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provided by Elsevier - Publisher Connector Current Biology, Vol. 15, 1257–1265, July 26, 2005, ©2005 Elsevier Ltd All rights reserved. DOI 10.1016/j.cub.2005.06.042 Positive Selection on a High-Sensitivity Allele of the Bitter- Receptor TAS2R16

Nicole Soranzo,1,* Bernd Bufe,3 Pardis C. Sabeti,4 by the N172 allele may have driven the signal of selec- James F. Wilson,1,5 Michael E. Weale,2 tion at an early stage of human evolution. Richard Marguerie,1 Wolfgang Meyerhof,3 and David B. Goldstein1,6 Introduction 1Department of Biology 2 Galton Institute for Molecular Medicine The chemical senses of taste and smell control our in- University College London teraction with the chemical environment and influence Gower Street, London WC1E 6BT our food preference [1]. Among the five different taste United Kingdom modalities recognized by —bitter, sweet, sour, 3 German Institute of Human Nutrition Potsdam- umami (monosodium glutamate), and salty—bitter taste Rehbruecke plays an important protective role because many plant Department of Molecular Genetics toxins have a bitter taste [1]. The ability to recognize Arthur-Scheunert-Allee 114-116 harmful compounds through taste may therefore influ- 14558 Nuthetal ence survival, at least in certain environments [2]. Hu- Germany mans can detect bitter compounds at very low concen- 4 Program in Medical and Population Genetics trations and show averse reactions toward foods that Whitehead/Massachusetts Institute for Technology are considered as excessively bitter [1]. Center for Genome Research The initiation of bitter-taste signaling is mediated by Cambridge, Massachusetts 02142 a highly variable family of seven-transmembrane G pro- tein-coupled receptor (TAS2Rs or T2Rs), com- prising 25 putatively functional genes and eight pseu- dogenes in humans [3–5]. TAS2R receptors are Summary encoded by small intronless genes that are 876–1020 bp [6] and are selectively expressed in Background: During periods of human expansion into cells of the tongue and palate epithelium [3]. Several new environments, recognition of bitter natural toxins TAS2R receptors having different substrate specificities through taste may have conferred an important selective are generally coexpressed within the same taste cells advantage. The G protein-coupled receptor encoded by [4]. This coexpression may explain why structurally un- TAS2R16 mediates response to salicin, amygdalin, and related compounds are often perceived by humans as many bitter β-glucopyranosides. β-glucopyranosides having a uniformly bitter taste [4]. are ubiquitous in nature, with many having a highly Mammals display considerable variation in their toxic cyanogenic activity. TAS2R repertoires [5, 7]. The evolution of the fam- Results: We examined evidence for natural selection ily along the mouse-human lineage took place through on the human receptor TAS2R16 by sequencing the en- gene duplication and adaptive diversification [5, 7]. In tire coding region, as well as part of the 5# and 3# UTRs, higher primates, comparative analyses of TAS2R genes in 997 individuals from 60 human populations. We de- have demonstrated overall hallmarks of neutral evolu- tected signatures of positive selection, indicated by an tion, with ongoing pseudogenization and loss of selec- excess of evolutionarily derived alleles at the nonsyn- tive constraints [5, 6]. However, there are some excep- onymous site K172N and two linked sites and signifi- tions to this overall trend. High levels of variability in cant values of Fay and Wu’s H statistics in 19 popula- the intercellular and C-terminal domains mediating tions. The estimated age range for the common substrate specificity and downstream signaling are ancestor of the derived N172 variant is 78,700–791,000 thought to ensure a wide spectrum of phenotypic re- years, placing it in the Middle Pleistocene and before sponse [5, 7, 8]. Segregation of different functional al- the expansion of early humans out of Africa. Using cal- leles in TAS2R receptors has been shown to contribute cium imaging in cells expressing different receptor vari- to variability in bitter-taste response in both humans ants, we showed that N172 is associated with an in- and mice [4, 9, 10]. One previous study documents nat- creased sensitivity to salicin, arbutin, and five different ural selection acting on functional TAS2R38 alleles [11]. cyanogenic glycosides. Diversification of TAS2R functions during phyloge- Conclusion: We have detected a clear signal of positive netic evolution is thought to reflect, at least in part, selection at the bitter-taste receptor gene TAS2R16.We adaptation to changing nutritional environments. The speculate that the increased sensitivity that is shown known substrates of TAS2R receptors include structur- toward harmful cyanogenic glycosides and conferred ally different compounds [9, 10, 12–15], some of which are potent natural toxins [12, 14]. The overall loss of *Correspondence: [email protected] evolutionary constraints in chimpanzees and in humans 5 Present address: Public Health Sciences, University of Edinburgh, [6] may be linked to the introduction of new diet and Teviot Place, Edinburgh, EH8 9AG Scotland. lifestyle practices that reduced the importance of bitter- 6 Present address: Center for Population Genomics and Pharmaco- genetics, Duke Institute for Genomic Sciences and Policy, Duke taste recognition as a means of toxin avoidance; for University, DUMC Box 3471, 4006 GSRB II, 103 Research Drive, instance, such practices include the use of cooking to Durham, North Carolina. inactivate toxins or increased meat and fruit dietary in- Current Biology 1258

Table 1. Summary of TAS2R16 Polymorphisms

Mean Frequency of Ancestral Allele (%)

a b dbSNP ID Nucleotide Change Amino Acid Change Ancestral Allele African Non-African FST rs2233988 C300T T100T C 85.6 99.1 0.4 ss32475769 G301A V101M G 100 w100 w0 rs2692396 C303G V101V C 13.7 0.8 0.38 ss32475771 C321G S108S C 97.1 w100 0.08 ss32475772 C333T T111T C w100 100 w0 ss32475773 A340G I114V A 96.8 w100 0.08 ss32475774 T347C L116P T 100 w100 w0 rs2233989 T460C L154L T 93 99.7 0.24 ss32475776 C481T P161S C 98.4 100 0.11 rs846664 G516T K172N G 15.6 0.9 0.45 ss32475778 A530C Q177P A 100 w100 w0 ss32475779 A646G N216D A 100 w100 w0 ss32475780 C662T A221V C 97.3 100 0.06 rs860170 A665G H222R A 96.4 69 0.21 ss32475782 G703A V235M G 98.8 100 w0 ss32475783 T718G F240V T 100 w100 w0 rs1204014 A846G T282T A 13.1 1.6 0.29 a rs dbSNP IDs indicate the six common alleles, and ss IDs indicate dbSNP accession numbers for variants discovered in this study. b Values calculated for the 52 populations of the HDP only are given.

take [16, 17]. It is likely, however, that TAS2R receptors derlined in Table 1; Table S1); the remaining 11 were mediating recognition of particularly harmful and ubiq- singletons or rare. Among the six high-frequency vari- uitous compounds may have been under stronger se- ants, four were synonymous changes and two were lective pressures, at least in some stages of human nonsynonymous changes. These two nonsynonymous evolution. changes are particularly important, because they may The TAS2R16 receptor is encoded by TAS2R16 (Hugo affect receptor function. The first, G516T (rs846664), Approved Gene Symbol [18], also known as hT2R16 leads to a Lys-Asn substitution at position 172 of the [3]). TAS2R16 mediates recognition of β-glucopyrano- protein (K172N). A665G (rs860170) causes a His-Arg sides [12], a class of compounds ubiquitous in nature substitution at amino acid 222 (H222R). [19], including some with a highly toxic cyanogenic ac- The polymorphic sites define four common haplo- tivity [1]. We investigated natural selection by rese- types (Table 2 and Figure 1A). Two main haplotypes, quencing the gene in a worldwide sample of 60 human HAP-A (N172/H222) and HAP-B (N172/R222), are both populations. We detected evidence of positive selec- characterized by evolutionarily derived alleles at the tion at TAS2R16, likely acting on the high-sensitivity al- nonsynonymous site K172N (N172). In addition, HAP-A lele N172. Overall, our data suggest that the improved carries the ancestral A allele at the site H222R (H222), sensitivity toward β-glucopyranosides has been advan- and HAP-B carries the derived G allele (R222). HAP-A tageous in the early stages of human evolution. and HAP-B are very frequent throughout the world and jointly account for 77.6% of all African and 98% of non- Results African haplotypes (Table 2). HAP-C (K172/H222) and HAP-D (K172/H222) are defined by ancestral states at Patterns of TAS2R16 Diversity K172N and H222R, and they are further differentiated in Human Populations by a synonymous substitution at locus T460C (L154L). We sequenced the entire coding sequence of TAS2R16 The two ancestral haplotypes HAP-C and HAP-D ac- (876 bp) and 203 bp of noncoding 5# and 3# UTR se- count jointly for 13.8% of all African and only 0.5% of quence in 997 individuals from a global sample. The non-African . population sample includes 52 populations of the hu- man diversity panel [20] and eight additional popula- Signatures of Positive Selection at TAS2R16 tions chosen to cover geographic regions poorly repre- The geographic distribution of TAS2R16 variants is sented in the human diversity panel (see Table S1 in the atypical of the majority of human alleles. Under neutral- Supplemental Data available with this article online). ity, evolutionarily derived variants will generally display We found 17 polymorphic sites in humans (Table 1), lower frequencies in populations than ancestral vari- all contained within the terminal two-thirds of the gene- ants do. A positive selection event, however, may act coding sequence (π = 6.1 × 10−4, q =1.9×10−3). For to rapidly increase the frequency of an allele and the these loci, we defined evolutionarily ancestral alleles by linked sites through the process of hitch-hiking [21]. comparison with the homologous primate sequences; The resulting decrease in linked variation represents a we defined evolutionarily derived alleles as the alterna- characteristic signature of positive selection and can tive alleles at these loci, or as the alleles newly arisen be detected with appropriate tests such as Fay and in humans. Out of the 17 polymorphic loci, six had Wu’s H statistic [22]. moderately high frequencies at least in Africa (un- We found that the three sites C303G (V101V), G516T Positive Selection on TAS2R16 1259

Table 2. Summary of TAS2R16 Haplotypes

Number of Chromosomes and Frequency (%, in Brackets)c Amino Acid at K172N/ Central/ Middle Haplotype H222R Functional Effect Africa America South Asia East Asia Europe East Oceania HAP-C/D KHa low sensitivity 34 (13.8) — 1 (0.2) — — 7 (2.4) — HAP-A NH high sensitivity 188 (76.4) 154 (80.2) 272 (66.7) 275 (58.5) 158 (65.8) 202 (70.1) 133 (88.7) HAP-B NR high sensitivity 3 (1.2) 36 (18.8) 126 (30.9) 193 (41.1) 74 (30.8) 75 (26) 16 (10.7) Otherb 21 (8.5) 2 (1) 7 (1.7) 2 (0.4) 8 (3.3) 4 (1.4) 1 (0.7) Total 246 192 408 470 240 288 150 a Ancestral state. b Haplotypes containing rarer/singleton variants and not tested in the functional analysis. c Regions defined in Table S1.

(K172N), and A846G (T282T) had very high derived- the TAS2R16 sequence data. We obtained highly signif- allele frequencies (Table 1), a pattern compatible with icant negative values of Fay and Wu’s H statistics in 19 positive selection acting on this gene. To test this hy- populations (Table S2), a deviation consistent with the pothesis, we applied Fay and Wu’s test of selection on hypothesis of positive selection acting on TAS2R16.

Figure 1. TAS2R16 Allele and Haplotype Fre- quency Distribution in the Worldwide Sample (A) Haplotype network for the four common haplotypes; the size of each pie chart is pro- portional to the total number of haplotypes; pie segments indicate main continents. (B and C) Worldwide frequency distribution of the two ancestral variants K172 (B) and H222 (C) are shown. Current Biology 1260

Three African populations (Biaka Pygmy, South African detects only recent selection events (roughly within the Bantu, and Yoruba) had borderline significant H values last 10,000 years [26]), this result does not exclude that (0.05 % p-value % 0.1); these populations had relatively selective events may have taken place at this locus in high ancestral-allele frequencies at these three sites in more ancient times. comparison to all the other populations. In all the re- maining populations, Fay and Wu’s test was not signifi- Age of the K172N Allele cant, as a result of the fact that the derived alleles at We calculated the posterior estimates of the genealogi- N172 and the two other linked variants were completely cal nodes immediately descendent from K172N on the fixed (Table S2). These populations therefore also con- Yoruba sample by using the method of Wilson et al. form to the hypothesis of a global selective event and [27]. Under the model of constant growth, the posterior exhibit an even more extreme genetic pattern than the 95% credible interval for the age of the node is 4,141– populations with significant H values. 39,571 generations, or 82,824–791,420 yr (assuming 20 The excess of linked derived alleles is the principal years per generation). Under the coalescent-with- indicator of selection here. We did not detect signifi- growth model, the estimate of the age for the descend- cant evidence for selection with other tests of selection ent node is 3,887–34,269 generations, corresponding based on the allelic spectrum; these tests included to 77,751–685,380 yr. Although they have wide credible both tests sensitive to ancestral states (Fu and Li’s F intervals, even the lower range of these estimates and D) and tests that are not sensitive to ancestral places the age of the mutation prior to the Neolithic states (Tajima’s D, Fu and Li’s F* and D*) (Table S2). revolution and prior to the expansion out of Africa un- Failure to detect natural selection here is not alarming der some models [28]. because these latter tests of selection are expected to be less sensitive than Fay and Wu’s H, particularly Functional Characterization of the K172N those not drawing on information about ancestral states. and H222R Alleles TAS2R16 appears to differ from the majority of hu- K172N lies in the second extracellular loop domain of man loci with respect also to population differentiation the TAS2R16 receptor. The K>N exchange at this site and extended linkage disequilibrium [23]. We estimated may affect interaction between the receptor and its li- the value, F , of the population-differentiation statis- ST gands. The other nonsynonymous site, H222R, lies tics for the 17 TAS2R16 sites in a subset of the data within the third intracellular loop domain. The H>R ex- corresponding to the 52 populations of the human di- change at this locus creates a putative protein kinase versity panel (Table 1). K172N had the highest F value ST A site and could influence receptor desensitization. We among all TAS2R16 sites (0.45). We then compared the analyzed the function of these two nonsynonymous F at K172N to an empirical F distribution of 210 ST ST variants by using heterologous expression and calcium short indels typed in the same sample. We found that imaging. To this end, we transiently transfected plas- only 11 out of 210 indels had an F higher than that of ST mids containing different permutations of the K172N K172N, corresponding to an empirical p-value of 0.057 and H222R variants into HEK293 cells containing the (=12/211). All the other TAS2R16 variants had lower F ST chimeric G protein G16-Gust44. values (0.21–0.4) than K172N’s. We first tested whether the K172N substitution alters We also analyzed a 222 kb interval surrounding receptor function. We challenged cells transfected with TAS2R16 and detected no apparent decay of linkage the N172/R222 and the K172/R222 constructs with disequilibrium, as measured by the D’ statistics [24], seven structurally divergent glycosides. The com- with physical distance (Figure S1). We carried out an pounds tested are all natural compounds, and they in- exact test of linkage disequilibrium for all pairwise sin- clude salicin (found in willow trees), arbutin (bearberry), gle nucleotide polymorphism (SNP) comparisons within the region. When we considered all SNPs, 86% of all amygdalin (bitter almonds), linamarin (manioc), pru- possible pairwise comparisons had significant p-values nasin (almonds), osmaronin epoxide (Rosaceae), and (p % 0.05); 97% of the comparisons were significant if 4-glucosyloxymandelonitrile (Berberidaceae) [12]. We loci with minor allele frequencies of 0.2 or higher were found that upon stimulation with all agonists, cells con- considered. We note, however, that extended linkage taining the TAS2R16 variants responded with a robust disequilibrium may result either from positive selection transient increase of the intracellular-calcium levels, or from locally decreased recombination rates [23, 25]; whereas mock-transfected control cells did not re- therefore, extended linkage disequilibrium alone does spond (Figure 2A). Notably, at low concentrations, the not provide evidence for selection, although compati- K172/R222 receptor variant showed a weaker response ble it is with it. Finally, we cannot exclude that a variant to all agonists than the N172/R222 receptor did (Fig- outside the sequenced region may be driving the signal ure 2A). of positive selection at TAS2R16, although we also note To test whether the observed functional difference is that the two closest genes are located at very large due to altered agonist sensitivity, we next challenged distances (around 100 kb) at the limit of the region of the K172/R222 and N172/R222 variants with three struc- high linkage disequilibrium. turally divergent TAS2R16 agonists (salicin, arbutin, and In addition to K172N, we also investigated the extent amygdalin) and calculated the relative concentration- to which positive selection may have influenced the ge- response curves (Figure 2B). We found that the half- ographical distribution of the second nonsynonymous maximal effective concentrations (EC50) for N172/R222 variant (H222R) by using the long-range haplotype test were 0.8 ± 0.2 mM for salicin, 5.5 ± 1.9 mM for arbutin, (LRH, [26]) (Supplemental Data). The LRH test did not and 6.0 ± 1.5 mM for amygdalin. The EC50 values of show evidence of recent positive selection at TAS2R16, K172/R222 were right-shifted to higher concentrations, and in particular at the H222R site. Because the test i.e., 2.6 ± 0.9 mM for salicin, 10.8 ± 3.7, mM for arbutin, Positive Selection on TAS2R16 1261

Figure 2. Functional Analyses of the TAS2R16 Variants in HEK293 Cells (A) Intracellular-calcium traces of the TAS2R16 constructs N172/R222 and K172/R222, as well as mock-transfected control cells recorded in the FLIPR experiment upon stimulation with seven structurally divergent compounds, are shown. The scale bar denotes 2000 relative light units and 1 min. (B) Concentration-response relations of cells transfected with different TAS2R16 constructs upon stimulation with salicin, arbutin, or amygda- lin are shown.

and 9.9 ± 2.2 mM for amygdalin. Thus, the comparison TAS2R16 receptors that contained the K172 or the of the EC50 values demonstrates a clear functional dif- N172 variant. ference between receptor variants containing either the Next, we analyzed the cell-surface expression of all K172 or the N172 allele, with the receptor variant K172/ receptor variants by using confocal microscopy. The re- R222 being about 2-fold less sensitive compared to sults clearly show that all four TAS2R16 variants are N172/R222. equally well targeted to the cell surface (Figure S3D). We also tested whether the amino acid substitution To control whether variations in the transfection rates H222R affects the receptor function by challenging the could account for the observed differences, we trans- receptor variants N172/H222 and K172/H222 with sali- fected the cells with different dilutions of TAS2R16 cin, arbutin, and amygdalin (Figure 2B). The EC50 values N172/R222 (Figure S3E) and determined the EC50 val- of N172/H222 were 1.0 ± 0.2 mM for salicin, 5.4 ± 1.5 ues in response to salicin. There was no shift in the mM for arbutin, and 6.1 ± 1.2 mM for amygdalin, and EC50 values between undiluted and 1:5 diluted DNA. the EC50 values of K172/H222 were 1.9 ± 0.2 mM for Even at a 1:10 dilution, the effects on the EC50 were salicin, 11.5 ± 7.4 mM for arbutin, and 10.1 ± 1.1 mM very moderate. On the basis of these experiments, we for amygdalin. The comparison of the EC50 values of can conclude that the observed differences between these variant receptors again revealed a clear func- the TAS2R16 variants are not caused by differences in tional difference. The receptor variant K172/H222 be- the expression rates, cell-surface targeting, or varia- haved like K172/R222 and showed the same 2-fold shift tions in the transfection protocol. in EC50 in comparison to N172/H222, which behaved In summary, we found that the K172 allele alone cre- like N172/R222 (Figure 2B). These data show that both ates a 2-fold-less-sensitive receptor, whereas we did receptors containing the K172 allele are less sensitive not observe any functional effect of the H222R ex- to all tested agonists than are the receptors that con- change. We have previously demonstrated (B.B. and tain the N172 allele and that this effect is independent W.M.) a strong correlation between in vitro dose- of the amino acid substitution at amino acid position response measurements for this receptor and response 222. in humans [12]. To examine whether the observed functional differ- ences were caused by variations in expression rates, Discussion we analyzed the number of cells expressing a given TAS2R16 variant by immunocytochemistry and West- We detected a clear signature of positive selection at ern blot (Supplemental Data). All TAS2R16 variants the bitter-taste receptor gene TAS2R16. This positive- showed a similar expression level (Figure S3A–S3C), selection event produced (or was apparent as) an ex- and no obvious difference was observed between the cess of high-frequency derived alleles at three neigh- Current Biology 1262

Table 3. K172 Allele Frequency and Current Malaria Risk in African Populations

Population Country N Individuals K172 Frequency (%, G Allele) Malaria Riska Tunisian Tunisia 17 0 0 Mozabite Algeria 26 9.6 0 Bantu South Africa 28 21 0.2 Amhara Ethiopia 20 7.5 0.4 Bantu Kenya 10 10 0.53 Cameroonian Cameroon 12 33 0.94 Mbuti Pygmies Democratic Republic of Congo 12 17 1 Mandenka Senegal 22 18 1 Biaka Pygmies Central African Republic 31 24 1 Yoruba Nigeria 18 33 1 a Percentage of population living in areas at risk for malaria transmission (endemic risk). Source: Africa Malaria Report 2003 (http:// www.rbm.who.int/amd2003/amr2003/table6.htm); for Tunisia and Algeria, the source is http://www.cdc.gov/travel/regionalmalaria/nafrica.htm.

boring loci (V101V, T282T, and K172N). Although any family also includes various toxic cyanogenic glyco- of these three loci could in theory drive the signal of sides. Cyanogenic glycosides are toxic as a result of selection, only the nonsynonymous variant K172N has the release of cyanide ions (CN−) during the enzymatic a clear functional effect and is to date the most likely breakdown in the intestine; CN− is rapidly absorbed target of positive selection. K172N lies in the second into the bloodstream, where it combines with Fe2+ to extracellular loop domain of the TAS2R16 receptor, a produce cyano-hemoglobin [32]. A single dose of cya- region implicated in receptor-ligand interaction. We nide of 1 mg kg−1 body weight is lethal for most verte- demonstrated with functional assays based on a heter- brates [33]. However, chronic exposure to sublethal ologous cell system that the N172 allele confers in- concentrations is tolerated, particularly in conditions of creased sensitivity for a range of seven structurally dif- high dietary intake of protein [17], where cyanide is ferent glycosides, some with a beneficial effect in combined with sulfur-containing amino acids to pro- humans (salicin, arbutin) and others toxic (amygdalin, duce the less-toxic thiocyanate [34]. linamarin, prunasin, osmaronin epoxide and 4-glucosy- One intriguing observation was that the low-sensitiv- loxymandelonitrile). The second common nonsynony- ity ancestral K172 allele was retained at moderately mous site (H222R) did not appear to affect receptor high frequencies throughout central Africa, with a distri- function and was not associated with an overall reduc- bution (Figure 1B) that appeared remarkably similar to tion or increase in fitness. those of some malaria-resistance alleles [35]. Chronic We estimated that K172N arose between 77,700 and low-level ingestion of cyanogenic foods has been linked 791,000 yr ago. This places the origin of the mutation with an increased protection against malaria [36]; this well before the Neolithic revolution and approximately increase in protection may take place through various in the Middle Pleistocene, a stage of human evolution mechanisms including cyanide-induced sickle cell ane- characterized by an active hunter-gatherer lifestyle [29]. mia, cyanide-induced oxidative stress, and inhibition of Given the functional significance of this mutation, it is Plasmodium respiratory chain [36–39]. One speculative reasonable to suppose that selective forces acted upon explanation for this distribution could therefore be that it as soon as it occurred (although it is not possible for K172 has conferred a modest selective advantage in our genetic analyses to demonstrate this with cer- malaria-infested areas through favoring chronic low- tainty). The worldwide geographical distribution of the level ingestion of cyanogenic glycosides. This hypo- variant also suggests that the selective sweep occurred thetical malaria effect would act antagonistically to the before the human expansion out of Africa 50,000– selective sweep to maintain K172 at high frequencies 100,000 yr ago [30], although the differentiation for in Central Africa. Under this hypothetical scenario, the K172N suggests that selection has continued after the global pattern of variation at the TAS2R16 gene results separation of major human population groups. from a balance between protection against malaria and We speculate that N172 may be driving the signal protection against toxins in malaria-free zones. of positive selection at TAS2R16 through an increased Testing the above hypothesis would require demon- protection against cyanogenic plant foods and natural strating experimentally a functional role for K172 as a toxins in general. Because the functional effect of the malaria-resistance allele. To obtain a first qualitative in- N172 variant is similar for all the compounds tested, it dication, we retrieved a general index of malaria risk is difficult to establish whether a specific compound (the percentage of population living in areas at risk for underlies the selective sweep. In the absence of paleo- endemic malaria transmission for each country) and botanical and archaeological data supporting the role compared it to the frequency of the K172 allele in the of a specific food source, we suggest conservatively African populations (Table 3). We found that popula- that a generalized response to β-glucopyranosides is a tions with high malaria risk (>0.7) had higher average likely cause. K172 allele frequencies (mean 25%, range 17%–33%) The natural bitter agonists of TAS2R16, β-glucopyra- compared to populations with intermediate or low ma- nosides [12], are a wide class of natural compounds laria risk (mean 10%, range 0%–21%), indicating a good synthesized by over 2,500 plant and insect species as overall correlation between the two variables. We did a protection against predators [19, 31]. This compound not test this correlation quantitatively because any Positive Selection on TAS2R16 1263

quantitative comparison would be affected by fluctua- Center (http://www.dpz.gwdg.de/). The following 13 individuals tions in malaria risk since the time of the selective were excluded from the analysis: HGDP00813, HGDP01233, sweep [35] and historical movements of people [40]. HGDP00762, HGDP00111, HGDP01149, HGDP00583, HGDP00650, HGDP00658, HGDP00657, HGDP00660, HGDP00472, HGDP00452, For instance, the South African Bantus are thought to and HGDP00457. have reached South Africa from Central Africa at around 500–1000 BC [40], well after the selective event. Resequencing of the TAS2R16 Gene Remarkably, we found that in this population, K172 had The entire coding region of TAS2R16 (876 bp) and 203 bp of 5# and the highest frequency (21%) among populations in 3# UTR sequence were amplified with the following primers: for- areas of low malaria risk, possibly reflecting the Central ward 5#-TAGCAAACCAGCAAGCAAAG-3#; reverse 5#-CGAAGATGG # African origin. AAGGGAATGAG-3 . Amplified PCR products were sequenced with Big Dye Terminator chemistry (Applied Biosystems) with 10 pmoles In a recent study, Wooding and colleagues suggested forward/reverse primer. Sequence analysis and contig assembly a role for balancing selection in maintaining three func- were performed with Sequencher software (v. 4.0.5, Genecodes). tional alleles of TAS2R38 throughout human evolution Sequence traces were scored manually twice, and the genotype [11]. Our study suggests nonneutral evolution for a sec- data were checked by random resequencing of 10% of the sam- ond bitter-taste gene in humans, indicating that posi- ples. The estimated error rate was 0.5%. tive as well as balancing selection has contributed to diversification of TAS2R function. Wang and colleagues Genetic Analyses Ancestral character states for the TAS2R16 loci were inferred on recently suggested overall neutral patterns of evolution the aligned human-primate sequences with the DnaML program for human TAS2R genes, including TAS2R16 [6]. As the contained in the PHYLIP software package [41]. The maximum- authors themselves stated, a neutral signal of selection likelihood character states at each node of an inferred phylogeny for the family does not exclude differential selection were all determined with likelihood > 95%. Haplotype inference on acting on specific TAS2R functions. Detecting such se- the 17 TAS2R16 polymorphic sites was carried out with the expec- lection events may require the application of more-sen- tation-maximization algorithm implemented in ARLEQUIN [42]. Nu- cleotide-diversity statistics and tests of selection were carried out sitive tests such as Fay and Wu’s statistics. with the TAS2R16 sequence data and the DnaSP software (version The proposed selective sweep at K172N may have 4) [43]. Calculation of the among-population differentiation statis- β consequences for modern human health. -glucopyra- tics FST was carried out with analysis of molecular variance by hier- nosides are a heterogeneous class of compounds, archically grouping the populations in an African and a non-African some of which are toxic and some of which lower the sample. FST values were compared to an empirical distribution of risk of cancer and cardiovascular disease [1]. As a re- 210 genome-wide short insertion/deletion polymorphisms ampli- fied in the same individuals of the human diversity panel (data sult of their bitter taste, these compounds cause aver- available at http://research.marshfieldclinic.org/genetics/indels/). sion in the consumer and are removed routinely by the Empirical p-values for TAS2R16 loci were calculated as the fraction food industry [1]. Aversion to bitter compounds is cur- of all indels having a FST higher than the observed value. Analysis rently a main limitation toward increasing the phytonu- of linkage disequilibrium surrounding TAS2R16 was carried out on trient content of plant foods as a dietary option for dis- genotype data retrieved from the HapMap project (www.hapmap. ease prevention [1]. Although the N172 allele may have org) and spanning 221,998 bp (SNP rs981696 to rs2470984); TAS2R16 sites were resequenced in our laboratory with the same been advantageous in our past through avoidance of trio DNAs. Haplotype inference from trio data and estimation of natural toxins, it may now contribute to increasing dis- linkage disequilibrium were obtained with the TagIT software [44]. ease risk through lowered intake of such beneficial compounds. The identification of genetic polymor- Estimation of Allele Age phisms affecting receptor function may have relevance Estimation of the age of the K172N mutation was carried out with in terms of pharmacological manipulation of bitter- the BATWING Bayesian MCMC program [27], with models similar taste receptors. to those described for the analysis of β globin data in that paper. We used data from the Yoruba sample only, in order to reduce the problems of recent demographic stratification. Analysis was car- Conclusions ried out assuming no current mutation or recombination (thus, all Our study provides evidence for positive selection on six polymorphic SNPs in this region are assumed to be UEP [unique the bitter-taste gene TAS2R16. The selective event is event polymorphisms]), and three recombinant individuals were likely to be explained by an increased fine-control of dropped from the dataset in order to satisfy this condition. The root haplotype of the genealogical tree linking the remaining 32 ingestion of cyanogenic compounds conferred by the chromosomes was assumed unknown. The per sequence per gen- N172 allele. As humans, we display overall reduced eration mutation rate ␮ was assumed to be uniform throughout the sensory characteristics in comparison to other mam- 1079 bp sequence. A prior distribution for ␮ was found as follows. mals and primates. However, our study suggests that We started with a gamma(3,105) preprior for ␮, based on a genome- −8 preservation of specific sensory functions through pos- wide substitution rate of 10 per 1079 bases. From our compari- itive selection may have been particularly important, at son with chimpanzee sequence, we found two human-chimp fixed differences in 1079 bp of sequence. Assuming there have been least in the earlier stages of human evolution. about 2.5 × 105 generations since the human-chimpanzee split, this leads to a gamma(3,6 × 105) prior distribution for ␮. For the effec- Experimental Procedures tive population size, we assumed gamma(5,2.5 × 10−4) and gamma(3,2.5 × 10−4) prior distributions under the standard (no Population Samples growth) coalescent and coalescent-with-growth models, respec- DNA for 52 populations of the human diversity panel [20] and the tively (the latter is a two-stage model, with no growth in Stage 1 trios used by the HapMap project were obtained from Dr. Cann and exponential growth from the initial population size in Stage 2). (Fondation Jean Dausset-CEPH). Eight additional populations cov- The coalescent-with-growth model also requires priors for the per- ering geographic regions poorly represented in the human diversity generation growth rate [here, set at gamma(2,400)] and the log- panel (Table S1) were donated to D.B.G. through previous collabo- ratio of current to initial effective population size [here, set at rations. Primate DNAs were obtained from the German Primate gamma(5,1)]. These priors are fairly broad and are justified further Current Biology 1264

in Wilson et al. [27]. Posterior estimates from BATWING (of the ge- practical tips; Dr. Mark Thomas for drawing the allele frequency nealogical nodes immediately ancestral and descendent from maps; Professor C.G. Lee and K. Tang (National University of Sin- K172N) were checked for convergence and mixing via the methods gapore, Singapore) for help with data analysis; and Gianpiero Ca- described in Wilson et al. [27]. valleri, Anna Need, and four anonymous reviewers for comments on an earlier version of the manuscript. Support was provided by Generation of the TAS2R16 Variants and Functional the Leverhulme Trust (N.S. and D.B.G.), the German Science Foun- Expression Analysis dation (Me 1024/2, W.M.), Damon Runyan Cancer Foundation, and N172/H222 (HAP-A) was cloned from genomic DNA into a pcDNA5 the L’Oreal for Women in Science Fellowship (P.C.S.). D.B.G. is a FRT expression vector (Invitrogen) and fused with the first 45 N-ter- Wolfson Merit Award Holder. minal amino acids of the 3 (sst-tag) to facili- tate membrane targeting and a C-terminal HSV-epitope to allow Received: February 24, 2005 immunocytochemical detection as described earlier [12]. The se- Revised: May 31, 2005 quence variants N172/R222 (HAP-B), K172/H222 (HAP-C), and Accepted: June 14, 2005 K172/R222 were created by inserting point mutations with the Published: July 26, 2005 PCR-based Quick Change protocol (Stratagene) according to the manufacturer’s recommendations. The cloned TAS2R16 gene vari- ants were sequenced to verify the mutation and to exclude amplifi- References cation errors during the PCR reaction. All plasmids containing the 1. Drewnowski, A., and Gomez-Carneros, C. (2000). Bitter taste, receptor variants were transiently transfected into HEK-293T cells phytonutrients, and the consumer: A review. Am. J. Clin. 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Genetic To investigate a correlation between K172 and malaria risk, we ge- analysis of a complex trait in the Utah Genetic Reference Pro- notyped K172 in three additional African samples available in our ject: A major locus for PTC taste ability on 7q laboratory: These included additional central (Cameroon) and and a secondary locus on chromosome 16p. Hum. Genet. 112, northern (Tunisia) African populations and additional Biaka Pyg- 567–572. mies samples (Central African Republic). Malaria-risk data was re- 10. Kim, U.K., Jorgenson, E., Coon, H., Leppert, M., Risch, N., and trieved from the Africa Malaria Report 2003 (http://www.rbm.who. Drayna, D. (2003). Positional cloning of the human quantitative int/amd2003/amr2003/table6.htm); for Algeria and Tunisia, malaria- trait locus underlying taste sensitivity to phenylthiocarbamide. risk indexes were set to zero to reflect current risk (http://www.cdc. 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