Prevalence of CYP2C9 and VKORC1 mutation in patients with valvular heart disease in northern Thailand.

Abstract

Warfarin has been widely used for the prevention and treatment of thromboembolism. Warfarin therapy depends on interaction between physiological, environmental, and genetic factors. Vitamin K epoxide reductase (VKORC1) and cytochrome P450 2C9 (CYP2C9) enzyme conjointly determine the warfarin maintenance dose. The prevalence of CYP2C9 and VKORC1 variants varies among ethnic groups. The purpose of the present study was to investigate the prevalence of CYP2C and VKORC1 in the Northern Thai population.
Patients with valvular heart disease who regularly took a steady maintenance warfarin dose for at least one month were recruited into the present study. Patients who had taken amiodarone or an anti-inflammatory drug were excluded Clinical data were obtained from medical records. Five milliliters of whole blood was drawn from each patient for gene analysis and prothrombin time with international normalized ratio (INR) measurement.
From 242 patients, CYP2C9 *1/*1 was found in 230 patients (95%) and CYP2C9 *1/*3 was found in 12 patients (5%). Neither mutant CYP2C9*2 allele nor individuals homozygous for CYP2C9*3 were observed. Regarding VKORC1, haplotype AB was found in 83 patients (34.3%) and haplotype AA was found in 154 patients (63.6%). Haplotype BB (wild type) was found in five patients (2.1%).
The prevalence of CYP2C9 *1/*1 is high while the prevalence of CYP2C9*2 and CYP2C9*3 is very low. VKORC1 haplotype AA is the most common among the Northern Thai population. Further study regarding pharmacogenetic and non-genetic factors to develop warfarin-dosing algorithm is warranted

Full text

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J Med Assoc Thai Vol. 92 No. 12 20091597
Prevalence of CYP2C9 and VKORC1 Mutation in Patients
with Valvular Heart Disease in Northern Thailand
Srun Kuanprasert MD*,
Sakchai Dettrairat MSc**, Poonsub Palacajornsuk PhD***,
Warunee Kunachiwa MSc***, Arintaya Phrommintikul MD*
* Department of Internal Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
** Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences,
Chiang Mai University, Chiang Mai, Thailand
*** Division of Transfusion Sciences, Department of Medical Technology, Faculty of Associated Medical Sciences,
Chiang Mai University, Chiang Mai, Thailand
Background: Warfarin has been widely used for the prevention and treatment of thromboembolism. Warfarin
therapy depends on interaction between physiological, environmental, and genetic factors. Vitamin K
epoxide reductase (VKORC1) and cytochrome P450 2C9 (CYP2C9) enzyme conjointly determine the
warfarin maintenance dose. The prevalence of CYP2C9 and VKORC1 variants varies among ethnic groups.
The purpose of the present study was to investigate the prevalence of CYP2C and VKORC1 in the Northern
Thai population.
Material and Method: Patients with valvular heart disease who regularly took a steady maintenance
warfarin dose for at least one month were recruited into the present study. Patients who had taken amiodarone
or an anti-inflammatory drug were excluded. Clinical data were obtained from medical records. Five milliliters
of whole blood was drawn from each patient for gene analysis and prothrombin time with international
normalized ratio (INR) measurement.
Results: From 242 patients, CYP2C9 *1/*1 was found in 230 patients (95%) and CYP2C9 *1/*3 was found
in 12 patients (5%). Neither mutant CYP2C9*2 allele nor individuals homozygous for CYP2C9*3 were
observed. Regarding VKORC1, haplotype AB was found in 83 patients (34.3%) and haplotype AA was found
in 154 patients (63.6%). Haplotype BB (wild type) was found in five patients (2.1%).
Conclusion: The prevalence of CYP2C9 *1/*1 is high while the prevalence of CYP2C9*2 and CYP2C9*3 is
very low. VKORC1 haplotype AA is the most common among the Northern Thai population. Further study
regarding pharmacogenetic and non-genetic factors to develop warfarin-dosing algorithm is warranted.
Keywords: Pharmacogenomics, Polymorphisms, Warfarin, Anticoagulant
Correspondence to: Phrommintikul A, Department of Medicine,
Faculty of Medicine, Chiang Mai University, Chiang Mai 50210,
Thailand. Phone: 053-945-486, Fax: 053-945-486. E-mail:
apromint@mail.med.cmu.ac.th.
Warfarin has been widely used for the
prevention and treatment of thromboembolism.
Warfarin treatment is dependent upon interaction
between physiological, environmental, and genetic
factors(1). Several studies have suggested clinical
application of genotypic information on warfarin
dosing adjustment(2, 3). The US Food and Drug
Administration has recently revised product
labeling of warfarin on the potential effect of
genetic polymorphism on drug dosing(4). Recently, a
pharmacogenetic guidance warfarin dosing study
was shown to have better accuracy and efficiency of
warfarin dose initiation(5).
Vitamin K epoxide reductase (VKORC1) is the
key enzyme of the vitamin K cycle and the molecular
target of warfarin. Warfarin interferes with clotting
factor synthesis by inhibition of the C1 subunit of the
VKORC1 enzyme complex, which results in reduction
J Med Assoc Thai 2009; 92 (12): 1597-601
Full text. e-Journal: http://www.mat.or.th/journal

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1598J Med Assoc Thai Vol. 92 No. 12 2009
of vitamin K1 epoxide(6). The degree of suppression
depends upon the patient’s haplotype. There are
three haplotypes (AA, AB, and BB) of VKORC1, all of
which are associated with warfarin dose requirements.
Patients with haplotype B require a higher maintenance
dose of warfarin than those with haplotype A(7).
Warfarin is a mixture of S-warfarin and R-warfarin.
S-warfarin is 5 times more potent than R-warfarin.
Metabolism of S-warfarin occurs via the cytochrome
P450 2C9 (CYP2C9) enzyme, whereas R-warfarin is
metabolized by cytochrome P450 1A2 (CYP1A2) and
cytochrome P450 3A4 (CYP3A4). Three genetic
variants (*1, *2 and *3 allele) have been identified
in CYP2C9 and are associated with different enzyme
activity on warfarin metabolism. The *2 allele and
*3 allele variants can cause in vitro reduction in
enzymatic activity by 30 and 80% respectively and
had led to increased anticoagulant efficacy of
warfarin, thereby decreasing the required warfarin
dosage for therapeutic range maintenance(8). The
importance of CYP2C9 and VKORC1 to patient-
specific dose requirement has been established in
several studies(5,7,8).
The prevalence of CYP2C9 and VKORC1
variants correlates with membership of ethnic groups.
The purpose of the present study was to investigate
the prevalence of CYP2C and VKORC1 in Thai
patients with valvular heart disease requiring warfarin
treatment.
Material and Method
The study population was patients with
valvular heart disease from the cardiology outpatient
department of Maharaj Nakorn Chiang Mai hospital
who regularly took a steady maintenance warfarin dose
for at least one month. Patients who currently took oral
amiodarone or anti-inflammatory agents (i.e. NSAIDs
or steroid) at the time of the present study were
excluded. After obtaining written informed consent,
247 patients were recruited into the present study.
Clinical data were obtained from medical records.
Five milliliters of whole blood was drawn from each
patient for gene analysis and prothrombin time with
international normalized ratio (INR) measurement.
INR was measured by CoaguChek system (Roche
Diagnostic, USA).
DNA samples
DNA samples were extracted using the High
Pure PCR Template Preparation Kit protocol (Roche
Applied Science, Mannheim, Germany).
Preparation of the PCR Mix for CYP2C9*2 and
CYP2C9*3
Polymerase Chain Reaction (PCR) contained
5 ml of DNA (~100 ng), 4 ml of reagent mix containing
primers and probes of CYP2C9*2 and CYP2C9*3, 3 ml
of Roche master containing FastStart Taq DNA
polymerase, reaction buffer, MgCl2 and dNTPs, Final
volume was adjusted with sterile distilled water to 20 ml.
Control DNA samples (wild type, heterozygous and
mutant) for human CYP2C9*2 and CYP2C9*3 and
negative control sample were run with the patient
samples.
Preparation of the PCR Mix for VKORC1 C1173T
and VKORC1 G-1639A
PCR contained 5 ml of DNA (~100 ng), 4 ml
of reagent mix containing primers and probes of
VKORC1 C1173T and VKORC1 G-1639A and 3 ml of
Roche master containing FastStart Taq DNA
polymerase, reaction buffer, MgCl2 and dNTPs and
final volume was adjusted with sterile distilled
water up to 20 ml. Control DNA samples (wild type,
heterozygous and mutant) for human VKORC1 C1173T
and VKORC1 G-1639A and negative control samples
were run with the patient samples. PCR reactions
were performed in a Real-time PCR thermal cycler as
described above for CYP2C9 genotyping reactions.
Tm analysis
374 bp and a 180 bp fragments of the CYP2C9
gene, and 176 bp and a 289 bp fragments of the human
VKORC1 gene were amplified with specific primers.
Human CYP2C9*2 and VKORC1 C1173T were detected
with a SimpleProbe® (detected in channel 530) and
human CYP2C9*3 and VKORC1 G-1639A were analyzed
with LighCycler® Red 640 labeled hybridization probes
(detected in channel 640). Hybridization of the probes
to the target DNA resulted in fluorescence resonance
energy transfer (FRET) between two fluorophores.
During melting of the final product, the sequence
alteration was detected as a change in the melting
temperature (Tm) of the sensor probe. In the presence
of the wild type DNA, hybridization probes formed an
exact match with the target, resulting in a higher stability
of the complex and the highest melting peak
temperature. Whereas, in the presence of the mutant
DNA, hybridization probes formed a mismatch with
the target, resulting in a lower stability of the complex
and the lowest melting peak temperature. Therefore,
the heterozygous samples showed two distinct melting
peaks of wild type and mutant DNA. The melting peak

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J Med Assoc Thai Vol. 92 No. 12 20091599
temperature of CYP2C9*2, CYP2C9*3, VKORC1 1173
and VKORC1-1639 genotypes was analyzed using Tm
Calling mode of the LightCycler® Software 4.05.
Statistical analysis
Continuous variables with a Gaussian
distribution were expressed as the mean + SD and
compared by means of student t-test. Categorical
variables were expressed as percentages of the
study population and compared by the χ2 test. All
calculations were performed using SPSS software
version 14.0 statistical package (Chicago, IL). Statistical
significance was considered for p < 0.05.
Results
From 247 valvular heart patients, five patients
were excluded from the present study due to absent
fluorescence peak after gene amplification. The mean
age was 51 + 10 years old and 160 (66%) were female.
Ninety-one (37%) patients had a mechanical valve.
Among 242 patients, CYP2C9*1/*1 was found in 230
patients (95%) and CYP2C9*1/*3 was found in 12
patients (5%). Neither mutant CYP2C9*2 allele nor
individuals homozygous for CYP2C9*3 were found in
this study population (Table 1). Regarding VKORC1,
haplotype AB of VKORC1 was found in 83 patients
(34.3%) and haplotype AA was found in 154 patients
(63.6%). Haplotype BB (wild type) was found in five
patients (2.1%). The distribution of VKORC1 genotype
was similar among CYP2C9 genotype *1/*1 and *1*3.
Discussion
From this first pharmacogenetic study of
warfarin in Northern Thailand, the authors found a
similar prevalence of genetic variations of CYP2C9 in
this population compared to a Korean population(9).
Neither Mutant CYP2C9*2 allele nor homozygous
CYP2C9*3 were found in the present study, which is
consistent with a rare prevalence of these genotype in
the other studies of East Asian populations(10,11). The
prevalence of mutant CYP2C9*2 allele and homogenous
CYP2C9*3 were reported to be higher in Caucasians(2).
The ethnic difference of CYP2C9 variant frequency
has been demonstrated in various studies(9,12) where
the variant of CYP2C9 in Asians was significantly
lower than in Westerners. Furthermore, the gene-dose
effect of defective CYP2C alleles on the in vivo CYP2C9
activity was different among Asian and Westerners.
Takahashi and colleague demonstrated that the
unbound clearance of warfarin was significantly
lower in Japanese patients with homozygous and
heterozygous of CYP2C9*3 than patients with
homozygous CYP2C9*1(13) while there was no
difference among Caucasian patients with CYP2C9*1
homozygous and patients with heterozygous
CYP2C9*2 or CYP2C9*3 genotypes. The difference
of warfarin clearance in genotype-matched has
shown the significantly higher warfarin clearance of
Japanese than Caucasian patients with homozygous
CYP2C9*1(14).
The prevalence of VKORC1 haplotype AA
and AB in the presented population was 97.9%,
which was higher than the prevalence reported in the
Westerners population(7,15). Similarly, Reider et al
demonstrated that Asian-Americans had a significantly
higher proportion of group A haplotype than in
European-Americans(7).
Although the pharmacogenomic effects of
CYP2C9 and VKORC1 on warfarin dosage have been
addressed in several studies(3,7,16,17), the non-genetic
factors also play significant roles on warfarin dose.
These factors include age, gender, body size, diet,
and the presence of co-morbidities and medications,
which can account for variation of warfarin dose,
VKORC1 haplotype CYP2C (genotype)
Rapid metabolizerIntermediate metabolizer Poor metabolizer
*1/*1 *1/*2*1/*3 *2/*2*2/*3*3/*3
High (BB)
Medium (AB)
Low (AA)
2%
33%
60%
-
-
-
0.5%
1.0%
3.5%
-
-
-
-
-
-
-
-
-
Data presented in number (%) of the patients
Table 1. The allelic frequency distribution of variant CYP 2C9 and VKORC1 genotype

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1600J Med Assoc Thai Vol. 92 No. 12 2009
ranging from 4.7 to 14.6%(17). Dosing algorithm
using both pharmacogenetic and non-genetic factors
could account for 54.2-62% of the warfarin dose
variability(2,19,20). Furthermore, time to steady state of
warfarin varies by genotype; 3-5 days for CYP2C9*1/
*1 to 12-15 days for CYP2C9*1/*3. This variation
knowledge may guide the frequency of dose adjust-
ment.
Until now, the selection of warfarin dosage
has relied upon an iterative change in daily dosage
until the desired therapeutic effect is achieved.
During this process of iteration towards an optimal
INR the patients are still at risk from thromboembolism
and bleeding, some of which could be fatal. Warfarin
dosing guided by pharmacogenetics and non-pharma-
cogenetic factors could be beneficial for a more
proper starting dose and this could lead to a faster
achievement of the best maintenance dose to minimize
the patient risk.
The present study has some limitations. It
recruited only patients with valvular heart disease,
mostly from Northern Thailand. Therefore, the
results may not be applied to other population. The
present study regarding the genetic, clinical, and
environmental factors that may affect warfarin dose in
larger populations to develop the dose algorithm for
warfarin are warranted.
Acknowledgement
The authors wish to thank Prof. Apichard
Sukonthasarn for helping in manuscript preparation.
We also wish to thank Mrs. Pornapa Thitasarn for
research assisting.
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ความชุกของ CYP2C9 และ VKORC1 mutation ในผู้ป่วยโรคลิ้นหัวใจในภาคเหนือของประเทศไทย
ศรัณย์ ควรประเสริฐ, ศักดิ์ชัย เดชตรัยรัตน, พูนทรัพย์ ผลาขจรศักดิ์, วารุณี คำสายใย, อรินทยา พรหมินธิกุล
ภูมิหลัง: ยาวาร์ฟาริน เป็นยาที่ใช้อย่างแพร่หลายในการป้องกันและรักษาภาวะลิ่มเลือดอุดตัน โดยการใช้ยาวาร์ฟาริน
ต้องคำนึงถึงปัจจัยด้านพยาธิสภาพสิ่งแวดล้อม และ พันธุกรรม เอนไซม์ Vitamin K epoxide reductase (VKORC1)
และ cytochrome P4502C9 (CYP2C9) มีความสำคัญต่อขนาดยาที่ใช้ความชุกของจีน CYP2C9 และ VKORC1
มีความแตกต่างกันไปในแต่ละเชื้อชาติ การศึกษานี้เพื่อหาความชุกของจีนทั้ง 2 ชนิด ในประชากรภาคเหนือของ
ประเทศไทยรวมถึงผลของจีนต่อขนาดของยาวาร์ฟาริน
วัสดุและวิธีการ: ผู้ป่วยโรคลิ้นหัวใจที่ได้รับยาวาร์ฟารินในขนาดคงที่อย่างน้อย 1 เดือน จะได้รับเข้าร่วมการศึกษา
ผู้ที่ได้รับ amiodarone และ ยาในกลุ่ม anti-inflammatory drug จะถูกคัดออก ข้อมูลทางคลินิกได้จากทะเบียนประวัติ
ผู้ป่วยจะได้รับการตรวจเลือดสำหรับวิเคราะห์จีน และ prothrombin time ร่วมกัย INR
ผลการศึกษา: จากผู้ป่วย 242 รายพบว่ามี CYP2C9 *1/*1 230 ราย (ร้อยละ 95) และพบ CYP2C9 *1/*3 12 ราย
(ร้อยละ 5) ไม่พบ mutant CYP2C9*2 หรือ hemozygous CYP2C9*3 ในแง่ของ VKORC1 พบ haplotype AB
ในผู้ป่วย 83 ราย (ร้อยละ 34.3) haplotype AA 154 ราย (ร้อยละ 63.6) และ haplotype BB 5 ราย (ร้อยละ 2.1)
สรุป: ความชุกของจีน CYP2C9 *1/*1 สูง แต่ CYP2C9 *2 และ CYP2C9 *3 มีความชุกต่ำความชุกของ VKCRC1
นั้นพบ haplotype AA มากที่สุด การศึกษาการใช้ปัจจัยพันธุกรรมและปัจจัยอื่น ๆ เพื่อสร้างวิธีการปรับขนาด
ของยาวาร์ฟารินควรจะจัดเป็นการศึกษาขั้นต่อไป