pharmacogenetics: from dna to dosage – just a click away cindy l. vnencak-jones, phd, facmg...
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Pharmacogenetics: From DNA to Dosage –
Just A Click Away Cindy L. Vnencak-Jones, PhD, FACMGVanderbilt University Medical Center
April, 2011
DISCLOSURE INFORMATION
Cindy L Vnencak-Jones, PhD FACMG
No relationships to disclose
Pharmacogenetics: From DNA To Dosage –
Just A Click Away
Pharmacogenetics: •influence of genetic variation on an individual’s response to pharmacologic agents •Pharmacogenetics testing is not routinely used in clinical practice •when ordered, is done “as needed” preventing usefulness for initial dosing•many drugs, many genes, many studies result in information overload for the provider
PREDICT:Pharmacogenomic Resource for Enhanced Decisions In Care and Treatment
Pharmacogenetics: From DNA To Dosage –
Just A Click Away
RationaleProvide real-time decision support thereby facilitating individualized drug therapy to maximize efficacy, minimize adverse drug reactions, and reduce health care costs
PREDICT Initiative
Assemble multidisciplinary, multidepartment teamPathology, Informatics, Pharmacy, Clinicians, Ethics, Legal, Regulatory
Proof of ConceptWhich drug/gene relationship should test the model?
GenotypingWhich methodology? Research or CLIA lab?
InformaticsData management, Electronic health record, decision support
Implementation – 9/15/2010Assessment of the initiative – ongoingMeasure utility of decision support and clinical impact of genotyping
PREDICT Initiative
PREDICTPREDICT
Office of Personalized
Medicine
Vanderbilt UniversityVanderbilt University VanderbiltInformatics
Center
Pharmacy and
Therapeutic Committee
VUMCComputational Genetics Core
Clinicians
Optimize Patient ManagementOptimize Patient Management
Molecular Diagnostics Lab
Ethics/Legal/ Regulatory
PREDICT Initiative
PREDICT Process – Phase I
• Consent process– Adult Admitting & ED Registration– “CONSENT FOR ROUTINE TESTS, MEDICAL
TREATMENT, AND GENETIC TESTS TO GUIDE DRUG THERAPY…”
• Provider discusses genotyping studies– Blood drawn
• Sample arrives in laboratory– DNA extracted (day 1) – Assay performed (day 2) – Results reviewed and released (day 3)
PREDICT Process – Phase I
• Raw data converted to drug genome interaction fact for computerized decision support in electronic health record (EHR)
• Provider accesses EHR; alerted to results• Provider receives decision support regarding
dosing or alternative medications• Provider optimizes patient management
utilizing information provided by genotyping studies
Clopidogrel (PLAVIX) –CYP2C19•FDA issued a “black box” warning regarding the clinical relevance of genotype analysis•Widely prescribed to patients at our medical facility•Could provide decision support and measure the change in prescribing behavior of the provider based on the given decision support•Targeted patient population to launch model – the cardiac catheterization lab
PREDICT Model
WARNING: DIMINISHED EFFECTIVENESS IN POOR METABOLIZERS
•Effectiveness of Plavix depends on activation to an active metabolite by the cytochrome P450 (CYP) system, principally CYP2C19. •Poor metabolizers treated with Plavix at recommended doses exhibit higher cardiovascular event rates following acute coronary syndrome (ACS) or percutaneous coronary intervention (PCI) than patients with normal CYP2C19 function. •Tests are available to identify a patient's CYP2C19 genotype and can be used as an aid in determining therapeutic strategy. •Consider alternative treatment or treatment strategies in patients identified as CYP2C19 poor metabolizers.
FDA – Black Box WarningIssued March 12, 2010
Simon T. et al, N Engl J Med 2009
Clopidogrel - PLAVIX• Requires gastro-intestinal
absorption and hepatic biotransformation
• Is an inhibitor to the P2RY12 receptor thereby preventing binding of ADP
• Increases risk of bleeding; especially GI bleeding when combined with warfarin and nonsteroidal anti-inflammatory drugs
CH3
CH3 • Antiplatelet therapy, often prescribed in combination with aspirin
• Initial dose 300 mg followed by 75 mg daily
• Indications for use: acute coronary syndrome; recent myocardial infarction or stroke; peripheral arterial disease; or patients managed following angioplasty, bypass surgery or stent placement
Prodrug
Active
Clopidogrel - PLAVIX
Evans and Relling, Science 1999
Phase IModification of
functional groups:
HydrolysisOxidation
DealkylationDehydrogenation
ReductionDeaminationDesulfuration
Phase IIConjugation
with endogenous substituents
to form:
GlucuronideAcetate
GlutathioneSulfate
Methionine
Drug Metabolizing Enzymes
VeraCode ADME Core Panel
• Absorption
• Distribution
• Metabolism
• Excretion
ADME Core (34 genes, 185 markers)
ABCB1 CYP2C9 NAT1 SULT1A1
ABCC2 CYP2D6 NAT2 TPMT
ABCG2 CYP2E1 SLC15A2 UGT1A1
CYP1A1 CYP3A4 SLC22A1 UGT2B15
CYP1A2 CYP3A5 SLC22A2 UGT2B17
CYP2A6 DPYD SLC22A6 UGT2B7
CYP2B6 GSTM1 SLCO1B1 VKORC1
CYP2C19 GSTP1 SLCO1B3
CYP2C8 GSTT1 SLCO2B1Illumina
Multiple polymorphic sites with clinical significance
Location: 10q24.1 – q24.3Gene: 90,209 bases
mRNA: 1,473Protein: 490 amino acid
5’ 3’
CYtochrome P450Family 2
Subfamily Cpolypeptide 19
*17
*4 *6 *2
*7
*8
*3*12
*5
g.-806C>T
W120Rc.358T>C
W212Xc.636G>A
c.1A>GATG>GTG
c.395G>AR132Q
g.19294T>A
c.681G>AP681P c.1297C>T
R433W
X491Cc.1473A>C
splicingpromoter
truncation
initiation codon
missense
insertion
CYP2C19
CYP2C19 – Clopidogrel
Patients with reduced function alleles have:– significantly lower levels of the active
metabolite–diminished platelet inhibition and higher
rate of platelet aggregation–higher rate of major adverse
cardiovascular events and higher risk of stent thrombosis
ADME Assay Design
CCCTACACAGATGTGGTGCACGAGGTCCAGAGATACATTGACCTTCTCCCCACCAGCCTGCCCCATGCGGGATGTGTCTACACCACGTGCTCCAGGTCTCTATGTAACTGGAAGAGGGGTGGTCGGACGGGGTACG
Gene 1:SNP-2
Gene 1:SNP-1
AT
Gene 1:SNP-3
SNPs Optimized in 3 pools
SNP-3 SNP-2 SNP-1
Adapted from Illumina
Patient 1Patient 30
+ control
- control
Universal PCR ForwardSequences (1, 2)
GENOMIC DNA TEMPLATE
SNP
(1-20 nt gap)IllumiCode ™Sequence tagidentifies bead
Universal PCR ReverseSequence 3
Locus Specific Oligos
5’3’
5’3’
Locus Specific Oligo
A
G
A/G
Adapted from Illumina
SNP
Assay – Primer Design
Polymerase
TGENOMIC DNA
AUniversal PCR Sequence 1 IllumiCode
Sequence Tag
Universal PCR Sequence 3
Ligase
GUniversal PCR Sequence 2
Adapted from Illumina
SNP specific primer binds and is extended
Assay – Allele Specific Extension and Ligation
Cy3
Universal Primer 1
Universal Primer 2
Cy5
PolymeraseUniversal PCR Sequence 1
BiotinUniversal PCR Sequence 3
A
IllumiCode Sequence Tag
Adapted from Illumina
Primer specific for Gwith red dye does not bind
Assay – PCR Amplification
VeraCode Technology – the glass microbead
• Cylindrical glass microbeads• 240 μm length x 28 μm diameter• Bar-coded for identification
Adapted from Illumina
A/A G/G
IllumiCode 2
IllumiCode 3
C/T
IllumiCode 1
SNP 1 SNP 2
SNP 3
GA
TC
Assay - Hybridization of PCR Products to VeraCode Beads
Homozygous
Heterozygous
Homozygous
Adapted from Illumina
Red and green signaldetection with the BeadXpress Reader
BeadXpress Reader
Adapted from Illumina
VeraCode Bead Loading & Detection
BEADS FALLINTO GROOVE
PLATE
CAPILLARY FORCE ATTRACTS BEADS INTO GROOVES
BEADS ALIGN TIGHTLY FOR OPTIMAL SCANNING EFFICIENCY Adapted from Illumina
VeraCode Bead Plate Scanning
Reports with Automatic Translation
Visualization of the Results
Samples with call rates >97.34% “Pass”
PREDICT Database
Electronic Health Record
Currently, CYP2C19 results sent to EHR, all other data is stored but can be sent to EHR in the future when drug genome interactions decisions become “actionable”
Electronic Health Record
Electronic Prescription Order
Electronic Prescription Order
Clopidogrel Response
• Genetic Factors– Polymorphisms in CYP2C19
and other CYPs, as well as SNPs in P2RY12,GpIIb/IIIa
• Cellular Factors– P2RY12 and non P2Y
pathways
• Clinical Factors– Drug-drug interactions,
elevated body mass, smoking, diabetes, poor compliance
RACE *2*2 *2 HET WT
Caucasian 5% 21% 74%
African American 3% 24% 73%
Asian 7% 43% 50%
CYP2C19 Genotype
ADME QA/QC
• Allele frequencies of all genotypes• Discordant results: controls and repeat
patients (which SNPs and frequency) • Assay performance: # of samples per plate
with average call rates <97.30% (7/185 SNPs no call)
• Locus performance (<95% call rates)
*1*1*1*17
*1,*2*1,*3*1,*4*1*5
*1*7*1*8*1*12*2*17
*17*17
*5*5*4*4*3*3*2*2
1419 patients
9/15/10 - 4/4/11
PREDICT Results
Assay Accuracy
ControlsAverage
Concordance
Paragon Control Cell Lines
99.58%
Coriell Control Cell Lines
98.34%
Assay Reproducibility
150 patients repeated
# of plates
Paragon controls
ADME QA/QC
Locus Performance (<95% call rates)
80 plates
Summary
• Implemented a mid-throughput assay to screen 34 genes (185 SNPs) involved in drug absorption, distribution, metabolism and excretion
• Detected polymorphisms similar in frequencies to that previously reported
• Established QA/QC parameters for assay • Developed a process to enable decision support to
providers for drug dosing based on DNA findings which will facilitate genetically informed medicine
Summary
• Implemented a scalable process to allow expansion to other actionable SNPs with associated decision support rules
• Process enables retrospective auto-population of stored data in patients EHR for future without the need for repeat testing
• Measure clinical utility and impact of genotyping data and decision support services
• Phase II - system permits identification of “at risk” patient populations for preemptive genotyping
AcknowledgementsVanderbilt University
Nicholas Zeppos - Chancellor
Jeff Balser, MD, PhD – Vice Chancellor VUMC
Gordon Bernard, MD – Vice Chancellor Research
Office of Personalized Medicine
Dan Roden, MD
PREDICT Implementation Team
Jill Pulley, MBA
Russ Wilke, MD
Jim Jirjis, MD
Josh Peterson, MD
John McPherson, MD
Andrea, Ramirez, MD
Mike Laposata, MD, PhD
Center for Biomedical Ethics and Society
Ellen Clayton, MD, JD
Kyle Brothers, MD
Molecular Diagnostics LabGladys Garrison, MSJennifer Carter, PhDLisa RochaSonia ByonVickie Fraser
VUMC Computational Genetics CoreHolli Dilks, PhDDoug SelphBrad Winfrey
Vanderbilt Informatics CenterDan Masys, MDJoshua Denny, MDEd Shultz, MDMarc Beller
Pharmacogenetics: From DNA to Dosage –
Just A Click Away Cindy L. Vnencak-Jones, PhD, FACMGVanderbilt University Medical Center
April, 2011