Using genetics for drug prescribing: will it happen? Hype and hope Relating DNA polymorphisms to variable human physiology and drug responses: examples A view to the future

Take-home messages Diversity among human genomes can explain variability in human physiology, and its response to the environment. Pharmacogenetic information is now affecting drug development, and will enter clinical practice (a personal opinion). “Proving” genotype-phenotype relations requires collaborations among clinical investigators, basic biologists, information managers and statisticians, systems biologists, technologists, ethicists…

Case presentations A 68 year old man presents with a large anterior myocardial infarction, and has a VF arrest in the Emergency Room. Grade 4 diarrhea occurs in a 65 year old man receiving irinotecan for colon cancer. A 70 year old woman starts coumadin 5 mg/day for atrial fibrillation. One week later, her INR is 12. A 78 year man develops a QT interval of 700 msec and Torsades de Pointes a day after starting dofetilide. Life-threatening sepsis arises in a 9-year-old girl after 6-MP therapy for ALL.

“idiosyncratic” drug response understand the biology identify causative DNA variants “idiosyncratic” response now predictable and avoidable Pharmacogenetics: Large single gene effects

cytotoxic 6-TGN concentrations 6-thioguanines (6-TGNs) LL *3A/*3A (mutant) HL *1/*3A (heterozygote) HH *1/*1 (wild-type) 1/300 *3A/*3A *1/*3A *1/*1 “single nucleotide polymorphisms” (SNPs) azathioprine

An “idiosyncratic” drug response AR, 78 year old man Chronic heart disease with history of bypass surgery and valve replacement Cardiac arrest 4 days after major abdominal surgery; placed on dofetilide (a potent and highly selective blocker of a potassium current called I Kr ) to prevent recurrence. 2 days later: Torsades de Pointes Also characteristic of the congenital long QT syndrome Drug-induced QT prolongation and torsades is the single commonest cause for drug withdrawal or relabeling in the past decade.

AR: An “idiosyncratic” drug response DNA variant resulting in R583C identified in KCNQ1, a gene controlling QT duration and mutated in a common form of the congenital Long QT Syndrome. R583C alters protein function in vitro: reduces I Ks. Absent in >1000 controls  “mutation”. This man has the congenital long QT syndrome, that remained asymptomatic for 78 years.

How did AR avoid arrhythmias for 2,000,000,000 heart beats? The concept of reduced repolarization reserve Redundancy (“reserve”) in cardiac repolarization allowed him to maintain a normal QT until other lesions (heart disease, dofetilide) were superimposed. Patient 1 Patient 2 Same QT-prolonging drug

Drug A TPMT inactive metabolite Drug B CYP2C9 inactive metabolite Drug C renal excretion Drug D CYP3A4 inactive metabolite transport by drug efflux pump Redundancy in physiologic systems: “high risk pharmacokinetics” 6-MP warfarin digoxin terfenadine (Seldane) inhibitor drug low margin between doses needed for efficacy and doses producing toxicity (therapeutic index) a single pathway for drug elimination that is genetically variable or subject to inhibition by interacting drugs PLUS

Drug A TPMT inactive metabolite Drug B CYP2C9 inactive metabolite Drug C renal excretion Drug D CYP3A4 inactive metabolite Drug E CYP2C9 inactive metabolite transport by drug efflux pump Redundancy in physiologic systems: “high risk pharmacokinetics” transporter renal excretion CYP3A4 inactive metabolite TPMT inactive metabolite 6-MP warfarin digoxin terfenadine (Seldane) inhibitor drug Drug E will be an especially attractive agent if it also has a high therapeutic index

Redundancy in physiologic systems as a protective mechanism arrhythmia susceptibility susceptibility to adverse drug reactions and interactions “multi-hit” requirement for carcinogenesis

“idiosyncratic” drug response understand the biology identify causative DNA variants “idiosyncratic” response now predictable and avoidable Pharmacogenetics: Large single gene effects unusually variable drug response identify associated DNA polymorphisms adjust dose or change drugs discover new biology and new drug targets Pharmacogenomics: Discovering new biology

PHARMACOGENETICS PHARMACOGENOMICS Single gene Small number of genes Complex biologic pathway Large single variant effect Smaller effect; multiple variants Whole genome

PHARMACOGENOMICS (large populations) Single gene Small number of genes Complex biologic pathway Large single variant effect Smaller effect; multiple variants Whole genome Rare coding region variants (polymorphisms or mutations) Commoner coding and regulatory variants Detectible effects of polymorphisms in >1 gene Pathway analysis and whole genome approaches PHARMACOGENOMICS PHARMACOGENETICS (snlall groups)

Variants in the warfarin target

2 5 = 32 possible combinations Haplotype A: CCGATCTCTG Haplotype B: TCGGTCCGCG Variants in the warfarin target – 2 Single Nucleotide Polymorphisms (SNPs) in the VKORC1 promoter CGTC C T A G T C C G T C G Reider et al. NEJM 2005

Variants in both drug metabolism (CYP2C9) and drug target (VKORC1) genes affect warfarin dose requirement 554 patients on chronic warfarin; early dropouts not included CYP2C9 genotype predicts 9% of dosage variability VKORC1 haplotype accounts for 23% Illustrates Multi-gene effects can be detected Understanding mechanisms in rare syndromes can inform the study of common biologic problems Increasing importance of haplotypes Reider et al., NEJM 2005

A 68 year old man presents with a large MI, and has a VF arrest in the ER Patient 1 Patient 2 Same acute MI ?“reduced antifibrillatory reserve”

Reduced cardiac sodium current predisposes to serious arrhythmias Hypothesis: Variable sodium channel expression is a candidate mechanism for variability in basal conduction velocity, and in susceptibility to slowed conduction with exogenous stressors (drugs, myocardial ischemia). Slow conduction predisposes to sudden death due to VF. Loss of function mutations in the sodium channel gene cause a distinctive ECG and ↑↑ risk of VF Brugada syndrome Sodium channel blocking drugs increase mortality Days from randomization % survival encainide or flecainide (n=730) Placebo (n=725)

6 sodium channel promoter variants, found only in Asians intron1promoter bp1000 T C T C T - 847G - 835insGC G - 354C C287T exon 1 (non-coding) intron1promoter bp bp1000 T C T C T - 847G - 835insGC G - 354C C287T exon 1 (non-coding) exon 1 (non-coding) 6 variants: 2 6 = 64 possible combinations

6 sodium channel promoter variants, found only in Asians, in very tight linkage disequilibrium intron1promoter bp1000 T C T C T - 847G - 835insGC G - 354C C287T exon 1 (non-coding) CCGinsCTCCG CT TTT---GCTTT GC 75.5%HaplotypeA 24%HaplotypeB CTT---CCCTT CC 0.5%HaplotypeC Frequency* intron1promoter bp bp1000 T C T C T - 847G - 835insGC G - 354C C287T exon 1 (non-coding) exon 1 (non-coding) CCGinsCTCCG CT TTT---GCT 75.5% 24% CTT---CCCTT CCCTT CCCTT CC Frequency

Haplotype B  ↓↓ promoter activity Wild Type6-change haplotype Wild Type6-change haplotype Fold activity CHO cellsCardiomyocytes n=13, p=0.04n=9, p=0.006 ABAB

AAABABBB AAABABBB normals 71 probands with Brugada Syndrome QRS duration (msec) Ventricular conduction is slower with the reduction of function allele

QRS duration (msec) Genotype-dependent incremental conduction slowing by sodium channel blockers AAABABBB msec +17 msec +29 msec Who has the longest QRS duration? Brugada Syndrome mutation + drug exposure + BB haplotype “Genes load the gun, environment pulls the trigger”

“idiosyncratic” response now predictable and avoidable “idiosyncratic” drug response understand the biology identify causative DNA variants Pharmacogenetics: Large single gene effects 1950 unusually variable drug response identify associated DNA polymorphisms adjust dose or change drugs discover new biology and new drug targets Pharmacogenomics: Discovering new biology Routine patient visit identify or look up DNA variants in that patient adjust dose or change drugs The future Moving to widespread practice

Nature Oct. 5, 2005

Genotypes: CYP2D6: *4/*4 CYP2C9: wt/*2 NAT: slow TPMT: wt/wt UGT1A1: 6/6 ACE: ID CETP: BB BRCA1: negative  1 AR: S49/G389  2 AR: R16/G27 KCNQ1: R583C HERG: wt/wt KCNE1: wt/wt KCNE2: wt/wt Apo  : 2/3 ABCA1: wt/wt

Genotypes: CYP2D6: *4/*4 CYP2C9: wt/*2 NAT: slow TPMT: wt/wt UGT1A1: 6/6 ACE: ID CETP: BB BRCA1: negative  1 AR: S49/G389  2 AR: R16/G27 KCNQ1: R583C HERG: wt/wt KCNE1: wt/wt KCNE2: wt/wt Apo  : 2/3 ABCA1: wt/wt