Recent Genetic Advances in Cardiovascular Disease

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Recent Genetic Advances in Cardiovascular Disease

• Linnea M. Baudhuin, Ph.D.
• Mayo Clinic
• Dec. 6, 2008

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DISCLOSUREInformation

• Relevant Financial Relationships
• None
• Off Label Usage
• None

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Recent Developments in CV Genetics

• Mendelian genetics
• Autosomal Dominant Hypercholesterolemia
• LDLR, APOB, PCSK9
• Familial Thoracic Aortic Aneurysm and Dissection
  syndromes
• FBN1, TGFBR1, TGFBR2, ACTA2, MYH11, NOTCH
• Genetic markers for CAD
• Chromosome 9p21/ANRIL
• KIF6
• Pharmacogenetics
• Warfarin sensitivity
• Statin efficacy

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Coronary Artery Disease

• Unable to predict significant portion of CAD
• 35 of CAD occurs in population with TClt200 mg/dL
• CAD could be eliminated in 21st century by
  treating all risk factors
• R. Roberts, Genetics of Premature Myocardial
  Infarction, Curr Ather Reports, 2008, 10 186-193
• Atherosclerotic CVD has strong heritable
  component
• Estimates as high as gt50
• Families with early CAD (14 of population)
  account for 72 of early CAD cases
• Most of the risk factors have predominant genetic
  component
• Need for identifying genetic modifiers and their
  function

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Complex Genetics of CAD

• Minority are single-gene disorders
• FH, FDB, ARH, Tangier, Sitosterolemia, etc.
• What about the majority of CAD?
• Whats going on genetically?

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Early stages of atherosclerosis
Late stages of atherosclerosis
Lusis et al., Ann Rev Gen Hum Gen, 2004, 5189-218
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Genes contributing to CAD susceptibility
Lusis A, Trends Cardio Med, 2003 13309-16.
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How can we identify genetic markers for complex
disease?

• Family studies
• Genetic variants that segregate with disease in
  affected vs. non-affected family members
• Linkage analysis
• Population studies
• Genetic variants that segregate with disease in
  cases vs. controls
• Candidate genes
• Localized genomic regions
• Genome-wide
• Association studies

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CAD Linkage Studies

• At least 7 family studies
• Occurrence of MI or subclinical atherosclerosis
• Linkage Chr 1, 2, 3, 13, 14, 15, 16, X
• Lack of replication
• Genes identified
• MEF2A (Chr 15q26)
• ALOX5AP
• LTA4

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Candidate Gene Association Analyses in CAD

• Generally single gene/single polymorphism
• Small cohorts
• Inability to replicate findings in additional
  studies
• Genetic analysis of 103 candidate genes (n1400,
  with follow-up n 806) unable to replicate
  findings (Pare et al, Am Hum Genet, 2007,
  80673-682)
• Similar results in n811 ACS patients, 70 genes
  (Morgan et al, JAMA, 2007, 297 1551-1561

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Why are the results of Linkage and Candidate Gene
studies so disappointing?

• Inadequately powered
• Phenotypic heterogeneity and imprecise definition
  of phenotypes
• Bias (e.g. population stratification)
• Multiple genes each contribute to small
  percentage of phenotype (lt5-10)
• A single gene is insufficient to induce disease
• Information from multiple polymorphisms should be
  integrated to become clinically useful
• Multiple possible combinations of genotypes
• Population and disease heterogeneity

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What about Genome Wide Association Studies?
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Genome-wide association studies

• Previously single SNP/single cohort associations
  with little replication
• Now large international consortium studies of
  tens of thousands of individuals and hundreds of
  thousands of SNPs
• Technology
• Ultra high-throughput genotyping with ability to
  analyze 500K to gt1M SNPs in each individual

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Topol, EJ et al., JAMA, 2007 298218-221
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Multiple GWASs confirming 9p21 in Caucasian
populations

• McPherson et al, Science (316), June 8 2007,
  1488-91
• Initial study, 322 cases vs 312 controls 73K
  SNPs
• Confirmed in 5 independent Caucasian populations
  (n23,000)
• Helgadottir et al, Science (316), June 8 2007,
  1491-3
• Initial study, 1607 cases vs 6728 controls 306K
  SNPs
• Replicated in
• 665 cases and 3533 controls
• Three other case control populations
• Philadelphia, Atlanta, Durham, NC
• 4587 cases, 12,767 controls
• Wellcome Trust Case Consortium, Nature,
  447661-678, 2007
• Samani et al, NEJM (357), Aug 2 2007, 443-53
• PROCARDIS Consortium (Broadbent et al, Hum Mol
  Gen, 2008, 17)

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Chromosome 9p21

• 9p21 confined to 58K bp region
• Risk associated with MI and CAD independent of
  known risk factors
• 50 of individuals heterozygous for allele
• 15-20 increased risk of CAD
• 25 of individuals homozygous for allele
• 30-40 increased risk of CAD
• Odds ratio
• Heterozygous 1.3-1.5
• Homozygous 1.8-2.1

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9p21 and ANRIL

• High-risk CAD 9p21 haplotype overlaps with exons
  13-19 of ANRIL
• Newly annotated gene, encodes large antisense
  non-coding RNA (ncRNA)
• Identified through deletion analysis of French
  family with hereditary melanoma-neural system
  tumors
• Expressed in atheromatous human vessels
• Comparable cell type profiles to atherosclerotic
  arteries
• Expressed in vascular endothelial cells,
  monocyte-derived macrophages, and coronary smooth
  muscle cells
• Very little known about function
• Gene class is thought to be involved in
  transcriptional control

Broadbent, et al, Human Mol Gen, 2008, 17 806-14
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KIF6 Variant and CAD

• CARE Cholesterol and Recurrent Events
• WOSCOPS West of Scotland Coronary Prevention
  Study

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Association of KIF6 with CAD 2 Prospective
Trials of Pravastatin

• CARE Cholesterol and Recurrent Events
• Secondary prevention in patients with a prior MI
• 40 mg pravastatin vs placebo
• 87 men and 13 women
• LDL-C 115 to 174 mg/dL at baseline
• 2,697 participants studied
• WOSCOPS West of Scotland Coronary Prevention
  Study
• Primary prevention
• 40 mg pravastatin vs placebo
• Men aged 45 to 64
• LDL-C 174 to 232 mg/dL at baseline
• 1,527 participants studied

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Risk of CAD in CARE Placebo ArmKIF6 Variant and
Traditional Risk Factors
Smoking
Diabetes
KIF6 Variant
Age 55
HDL-C lt 40
Hypertension
LDL-C 130
Adjusted Hazard Ratio

• Magnitude of risk predicted by KIF6 variant was
• Similar to that of traditional risk factors
• Independent of traditional risk factors

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Risk of CAD in WOSCOPS Placebo ArmKIF6 Variant
and Traditional Risk Factors
Diabetes
KIF6 Variant
HDL-C lt 40
LDL-C 189
Hypertension
Adjusted Risk Ratio

• Magnitude of risk predicted by KIF6 variant was
• Similar to that of traditional risk factors
• Independent of traditional risk factors

Case and control patients were matched for age
and smoking all were men Median level in
placebo arm
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KIF6 Variant is Associated with CAD 5
Prospective Studies
CARE
Untreated patients
WOSCOPS
CHS
Some treatment
ARIC
WHS
Hazard Ratio

• KIF6 variant predicts risk of CAD
• Up to 55 increased risk in untreated populations

Adjusted for traditional risk factors
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Recent Developments in CV Genetics

• Mendelian genetics
• Autosomal Dominant Hypercholesterolemia
• LDLR, APOB, PCSK9
• Familial Thoracic Aortic Aneurysm and Dissection
  syndromes
• FBN1, TGFBR1, TGFBR2, ACTA2, MYH11, NOTCH
• Genetic markers for CAD
• Chromosome 9p21/ANRIL
• KIF6
• Pharmacogenetics
• Warfarin sensitivity
• Statin efficacy

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• PROVE IT - TIMI 22 study
• Pravastatin or Atorvastatin Evaluation and
  Infection Therapy, Thrombolysis in MI
• Patients hospitalized within 10 days after an
  acute coronary syndrome

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Prevention of CHD Events by Statins Carriers of
the KIF6 Variant Benefit the Most
WOSCOPS
PROVE IT
Pravastatin 40 mg
Atorvastatin 80 mg
Non-carriers
Non-carriers
Carriers
Carriers
All
All
Absolute Risk Reduction ()
3.5
10.0
6.2
0.1
0.8
5.5

• KIF6 carriers received significant risk reduction
  from atorvastatin in PROVE IT, suggesting KIF6
  may also predict response with other statins
• KIF6 carriers also received significantly greater
  benefit than noncarriers (p0.003 for WOSCOPS and
  0.018 for PROVE IT)

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Statin Intensity and CHD Event ReductionAccording
to KIF6 719Arg Carrier Status
Noncarriers
KIF6 Carriers
Pravastatin
Pravastatin
P1.0
p0.001
Death or major CV events
Atorvastatin
Atorvastatin
Months of follow-up
Months of follow-up

• KIF6 carriers received greater benefit from 80 mg
  atorvastatin, compared with 40 mg pravastatin,
  than did noncarriers
• Number Needed to Treat with atorvastatin for 2
  years to prevent 1 event was 10 for KIF6 carriers
  and 125 for noncarriers

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KIF6 SummaryRisk for CAD and Risk Reduction from
Statins

• KIF6 carriers, 60 of the population, have
  greater risk for CAD
• Up to 55 increased risk of CAD
• Independent of traditional risk factors, but
  similar in magnitude of risk
• Associated with CAD risk in men (WOSCOPS), women
  (WHS), the middle aged (ARIC) and the elderly
  (CHS)
• KIF6 carriers treated with pravastatin or
  atorvastatin had a greater reduction of coronary
  events than noncarriers

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Warfarin PGx

• Warfarin is commonly prescribed drug
• Variants in CYP2C9 and VKORC1 account for high
  percentage of variability of warfarin response
• FDA relabeled warfarin in Aug. 2007 to encourage
  pharmacogenetic testing
• Utility still remains low
• Controversial topic
• Unanswered questions

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Warfarin PGx Ready for Prime Time?

• How is the information applied to clinical
  practice?
• Can genetic testing be used to determine the
  right warfarin dosage?
• Does it add significantly to the information
  discerned by careful INR monitoring and other
  factors (e.g. age, underlying disease state,
  drug-drug interactions)?
• Will it really reduce clinical complications?
• Will it result in shorter time to stable INR?
• Is it cost-effective?
• Should genotyping be ordered on all patients
  taking Warfarin?
• Can the information be obtained in a timely
  fashion?

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Warfarin

• Widely prescribed anticoagulant
• 12th most commonly prescribed drug

45 increase from 1998 to 2004
Wysowski, D. K. et al. Arch Intern Med
20071671414-1419.
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Warfarin

• Challenges in regulating warfarin dosing
• Prothrombin time (INR) must remain in narrow
  therapeutic range
• Elevated INR risk for major bleeding
  complications
• Risk for serious bleeding increases with INR gt
  4.0
• Most likely to occur within first few weeks of
  initiating treatment
• Subtherapeutic INR thrombotic complications

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Warfarin is the Most Commonly Implicated
Medication in U.S. ED Visits
Furosemide
Bactrim
Lisinopril
Levofloxacin
Glipizide
Phenytoin
Vicodin
Glyburide
Metformin
Digoxin
Clopodigrel
Aspirin
Insulin
Warfarin
0
5
10
15
20
Frequency
Budnitz et al, Ann Intern Med 2007147755-765.
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Wide Range in Warfarin Dose Requirements
Warfarin Sensitivity
Warfarin Resistance
James AH. J Clin Path 199245704-06
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Sources of variability in Warfarin dosing
VKORC1 25
Other 28
CYP2C9 17
Drugs 12
CYP4F2 2
Age 7
Weight 9
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Warfarin Pharmacology

• Racemic mixture of R- and S-enantiomers
• S-Warfarin approx. 7-10X as potent as R-
• Majority of in vivo activity of warfarin resides
  in S-warfarin
• Metabolized mainly through CYP2C9
• Targets VKORC1 (Vitamin K epoxide reductase
  complex subunit 1)
• Interferes with recycling of Vit K in the liver
• Reduced activation of clotting factors

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Pharmacodynamics
Pharmacokinetics
(hepatocyte)
(hepatocyte)
S-Warfarin
R-Warfarin
S-Warfarin
R-Warfarin
CYP2C19 CYP3A4 CYP1A1 CYP1A2 CYP2C8 CYP2C9
CYP2C9
VKOR
inactive
4-hydroxywarfarin
6-hydroxywarfarin
Vitamin K (oxidized)
Vitamin K (reduced)
6-hydroxywarfarin
7-hydroxywarfarin
7-hydroxywarfarin
8-hydroxywarfarin
GGCX
10-hydroxywarfarin
Inactive Vitamin K- dependent clotting factors
Active Vitamin K- dependent clotting factors
Elimination
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Warfarin PGx

• CYP2C9
• CYP2C92 variant
• Arg144Cys
• 30 decrease in enzymatic activity
• CYP2C93 variant
• Ile359Leu
• 70-95 decreased enzymatic activity
• VKORC1
• Promoter SNP -1639GgtA
• 44 decrease in promoter activity

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Allele Frequencies
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Genotype-guided Warfarin Dosing
McClain, et al., Gen Med, 2008, 10(2)
89-98 www.warfarindosing.org
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Can Pharmacogenomics Assist Warfarin Dosing?
Warfarin Dosing
Initiation
Stabilization
PGx- Informed (CYP2C9 only)
185 Patients
Algorithm Guided (Ageno et al, 2000)
Caraco, Clin Pharm Ther, 2008
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CYP2C9 Genotype-guided Warfarin Prescribing
Efficacy and Safety
Time to First Therapeutic INR
Plt0.001
1.0
0.8
PGx-informed
Controls
0.6
Proportion of patients with INRgt2
0.4
0.2
Time to therapeutic INR 2.73 days earlier
0.0
0
5
10
15
20
25
Days
Caraco, Clin Pharm Ther, 2008
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CYP2C9 Genotype-guided Warfarin Prescribing
Efficacy and Safety
Time to First Stable Anticoagulation
1.0
Plt0.001
0.8
PGx-informed
Controls
0.6
Proportion of patients at stable anticoagulation
0.4
Time to stable anticoagulation 18 days
earlier (227 vs 4021 days)
0.2
0.0
0
25
50
75
100
125
Days
Caraco, Clin Pharm Ther, 2008
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CYP2C9 Genotype-guided Warfarin Prescribing
Efficacy and Safety

• Time spent in the therapeutic range was higher
• (80.4 vs. 63.4)
• Bleeding incidence was lower (3.2 vs. 12.5)
• Patients with 1 or 2 variant alleles required 77
  and 52 of dose used by 1/1, respectively

Caraco, Clin Pharm Ther, 2008
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Using genotyping results to make Warfarin dosing
decisions

• No clinically validated algorithm
• A work in progress
• Several large multi-center studies being
  undertaken to address this question
• www.warfarindosing.org

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Warfarin PGx Summary

• Some studies suggest that warfarin PGx testing is
  beneficial
• Reduced time to stable INR
• Reduced adverse events
• Large multi-center studies currently underway
• Clinical outcomes
• Validated dosing algorithms
• New technology allowing for more rapid results
• Genetic variants explain 40-45 of variability in
  response to warfarin
• Important to realize the impact of the compound
  effects of
• polymorphisms in CYP2C9 AND VKORC1
• Drug-drug interactions, co-morbidities, age, BMI

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Recent Developments in CV Genetics

• Mendelian genetics
• Autosomal Dominant Hypercholesterolemia
• LDLR, APOB, PCSK9
• Familial Thoracic Aortic Aneurysm and Dissection
  syndromes
• FBN1, TGFBR1, TGFBR2, ACTA2, MYH11, NOTCH
• Genetic markers for CAD
• Multiple genes contribute to small percentage of
  phenotype
• Chromosome 9p21/ANRIL
• KIF6
• Pharmacogenetics
• Controversial, but gathering speed
• Warfarin sensitivity
• Statin efficacy

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Thank You!!