Recent Genetic Advances in
Cardiovascular Disease
Linnea M. Baudhuin, Ph.D.
Mayo Clinic
Dec. 6, 2008
DISCLOSURE
Information
Relevant Financial Relationships
None
Off Label Usage
None
Recent Developments in CV Genetics

Mendelian genetics

Autosomal Dominant Hypercholesterolemia


Familial Thoracic Aortic Aneurysm and Dissection
syndromes:



LDLR, APOB, PCSK9
FBN1, TGFBR1, TGFBR2, ACTA2, MYH11, NOTCH
Genetic markers for CAD

Chromosome 9p21/ANRIL

KIF6
Pharmacogenetics

Warfarin sensitivity

Statin efficacy
Coronary Artery Disease

Unable to predict significant portion of CAD


“CAD could be eliminated in 21st century by treating all risk
factors”



35% of CAD occurs in population with TC<200 mg/dL
R. Roberts, Genetics of Premature Myocardial Infarction, Curr
Ather Reports, 2008, 10: 186-193
Atherosclerotic CVD has strong heritable component

Estimates as high as >50%

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
Complex Genetics of CAD

Minority are single-gene disorders



FH, FDB, ARH, Tangier, Sitosterolemia, etc.
What about the majority of CAD?
What’s going on genetically?
Early stages of
atherosclerosis
Late stages of
atherosclerosis
Lusis et al., Ann Rev Gen Hum Gen, 2004, 5:189-218
Genes
contributing to
CAD
susceptibility
Lusis A, Trends
Cardio Med, 2003;
13:309-16.
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
CAD Linkage Studies

At least 7 family studies


Linkage: Chr 1, 2, 3, 13, 14, 15, 16, X


Occurrence of MI or subclinical atherosclerosis
Lack of replication
Genes identified:



MEF2A (Chr 15q26)
ALOX5AP
LTA4
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 (n=1400,
with follow-up n= 806) unable to replicate findings
(Pare et al, Am Hum Genet, 2007, 80:673-682)

Similar results in n=811 ACS patients, 70 genes
(Morgan et al, JAMA, 2007, 297: 1551-1561
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 (<5-10%)


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
What about
Genome Wide
Association
Studies?
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 >1M SNPs in each individual
Topol, EJ et al., JAMA, 2007; 298:218-221
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 (n=23,000)
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, 447:661-678, 2007

Samani et al, NEJM (357), Aug 2 2007, 443-53

PROCARDIS Consortium (Broadbent et al, Hum Mol Gen, 2008, 17)
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


25% of individuals homozygous for allele


15-20% increased risk of CAD
30-40% increased risk of CAD
Odds ratio

Heterozygous: 1.3-1.5

Homozygous: 1.8-2.1
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
KIF6 Variant and CAD

CARE: Cholesterol and Recurrent Events

WOSCOPS: West of Scotland Coronary Prevention Study
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
Risk of CAD in CARE Placebo Arm
KIF6 Variant and Traditional Risk Factors
Smoking
Diabetes
KIF6 Variant
Age ≥ 55
HDL-C < 40
Hypertension
LDL-C ≥130
0.5
1
1.5
2
2.5 3
Adjusted Hazard Ratio
 Magnitude of risk predicted by KIF6 variant was:
 Similar to that of traditional risk factors
 Independent of traditional risk factors
Risk of CAD in WOSCOPS Placebo Arm
KIF6 Variant and Traditional Risk Factors
Diabetes
KIF6 Variant
HDL-C < 40
LDL-C ≥ 189*
Hypertension
0.5
1
1.5
2
2.5 3
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
KIF6 Variant is Associated with CAD
5 Prospective Studies
CARE
Untreated patients
WOSCOPS
CHS
Some treatment
ARIC
WHS
0.5
1
1.5
2
2.5
Hazard Ratio*


KIF6 variant predicts risk of CAD
Up to 55% increased risk in untreated populations
*Adjusted for traditional risk factors
Recent Developments in CV Genetics

Mendelian genetics

Autosomal Dominant Hypercholesterolemia


Familial Thoracic Aortic Aneurysm and Dissection
syndromes:



LDLR, APOB, PCSK9
FBN1, TGFBR1, TGFBR2, ACTA2, MYH11, NOTCH
Genetic markers for CAD

Chromosome 9p21/ANRIL

KIF6
Pharmacogenetics

Warfarin sensitivity

Statin efficacy

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
Prevention of CHD Events by Statins
Carriers of the KIF6 Variant Benefit the Most
WOSCOPS
PROVE IT
Pravastatin 40 mg
Atorvastatin 80 mg
All
Carriers
Noncarriers
All
NonCarriers carriers
0
2
4
Absolute Risk
Reduction
(%)
6
8
10
12
14
3.5%
5.5%
0.1%
6.2%
10.0%
0.8%

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
(p=0.003 for WOSCOPS and 0.018 for PROVE IT)
Statin Intensity and CHD Event Reduction
According to KIF6 719Arg Carrier Status
Noncarriers
Death or major CV events
KIF6 Carriers
40
40
Pravastatin
30
30
p≤0.001
20
20
Atorvastatin
10
Pravastatin
10
0
0
0
3
6
9
12
15
18
21
Months of follow-up
24
27
30
0
3
6
Inclusion
 Hospit
 Total c
 Stabili
P=1.0
Major Exc
 Co-mo
Atorvastatin
 Curren
 Need f
9
12
15
18
21
24 CABG
27
30

Months of follow-up  Liver d
 Strong
 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
KIF6 Summary
Risk 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
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
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?
Warfarin


Widely prescribed anticoagulant
12th most commonly prescribed drug
Annual Number of Outpatient Warfarin
Prescriptions, 1998-2004
35000000
45% increase from
1998 to 2004
30000000
Annual
Number of
Prescriptions
25000000
20000000
15000000
10000000
5000000
0
1998
1999
2000
2001
2002
2003
2004
Year
Wysowski, D. K. et al. Arch Intern Med 2007;167:1414-1419.
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 > 4.0
Most likely to occur within first few weeks of initiating
treatment
Subtherapeutic INR: thrombotic complications
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
Frequency
15
20
Budnitz et al, Ann Intern Med 2007;147:755-765.
Wide Range in Warfarin Dose Requirements
Variability in Warfarin Dose Requirements, N = 2305
800
780
Number of Patients
750
700
600
500
Warfarin
400Sensitivity
390
300
200
Warfarin Resistance
180
180
100
70
30
0
25
Daily Dose, mg
James AH. J Clin Path 1992;45:704-06
12
Sources of variability in Warfarin dosing
VKORC1 25%
Other 28%
CYP2C9 17%
Drugs 12%
CYP4F2 2%
Age 7%
Weight 9%
Warfarin Pharmacology

Racemic mixture of R- and S-enantiomers


S-Warfarin approx. 7-10X as potent as RMajority 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
Pharmacodynamics
Pharmacokinetics
(hepatocyte)
R-Warfarin S-Warfarin
CYP2C19
CYP3A4
CYP1A1
CYP1A2
CYP2C8
CYP2C9
(hepatocyte)
R-Warfarin S-Warfarin
CYP2C9
VKOR
inactive
4-hydroxywarfarin
6-hydroxywarfarin
6-hydroxywarfarin
7-hydroxywarfarin
Vitamin K
(oxidized)
Vitamin K
(reduced)
7-hydroxywarfarin
8-hydroxywarfarin
GGCX
10-hydroxywarfarin
Elimination
Inactive Vitamin Kdependent clotting
factors
Active Vitamin Kdependent clotting
factors
Warfarin PGx

CYP2C9

CYP2C9*2 variant



CYP2C9*3 variant



Arg144Cys
30% decrease in enzymatic activity
Ile359Leu
70%-95% decreased enzymatic activity
VKORC1


Promoter SNP -1639G>A
44% decrease in promoter activity
Allele Frequencies
CYP2C9 CYP2C9 VKORC1
*2
*3
-1639G>A
Caucasian
8-13%
6-10%
42%
Asian
2-5%
<1%
89%
African American
<1%
1-4%
8%
Genotype-guided Warfarin Dosing
CYP2C9 Genotype
Rapid
Intermediate
*1/*1
*1/*2
*1/*3
*2/*2
*2/*3
*3/*3
Low Sensitivity
(GG)
140%
113%
94%
92%
75%
63%
Med Sensitivity
(AG)
100%
81%
67%
67%
54%
46%
High Sensitivity
(AA)
73%
58%
48%
48%
40%
33%
VKORC1
Genotype
Slow
McClain, et al., Gen Med, 2008, 10(2): 89-98
www.warfarindosing.org
Very Slow
Can Pharmacogenomics Assist
Warfarin Dosing?
Warfarin Dosing
185 Patients
Initiation
Stabilization
PGxInformed
(CYP2C9 only)
Algorithm
Guided
(Ageno et al, 2000)
Caraco, Clin Pharm Ther, 2008
CYP2C9 Genotype-guided Warfarin
Prescribing Efficacy and Safety
Time to First Therapeutic INR
P<0.001*
Proportion of patients
with INR>2
1.0
0.8
PGx-informed
Controls
0.6
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
CYP2C9 Genotype-guided Warfarin
Prescribing Efficacy and Safety
Proportion of patients
at stable anticoagulation
Time to First Stable Anticoagulation
1.0
P<0.001*
0.8
PGx-informed
Controls
0.6
0.4
*Time to stable anticoagulation 18 days earlier
(22+7 vs 40+21 days)
0.2
0.0
0
25
50
Days
75
100
125
Caraco, Clin Pharm Ther, 2008
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
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
Warfarin PGx: Summary

Some studies suggest that warfarin PGx testing is beneficial



Large multi-center studies currently underway




Reduced time to stable INR
Reduced adverse events
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
Recent Developments in CV Genetics

Mendelian genetics

Autosomal Dominant Hypercholesterolemia


Familial Thoracic Aortic Aneurysm and Dissection
syndromes:


FBN1, TGFBR1, TGFBR2, ACTA2, MYH11, NOTCH
Genetic markers for CAD


LDLR, APOB, PCSK9
Multiple genes contribute to small percentage of phenotype

Chromosome 9p21/ANRIL

KIF6
Pharmacogenetics

Controversial, but gathering speed

Warfarin sensitivity

Statin efficacy
Thank You!!