Bitter Taste Genetics: PTC and Other Stories Lynn Jorde and Steve Wooding Department of Human Genetics University of Utah School of Medicine

Overview PTC: evidence for balancing selection PTC in chimps and humans The other bitter taste receptor genes

Michael Bamshad, MD Stephen Wooding, PhD

Molecules involved in taste perception Sour Ion channels Salty Sweet TAS1R family of G-protein coupled receptors Umami Bitter TAS2R family (25 functional members)

Sweet and Umami (T1R) 1 2 3 T1R1 T1R2 T1R3 Cell types observed in vivo High-affinity sugar receptor High-affinity umami receptor Low-affinity umami receptor Cell types observed in vivo

The Bitter Draught Adriaen Brouwer, 1635

Many plants contain toxins Ricin Digitoxin Ricin: castor bean; Digitoxin: foxglove; strychnine: member of Strychnos genus (“monkey orange”); Abrin: pea plant (10,000 x more toxic than ricin) Abrin Strychnine

Many plant toxins taste bitter Quinine Nicotine Note that some plant toxins actually have a bitter taste – Quinine was extracted from the bark of the South American cinchona tree Nicotine from tobacco plants; salicin from willow; papaverine from opium poppy Salicin Papaverine

Biomedical relevance of bitter taste Bitter-taste sensitivity Smoking Diet Choice Cancer Body Mass Index

Bitter-taste (TAS2R) Receptors Note: these receptors ALSO expressed in gut, possibly to signal hormone changes, upregulate detox genes, etc.

Bitter-taste (TAS2R) Receptors x TAS2R Ga Ca+ Ca+ Ca+ Ca+

Bitter-taste receptors bind plant toxins L’absinthe Edgar Degas, 1876 TAS2R10 strychnine TAS2R14 a-thujones TAS2R16 salicin, cyanogenic compounds Alpha-Thujone comes from wormwood, used in flavoring absinthe. Note it is a GABA-receptor blocker, decreasing inhibition of neurons – increased firing can lead to hallucinations and psychosis Bufe et al., Nat. Gen., 2002; Behrens et al., BBRC, 2004

Phenylthiocarbamide (PTC) Synthetic Bitter or tasteless Fox 1932 Variable sensitivity Similar in structure to plant compounds Blakeslee 1932 Taste blindness is Mendelian recessive Fisher et al., 1939 Variable sensitivity in chimp Thousands of individuals have been typed for PTC taster status

How have natural selection and population history PTC Gene Drayna et al., 2003 Genome-wide linkage analysis Utah CEPH families PTC gene localized to chromosome 7 and subsequently cloned PTC Gene (TAS2R38) Haplotypes strongly associated with phenotype Accounted for ~50-85% of phenotypic variance How have natural selection and population history affected the PTC gene?

PTC resequencing in humans Sequenced entire coding region (1,002 bp) 174 individuals (348 chromosomes) 40 African 69 Asian 55 European 10 Native American Not selected for PTC sensitivity “Anthropological” sampling Wooding et al., 2004, Am. J. Hum. Genet. 74: 637-46

Diversity in PTC 6 nucleotide substitutions in humans

Diversity in PTC 6 nucleotide substitutions 7 haplotypes PAV AVI

Diversity in PTC 6 nucleotide substitutions 7 haplotypes Taster Allele PAV: AVI: Taster Allele Nontaster Allele

Allele frequencies T t Native American African Asian European FST = 0.05

Has selection been acting? Neutrality Not Rejected, p > 0.06

Has selection been acting? Neutrality rejected Exceeds 99.5% of D values reported for >1,600 genes Wooding et al., Am. J. Hum. Genet., 2004

How has selection acted? Nontaster alleles are not premature stop codons or frameshifts 1977: fruit (antidesma bunius) is bitter to nontasters. Compound not yet identified. HYPOTHESIS: Nontaster allele is a functional receptor that binds ligands other than PTC. Tt +

What happened in chimps?

37 Wild-born Chimpanzees TAS2R38 Resequencing in 37 Wild-born Chimpanzees ATG Start AGG Start Note troglodytes is monomorphic for tasting, schwein… for tasting (small samples, though) Wooding et al., 2006, Nature 440: 930-4

Hypothesized PTC Tasting in Chimpanzees “Taster” “Non-taster” Wooding et al., 2006, Nature 440: 930-4

How does AGG make a nontaster allele? ATG AGG Full-length Protein: PTC response Truncated Protein: no PTC response

Phenotyping Chimpanzees

Is AGG the nontaster allele in chimps? Genotype-phenotype association analysis 40 chimpanzee subjects Genotyped Phenotyped Apples soaked in H2O or 4.0 mM PTC Response on a 1-5 scale (1 = Readily Accepted; 5 = Strongly Rejected) Fisher’s Exact Test (2x2) Expected Taster Nontaster 23 1 Taster Observed 7 8 Nontaster Rejected no association (p < 10-3) AGG/AGG “broken start” chimps eat PTC apples

Humans versus Chimps Humans Chimps T, t alleles T, t alleles 50:50 frequencies 50:50 frequencies 3 amino acid changes One start codon change Same gene, same phenotype, same allele frequencies… different mechanism.

Fisher’s Hypothesis Hu Ch Hu Ch T t T t “Taster” “Non-Taster” Human Chimpanzee Hu Ch Hu Ch T t T t Chimp-Human Divergence Ancestral Gene Ancestral Gene

Resequencing all 25 TAS2R genes in 55 humans Do all TAS2Rs have two clusters (i.e., T and t)? No – some have very low levels of variation. Are all TAS2R alleles found at similar frequencies across populations? No – Some have dramatically different frequencies On average populations differ more at TAS2Rs than at other genes TAS2R49 FST = 0.33 Kim et al., 2005, Hum. Mutation 26: 199-204

Population Differentiation Implies Local Adaptation PTC Observed Simulated FST TAS2Rs (p < 0.01) Kim et al., 2005, Hum. Mutation 26: 199-204

TAS2R genes are involved in the perception of many substances Alcohol Subjects with PTC taster haplotype experience more bitterness and have lower alcohol intake (Duffy et al., 2004, Alcohol Clin. Exp. Res. 28: 1629-37) Variant in TAS2R16 is strongly associated with risk of alcohol dependence (Hinrichs et al., 2006, Am. J. Hum. Genet. 78: 103-11) Cigarette smoke Some evidence that PTC tasters are aversive to cigarette smoke and less likely to become smokers (Enoch et al., 2001, Addict. Behav. 26: 399-404) Saccharin and acesulfame K Activate TAS2R43 and TAS2R44 bitter taste receptors (Kuhn et al., 2004, J. Neurosci. 10: 10260-5)

Summary Polymorphism in the human PTC gene is maintained by balancing natural selection Humans and chimps both have taster and non-taster alleles, but these alleles arose independently Substantial inter-population differentiation of TAS2R genes exists, consistent with local adaptation to environmental toxins

Acknowledgments University of Utah: Mike Bamshad, Steve Wooding, Diane Dunn, Bob Weiss, Mike Howard NIH: Dennis Drayna, U. Kim Potsdam: Bernd Bufe, Wolfgang Meyerhof University of Arizona: Anne Stone

How has selection acted?

How does AGG make a nontaster allele? ATG AGG DNA TAC TCC Transcription AUG AGG RNA Translation Met Met Met Protein

Has selection been acting? Tajima’s D statistic Compares S S = Number of nucleotide substitutions

Has selection been acting? Tajima’s D statistic Compares S and P P = Mean pairwise nucleotide difference

Has selection been acting? Tajima’s D statistic Compares S and P Affected by “shape” of haplotype network

Has selection been acting? Tajima’s D statistic Compares S and P Affected by “shape” of haplotype network S = 6 S = 6

Has selection been acting? Tajima’s D statistic Compares S and P Affected by “shape” of haplotype network S = 6 S = 6 P = 0.1 P = 3.0

Has selection been acting? Tajima’s D statistic Compares S and P Affected by “shape” of haplotype network S = 6 S = 6 P = 0.1 P = 3.0 D = -2.0 D = 3.6

Has selection been acting? Tajima’s D statistic Compares S and P Affected by “shape” of haplotype network Positive selection Balancing selection or Local adaptation

Human Variation

Habitats

Genes and diet Gene Phenotype Hemochromatosis Iron absorption Angiotensinogen Sodium retention Lactase Lactose tolerance Alcohol dehydrogenase Ethanol metabolism

Environmentally Responsive Genes Genes controlling the active interface between the human body and its environment Genes involved in: Sensory perception Immune system Toxin metabolism Bitter-taste sensitivity