Pharmacogenetics

1
Pharmacogenetics

• Definition and introduction
• Relevance to medicine
• Post-genomic approaches to studying
  pharmacogenetics
• SNP detection

2
Promise of Pharmacogenetics in Post-Genome Era

• Completion of human genome variation between
  individuals are lt0.1 (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

3
Pharmacogenetics

• 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)

4
Pharmacogenetics in Nature

• Environmental toxicogenetics
• Insecticide/herbicide resistance in agriculture
• Antibiotic resistance in microbes

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. Haldan
e, 1949
6
Human exposure to natural toxins

• St. Anthonys 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

7
Resistance of DTT in houseflies

• 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

8
Antibiotic resistance in bacteria

• 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

9
Biological cost of variation

• 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

10
Balance polymorphisms Sickle cell anemia

• 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

11
Neutral mutations vs. balanced polymorhphisms

• 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

12
Types of SNPs and their frequency
Type Description Number (in
1000s) 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 Oth
er non-coding gt1000
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
Classical Pharmacogenetics enzymology,
biochemistry and population genetics

• 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)

15
Molecular Pharmacogenetics Cytochrome P450

• 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

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 Alzheimers disease ApoE4,
ApoE2 (19q13), 12q
17
SNP Mutation vs. Polymorphism

• Definitions are arbitrary
• Gene mutations are rare, lt1 of population
• Gene polymorphisms exist in gt1
• SNPs exists about every 1000 bases, ie.
  3,000,000/genome

18
How to use SNPs to measure drug responses?

• SNP linkage disequilibrium profiles
• With no prior knowledge of genetic involvement,
  analysis of 500,000 SNPs is required for whole
  genome association study

19
Functional Genomics Approaches to Pharmacogenetics

• 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

20
SNP Identification

• 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

21
SNP Detection Standard Methods

• 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 100s-1000s per day

22
SNP Detection High throughput PCR

• 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 1,000s per day

23
SNP Detection Microarrays

• 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

24
SNP Detection Minisequencing/SBE on microarray

• 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

25
SNP detection SBE on Tag arrays

• 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)

26
SNP Detection Bead-based Fiber Optics Arrays

• 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

27
Bead-based Fiber Optics Arrays

• 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

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 (gt100 times)
• Sensitivity 100 better lower
• limit than microarrays, no PCR
• Small sample size, dip fibre into plate with lt5
  ul
• Scalability 1ml bead preparation of 20 slurry
  contains 1010 beads
• Fibre threads, up to 50,000

29
How will SNPs improve drug development

• 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

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
Applications of SNP analysis

• Genetic linkage analysis for disease
  susceptibility
• A individuals genotype is stable
• Limitations influence of environmental factors
  are not reflected in genome/SNPs

32
Summary

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