Clinical Pharmacogenetics David A Flockhart MD, PhD Chief, Division of Clinical Pharmacology Professor of Medicine, Genetics and Pharmacology Indiana.
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Clinical Pharmacogenetics
David A Flockhart MD, PhD Chief, Division of Clinical Pharmacology
Professor of Medicine, Genetics and Pharmacology
Indiana University School of Medicine
Pharmacogenetics, 1990
Pharmacogenomic Journals, 2007
Ethical, legal and policy issues within pharmacogenetics
• Risk of Loss of Patient Confidentiality– Need for anonymized DNA storage systems
• Risk that existing patents will stifle progress– Need for careful interpration of Bayh-Dole
• Untangling the relationship between genetics and self-described ethnicity
Role Models for Pharmacogenetics
• Concorde?
• Nuclear Power?
• The Longitude Problem?
SNP Variability in The Human Genome December 2007
• 2.85 billion base pairs• ~22,000 genes • 1.7% of the genome codes for protein• 3.3% of the genome is as conserved as the 1.7% that
codes for protein• On average 1 SNP/1.2kb• 10 - 15 million SNPs that occur at > 1% frequency• ~450,000 SNPs in MCS (Multiply Conserved Regions)
SNP Variability In Exons
• ~150,000 SNPs in known exons
• 48,451 non-synonymous SNPs
• 1113 introduce a stop codon
• 104 disrupt an existing STOP
SNPs that change clinical outcome
SNPs that change drug response
SNPs that change pharmacokinetics
SNPs that change activity in vitro
Non-conservative amino acid changes
Non-synonymous SNPs in exons
Exon-based changes
All SNPs
Hierarchy of Pharmacogenetic Information from Single Nucleotide Polymorphisms (SNPs)
Current Clinical Ability to Predict Response
Clinical
Value
of a
Pharmacogenetic
Test
Pharmacogenetic Principle 1:
Value Decreases when Current Predictive Ability is High
Meyer UA and Flockhart DA, 2005
Azathioprine/TPMT
β-blockade/β Receptor
Cancer Chemotherapy
Antidepressants/5HTR
Methods in Pharmacogenetics• SNP discovery:
– Candidate gene approach
– Pathway approach
– Genome Wide Arrays
• Identification of gene and variants• Development of a genetic test for DNA variants • Correlation between genotype and phenotype• Validation
• Application in Clinical Practice
Polymorphic Distribution
0
10
20
30
40
50
60
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Activity (Phenotype)
Fre
qu
en
cy
Antimode
The Value of Normit Distribution Plots:
Population Distribution of CYP2C19 phenotype
Flockhart et al: Clin Pharmacol Ther 1995;57:662-669
Skewed Distribution
0
10
20
30
40
50
60
1 2 3 4 5 6 7 8 9 10 11 12 13
Activity (Phenotype)
Fre
qu
ency
Example 1 of a Skewed Distribution: Heterogeneity in response to Inhaled
Corticosteroids
Weiss ST et al. Hum Molec Genetics 2004; 13:1353-1359
Using the extremes of a phenotypic distribution as a strategy to identify
pharmacogenetic predictors
Example: Iressa™ and the EGF receptor
Lessons
• Germline genetic variation matters, but so do somatic mutations in tumors
• Extremes of phenotype are often viewed as “discardable data”, but outliers should be viewed as important research stimuli
Genetics and Drug Absorption
Enterocyte GI Lumen
ATP
ADP
P-gp Transport
Passive Diffusion
Digoxin Transport across the GI lumen
Fig. 3. Correlation of the exon 26 SNP with MDR-1 expression. The MDR-phenotype (expression and activity) of 21 volunteers and patients was determined by Western blot analyses. The box plot shows the distribution of MDR-1 expression clustered according to the MDR-1 genotype at the relevant exon 26 SNP. The genotype-phenotype correlation has a significance of P = 0.056 (n = 21).
P-Glycoprotein Pharmacogenetics :
Effect of a “wobble” (no coding change) SNP in exon 26
Eichelbaum et al. Proc Nat Acad Sci March, 2000.
Eichelbaum et al, Proc Nat Acad Sci, 2000:March
0.25 mg of digoxin po at steady state
Brain Blood
ATP
ADP
P-gp Transport
Passive Diffusion
Digoxin Transport across the Blood-Brain Barrier
Genetics and Drug Elimination
Cytochrome P450 2D6• Absent in 7% of Caucasians• Hyperactive in up to 30% of East Africans• Catalyzes primary metabolism of:
• propafenone• codeine -blockers• tricyclic antidepressants
• Inhibited by:• fluoxetine• haloperidol• paroxetine• quinidine
CYP2D6 Pharmacogenetics
120
80
40
00.01 0.1 1 10 100
1011 Subjects
Debrisoquine/4-HydroxydebrisoquineMetabolic Ratio
UMsEMs
PMs
cutoff
Nu
mb
er o
f S
ub
ject
s
New CYP2D6 variants continue to appear….
From: Zanger et al: Clin Pharmacol Ther. 2004 Aug;76(2):128-38.
CYP2D6 Alleles
• 67 as of December, 2007• 24 alleles have no activity• 6 have decreased activity• *1, *2, *4 and many others have
copy number polymorphisms• The *2 variant can have 1,2,3,4,5 or
13 copies i.e increased activity
Oligonucleotide array for cytochrome P450 genotesting
From: Flockhart DA and Webb DJ. Lancet End of Year Review for Clinical Pharmacology, 1998.
From: Dalen P, et al. Clin Pharmacol Ther 63:444-452, 1998.
Paroxetine and CYP2D6 genotype change the plasma concentrations of
endoxifen
Stearns, Flockhart et al. J Natl Cancer Inst. 2003;95(23):1758-64.
Endoxifen
0
10
20
30
40
50
60
70
Before After
En
do
xife
n (
nM
)
p = 0.004
4-OH Tamoxifen
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Before After
4-O
H T
am
ox
ife
n (
nM
)
Inhibition of CYP2D6 Affects Endoxifen Concentrations
0
20
40
60
80
100
120
140
Jin Y et al: J Natl Cancer Inst 97:30, 2005
Wt/Wt, noinhibitor
Venlafaxine Sertraline Paroxetine *4/*4, noinhibitor
Plasma Endoxifen
(nM)
0
20
40
60
80
100
0 2 4 6 8 10 12
Relapse-free Survival
%%
Years after randomizationYears after randomization
P=0.009 (vs. 0.02 genotype only)P=0.009 (vs. 0.02 genotype only)
EMEM
IMIM
PMPM
2 year RFS: 2 year RFS:
EM 98%EM 98%
IM 92%IM 92%
PM: 68%PM: 68%
10 year NNT = 3
Lessons from CYP Pharmacogenetics
• Multiple genetic tests of one gene may be needed to accurately predict phenotype
• Gene duplication in the germline exists
• The environment in the form of Drug Interactions can mimick a genetic change
Vitamin K Carboxylase and CYP2C9 Genotypes altered Warfarin Dose
Rieder et al. N. Eng J. Med 2005;352: 2285-2293
Genetic alterations in Phase 2 enzymes with clinical consequences
UGT1A1 NAT-2
SULT1A1COMTTPMT
UGT1A1 TA repeat genotype altersirinotecan neutropenia/activity
35.7
16.3
8.6
0
5
10
15
20
25
30
35
40
45
50
6/6 6/7 7/7
P=0.007
UGT1A1 genotype
% g
rade
4/5
neu
trop
enia
N=524
41.9
33.8
14.3
05
1015202530354045
6/6 6/7 7/7
UGT1A1 genotypeO
bjec
tive
resp
onse
(%
)
P=0.045
McLeod H. et al, 2003.
N-Acetylation PolymorphismNAT-2
• Late 1940’s : Peripheral Neuropathy noted in patients treated for tuberculosis.
• 1959 : Genetic factors influencing isoniazid blood levels in humans. Trans Conf Chemother Tuberc 1959: 8, 52–56.
NAT-2 substrates(All have been used as probes)
• Caffeine
• Dapsone
• Hydralazine
• Isoniazid
• Procainamide
Incidence of the Slow Acetylator NAT-2 phenotype
• 50% among Caucasians
• 50% among Africans
• 20% among Egyptians
• 15% among Chinese
• 10% among Japanese
0
20
40
60
80
100
120
0 20 40 60 80 100
Duration of Therapy (months)
% o
f p
ts w
ith
lu
pu
s
Slow Acetylators
Fast Acetylators
Onset of Positive ANA Syndrome with Procainamide.
Woosley RL, et al. N Engl J Med 298:1157-1159, 1978.
Thiopurine Methyl Transferase
• Homozygous mutants are 0.2% of Caucasian Populations
• Heterozygotes are ~ 10%
• Homozygous wild type is 90%– Metabolism of Azathioprine– 6-Mercaptopurine
Thiopurine Methyl Transferase Deficiency
From: Weinshilboum et al. JPET;222:174-81. 1982
Effect of TPMT genotype on duration of Azathioprine therapy.
From: Macleod et al: Ann Int Med 1998;
Examples of Human Receptors shown to be genetically
polymorphic with possible alterations in clinical phenotype
• G-proteins• Angiotensin II receptor and angiotensinogen• Angiotensin converting enzyme 2receptor• Dopamine D4 receptor • Endothelial NO synthase• 5HT4receptor
Ser Arg
Ser Gly
Gly Arg
Gly Gly
Ser Arg
Ser Arg
Ser Gly
Ser Arg
Gly Arg
Ser Arg
Gly Gly
Ser Arg
Gly Gly
Ser Gly
Gly Arg
Ser Gly
Ser Gly
Ser Gly
Gly Gly
Gly Arg
Gly Arg
Gly Arg
Haplotypes Diplotypes
Ying-Hong Wang PhD,
Indiana University School of Medicine
Gly Gly
Gly Gly
2SNPs: 10 possible hapoltypes
Observed 1AR Haplotypes in Caucasians and African American Women (WISE study)
Terra et al. Clin. Pharmacol. Ther. 71:70 (2002)
Ser Arg
Ser Arg
Ser Gly
Ser Arg
Gly Arg
Ser Arg
Gly Arg
Ser Gly
Gly Gly
Ser Arg
Gly Gly
Ser Gly
Ser Gly
Ser Gly
Gly Gly
Gly Arg
Gly Arg
Gly Arg
Haplotypes Diplotypes
SR/SR SR/SG SR/GR
SG/GR
Ser Arg
Ser Gly
Gly Arg
Gly Gly
Ying-Hong Wang PhD, Indiana University School of Medicine
Of 10 theoretical diplotypes, only 4 were present in the study population
Johnson et al. Clin Pharmacol & Ther. 2003,74:44-52.
Diplotype predicts Beta-blocker effect
Lesson: Diplotype may be a better predictor of effect than
Genotype
A SNP that tags a Haplotype (tagSNP) may be an economical
means of screening
Lanfear DE et al. JAMA September 28th, 2005;294:1526-1533.
β2 receptor Gln27Glu (79CG) genotype predicts mortality
during β-blockade after MI.
Non-synonymous coding region polymorphisms in long QT disease genes
NH2
NH2
NH2
COOH
extracellular
extracellular
intracellular
intracellular
COOH
COOH
NH2
NH2
COOH
COOH
SCN5AINa
IKs
IKr
KvLQT1
minK
HERG MiRP1
K897T(10%)
T8A(1.5%)
D85N(1.5%)
S38G(38%)
H558R(24%)
Vanderbilt
R34C (4%)
P1090L(4%)
G643S(9%)
Japan only
P448R(20%[?])
Dan Roden MD, October 2003.
A Genetic Effect on Hydrochlorothiazide Efficacy
SNPs that change clinical outcome
SNPs that change drug response
SNPs that change pharmacokinetics
SNPs that change activity in vitro
Non-conservative amino acid changes
Non-synonymous SNPs in exons
Exon-based changes
All SNPs
Hierarchy of Pharmacogenetic Information from Single Nucleotide Polymorphisms (SNPs)
SNPs that change clinical outcome
SNPs that change drug response
SNPs that change pharmacokinetics
SNPs that change activity in vitro
Non-conservative amino acid changes
Non-synonymous SNPs in exons
Exon-based changes
All SNPs
Hierarchy of Pharmacogenetic Information from Single Nucleotide Polymorphisms (SNPs)
Current Methods for genetic testing
• By phenotype: metabolic probe drug or Western blot or Immunohistochemistry
• By PCR with mutation-specific endonuclease
• By PCR and allele-specific hybrization
• By oligonucleotide chip hybridization
• By laser lithography - guided oligonucleotide chip hybridization.
• By rapid throughput pyrosequencing
• Taqman probe screening
• By rapid and high throughput full sequencing
Medication History:AVOID Mistakes
Allergies? : Is there any medicine that we should not give you for any reason?
Vitamins and Herbs?Old drugs? …..as well as currentInteractions?Dependence?Mendel: Family Hx of benefits or problems
with any drugs?
Pharmacogenetics Websites
• www.pharmgkb.org
• The SNP consortium: http://brie2.cshl.org
• The Human Genome:www.ncbi.nlm.nih.gov/genome/guide/H_sapiens.html
• CYP alleles: www.imm.ki.se/CYPalleles/
• Drug Interactions: www.drug-interactions.com
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