Bitter Taste Genetics: PTC and Other Stories

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

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

3. Michael Bamshad, MD Stephen Wooding, PhD

4. Molecules involved in taste perception

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

6. The Bitter Draught Adriaen Brouwer, 1635

7. Many plants contain toxins Digitoxin Ricin Abrin Strychnine

8. Many plant toxins taste bitter Quinine Nicotine Salicin Papaverine

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

10. Bitter-taste (TAS2R) Receptors

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

12. L’absinthe Edgar Degas, 1876 Bitter-taste receptors bind plant toxins TAS2R10 strychnine TAS2R14 a-thujones TAS2R16 salicin, cyanogenic compounds Bufe et al., Nat. Gen., 2002; Behrens et al., BBRC, 2004

13. 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

14. 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?

15. 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

16. Diversity in PTC • 6 nucleotide substitutions in humans

17. Diversity in PTC • 6 nucleotide substitutions • 7 haplotypes PAV AVI

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

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

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

21. 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

22. Tt + How has selection acted? Nontaster alleles are not premature stop codons or frameshifts HYPOTHESIS: Nontaster allele is a functional receptor that binds ligands other than PTC.

23. What happened in chimps?

25. TAS2R38 Resequencingin 37 Wild-born Chimpanzees AGG Start ATG Start Wooding et al., 2006, Nature 440: 930-4

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

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

28. Phenotyping Chimpanzees

29. 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 1 23 Taster Observed 7 8 Nontaster • Rejected no association (p < 10-3) • AGG/AGG “broken start” chimps eat PTC apples

30. 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.

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

32. 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

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

34. 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)

35. 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

36. 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

38. How has selection acted?

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

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

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

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

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

44. 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

45. 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 = 3.6 D = -2.0

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

47. Human Variation

48. Habitats

49. Genes and diet

50. 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