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

5. 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

6. The Bitter Draught
Adriaen Brouwer, 1635

7. 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

8. 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

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

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

11. Bitter-taste (TAS2R) Receptorsx
TAS2R Ga
Ca+
Ca+
Ca+
Ca+

12. 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

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. 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?

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:

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 Taster Allele
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. 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 +

23. What happened in chimps?

25. 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

26. Hypothesized PTC Tasting in Chimpanzees
“Taster”
“Non-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 23 1
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 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

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:

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

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: )
Variant in TAS2R16 is strongly associated with risk of alcohol dependence (Hinrichs et al., 2006, Am. J. Hum. Genet. 78: )
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: )
Saccharin and acesulfame K
Activate TAS2R43 and TAS2R44 bitter taste receptors
(Kuhn et al., 2004, J. Neurosci. 10: )

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
TAC
TCC
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 = -2.0
D = 3.6

46. 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

47. Human Variation

48. Habitats

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

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