Higashi MK, Veenstra DL, Kondo LM, Wittkowsky AK, Srinouanprachanh SL, Farin FM, Rettie AE. Association Between CYP2C9 Genetic Variants and Anticoagulation-Related Outcomes During Warfarin Therapy. JAMA. 2002;287(13):1690-1698. doi:10.1001/jama.287.13.1690
Author Affiliations: Pharmaceutical Outcomes Research and Policy Program (Drs Higashi and Veenstra), Institute for Public Health Genetics (Dr Veenstra), and Department of Pharmacy Services (Drs Kondo and Wittkowsky), University of Washington Medical Center; Molecular Biomarkers Laboratory, Center for Ecogenetics and Environmental Health (Ms Srinouanprachanh and Dr Farin); and Department of Medicinal Chemistry (Dr Rettie), University of Washington, Seattle.
Context Warfarin is a commonly used anticoagulant that requires careful clinical
management to balance the risks of overanticoagulation and bleeding with those
of underanticoagulation and clotting. The principal enzyme involved in warfarin
metabolism is CYP2C9, and 2 relatively common variant forms with reduced activity
have been identified, CYP2C9*2 and CYP2C9*3. Patients with these genetic variants have been shown to require
lower maintenance doses of warfarin, but a direct association between CYP2C9
genotype and anticoagulation status or bleeding risk has not been established.
Objective To determine if CYP2C9*2 and CYP2C9*3 variants are associated with overanticoagulation and bleeding
events during warfarin therapy.
Design and Setting Retrospective cohort study conducted at 2 anticoagulation clinics based
in Seattle, Wash.
Participants Two hundred patients receiving long-term warfarin therapy for various
indications during April 3, 1990, to May 31, 2001. Only patients with a complete
history of warfarin exposure were included.
Main Outcome Measures Anticoagulation status, measured by time to therapeutic international
normalized ratio (INR), rate of above-range INRs, and time to stable warfarin
dosing; and time to serious or life-threatening bleeding events.
Results Among 185 patients with analyzable data, 58 (31.4%) had at least 1 variant
CYP2C9 allele and 127 (68.6%) had the wild-type (*1/*1)
genotype. Mean maintenance dose varied significantly among the 6 genotype
groups (*1/*1 [n = 127], *1/*2 [n = 28], *1/*3 [n = 18], *2/*2 [n = 4], *2/*3 [n = 3], *3/*3 [n = 5]) (by Kruskall-Wallis test, χ25
= 37.348; P<.001). Compared with patients with
the wild-type genotype, patients with at least 1 variant allele had an increased
risk of above-range INRs (hazard ratio [HR], 1.40; 95% confidence interval
[CI], 1.03-1.90). The variant group also required more time to achieve stable
dosing (HR, 0.65; 95% CI, 0.45-0.94), with a median difference of 95 days
(P = .004). In addition, although numbers were small
for some genotypes, representing potentially unstable estimates, patients
with a variant genotype had a significantly increased risk of a serious or
life-threatening bleeding event (HR, 2.39; 95% CI, 1.18-4.86).
Conclusions The results of our study suggest that the CYP2C9*2 and CYP2C9*3 polymorphisms are associated
with an increased risk of overanticoagulation and of bleeding events among
patients in a warfarin anticoagulation clinic setting, although small numbers
in some cases would suggest the need for caution in interpretation. Screening
for CYP2C9 variants may allow clinicians to develop dosing protocols and surveillance
techniques to reduce the risk of adverse drug reactions in patients receiving
Warfarin is an anticoagulant agent used for the prevention of thromboembolic
events in patients with chronic conditions such as atrial fibrillation, and
is prescribed to more than 1 million patients in the United States annually.1 Because warfarin has a narrow therapeutic range and
may increase the risk of bleeding events, therapy is individualized by monitoring
the prothrombin time international normalized ratio (INR), a measure of anticoagulation
status. The management of warfarin therapy is challenging because of variability
in patient response due to a multitude of factors including drug, diet, and
disease-state interactions.1 In addition, genetic
variation of the hepatic microsomal enzyme CYP2C9, the activity of which constitutes
the primary pathway for the metabolism of S-warfarin, may lead to significant
differences in patient response to warfarin.
Two common variant alleles (polymorphisms) of CYP2C9 have been identified.2- 10
The *2 allele (R144C) and
the *3 allele (I359L) cause
decreased enzymatic activity of 30% and 80%, respectively.11,12
The frequencies of the *2 and *3 alleles have been estimated at 11% and 7%, respectively.13
Several studies have evaluated the association of these polymorphisms
with clinical phenotypes in patients treated with warfarin. Aithal et al14 compared individuals having at least 1 variant CYP2C9
allele with individuals having the wild-type (*1/*1)
genotype. They reported significant associations between variant CYP2C9 genotype
and low-dose requirements for warfarin, and between low-dose warfarin requirements
(but not genotype) and major but not minor bleeding events. Taube et al15 also found a significantly lower maintenance dose
in patients with a variant allele vs patients having the wild-type genotype,
but did not find evidence of an association between genotype and anticoagulation
status (as assessed via INR). Loebstein et al,16
using multiple regression analysis, reported that CYP2C9 genotype was independently
associated with warfarin maintenance doses. Thus, although the *2 and *3 polymorphisms have been associated
with lower warfarin dose requirements, a direct association with anticoagulation
status or bleeding events has not been reported.
The current study differs from prior studies in that it was designed
to evaluate the association between variant CYP2C9 alleles and clinical outcomes
such as anticoagulation status and bleeding events. We conducted a retrospective
cohort study of patients managed in a university hospital–based anticoagulation
clinic. In the study, strict inclusion criteria and direct genetic sequencing
were used, and adjustments made for potential confounders. Although medical
management practices are crucial in determining anticoagulation state, we
hypothesized that variant CYP2C9 alleles would also play a role and result
in a longer time to therapeutic INR, a higher rate of out-of-range INRs, a
longer time to achieve stable dosing, and a higher risk of serious or life-threatening
This study was approved by the Human Subjects Review Committee at the
University of Washington. The study was conducted at the pharmacist-run anticoagulation
clinics affiliated with the University of Washington Medical Center (UWMC),
Seattle. Five hundred twenty-six patients attend these clinics at regular
intervals of 2 to 6 weeks. The mean age of patients in the clinic is 60 years,
54% are male, and the distribution of ancestry is 91.0% European, 4% Asian,
3% African, and 2% Hispanic.
Patient care is managed via a physician-approved prescriptive authority
protocol that provides a standardized approach to dosing adjustments based
on INR results, management of overanticoagulation and underanticoagulation,
and frequency of follow-up. For patients in whom warfarin therapy was initiated
after enrollment in clinic, dosing was adjusted based on a protocol developed
by Harrison and colleagues.17 Dosing adjustments
during maintenance therapy were made according to previously published protocols.18 Once receiving maintenance therapy, patients requiring
dosage adjustments were reevaluated within a maximum of 2 weeks. Routine follow-up
of medically stable and reliable patients occurred every 4 to 6 weeks.
Retrospective clinical data were abstracted from medical charts, anticoagulation
records, and prescription records. Genotype data were collected from patients
currently attending the anticoagulation clinics. The investigators were blinded
to genotype information, as DNA analysis was not conducted until data abstraction
Power calculations were performed using Egret Sample Size Module v1.01.03
(Statistics and Epidemiology Research Corp, Seattle, Wash). Analysis of the
UWMC clinic data revealed an above-range INR rate of 0.32 per patient-year
and mean follow-up time of 2 years per patient. A study size of 200 patients
was estimated to have 80% power to detect an above-range INR hazard of 2.0
at an α of .05 (2-tailed).
Eligibility criteria included patients with a confirmed index date of
first warfarin exposure, patients currently undergoing anticoagulation therapy,
and patients older than 18 years. Our goal was to determine the genotype of
patients with accurate warfarin initiation information in order to include
the effect of CYP2C9 genotype following the postinitiation period when the
risk of overanticoagulation is highest and the subsequent bleeding risk is
also highest.19,20 Patients attending
the clinic at some point during April 3, 1990, to May 31, 2001, were eligible
for the study.
Exclusion criteria were (1) patients of Asian or African descent (n
= 36), (2) patients managed via telephone rather than in person (n = 185),
(3) inability to obtain verbal and written consent (non–English-speaking)
(n = 5), (4) inability to draw blood (n = 3), and (5) inability to ascertain
the index date (n = 11). We excluded patients of known Asian and African descent
because CYP2C9 allele frequencies are associated with ethnicity and the UWMC
anticoagulation clinic population, being predominantly (91.0%) white, could
not provide a sample of sufficient size to control for potential confounding
in a regression model.3,5,21- 23
The study base was restricted to patients currently attending the local anticoagulation
clinics because a blood sample is required for the genotyping portion of the
analysis. The study base was further restricted to patients who were at least
18 years of age at the index date due to age criteria specified by the Human
Subjects Review Committee. There was no restriction on the amount of follow-up
time required for each patient. Patients were screened to determine their
eligibility and enrolled if informed consent was obtained. Two hundred eighty-six
patients (54.4%) were determined eligible to participate.
Data collection consisted of a review of inpatient and outpatient medical
records and a venous blood sample from consenting participants. Two trained
abstractors collected data using standardized abstract forms.
We used the UWMC anticoagulation database to obtain information on INR
measurements, warfarin daily dose, prescription drugs, over-the-counter drugs,
and vitamin use. This database has been used since 1994 by all UWMC-affiliated
clinics to maintain records for patients receiving long-term warfarin therapy.
Data for patients before 1994 (n = 4) were obtained from the paper chart.
We used the electronic medical records database MINDscape (University
of Washington, Seattle) to obtain information on bleeding events, comorbid
conditions, and demographic variables. The MINDscape database has been used
by the UWMC and affiliated clinics to record medical records electronically
since 1994. Information obtained from this database was supplemented and confirmed
by reviewing the paper medical chart. For bleeding events, the concordance
between these 2 data sources was 94%.
Blood (5 mL) was drawn in a sodium citrate vacutainer during a regularly
scheduled blood draw. Genomic DNA isolated from whole blood using the Qiagen
blood kits (Qiagen Inc, Chatsworth, Calif) was amplified using intron-specific
primers for exon 3 and exon 7 of the gene CYP2C9,
yielding 490-base pair (bp) and 284-bp fragments, respectively (exon 3 [*2] sense: 5′-GCTGCA TGGATATGAAGCA-3′, antisense:
5′CCAAGAATGTCAGTAGAGAAGATAG-3′; exon 7 (*3)
sense: 5′-CTCCTTTTCCATCAGTTTTTACT-3′, antisense: 5′-GATACTATGAATTTGGGACTTC-3′).
Both exons were amplified by polymerase chain reaction (PCR) and subsequently
sequenced using the ABI Prism 377 and BigDye dye terminator cycle sequencing
(Applied Biosystems, Foster City, Calif). The PCR for the Arg144 to Cys144
variant in exon 3 was performed in 10× PCR buffer (Qiagen, Inc), 3.0
mM of MgCl2, 0.4 mM of each primer, 0.2 mM of dNTPs, 0.15 U of
HotStarTaq DNA polymerase (Qiagen Inc), and approximately 100 ng of genomic
DNA. The PCR for the Ile359 to Leu359 variant in exon 7 was performed in 10×
PCR buffer (Life Technologies, Rockville, Md), 1.6 mM of MgCl2,
0.24 mM of each primer, 0.2 mM of dNTPs, 0.02 U of Taq polymerase (Life Technologies),
and approximately 100 ng of genomic DNA. The BigDye-based reactions were performed
according to the manufacturer's protocol.
All CYP2C9*2 and CYP2C9*3 homozygotes detected during primary sequencing were confirmed by sequencing
the reverse complementary strand using a second PCR reaction. In addition,
randomly selected heterozygote variants (n = 19) and wild-type homozygotes
(n = 34) were confirmed using the same method.
The primary end point of this study was anticoagulation status, as measured
by INR in 3 ways: time to therapeutic INR, rate of above-range INRs, and time
to stable warfarin dosing (defined as 3 consecutive clinic visits for which
INR measurements were within therapeutic range for the same mean daily dose).14 Once a patient achieved stable dosing, we recorded
the maintenance dose in order to compare mean maintenance doses among the
various CYP2C9 genotypes. Therapeutic INR was defined as the first INR measured
within the optimal therapeutic range for a given indication. If the target
therapeutic range was 2.0 to 3.0,1 then INRs
between 2.0 and 3.0 were defined as within range. Above-range INRs were defined
as measurements of 4.0 or greater.14 Twelve
patients receiving warfarin therapy for prosthetic valve replacement were
anticoagulated at a higher target range (2.5-3.5).1
Consequently, these patients required an above-range INR definition of 4.5
or greater to account for their higher baseline level of anticoagulation.24 This approach was used to try to ensure that the
12 patients did not contribute a disproportionate number of events to the
Although INR values between 3.0 and 4.0 (3.5-4.5 for patients with prosthetic
valves) were not defined as "above-range," this does not mean that clinicians
necessarily consider these levels of anticoagulation "normal." However, in
given instances, these levels could be regarded as acceptable, factoring in
perceived thromboembolic risk, diet or drug changes, or compliance.
We recorded every INR value for all patient visits. In this data set,
there were 5 415 INR measurements obtained for patients with a 2.0 to
3.0 target range. For these patients, there were 676 observations (12.5%)
occurring between 3.01 and 3.99. For the 12 patients with prosthetic valves,
there were 850 INR measurements obtained. For these patients, there were 180
observations (21.2%) between 3.51 and 4.49.
The minimum cutoff for above-range INR was considered to be 4.0 because
INR measurements above 4.0 are less likely to be misclassified as above-range
when compared with measurements below 4.0.25
Also, INR values between 3.0 and 4.0 are not considered to be strong predictors
of bleeding risk.19,26 Levels
of 4.0 or greater have been previously used14
to define "above range."
The above-range INR model was modified to include recurrent events.
All 185 patients (see below) contributed exposure time to this analysis. The
sample size and exposure time would not change regardless of how above-range
INR is defined; it would only change the number of above-range events contributed
by each patient. A higher cutoff would result in fewer events contributed
by each patient and less power to detect an association. For this study, an
INR of 4.0 was considered to be the minimum clinically relevant event (see
The secondary end point was time until first serious or life-threatening
bleeding event. We used the criteria of Fihn et al20
to classify bleeding episodes as serious (requiring treatment or medical evaluation)
or as life threatening. Examples of serious bleeding included overt gastrointestinal
bleeding, occult gastrointestinal bleeding if endoscopic or radiographic studies
were performed, gross hematuria that prompted cystoscopy or intravenous urography
or lasted more than 2 days, and hemoptysis. Episodes involving blood transfusions
of 2 units or more were classified as serious bleeding events. We defined
life-threatening bleeding events as those leading to cardiopulmonary arrest,
surgical or angiographic intervention, or irreversible sequelae such as myocardial
infarction, neurologic deficit consequent to intracerebral hemorrhage, or
massive hemothorax. Bleeding was also considered to be life threatening if
it resulted in 2 of the following consequences: (1) loss of 3 or more units
of blood, (2) systolic hypotension (<90 mm Hg), or (3) critical anemia
(hematocrit of 20% or less).
The associations between CYP2C9 genotype and the primary and secondary
end points were evaluated using survival analysis techniques. Patients were
divided into 2 groups based on genotype: wild type (CYP2C9*1/*1 homozygotes) and variant (1 or more mutant alleles). For each analysis,
a hazard ratio (HR) and 95% CI comparing variant and wild-type genotype groups
were computed. We used Cox proportional hazards models to adjust for the potential
confounding effect of sex, age, warfarin indication, comorbid conditions,
prescription medications, and over-the-counter products (Table 1) and to increase the precision of the model.27,28
Any prescription medicine metabolized by CYP2C9 and used by at least 5% of
this clinic population was considered in the analysis. Covariates were added
to the model 1 at a time to assess potential confounding effects on the variant
genotype HR. A covariate was defined to have an important effect on the HR
if the HR changed by more than 5% upon inclusion of the covariate in the model.
Duration of warfarin therapy was measured in days. Patients were followed
up from the index date of first warfarin exposure until either the date of
an event observation or the end-of-study date (May 31, 2001), when all data
were subject to administrative right-censoring. Because this study required
a blood sample at enrollment followed by a retrospective chart review, no
patients were withdrawn or lost to follow-up.
To account for changes in the prescribed dose of warfarin, we programmed
mean daily dose as a time-varying covariate. When there was a change in warfarin
dose or a new INR value, we updated the regression model's covariates accordingly.
Consequently, the regression model always used patients' most recent mean
dose to adjust the HR for genotype.
Individuals could contribute more than 1 event to the above-range INR
outcome. We stratified the analysis on patients' history of above-range INR
values so that patients who experienced a recurrent above-range INR were always
compared with patients who had experienced the same number of prior above-range
INRs. In doing so, we also produced valid CIs for recurrent events that do
not violate the independence assumption.
In order to assess potential confounding, we fit each covariate (Table 1) to the model to determine changes
to the exposure coefficient. We also fit interaction terms to assess potential
effect modification of the genotype exposure by each covariate. We then conducted
model diagnostics and identified potentially influential cases, ie, the 12
patients having prosthetic valves with a higher target INR range (2.5-3.5).
An analysis was performed in which they were excluded to determine their effect
on the exposure HR. Statistical testing of the Schoenfeld residuals and graphical
assessment revealed no significant departure from the proportional hazards
Testing for deviation of genotype frequencies from Hardy-Weinberg equilibrium
was calculated by applying the Hardy-Weinberg model to our data.29
For comparing expected vs actual prevalence of each CYP2C9 genotype, because
of small samples for some genotypes, the χ2 goodness-of-fit
test could not be used. Thus, the likelihood ratio test was used, which is
appropriate for use with small sample sizes when testing for Hardy-Weinberg
equilibrium with rare allele frequencies.30,31
The likelihood ratio test statistic has an approximate χ2 distribution
with 3 df. With the general model, there are 6 parameters
(6 different genotype frequencies) and 1 constraint, giving 5 df. With the Hardy-Weinberg model, there are 3 parameters (3 allele
frequencies) and 1 constraint, giving 2 df. Thus,
the df for the χ2 test statistic is
equal to the dimensions of the null hypothesis of Hardy-Weinberg equilibrium
subtracted from the dimensions of the general model (5 − 2 = 3).
We calculated the HR for bleeding events during the first 3 months of
treatment and during the entire follow-up period. We did not calculate the
HR for only the maintenance phase because patients became "at risk" for a
bleeding event on the index date of first warfarin administration (time zero).
Using survival analysis techniques, we were able to examine the risk conferred
by genotype during initiation, and during initiation and maintenance phases
combined. However, we could not assess the risk during the maintenance phase
exclusively because this would involve recoding time zero at a later point,
which could introduce bias if persons with variant genotypes have more first
bleeding events during initiation and are thus censored, and would not allow
us to define the time until first bleeding event following warfarin administration.
An unadjusted incidence rate ratio was also calculated for bleeding events
by taking the ratio of the unadjusted bleeding rates in the wild-type and
variant genotype groups.
Of the 286 patients eligible for participation, 213 were randomly approached
regarding participation in this study. Thirteen patients declined to participate,
resulting in 200 enrolled patients. Two patients subsequently were excluded
due to African ancestry, 2 were excluded due to warfarin therapy prior to
their clinic initiation date, and 1 was excluded due to a liver transplant.
Ten samples were excluded because of difficulties in generating PCR products
for either exon 3 or exon 7. There were 185 patients available for analysis—127
with the wild-type genotype, and 58 with a variant genotype.
Table 1 summarizes the main
characteristics of the cohort. The mean age of all patients at the start of
therapy was 59.9 years, and 63.8% of the patients were men. White patients
comprised 100% of the sample with 3.8% of those classified as Hispanic ethnicity.
The majority of patients (51%) were receiving warfarin for atrial fibrillation.
The mean follow-up time was 818 days (2.24 years). Patients were seen a median
of 23 times over the follow-up period and for a median of 543 days. With the
exception of type 2 diabetes mellitus and use of vitamin E, there were no
significant differences in characteristics between patients with the wild-type
genotype (*1/*1) and patients with at least 1 variant
Table 2 summarizes the prevalence
of each genotype within the cohort. The allelic frequencies were similar to
those from a large study from Sweden.13 However,
the prevalence of the CYP2C9*3/*3 genotype was 4
times greater than expected based on Hardy-Weinberg calculations.29 In addition, we discovered that 2 of the CYP2C9*1/*1 patients had a novel polymorphism, a C/T at nucleotide
1003 (GenBank Accession NM 00771) resulting in an Arg→Trp substitution
at position 335 of exon 7. The mean daily dose of warfarin for these 2 patients
was 5.00 and 5.18 mg.
Table 3 summarizes the mean
maintenance dose of warfarin stratified by genotype. Maintenance dose was
significantly related to genotype. Furthermore, a possible gene-dose relationship
is suggested when comparing the *1/*1, *1/*2, and *1/*3 genotypes, with mean maintenance
doses of 5.63, 4.88, and 3.32 mg, respectively (Table 3).
The unadjusted incidence rate of bleeding complications (serious and
life threatening combined [n = 32]) was 7.72 per 100 patient-years (Table 4). For patients with a variant allele,
the rate of serious bleeding events (n = 14) was 10.92 per 100 patient-years,
and the rate of life-threatening bleeding events (n = 2) was 1.56 per 100
patient-years. For patients with the wild-type genotype, the rate of serious
(n = 14) and life-threatening (n = 2) bleeding was 4.89 and 0.70 per 100 patient-years,
respectively. Patients with the variant genotype experienced a significantly
higher bleeding rate. For variant vs wild-type genotype, the unadjusted incidence
rate ratio for serious and life-threatening bleeding combined was 2.23 (95%
Table 5 summarizes the main
findings of the study. For all 3 Cox models using INR measurements as end
points, the time-varying covariate, warfarin daily dose, was included in the
final model as a possible confounder. None of the covariates (sex, age, warfarin
indication, comorbid conditions, prescription medications, and over-the-counter
products) added in the stepwise model-fitting procedure produced remarkable
changes (ie, greater than 5%) to the HR estimate of bleeding risk. The exclusion
of 12 patients having prosthetic valves and a higher target INR range (2.5-3.5)
resulted in only trivial effects on the HR estimates. The Kaplan-Meier curves
for time to first therapeutic INR value did not differ significantly between
groups as evaluated by the log-rank test (P = .63)
(Figure 1, A). This finding did
not change after adjusting for other covariates with a Cox regression model
(Table 5). The curves showing
time until first above-range INR did not differ significantly between groups
(P = .10) (Figure
1, B). Similarly, the curves for a second event (P = .27) and third event (P = .38) did not
differ significantly (M.K.H., unpublished data, November 2001). However, when
we adjusted for warfarin daily dose in a Cox regression model, patients with
the variant genotype showed an increased risk of above-range INRs (HR, 1.40;
95% CI, 1.03-1.90). The variant group required more time to achieve stable
dosing compared with the wild-type group (P = .004)
(Figure 1, C), with a median difference
of 95 days. The slower rate of stabilization for the variant group was confirmed
in a Cox regression model (HR, 0.65; 95% CI, 0.45-0.94) (Table 5).
Patients with the variant genotype experienced a first bleeding event
sooner than patients with the wild-type genotype (P
= .01) (Figure 1, D). In the Cox
model, we examined the effect of variant genotype on bleeding risk during
the initiation phase of therapy by censoring the data at 90 days postinitiation.
Variant genotype increased the risk of bleeding during the initiation phase
(HR, 3.94; 95% CI, 1.29-12.06). When all the follow-up data were considered
(initiation and maintenance phase), variant genotype still conferred an increased
risk of bleeding (HR, 2.39; 95% CI, 1.18-4.86).
The results of this study suggest that CYP2C9 genotype is associated
with (1) warfarin maintenance dose, (2) time to stable warfarin dosing, (3)
rate of above-range INRs, and (4) bleeding events in patients taking warfarin.
During the initiation period of warfarin therapy, it appears that patients
with variant CYP2C9 alleles become overanticoagulated at a faster rate and
must undergo additional dose adjustments, thus translating into a longer time
until stable dosing is achieved. When these patients do become stable, their
daily maintenance dose of warfarin is significantly lower than that of patients
without genetic impairment of warfarin metabolism.
Patients with variant CYP2C9 alleles also experience a higher risk of
serious and major bleeding events, although numbers were small for some genotypes,
representing potentially unstable estimates, suggesting the need for caution
in interpretation. Fihn et al20 found that
recent initiation (first 90 days) of warfarin therapy compared with any time
thereafter was an independent predictor of first-episode serious bleeding,
with a relative risk of 5.9 (95% CI, 3.8-9.3). We found CYP2C9 genotype to
be an independent predictor of a first bleeding event during the initiation
phase of therapy (HR, 3.94; 95% CI, 1.29-12.06). This increased risk may be
caused by the administration of loading doses that are too high for patients
with genetic impairment of CYP2C9. Clinicians make rapid, downward dose adjustments
based on above-range INR values,18 so the increased
bleeding risk during initiation should rapidly dissipate. The effect of variant
genotype on bleeding holds over the entire course of therapy when comparing
rates (unadjusted incidence rate ratio, 2.23; 95% CI, 1.05-4.77) or using
a Cox proportional hazards model (HR, 2.39; 95% CI, 1.18-4.86); however, further
research is required to determine whether the variant genotype continues to
confer a bleeding risk once patients are stabilized with maintenance doses.
Serious and life-threatening bleeding episodes were combined as a single end
point due to the small number of observed events (n = 32). This study is unable
to assess the effect of genotype on life-threatening bleeding alone.
The incidences of serious (n = 28) and life-threatening (n = 4) bleeding
events in our study (6.8 per 100 person-years and 1.0 per 100 person-years,
respectively) are similar to those found in other studies (eg, 7.5 per 100
person-years and 1.1 per 100 person-years, respectively20),
suggesting the patients in our study were not at an unusually high risk of
bleeding events.32,33 These results
also suggest that the patient management practices in our clinic population
may be comparable to those of other anticoagulation clinics in the United
States, thus increasing the generalizability of our results.
The lack of a significant difference in the time to first therapeutic
INR is likely because the first therapeutic INR is often achieved within a
few days of warfarin initiation, may be transient, and is not reflective of
stable anticoagulation status. The median difference in time to stable warfarin
dosing (95 days) suggests that patients with the variant genotype require
more time to achieve stable anticoagulation status.
The frequencies of the *2 and *3 alleles (10.5% and 8.4%, respectively) were similar in our population
compared with previously published results. Yasar et al13
genotyped 430 Swedish volunteers and found *2 and *3 frequencies of 11% and 7%. However, we found 5 *3/*3 homozygotes in our population, or a prevalence of
2.7%. This result was 4 times higher than what we would expect by applying
the Hardy-Weinberg derivation to our data (Table 2).29 Two previous studies
in the United Kingdom did not detect any *3/*3 homozygotes
in patients receiving warfarin.14,15
Therefore, the current study is the first to analyze and describe the clinical
effect of this genotype in a stable, anticoagulated patient population.
There are several possible reasons why the prevalence of the *3/*3 genotype in our study is higher than expected. First, we used
direct sequencing of exons 3 and 7 to detect variant alleles. Prior studies
have relied on restriction enzyme analysis using NsiI
digestion of the exon 7 product to detect the 2C9*3
allele using a restriction site forced into the forward primer.13- 15
Direct sequencing is a more costly but more accurate approach to the detection
of these alleles, and previous studies may have underestimated the prevalence
of the *3 allele, resulting in misclassification
bias. Second, Aithal et al14 and Taube et al15 cited selection bias as a possible explanation for
why these UK-based studies failed to detect the *3/*3
homozygote in their warfarin clinic population. The suggestion was that individuals
homozygous for this allele have such a low warfarin dose requirement that
stabilization is unsuccessful and warfarin treatment is abandoned. Different
approaches to maintenance therapy between UK-based and US-based practices
could explain a stronger effect of selection bias in the UK-based populations.
Finally, the UWMC anticoagulation population may be enriched with patients
of the *3/*3 genotype. Patients who are difficult
to stabilize (and more likely to carry the *3/*3
genotype) could be preferentially referred to the anticoagulation clinic for
maintenance therapy, although physicians may simply refer patients on the
basis of general convenience. Review of the clinical records of the 5 *3/*3 homozygotes did not reveal comments or observations
to suggest preferential referral but this possibility cannot be excluded.
Adjusting for warfarin dose as a time-varying covariate did not materially
change the results of any Cox model except for the above-range INR end point.
Although warfarin dose is in the causal pathway of predicting INR values,
we controlled for it as a confounder because dose and genotype are associated,14,15 and because dose influences INR values
independent of genotype.19
As genomic information becomes more readily available, it is likely
that clinicians will need to consider new guidelines for patient management,
especially when administering drugs with narrow therapeutic indexes such as
warfarin.34,35 Variant CYP2C9
genotype could be considered a "sensitivity factor" for low-dose requirements
when initiating warfarin therapy, and patients with a variant genotype could
be considered candidates for increased surveillance for bleeding risk. As
oral versions of direct thrombin inhibitors become available,36
CYP2C9 genotyping could identify patients with impaired warfarin metabolism
as potential candidates for these newer alternate therapies.
There are several additional research questions that should be addressed.
Our study was conducted in a relatively homogeneous population, and the presence
of other functionally important polymorphisms in ethnically diverse groups
has not been well studied. For example, the *2 and *3 alleles are relatively rare in African Americans, but
approximately 3% carry the recently reported *5 polymorphism
(Asp360Glu).23 A novel and relatively common
polymorphism (L208V; homozygous prevalence = 19%) with a reduced warfarin
dose requirement has also recently been reported in Chinese patients.37 Given the ethnically diverse population in the United
States, additional studies are needed to evaluate the prevalence and clinical
importance of these polymorphisms. Establishing the effectiveness of CYP2C9
genetic testing to reduce adverse bleeding events may require a controlled
clinical trial in which some patients are randomized to receive a CYP2C9 genetic
test at the time of warfarin initiation. Ideally, this study would be large
enough to make meaningful comparisons of the risk-benefit ratio of warfarin
therapy in genetic subgroups (eg, *2/*2, *3/*3). The prognostic specificity of CYP2C9 genotype will also be
an important factor in its clinical usefulness. Significant additional medical
care resources could be consumed unnecessarily by patients with variant genotypes
despite low risk of bleeding. It will be necessary to demonstrate the value
of the genotype information as being useful in the maintenance phase. For
example, it is unclear whether those with variant genotypes would require
a different target INR, as there may be substantial overlap in dose requirements
with those not having the genetic variant. Finally, the cost-effectiveness
of genotyping patients in an anticoagulation clinic must be considered.
Understanding the pharmacogenetics that contribute to variability in
the warfarin dose-response relationship may help in tailoring drug therapy
to patients in a safe and effective manner. This study confirms the dose-genotype
association found in previous studies and is the first to describe the *3/*3 genotype effect in a stable anticoagulated population.
We found that patients with a variant genotype experienced a higher rate of
above-range INRs, less stability on maintenance therapy, and a higher risk
of serious or life-threatening bleeding events. The use of CYP2C9 testing
may be a method to identify high-risk patients who are candidates for lower
warfarin doses, more frequent monitoring, or treatment with alternate therapies
as they become available.