Pharmacogenetics

1. Definition and introduction Relevance to medicine Post-genomic approaches to studying pharmacogenetics SNP detection Pharmacogenetics

2. Completion of human genome: variation between individuals are <0.1% (3M bp) Hypothesis: variations in genome account for individual responses to drugs: Optimized medicines for individuals Reduction in adverse drug reactions Expedient development of new drugs Promise of Pharmacogenetics in Post-Genome Era

3. Definition (modern): Study of variability in the responses of different patients to drugs and medicines Broad definition: “pharmakon”- magic charm, drug, poison Any biologically active material originating from outside of the body (xenobiotic vs. endobiotic) Pharmacogenetics

4. Environmental toxicogenetics Insecticide/herbicide resistance in agriculture Antibiotic resistance in microbes Pharmacogenetics in Nature

5. Pharmacogenetics “It is an advantage to a species to be biochemically diverse … for such species will contain at least some members capable of resisting any particular pestilence.” Haldane, 1949

6. “St. Anthony’s Fire” in Middle Ages Ergot intoxification, gangrene of hands and feet Mold poisoning in England, 1500-1830 Low fertility, high mortality After 1830, diet changed from grains to potatoes Unlike viral or bacterial outbreaks, adaptive immunity is not acquired; resistance to toxins require genetic changes over generations Human exposure to natural toxins

7. DTT led to resistance within a few generations DTT resistance associated with slower development of larvae Resistant strains were lost without DTT selection Genetically stable DTT resistance required 30 generations of selection Resistance of DTT in houseflies

8. Resistance detected to all clinically relevant antibiotics Resistance mechanisms conferred by chromosomal mutations/changes or more commonly, multi-resistance R-plasmids Most antibiotics are derived from fungal or bacterial products Antibiotic resistance in bacteria

9. If a mutation confers resistance to a toxin, why is it not maintained or expaanded in a population? Concept of Balanced polymorphisms Explanation for high frequency of certain genetic variants Gene variant may be detrimental in homozygous double dose but confers increased fitness in heterozygotes Frequency of variant reflects the balance between heterozygote advantagea and homozygote disadvantage Biological cost of variation

10. Most famous SNP, mutation in hemoglobin gene Children with homozygous mutation die early Heterozygous adult have enhanced survival to malaria Sickle gene variant only stays in areas with malaria Balance polymorphisms: Sickle cell anemia

11. If a new mutation is disadvantageous, it will tend to be eliminated quickly by selective forces It is only possible to expect new mutations to survive in a population if effects are neutral or mildy disadvantageous Neutral mutations represents the only viable mechanisms for mutations to be maintained as a reserve of variation against unforeseen needs Neutral mutations vs. balanced polymorhphisms

12. Types of SNPs and their frequency Type Description Number (in 1000’s) I Coding, non- synonymous, non-conservative 60-100 II Coding, non- synonymous, conservative 100-180 III Coding, synonymous 200-240 IV Non-coding, 5’-UTR 140 V Non-coding, 3’-UTR 300 VI Other non-coding >1000

13. Pharmacogenetics: Practice of medicine “If it were not for the great variability among individuals Medicine might as well be a science and not an art.” Sir William Osler, 1892

14. Variability in patients’ responses could be related to differences in the activities of drug-metabolizing enzymes About 20-30 enzymes can interact with nearly every chemical to which the body is exposed Metabolizing enzymes, receptors, drug transport systems Variants of these enzymes may represent deficient or excessive metabolizing activities Variants often showed ethnic-biased distributions (Fig.1) Classical Pharmacogenetics: enzymology, biochemistry and population genetics

15. multi-gene family, CYP2D6 is one of best studied 70 variant alleles of CYD2D6 known Variants encode for non-functional enzymes, poor metabolisers, and ultra-rapid metabolisers Table 1 Molecular Pharmacogenetics: Cytochrome P450

16. Monogenic vs. Gaussian Variations Monogenic All-or-none function in affected gene Mendelian inheritance Examples: Sickle cell, CYP2D6, Cystic fibrosis, Rb Gaussian or polygenic Function of affection gene shows a range that is defined by ED50 Multiple gene variation involved Accounts for majority of variations in drug responses Disease Susceptibility Alzheimer’s disease ApoE4, ApoE2 (19q13), 12q

17. Definitions are arbitrary Gene mutations are rare, <1% of population Gene polymorphisms exist in >1% SNPs exists about every 1000 bases, ie. ~3,000,000/genome SNP: Mutation vs. Polymorphism

18. SNP linkage disequilibrium profiles With no prior knowledge of genetic involvement, analysis of ~500,000 SNPs is required for whole genome association study How to use SNPs to measure drug responses?

19. Proteomics, mass-spec., protein chips Microarrays for mRNA expression profiling Proteins, need to assay for function mRNA expression is indirect readout of underlying genomic variation, at best Functional Genomics Approaches to Pharmacogenetics

20. Genome sequencing of individuals Dense SNP maps develop around disease susceptibility loci SNP Consortium, ~800,000 identified by 2001 Many SNPs from genome efforts SNPs needed from different ethnic populations SNP Identification

21. All methods rely on PCR amplification of locus surrounding SNP Different methodologies used to identify the sequence at the SNP Direct sequencing SSCP, single-standed conformation polymorphism Throughput of 100’s-1000’s per day SNP Detection: Standard Methods

22. Realtime PCR using Taqman probes Realtime PCR using Molecular beacons Realtime PCR using melt curve analysis Closed tube, walk-away assays ABI7900, 384-well + robot feeder Throughput of 1,000’s per day SNP Detection: High throughput PCR

23. Affymetrix SNP-specific oligo arrays Multiplex of ~6000 PCR reactions of each sample Hybridize to arrays, high level of redunndancy Throughput of 6000 per day per sample SNP Detection : Microarrays

24. Multiplex PCR of each sample, T7 tagged Make cRNA Hybridize to oligo arrays Each oligo is allele-specific Single base extension (SBE) with RT using labeled nucleotide SNP Detection: Minisequencing/SBE on microarray

25. Multiplex PCR of each sample SBE with Taged-primer and allele-spefic labeled nucleotide Hybridize the labeled Tag-primer to Tag array Tag arrays have 32,000 unique tags (20mer) SNP detection: SBE on Tag arrays

26. Optical fibre has inner ring and outer ring/cladding Light transmission occurs by bouncing internally To produce a fibre-DNA sensor array: Etch core to create well Bundle fibres to form thread 5000-50,000 fibres, each 3-7 uM total diameter of 300-1000 um SNP Detection : Bead-based Fiber Optics Arrays

27. To create the beads/microspheres: Polystyrene beads, 3 um, must be registered or coded by entrapping unique proportions of 2 different dyes Add DNA-probe by: Direct synthesis of oligonucleotide for gene X on bead Coat with avidin and add biotin-DNA probe for gene X Repeat for each probe required Add pool of encoded beads to fibre optic array Bead-based Fiber Optics Arrays

28. Bead-based Fiber Optics Arrays • Disadvantages • Random Poisson distribution • of beads incorporated in an array • Every array is unique and must decoded each time • Each oligo and/or bead must be • synthesized separately, unlike • combinatorial synthesis • Advantages • High level of redundancy • Reusuable (>100 times) • Sensitivity 100 better lower • limit than microarrays, no PCR • Small sample size, dip fibre into plate with <5 ul • Scalability: 1ml bead preparation of 20% slurry contains 1010 beads • Fibre threads, up to 50,000

29. Optimized medicines for individuals Indicate best choice from different available drugs Reduction in adverse drug reactions Currently, ~6.7% of inpatients suffer adverse reaction 4th-6th leading cause of death for inpatients Expedient development of new drugs Successful drug may have efficacy of 20% How will SNPs improve drug development

30. Integration of SNP analysis with clinical trials Phase I Safety/Tolerance 500,000 SNPs Phase II Efficacy in small group 500,000 SNPs (20% response) Phase III Efficacy in large group 50,000 SNPs (20% response)

31. Genetic linkage analysis for disease susceptibility A individual’s genotype is stable Limitations: influence of environmental factors are not reflected in genome/SNPs Applications of SNP analysis

32. Genetic variability is essential for species survival Unprecedented depth of genetic analyses in post-genomic era Immediate challenges in building technological tools and bioinformatics Summary