Diversity and evolution at loci of pharmacogenetic interest. Silvia Fuselli University of Ferrara

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1 Diversity and evolution at loci of pharmacogenetic interest Silvia Fuselli University of Ferrara

2 Pharmacogenetics Same diagnosis Therapeutic failure Good Therapeutic effect Adverse drug reaction (ADR)?

3 The percentage of phase I and phase II metabolism of drugs that each enzyme contributes is estimated Drug transporters Drug-metabolizing enzymes (DMEs) Drug target proteins Evans WE & Relling MV Science 1999

4 High genetic variation 350 functionally different CYP alleles affecting the function of the gene product CYP2D6 75 CYP2A6 37 CYP2B6 29 CYP1B1 26

5 Cytochrome P450-2D6 (debrisoquine hydroxylase) Phenotypic outcomes UM: ultrarapid metabolizers EM: extensive metabolizers IM: intermediate metabolizers PM: poor metabolizers Meyer 2004

6 Ingelman-Sundberg et al. 2004

7 A human genome diversity cell line panel 52 populations 1064 individuals Pima Maya Colombian Karitiana Surui Russian Yakut Orcadian Oroqen Daur French North Italian Adygei Uygur Mongola French Basque Tuscan Xibo Hezhen Sardinian Kalash Palestinian Burusho Pathan Tu Druze Brahui Hazara Han Japanese Yizu Tujia Mozabite Balochi Bedouin Naxi Makrani Sindhi Miaozu Lahu She Mandenka Yoruba Biaka Pygmy San Bantu S.E. and S.W. Mbuti Pygmy Bantu N.E. Dai Cambodian Papuan NAN Melanesian Deposited at the Foundation Jean Dausset (CEPH) in Paris. LCLs collected by the Human Genome Diversity Project (HGDP) Cann et al. 2002

8 Cyp2D6 locus and typing chr C>T 1023C>T 1659G>A 1661G>C 1846G>A del 2850C>T 3183G>A 4180G>C 1707delT 2549delA 2988G>A multiplex single base extension (12 positions) Sistonen et al. 2005

9 Bayesian analysis of genetic differentiation between populations (BAPS) Orcadian Russian Yakut Pima Maya French French Basque Mandenka Sardinian Mozabite North Italian Tuscan Palestinian Druze Bedouin Adygei Uygur Xibo Kalash Burusho Tu Pathan Brahui Hazara Yizu Balochi Naxi Makrani Sindhi Lahu Mongola Dai Han Tujia Miaozu Daur She Oroqen Hezhen Japanese Colombian Yoruba Biaka Pygmy Mbuti Pygmy Bantu N.E. Cambodian Papuan Karitiana Surui NAN Melanesian San Bantu S.E. and S.W.

10 High frequency of low-null activity alleles: convergent evolution? Relaxation of selective pressure? Gene duplication = ultrafast metabolism : convergent evolution? Some intriguing questions Sistonen et al. 2007

11 Neolithic transition: a turning point in human evolution Patin and Quintana Murci 2008

12 Human amylase gene AMY Perry et al. 2007: significant correlation between AMY1 genotype (copy number polymorphism) and phenotype (amylase concentration in saliva). Populations with traditional starch-dense diets had a higher AMY1 copy number than populations with lowstarch diets, such as rainforest and arctic hunter-gatherers.

13 Lactase gene LCT LCTPersistence Tishkoff et al the cultural trait of milk consumption confers such a strong selective advantage in terms of human survival that lactase persistence alleles have emerged independently in several parts of the world.

14 Why CYP2D6? Drug metabolizing enzymes (DMEs) are mediators between the organism and the environment Detoxification of xenobiotics implies that variability at these genes is under selective pressures The chemical environment varies significantly with diet, climate, lifestyle, etc., we observe a great deal of interethnic differentiation for DME polymorphisms.

15 Samples Inari (15) Kolasaami (15) Russians (16) Finns (14) Xanty (15) Mansi (15) Food producers (FP) Bakaka (15) Ngoumba (14) Hunter-Gatherers (HG) Bakola (16) Baka (15)

16 4,245 bp Non synonymous Lifestyle Populations CYP2D6 No chromosomes S No haplotypes HD ±SD π ±SD (x 10-3 ) θ s ±SD (per site) (x 10-3 ) Tajima's D Fu's Fs Africa Western Eurasia HG FP HG FP BKL BAK NGO BKK LSA INA KHA MAN FIN RUS ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

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18 1. Globally, HG are significantly faster CYP2D6 metabolizers than FP (Mann-Whitney U test: p = ). HG: normal activity 73.3% FP: normal activity 57.7% reduced-activity 10.9% reduced activity 24.7%

19 AMOVA: 2. within continent the differences between HG and FP at CYP2D6 account for most of the variances between populations. between populations within groups between groups Population samples groups pops CYP2D6 mtdna 15STRs 783STRs a CYP2D6 mtdna 15STRs 783STRs a All HG-FP Africa HG-FP W. Eurasia HG-FP The patterns of divergence shown by CYP2D6 differ from the patterns observed at neutral markers a 783 STRs, Rosenberg et al. 2005

20 Neutrality tests: Positive values: population subdivision, balancing selection Negative values: population expansion, selective sweep Neutral = Stationarity Balancing selection = Pop. structure Selective sweep = Pop. expansion The confounding effect of the history of a population. ΔD (CYP-mt) and ΔFs (CYP-mt): result of a neutrality test when demographic effects are excluded.

21 4. HG values depend on the samples geographic location African and Western Eurasian FP show a clear trend towards positive values, as expected when two or more gene variants are maintained in the population. HG an FP are significantly different in both cases (Mann Whitney test)

22 If selection is acting on this gene, the mode and intensity of this process depend on the phenotypic effect of a mutation. 5. Only nucleotide changes resulting in amino acid replacements show a pattern that depends on lifestyle, disregarding the geographic origin of the samples

23 Several lines of evidence support the notion that variation at CYP2D6 was selected as a response to the new dietary habits introduced by the transition to agriculture. 1. different patterns of genetic diversity at the CYP2D6 locus in HG and FP populations are observed AMOVA results 2. ΔD and ΔFs: only FP consistently show high and positive values, mainly due to differences at nonsynonymous sites (3), which in turn produce metabolic changes in FP from different continents (4)

24 Which type of selection is a plausible explanation? Internal genetic diversity at CYP2D6 are among the highest observed for coding autosomal loci. FP are less divergent at CYP2D6 (Фst: 4%) than HG (Фst:11%), the latter showing values usually found for neutral autosomal loci. More than one CYP2D6 variant is maintained at high frequency in each geographic region. Balancing selection CYP2D6 enzymes with reduced function (FP) show substrate-dependent catalytic properties and enzyme inhibitor affinities, both conferring a wider spectrum of metabolic responses. Reduced function enzymes do not activate toxic molecules possibly present in the new dietary regime

25 Demography clearly contributed to shaping patterns of genetic diversity observed at CYP2D6. However demography without selection requires a very complex and unlikely scenario whereby the combination of continent-specific populations history and demographic effects due to two different subsistence economies gave rise to the same trend in African and non-african populations.