affect susceptibility to BEN, with individuals carrying functional forms of the receptor being protected by an ability to detect tainted foods. METHODS: To determine whether genetic variation in TAS2R43 predicts BEN susceptibility, we examined genotype-phenotype associations in a case-control study. A cohort of 88 affected and 99 control subjects from western Bulgaria were genotyped with respect to two key missense variants and a polymorphic whole-gene deletion of TAS2R43 (W35S, H212R, and wt/Δ), which are known to affect taste sensitivity to aristolochic acid. Tests for association between haplotypes and BEN status were then performed. RESULTS: Three major TAS2R43 haplotypes observed in previous studies (TAS2R43-W35/H212, -S35/R212 and -Δ) were present at high frequencies (0.17, 0.36, and 0.47 respectively) in our sample, and a significant association between genotype and BEN status was present (P = 0.020; odds ratio 1.18). However, contrary to expectation, BEN was positively associated with TAS2R43-W35/H212, a highly responsive allele previously shown to confer elevated bitter sensitivity to aristolochic acid, which should drive aversion but might also affect absorption, altering toxin activation. CONCLUSIONS: Our findings are at strong odds with the prediction that carriers of functional alleles of TAS2R43 are protected from BEN by an ability to detect and avoid aristolochic acid exposure. Evidence for a positive association between high-sensitivity alleles and BEN status suggests instead that possession of toxin-responsive receptor variants may paradoxically increase vulnerability, possibly by shifting attractive responses associated with low-intensity bitter sensations. The broad-spectrum tuning of the ~25-member TAS2R family as a whole toward xenobiotics points to a potentially far-reaching relevance of bitter responses to exposure-related disease in both individuals and populations.