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Impact of CYP2C19 gene polymorphism on warfarin maintenance doses in patients with non-valvular atrial fibrillation
Impact of CYP2C19 gene polymorphism on warfarin maintenance doses in patients with non-valvular atrial fibrillation Hongshen Zhang, Kezhong Ma, Wenwei Liu, Feng Yang, Jianfei Liu, Hanyun Zhou PII: DOI: Reference:
S0378-1119(16)30509-1 doi: 10.1016/j.gene.2016.06.046 GENE 41416
To appear in:
Gene
Received date: Revised date: Accepted date:
15 April 2016 30 May 2016 22 June 2016
Please cite this article as: Zhang, Hongshen, Ma, Kezhong, Liu, Wenwei, Yang, Feng, Liu, Jianfei, Zhou, Hanyun, Impact of CYP2C19 gene polymorphism on warfarin maintenance doses in patients with non-valvular atrial fibrillation, Gene (2016), doi: 10.1016/j.gene.2016.06.046
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ACCEPTED MANUSCRIPT Impact of CYP2C19 gene polymorphism on warfarin maintenance doses in patients with non-valvular atrial fibrillation
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Running title: CYP2C19 gene polymorphism and warfarin doses
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Hongshen Zhang, Kezhong Ma, Wenwei Liu, Feng Yang, Jianfei Liu, Hanyun Zhou * Department of Cardiology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and
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Science, Xiangyang 441021, P.R. China
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* Correspondence to: Dr. Hanyun Zhou, Department of Cardiology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, No. 39 Jingzhou street, Xiangcheng
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Tel. /fax: +86-0710-3253216
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E-mail: [email protected]
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District, Xiangyang 441021, P.R. China
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ACCEPTED MANUSCRIPT Abstract Background: Pharmacogenetics has provided compelling evidence towards the influence of gene polymorphisms on warfarin therapies. This study aimed to determine the impact of CYP2C19 gene
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polymorphism on warfarin maintenance doses in patients with non-valvular atrial fibrillation for the
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progress in overcoming obstacles facing warfarin pharmacogenetics.
Methods: In this study, we utilized polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to investigate the distribution of CYP2C19*2 and *3 gene
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polymorphism in patients with NVAF. In order to exclude the interference of basic indexes, we compared the association between different genotypes and warfarin maintenance doses. And the
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comparisons among extensive metabolizer, intermediate metabolizer and poor metabolizer were performed.
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Results: CYP2C19 mutation accounted for 88.07% of in total NVAF patients, which was 7.38 times
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of CYP2C19*1/*1 (11.93%). No significant association was observed between different genotypes
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and basic indexes. The warfarin maintenance dose of patients with CYP2C19*1/*1 was significantly higher than those with other five genotypes (all P < 0.05). Besides, the warfarin maintenance doses of patients with CYP2C19*1/*2 and *1/*3 were remarkably higher than those
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with *2/*2 and *2/*3 (P < 0.05). The warfarin maintenance doses of patients with extensive metabolizer were dramatically higher than those with intermediate metabolizer and poor metabolizer (both P < 0.05), and also the patients with intermediate metabolizer had higher warfarin maintenance doses than those with poor metabolizer (P < 0.05). Conclusion: CYP2C19 gene polymorphism can affect maintenance dose of warfarin, with the amount of warfarin dose ranked among different genotypes as follow: CYP2C19*2/*2, CYP2C19*2/*3 and CYP2C19*3/*3 < CYP2C19*1/*2 and CYP2C19*1* < CYP2C19*1/*1.
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ACCEPTED MANUSCRIPT Abbreviations list Non-valvular atrial fibrillation (NVAF)
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International Normalized Ratio (INR)
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ethylenediaminetetraacetic acid (EDTA)
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Single Nucleotide Polymorphism (SNP)
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)
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bovine serum (BSA) ethidium bromide (EB)
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vitamin K epoxide reductase (VKOR)
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vitamin K epoxide reductase complex subunit 1 (VKORC1)
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Key words: CYP2C19; Gene polymorphism; Non-valvular atrial fibrillation; Warfarin; Extensive
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metabolizer; Intermediate metabolizer; Poor metabolizer
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ACCEPTED MANUSCRIPT Introduction Non-valvular atrial fibrillation (NVAF) is clinically defined as a usual heart arrhythmia, with 5% of incidence for people older than 65 years and 10% for people older than 75 years (Gattellari et
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al., 2008). The pathogenesis of NVAF is that due to loss of contraction function in heart atrium,
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thrombus caused by clogged blood in heart atrium can trigger fatal and disabling stroke which is the most serious complication of NVAF. NVAF has been implicated in approximately 15% of ischemic strokes (Hankey, 2001). Surprisingly, individuals with NVAF suffer from stroke at high risk up to
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18.2% annually (Gattellari et al., 2008). With warfarin as the mainstay of oral anticoagulation therapy with vitamin K antagonists, the risk of NVAF-associated stroke can be declined by 60% to
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70% per year (Kodani et al., 2015). To the best of our knowledge, warfarin maintenance dose is varied for patients with different gene background or gene polymorphisms (Yan et al., 2015).
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CYP2C19 gene is located at 10q24.1 ~ 24.3 in human and contains 9 exons and 5 introns, with
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a length of 1.473 kb encoding area. As a highly polymorphic gene, CYP2C19 encodes for a protein
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that takes charge of the metabolism of many clinical drugs, such as the antiplatelet therapeutic agent clopidogrel, anticonvulsants, as well as proton pump inhibitors (Gardiner and Begg, 2006). CYP2C19 constitutes 16% of the CYP2C subfamily and its genetic variation is commonly
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associated with therapeutic outcomes, adverse drug reactions along with treatment failures (Jin et al., 2016). Several CYP2C19 polymorphisms are shown to be related to reduction or even loss of enzyme activity. Most cases are attributed to either CYP2C19*2 with a mutation in exon 5 or CYP2C19*3 characterized by a point mutation in exon 4. However, CYP2C19*1 encodes normal enzyme activity of CYP2C19 (Peng et al., 2015). Interestingly, it was reported that the clopidogrel can be converted to an active metabolic product by CYP2C19, the loss of function alleles of which may be able to reduce clopidogrel responses in patients with coronary heart disease (Collet et al., 2009). Previous study also demonstrated that, among acute cerebral infarction patients treated by clopidogrel, those who have the loss-of-function CYP2C19*2 alleles have a lower platelet inhibition as well as a higher incidence of adverse cardiac events (Depta et al., 2015). Also, there are some 4
ACCEPTED MANUSCRIPT studies reporting the influence of CYP2C19 polymorphisms on the difference in warfarin maintenance dose, Chang M et al. demonstrated that CYP2C19 genotypes influenced the clearance of R-warfarin and contributed significantly to the variability in international normalized ratio/daily
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dose, indirectly indicating a clinical relevance of R-warfarin; Kim SY et al. reported that CYP2C19
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likely impacts R-warfarin metabolism and patient response to therapy, and CYP2C19 may
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contribute to S-warfarin metabolism in patients, especially when CYP2C9 activity is compromised due to drug interactions or genetic polymorphisms (Chang et al., 2015; Scordo et al., 2002).
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However, the influence of CYP2C19 polymorphisms on warfarin maintenance dose among NVAF patients was reported rarely, thus the present study is designed to investigate the association
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Materials and methods
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between the CYP2C19 gene polymorphisms and the clinical efficacy of warfarin for NVAF patients.
Subjects
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The study recruited 176 patients suffering from NVAF in between October 2011 and August 2014, including 42 males and 134 females aged from 20 to 88 years old, with the average age 58.6
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± 17.2 years. The present study was approved by the Academic Ethics Committee of Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, and all patients signed informed consents The inclusion criteria of subject enrollment were illustrated as follows: Chinese Han people; >18 years old; NVAF patients; taking the same warfarin more than three months; reaching the International Normalized Ratio (INR) of 2.0~3.0. The following conditions were excluded: acute diseases, stroke, myocardial infarction or receiving surgery within 30 days; moderate or severe heart valve diseases; artificial heart valve; congenital non-triggered venous or arterial thrombosis; receiving warfarin therapy unrelated to massive hemorrhage; liver and kidney dysfunction; malignant tumor; receiving estrogen or progesterone therapy or pregnant; taking 5
ACCEPTED MANUSCRIPT antiplatelet drugs and non-steroidal anti-inflammatory drugs for a long time. Specimen Collection All subjects maintained an empty stomach for 10 to 12 hours. Peripheral venous blood (10 mL)
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was collected in ethylenediaminetetraacetic acid (EDTA)-contained tube in the morning. After one
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hour, 5 ml of anti-coagulation was centrifuged at 3000 rpm for 10 minute to isolate peripheral blood
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mononuclear cells. The whole blood genomic DNA Extraction Kit (Beijing Tiangen Biotechnology Co. Ltd, batch number: DP318-03) was utilized to extract genomic DNA from peripheral blood
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leukocytes, and the 5ml anti-coagulation was used to detect INR. Single Nucleotide Polymorphism (SNP) Detection
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Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was adopted to detect CYP2C19*2 (G681A), CYP2C19*3 (G636A) polymorphisms. PCR primer was
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designed by Primer Premier 5.0 and synthesized by Shanghai Sangon Bioengineering Co. Ltd, the
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sequence and length of which were presented in Table 1. The PCR reaction system (50 uL)
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contained 10 × PCR buffer 5 uL, dNTPs (10 mmol/L) 1 uL, forward and reverse primers (20 mmol/L) 2 uL, Taq enzyme (5 u/uL) (TAKARA BIOTECHNOLOGY (DALIAN) CO., LTD.) 0.5 uL, template 1 uL, and double distilled water. The PCR cycle parameters involved 95°C pre-
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denaturation for 7 min and 94°C denaturation for 60 s and then annealing for 30 s with temperatures of 53°C, 54°C, 72°C extension for 60 s in 35 cycles and final 72°C extension for 10 minutes. The negative control group was also designed in PCR reaction, in which the template was replaced with double distilled water to ensure the reaction free from contamination. The PCR product was tested by 3% agarose gel electrophoresis and was recycled by DNA agarose gel extraction kit (Beijing Solarbio science and technology Co., Ltd, batch number: D1200). Then enzyme digested PCR product. CYP2C19*1 has Sam I and BamH enzyme digestion sites while CYP2C19*2 lacked Sam I and CYP2C19*3 lacked BamH I. Sam I enzyme digestion system (20 uL) consisted of Sam I 1 uL, 10 × T Buffer, 0.1% albumin from bovine serum 2 uL, DNA 10 uL, double distilled water 5 uL. BamH I enzyme digestion system (20 uL) contained BamH I 1 uL, 10 × K Buffer, DNA 12 uL, 6
ACCEPTED MANUSCRIPT double distilled water 5 uL. The reactants mentioned above were put in a constant-temperature incubator at 30°C. The enzyme digestion was stopped after 12 hours Sam I enzyme digestion and 3 hours BamH I enzyme digestion. The products were visualized in 3% agarose gel electrophoresis
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Observation Index
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and then stained with ethidium bromide (EB) to obtain results.
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The four channel blood coagulation analyzer (PUN-2048A) was applied to detect the INR of the venous blood samples by the transmission turbidity method. The maintenance dose of warfarin
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of subjects served was recorded, which was namely the daily dose of warfarin when the subjects maintained the stable condition (INR = 2.0 ~ 3.0). The height and weight of subjects were measured
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to figure out their body mass index (BMI) and body surface area (BSA). BMI = weight (kg)/height (m) 2; BSA (m2) = 0.0061 × height (m) + 0.0128 × weight (kg) - 0.01529.
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Statistical Analysis
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All data were analyzed by statistical software SPSS 18.0 (IBM Corporation, Somers, NY,
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USA). Measurement data were described by mean ± standard deviation. T-test and variance analysis were employed to compare measurement data, chi-square test to compare counting data, single factor variance analysis to determine the relationship between gene polymorphisms and basic
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indexes, covariance analysis to compare the INR value of different gene polymorphisms and warfarin maintenance dose after adjustment for age, gender and other confounding factors, and covariance analysis to compare warfarin maintenance dose in patients with different metabolizers after adjustment for age, gender and other confounding factors. All P values were tested in both sides. When P value is less than 0.05, the differences exhibited statistical significance.
Results Genotyping Results Different genotypes and the distribution frequency of CYP2C19 were shown in Table 2 and Table 3. According to the classification of CYP2C19 gene polymorphisms (Sukasem et al., 2013): 7
ACCEPTED MANUSCRIPT the results revealed that CYP2C19*2 and *3 gene polymorphisms were divided into three phenotypes, namely extensive metabolizer, CYP2C19*1/*1 with genotype frequency of 11.93%, intermediate metabolizers including CYP2C19*1/*2 with genotype frequency of 24.43% and
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CYP2C19*1/*3 with genotype frequency of 19.32%, and poor metabolizers including
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CYP2C19*2/*2 with genotype frequency of 28.98%, CYP2C19*2/*3 with genotype frequency of
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11.93% and CYP2C19*3/*3 with genotype frequency of 3.41%. The distribution frequency of CYP2C19*1, CYP2C19*2 and CYP2C19*3 were 33.81%, 47.16%, and 19.03%, respectively.
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CYP2C19*2 and *3 mutation frequency in patients with NVAF were 88.07% in total, 7.38 times than CYP2C19*1*1 (11.93%), and frequency of allele mutation were 66.16% in total, 1.96 times
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than CYP2C19*1.
Association between Basic Index and Genotype
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One-Way ANOVA analysis (Table 4) showed that there were no association between age,
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height, weight, BMI and BSA, and different genotypes of CYP2C19. Chi-square test indicated that
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different genotype distributions showed no statistical significance (P = 0.493), nor did the pairwise comparisons of all factors among different genotypes (all P > 0.05). Association between Basic Index and Warfarin Maintenance Dose
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The association between basic index and warfarin maintain dose is shown in Table 5. Results showed that age, gender, height, weight, BMI and BSA had no statistically significant differences, namely, there was no association between based indexes and warfarin maintain dose in NVAF patients (all P > 0.05). Association between Different Genotype and Warfarin Maintenance Dose As Table 6 demonstrated, the association between CYP2C19 gene polymorphism and warfarin maintenance dose would not be affected by basic index of patients. Covariance analysis showed that age, height, weight, BMI, BSA had on effect on warfarin maintenance dose in different genotypes (all P > 0.05). The INR value of patients with different genotypes maintained between 2.0 and 3.0 and comparison in pairs of them showed no statistical significance (all P > 0.05). The warfarin 8
ACCEPTED MANUSCRIPT maintenance doses of patients with *1/*1 were significantly higher than those with other five genotypes (all P < 0.05). What’s more, the warfarin maintenance doses of patients with *1/*2 and *1/*3 were remarkably higher than those with *2/*2 and *2/*3 (all P < 0.05) (Table 6).
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Association between Different Phenotype and Warfarin Maintenance Dose
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As shown in Table 7, the INR value of different metabolizers also maintained between 2.0 and
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3.0, and comparisons in pairs of INR values among different metabolizers demonstrated no statistical significance (all P > 0.05). The warfarin maintenance dose of patients with extensive
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metabolizer was dramatically higher than those with intermediate metabolizer and poor metabolizer (both P < 0.05). Also, the warfarin maintenance dose of patients with intermediate metabolizer was
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dramatically higher than those with poor metabolizer (P < 0.05).
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Discussion
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Warfarin anticoagulants can inhibit the action of the vitamin K epoxide reductase (VKOR),
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which resulted in inhibiting the synthesis of vitamin K into clotting factor in the human liver cells. It was mainly clinically adopted in patients with NVAF for antithrombotic therapies (Verhoef et al., 2014; Hirai et al., 2015; Senoo et al., 2015). Corresponding to our study, previous evidence revealed
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that the response to warfarin therapy exhibited a large inter-individual difference due to genetic polymorphisms (Li et al., 2015). Therefore, our study aimed to elucidate the influence of the CYP2C19 gene polymorphism on the clinical treatment dose of warfarin in NVAF patients. Recent studies had reported that CYP2C19 was highly polymorphic enzyme in the liver, and played an essential part in the metabolism of clinically distinctive drugs (Jiang et al., 2013a; Noai et al., 2016). Our results demonstrated that there was no association between different CYP2C19 genotypes and factors including age, height, weight, BMI value, BSA values. In addition, the relationship between CYP2C19 gene polymorphism and the dose of warfarin maintenance was not susceptible to basic indicators of patients. Further, no correlation between different basic indexes and warfarin maintenance dose was found in our study, suggesting that the basic indexes of patients 9
ACCEPTED MANUSCRIPT had no effect on warfarin maintenance dose. Consistent with our study, several studies pointed out that the distribution of CYP2C19*2 and CYP2C19*3 genotype between the group with < BMI values of 25 and the group with > BMI values of 25 was not statistically significant, suggesting that
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weight and height were unrelated with CYP2C19 genotype. And the age may not be the factors
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influencing CYP2C19 genetic polymorphisms (Jiang et al., 2013b; Lee et al., 2014). Consequently,
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our study confirmed that the differences in BSA value were not statistically significant among the genotypes according to the comparison of INR. Therefore, BSA value had no effect on CYP2C19
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gene polymorphism.
Our study also provided evidences that different CYP2C19 genotypes were related to warfarin
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maintenance dose. Recently, several studies have reported that CYP2C19*2 and CYP2C19*3 were the point of genetic mutations of CYP2C19 which were G681A in exon 5 and G636A in exon 4,
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respectively, with the 18% to 23% mutation frequency. The reason why warfarin had the functions
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of anticoagulant therapies and prevention of thrombosis in patients was that vitamin K epoxide reductase complex subunit 1 (VKORC1) can catalyze epoxy vitamin K into reduced vitamin K
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which was necessary cofactors of γ-glutamyl carboxylase. Besides, the premise of the anticoagulant activity of vitamin K-dependent coagulation factors containing glutamic acid residues must be
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carboxylated by γ-glutamyl carboxylase, and then a series of clotting cascade will emerge (Haraikawa et al., 2013; Mandic et al., 2015). Therefore, Warfarin, the most common oral anticoagulant that blocks the synthesis of clotting factors by inhibiting VKOR, is widely employed in the prevention as well as treatment of thrombosis (Tie et al., 2013). R-isomer warfarin was mainly metabolized by CYP2C19 in vivo, while CYP2C19 mutant alleles affect either the catalytic activity of the enzyme or enzyme expression, and its impact on the generation of active metabolites was more obvious (Chang et al., 2015). The mechanism of CYP2C19 involving warfarin indicated that CYP2C19 may inhibit the action of the VKOR in the process of blood coagulation. Consequently, genetic polymorphisms of CYP2C19 had a significant impact on the clinical effect of warfarin therapy in patients with NVAF. Furthermore, Sun et al. reported that CYP4F2 10
ACCEPTED MANUSCRIPT polymorphism had an effect on individual warfarin dose via vitamin K (Sun et al., 2016). Moreover, Kamali X et al. demonstrated that patients with CT and TT genotypes in GGCX gene rs259251 loci required for significantly higher warfarin dose than those with CC genotype; therefore, rs2592551
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polymorphism may one of the factors affecting the warfarin dose in patients with atrial fibrillation
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(Kamali et al., 2013).
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The study also found that CYP2C19 phenotypes exerted influences on different warfarin dose. The volume of warfarin maintenance dose in the extensive metabolizer was lowered than that in the
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intermediate metabolizers, which revealed that CYP2C19*2 mutation was the main reason for the dose of poor metabolizers. In addition, the CYP2C19 gene polymorphism accounted for inter-
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individual differences in warfarin dose requirements among different ethnicities (Sun et al., 2015). Mamiya et al introduced the CYP2C19 genetic polymorphism into population pharmacokinetics.
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Their research reported that the phenobarbital clearance rate in the poor metabolizers of CYP2C19
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was 18.8% lower than that in the extensive metabolizer (Mamiya et al., 2000).
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In conclusion, CYP2C19 genetic polymorphisms were highly associated with warfarin dose in patients with NVAF. Furthermore, CYP2C19 genotypes may be used as an indicator for clinically appropriate medication in patients with NVAF. However, this experiment still has the limitation
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which may be attributed to small genetic sample size, possibly leading to bias in our results. In addition, due to limitation in time and funding, other genes related to the metabolism and drug dose of warfarin in NVAF were not studied. Therefore, further study on the polymorphic CYP2C19 gene and other related genes, such as CYP2D6, CYP2C9, CYP2C19 and CYP1A2, as well as the improvement of genetic polymorphism mechanisms will promote clinical medicine rationalization of warfarin, which also plays a role in the prevention and treatment of patient with NVAF.
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ACCEPTED MANUSCRIPT Acknowledgements
Competing interests
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All authors in our study have no conflict of interest.
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The authors express their gratitude to the reviewers for their valuable advice.
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ACCEPTED MANUSCRIPT Table 1. The Primer Sequences Length SNP 169 bp
CYP2C19*3
233 bp
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CYP2C19*2
Primer sequences 5'-AATTACAACCAGAGCTTGGC-3' 5'-TATCACTTTCCATAAAAGCAAG-3' 5'-TATTATTATCTGTTAACTAATATGA-3' 5'-ACTTCAGGGCTTGGTCAATA-3'
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SNP, single nucleotide polymorphism.
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Table 2. Distribution Frequency of CYP2C19 Genotype Phenotypes Genotypes Cases/n Frequency/% Extensive metabolizer CYP2C19 * 1 * 1 21 11.93% Intermediate CYP2C19* 1* 2 43 24.43% metabolizer Intermediate CYP2C19* 1* 3 34 19.32% metabolizer Poor metabolizer CYP2C19 * 2 *2 51 28.98% Poor metabolizer CYP2C19* 2* 3 21 11.93% Poor metabolizer CYP2C19* 3* 3 6 3.41%
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ACCEPTED MANUSCRIPT Table 3. Distribution Frequency of CYP2C19 Allele Alleles Cases/n 119 166 67
33.81% 47.16% 19.03%
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CYP2C19* 1 CYP2C19* 2 CYP2C19* 3
Frequency/%
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ACCEPTED MANUSCRIPT *2*3 (n = 57.221) ± 14.5 21 (3/18) 162.5 ± 10.2 62.5 ± 5.9
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Table 4. Basic Indexes of Patients with Different Genotypes *1*1 (n = *1*2 (n = *1*3 (n = *2*2 (n = Indexes Age 59.521) ± 18.7 57.443) ± 16.3 56.334) ± 16.1 61.051) ± 19.8 21 (5/16) 43 (9/34) 34 (9/25) 51 (15/36) Gender (M/F) Height 163.3 ± 7.0 166.0 ± 7.8 164.7 ± 8.9 164.7 ± 7.9 60.7 ± 6.4 Weight 61.4 ± 5.9 62.2 ± 6.2 61.7 ± 7.2 BMI 22.8 ± 2.8 22.4 ± 2.4 23.5 ± 2.6 22.5 ± 2.5 BSA 0.77 ± 0.08 0.78 ± 0.08 0.81 ± 0.07 0.77 ± 0.08
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M, male; F, female; BMI, body mass index; BSA, body surface area.
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23.8 ± 2.8 0.79 ± 0.08
*3 *3 (n = 6)± 62.2 612.2 (1/5) 158.0 ± 10.1± 58.0 8.7 ± 23.3 3.4 ± 0.74 0.11
ACCEPTED MANUSCRIPT Table 5. Association between Different Basic Indexes and Warfarin Maintenance Doses Warfarin maintenance dose
85 91
3.44 ± 0.78 3.55 ± 0.74
42 134
3.60 ± 0.83 3.47 ± 0.74
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P > 0.05
> 0.05
> 0.05
3.57 ± 0.74 3.35 ± 0.80
122 54
> 0.05
3.53 ± 0.79 3.46 ± 0.72
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103 73
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4 115 57 92 84
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n
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Index Age (year) ≥60 < 60 Gender Male Female Height (cm) ≥ 60 < 160 Weight (kg) ≥ 60 < 60 BMI (kg/ m2) < 18.5 18.5~23.9 ≥. 24.0 BSA (m2) ≥ 0.77 < 0.77
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> 0.05
4.00 ± 0.94 3.46 ± 0.72 3.54 ± 0.82 > 0.05 3.54 ± 0.76 3.49 ± 0.76
ACCEPTED MANUSCRIPT Table 6. Warfarin Maintenance Dose of Patients with Different Genotypes INR value
Warfarin maintenance dose
*1 *1 (n = 21)
2.50 ± 0.18
4.46 ± 0.40
*1 *2 (n = 43)
2.50 ± 0.17
3.62 ± 0.67*
*1 *3 (n = 34)
2.47 ± 0.16
3.67 ± 0.78*
*2 *2 (n = 51)
2.54 ± 0.19
3.24 ± 0. 54*+
*2 *3 (n = 21)
2.45 ± 0.18
*3 *3 (n = 6)
2.37 ± 0.14
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Genotypes
3. 03 ± 0. 46*+ 2.14 ±0.06*+✿
, Compared with *1*1, P < 0.05; +, Compared with *1 *2 and *1 *3, P < 0.05; ✿, Compared
*
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with *2 *2 and *2 *3, P < 0.05; all P values werecorrected for age and gender by covariance analysis; INR: international normalized ratio.
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ACCEPTED MANUSCRIPT Table 7. Warfarin Maintenance Doses of Patients with Different Phenotypes Genotypes INR value Warfarin maintenance dose Extensive metabolizer (n = 21) Intermediate metabolizer (n = 77) Poor metabolizer (n = 78)
2.50 ± 0.18 2.49 ± 0.17 2.50 ± 0.19
4.46 ± 0.40 3.64 ± 0.72* 3.10 ± 0.58+
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*, compared with extensive metabolizer, P < 0.05; +, compared with intermediate metabolizer, P < 0.05; all P values were adjusted for age, gender, and other confounding factors by covariance analysis; INR, international normalized ratio.
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ACCEPTED MANUSCRIPT Highlights 1. First report about CYP2C19*2 and *3 polymorphism and warfarin maintenance dose. 2. The patients with CYP2C19*1/*1 had the lowest warfarin maintenance dose.
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3. CYP2C19*1/*2 and *1/*3 had lower warfarin dose than CYP2C19*2/*2 and *2/*3.
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4. Warfarin maintenance dose: extensive < intermediate < poor metabolizer.
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5. CYP2C19 genotype may be used as an indicator for medication in NVAF patients.
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S0378-1119(16)30509-1 doi: 10.1016/j.gene.2016.06.046 GENE 41416
To appear in:
Gene
Received date: Revised date: Accepted date:
15 April 2016 30 May 2016 22 June 2016
Please cite this article as: Zhang, Hongshen, Ma, Kezhong, Liu, Wenwei, Yang, Feng, Liu, Jianfei, Zhou, Hanyun, Impact of CYP2C19 gene polymorphism on warfarin maintenance doses in patients with non-valvular atrial fibrillation, Gene (2016), doi: 10.1016/j.gene.2016.06.046
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Impact of CYP2C19 gene polymorphism on warfarin maintenance doses in patients with non-valvular atrial fibrillation
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Running title: CYP2C19 gene polymorphism and warfarin doses
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Hongshen Zhang, Kezhong Ma, Wenwei Liu, Feng Yang, Jianfei Liu, Hanyun Zhou * Department of Cardiology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and
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Science, Xiangyang 441021, P.R. China
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* Correspondence to: Dr. Hanyun Zhou, Department of Cardiology, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, No. 39 Jingzhou street, Xiangcheng
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Tel. /fax: +86-0710-3253216
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E-mail: [email protected]
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District, Xiangyang 441021, P.R. China
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ACCEPTED MANUSCRIPT Abstract Background: Pharmacogenetics has provided compelling evidence towards the influence of gene polymorphisms on warfarin therapies. This study aimed to determine the impact of CYP2C19 gene
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polymorphism on warfarin maintenance doses in patients with non-valvular atrial fibrillation for the
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progress in overcoming obstacles facing warfarin pharmacogenetics.
Methods: In this study, we utilized polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to investigate the distribution of CYP2C19*2 and *3 gene
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polymorphism in patients with NVAF. In order to exclude the interference of basic indexes, we compared the association between different genotypes and warfarin maintenance doses. And the
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comparisons among extensive metabolizer, intermediate metabolizer and poor metabolizer were performed.
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Results: CYP2C19 mutation accounted for 88.07% of in total NVAF patients, which was 7.38 times
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of CYP2C19*1/*1 (11.93%). No significant association was observed between different genotypes
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and basic indexes. The warfarin maintenance dose of patients with CYP2C19*1/*1 was significantly higher than those with other five genotypes (all P < 0.05). Besides, the warfarin maintenance doses of patients with CYP2C19*1/*2 and *1/*3 were remarkably higher than those
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with *2/*2 and *2/*3 (P < 0.05). The warfarin maintenance doses of patients with extensive metabolizer were dramatically higher than those with intermediate metabolizer and poor metabolizer (both P < 0.05), and also the patients with intermediate metabolizer had higher warfarin maintenance doses than those with poor metabolizer (P < 0.05). Conclusion: CYP2C19 gene polymorphism can affect maintenance dose of warfarin, with the amount of warfarin dose ranked among different genotypes as follow: CYP2C19*2/*2, CYP2C19*2/*3 and CYP2C19*3/*3 < CYP2C19*1/*2 and CYP2C19*1* < CYP2C19*1/*1.
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ACCEPTED MANUSCRIPT Abbreviations list Non-valvular atrial fibrillation (NVAF)
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International Normalized Ratio (INR)
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ethylenediaminetetraacetic acid (EDTA)
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Single Nucleotide Polymorphism (SNP)
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)
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bovine serum (BSA) ethidium bromide (EB)
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vitamin K epoxide reductase (VKOR)
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vitamin K epoxide reductase complex subunit 1 (VKORC1)
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Key words: CYP2C19; Gene polymorphism; Non-valvular atrial fibrillation; Warfarin; Extensive
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metabolizer; Intermediate metabolizer; Poor metabolizer
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ACCEPTED MANUSCRIPT Introduction Non-valvular atrial fibrillation (NVAF) is clinically defined as a usual heart arrhythmia, with 5% of incidence for people older than 65 years and 10% for people older than 75 years (Gattellari et
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al., 2008). The pathogenesis of NVAF is that due to loss of contraction function in heart atrium,
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thrombus caused by clogged blood in heart atrium can trigger fatal and disabling stroke which is the most serious complication of NVAF. NVAF has been implicated in approximately 15% of ischemic strokes (Hankey, 2001). Surprisingly, individuals with NVAF suffer from stroke at high risk up to
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18.2% annually (Gattellari et al., 2008). With warfarin as the mainstay of oral anticoagulation therapy with vitamin K antagonists, the risk of NVAF-associated stroke can be declined by 60% to
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70% per year (Kodani et al., 2015). To the best of our knowledge, warfarin maintenance dose is varied for patients with different gene background or gene polymorphisms (Yan et al., 2015).
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CYP2C19 gene is located at 10q24.1 ~ 24.3 in human and contains 9 exons and 5 introns, with
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a length of 1.473 kb encoding area. As a highly polymorphic gene, CYP2C19 encodes for a protein
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that takes charge of the metabolism of many clinical drugs, such as the antiplatelet therapeutic agent clopidogrel, anticonvulsants, as well as proton pump inhibitors (Gardiner and Begg, 2006). CYP2C19 constitutes 16% of the CYP2C subfamily and its genetic variation is commonly
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associated with therapeutic outcomes, adverse drug reactions along with treatment failures (Jin et al., 2016). Several CYP2C19 polymorphisms are shown to be related to reduction or even loss of enzyme activity. Most cases are attributed to either CYP2C19*2 with a mutation in exon 5 or CYP2C19*3 characterized by a point mutation in exon 4. However, CYP2C19*1 encodes normal enzyme activity of CYP2C19 (Peng et al., 2015). Interestingly, it was reported that the clopidogrel can be converted to an active metabolic product by CYP2C19, the loss of function alleles of which may be able to reduce clopidogrel responses in patients with coronary heart disease (Collet et al., 2009). Previous study also demonstrated that, among acute cerebral infarction patients treated by clopidogrel, those who have the loss-of-function CYP2C19*2 alleles have a lower platelet inhibition as well as a higher incidence of adverse cardiac events (Depta et al., 2015). Also, there are some 4
ACCEPTED MANUSCRIPT studies reporting the influence of CYP2C19 polymorphisms on the difference in warfarin maintenance dose, Chang M et al. demonstrated that CYP2C19 genotypes influenced the clearance of R-warfarin and contributed significantly to the variability in international normalized ratio/daily
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dose, indirectly indicating a clinical relevance of R-warfarin; Kim SY et al. reported that CYP2C19
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likely impacts R-warfarin metabolism and patient response to therapy, and CYP2C19 may
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contribute to S-warfarin metabolism in patients, especially when CYP2C9 activity is compromised due to drug interactions or genetic polymorphisms (Chang et al., 2015; Scordo et al., 2002).
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However, the influence of CYP2C19 polymorphisms on warfarin maintenance dose among NVAF patients was reported rarely, thus the present study is designed to investigate the association
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Materials and methods
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between the CYP2C19 gene polymorphisms and the clinical efficacy of warfarin for NVAF patients.
Subjects
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The study recruited 176 patients suffering from NVAF in between October 2011 and August 2014, including 42 males and 134 females aged from 20 to 88 years old, with the average age 58.6
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± 17.2 years. The present study was approved by the Academic Ethics Committee of Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, and all patients signed informed consents The inclusion criteria of subject enrollment were illustrated as follows: Chinese Han people; >18 years old; NVAF patients; taking the same warfarin more than three months; reaching the International Normalized Ratio (INR) of 2.0~3.0. The following conditions were excluded: acute diseases, stroke, myocardial infarction or receiving surgery within 30 days; moderate or severe heart valve diseases; artificial heart valve; congenital non-triggered venous or arterial thrombosis; receiving warfarin therapy unrelated to massive hemorrhage; liver and kidney dysfunction; malignant tumor; receiving estrogen or progesterone therapy or pregnant; taking 5
ACCEPTED MANUSCRIPT antiplatelet drugs and non-steroidal anti-inflammatory drugs for a long time. Specimen Collection All subjects maintained an empty stomach for 10 to 12 hours. Peripheral venous blood (10 mL)
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was collected in ethylenediaminetetraacetic acid (EDTA)-contained tube in the morning. After one
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hour, 5 ml of anti-coagulation was centrifuged at 3000 rpm for 10 minute to isolate peripheral blood
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mononuclear cells. The whole blood genomic DNA Extraction Kit (Beijing Tiangen Biotechnology Co. Ltd, batch number: DP318-03) was utilized to extract genomic DNA from peripheral blood
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leukocytes, and the 5ml anti-coagulation was used to detect INR. Single Nucleotide Polymorphism (SNP) Detection
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Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was adopted to detect CYP2C19*2 (G681A), CYP2C19*3 (G636A) polymorphisms. PCR primer was
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designed by Primer Premier 5.0 and synthesized by Shanghai Sangon Bioengineering Co. Ltd, the
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sequence and length of which were presented in Table 1. The PCR reaction system (50 uL)
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contained 10 × PCR buffer 5 uL, dNTPs (10 mmol/L) 1 uL, forward and reverse primers (20 mmol/L) 2 uL, Taq enzyme (5 u/uL) (TAKARA BIOTECHNOLOGY (DALIAN) CO., LTD.) 0.5 uL, template 1 uL, and double distilled water. The PCR cycle parameters involved 95°C pre-
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denaturation for 7 min and 94°C denaturation for 60 s and then annealing for 30 s with temperatures of 53°C, 54°C, 72°C extension for 60 s in 35 cycles and final 72°C extension for 10 minutes. The negative control group was also designed in PCR reaction, in which the template was replaced with double distilled water to ensure the reaction free from contamination. The PCR product was tested by 3% agarose gel electrophoresis and was recycled by DNA agarose gel extraction kit (Beijing Solarbio science and technology Co., Ltd, batch number: D1200). Then enzyme digested PCR product. CYP2C19*1 has Sam I and BamH enzyme digestion sites while CYP2C19*2 lacked Sam I and CYP2C19*3 lacked BamH I. Sam I enzyme digestion system (20 uL) consisted of Sam I 1 uL, 10 × T Buffer, 0.1% albumin from bovine serum 2 uL, DNA 10 uL, double distilled water 5 uL. BamH I enzyme digestion system (20 uL) contained BamH I 1 uL, 10 × K Buffer, DNA 12 uL, 6
ACCEPTED MANUSCRIPT double distilled water 5 uL. The reactants mentioned above were put in a constant-temperature incubator at 30°C. The enzyme digestion was stopped after 12 hours Sam I enzyme digestion and 3 hours BamH I enzyme digestion. The products were visualized in 3% agarose gel electrophoresis
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Observation Index
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and then stained with ethidium bromide (EB) to obtain results.
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The four channel blood coagulation analyzer (PUN-2048A) was applied to detect the INR of the venous blood samples by the transmission turbidity method. The maintenance dose of warfarin
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of subjects served was recorded, which was namely the daily dose of warfarin when the subjects maintained the stable condition (INR = 2.0 ~ 3.0). The height and weight of subjects were measured
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to figure out their body mass index (BMI) and body surface area (BSA). BMI = weight (kg)/height (m) 2; BSA (m2) = 0.0061 × height (m) + 0.0128 × weight (kg) - 0.01529.
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Statistical Analysis
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All data were analyzed by statistical software SPSS 18.0 (IBM Corporation, Somers, NY,
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USA). Measurement data were described by mean ± standard deviation. T-test and variance analysis were employed to compare measurement data, chi-square test to compare counting data, single factor variance analysis to determine the relationship between gene polymorphisms and basic
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indexes, covariance analysis to compare the INR value of different gene polymorphisms and warfarin maintenance dose after adjustment for age, gender and other confounding factors, and covariance analysis to compare warfarin maintenance dose in patients with different metabolizers after adjustment for age, gender and other confounding factors. All P values were tested in both sides. When P value is less than 0.05, the differences exhibited statistical significance.
Results Genotyping Results Different genotypes and the distribution frequency of CYP2C19 were shown in Table 2 and Table 3. According to the classification of CYP2C19 gene polymorphisms (Sukasem et al., 2013): 7
ACCEPTED MANUSCRIPT the results revealed that CYP2C19*2 and *3 gene polymorphisms were divided into three phenotypes, namely extensive metabolizer, CYP2C19*1/*1 with genotype frequency of 11.93%, intermediate metabolizers including CYP2C19*1/*2 with genotype frequency of 24.43% and
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CYP2C19*1/*3 with genotype frequency of 19.32%, and poor metabolizers including
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CYP2C19*2/*2 with genotype frequency of 28.98%, CYP2C19*2/*3 with genotype frequency of
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11.93% and CYP2C19*3/*3 with genotype frequency of 3.41%. The distribution frequency of CYP2C19*1, CYP2C19*2 and CYP2C19*3 were 33.81%, 47.16%, and 19.03%, respectively.
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CYP2C19*2 and *3 mutation frequency in patients with NVAF were 88.07% in total, 7.38 times than CYP2C19*1*1 (11.93%), and frequency of allele mutation were 66.16% in total, 1.96 times
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than CYP2C19*1.
Association between Basic Index and Genotype
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One-Way ANOVA analysis (Table 4) showed that there were no association between age,
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height, weight, BMI and BSA, and different genotypes of CYP2C19. Chi-square test indicated that
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different genotype distributions showed no statistical significance (P = 0.493), nor did the pairwise comparisons of all factors among different genotypes (all P > 0.05). Association between Basic Index and Warfarin Maintenance Dose
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The association between basic index and warfarin maintain dose is shown in Table 5. Results showed that age, gender, height, weight, BMI and BSA had no statistically significant differences, namely, there was no association between based indexes and warfarin maintain dose in NVAF patients (all P > 0.05). Association between Different Genotype and Warfarin Maintenance Dose As Table 6 demonstrated, the association between CYP2C19 gene polymorphism and warfarin maintenance dose would not be affected by basic index of patients. Covariance analysis showed that age, height, weight, BMI, BSA had on effect on warfarin maintenance dose in different genotypes (all P > 0.05). The INR value of patients with different genotypes maintained between 2.0 and 3.0 and comparison in pairs of them showed no statistical significance (all P > 0.05). The warfarin 8
ACCEPTED MANUSCRIPT maintenance doses of patients with *1/*1 were significantly higher than those with other five genotypes (all P < 0.05). What’s more, the warfarin maintenance doses of patients with *1/*2 and *1/*3 were remarkably higher than those with *2/*2 and *2/*3 (all P < 0.05) (Table 6).
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Association between Different Phenotype and Warfarin Maintenance Dose
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As shown in Table 7, the INR value of different metabolizers also maintained between 2.0 and
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3.0, and comparisons in pairs of INR values among different metabolizers demonstrated no statistical significance (all P > 0.05). The warfarin maintenance dose of patients with extensive
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metabolizer was dramatically higher than those with intermediate metabolizer and poor metabolizer (both P < 0.05). Also, the warfarin maintenance dose of patients with intermediate metabolizer was
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dramatically higher than those with poor metabolizer (P < 0.05).
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Discussion
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Warfarin anticoagulants can inhibit the action of the vitamin K epoxide reductase (VKOR),
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which resulted in inhibiting the synthesis of vitamin K into clotting factor in the human liver cells. It was mainly clinically adopted in patients with NVAF for antithrombotic therapies (Verhoef et al., 2014; Hirai et al., 2015; Senoo et al., 2015). Corresponding to our study, previous evidence revealed
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that the response to warfarin therapy exhibited a large inter-individual difference due to genetic polymorphisms (Li et al., 2015). Therefore, our study aimed to elucidate the influence of the CYP2C19 gene polymorphism on the clinical treatment dose of warfarin in NVAF patients. Recent studies had reported that CYP2C19 was highly polymorphic enzyme in the liver, and played an essential part in the metabolism of clinically distinctive drugs (Jiang et al., 2013a; Noai et al., 2016). Our results demonstrated that there was no association between different CYP2C19 genotypes and factors including age, height, weight, BMI value, BSA values. In addition, the relationship between CYP2C19 gene polymorphism and the dose of warfarin maintenance was not susceptible to basic indicators of patients. Further, no correlation between different basic indexes and warfarin maintenance dose was found in our study, suggesting that the basic indexes of patients 9
ACCEPTED MANUSCRIPT had no effect on warfarin maintenance dose. Consistent with our study, several studies pointed out that the distribution of CYP2C19*2 and CYP2C19*3 genotype between the group with < BMI values of 25 and the group with > BMI values of 25 was not statistically significant, suggesting that
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weight and height were unrelated with CYP2C19 genotype. And the age may not be the factors
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influencing CYP2C19 genetic polymorphisms (Jiang et al., 2013b; Lee et al., 2014). Consequently,
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our study confirmed that the differences in BSA value were not statistically significant among the genotypes according to the comparison of INR. Therefore, BSA value had no effect on CYP2C19
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gene polymorphism.
Our study also provided evidences that different CYP2C19 genotypes were related to warfarin
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maintenance dose. Recently, several studies have reported that CYP2C19*2 and CYP2C19*3 were the point of genetic mutations of CYP2C19 which were G681A in exon 5 and G636A in exon 4,
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respectively, with the 18% to 23% mutation frequency. The reason why warfarin had the functions
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of anticoagulant therapies and prevention of thrombosis in patients was that vitamin K epoxide reductase complex subunit 1 (VKORC1) can catalyze epoxy vitamin K into reduced vitamin K
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which was necessary cofactors of γ-glutamyl carboxylase. Besides, the premise of the anticoagulant activity of vitamin K-dependent coagulation factors containing glutamic acid residues must be
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carboxylated by γ-glutamyl carboxylase, and then a series of clotting cascade will emerge (Haraikawa et al., 2013; Mandic et al., 2015). Therefore, Warfarin, the most common oral anticoagulant that blocks the synthesis of clotting factors by inhibiting VKOR, is widely employed in the prevention as well as treatment of thrombosis (Tie et al., 2013). R-isomer warfarin was mainly metabolized by CYP2C19 in vivo, while CYP2C19 mutant alleles affect either the catalytic activity of the enzyme or enzyme expression, and its impact on the generation of active metabolites was more obvious (Chang et al., 2015). The mechanism of CYP2C19 involving warfarin indicated that CYP2C19 may inhibit the action of the VKOR in the process of blood coagulation. Consequently, genetic polymorphisms of CYP2C19 had a significant impact on the clinical effect of warfarin therapy in patients with NVAF. Furthermore, Sun et al. reported that CYP4F2 10
ACCEPTED MANUSCRIPT polymorphism had an effect on individual warfarin dose via vitamin K (Sun et al., 2016). Moreover, Kamali X et al. demonstrated that patients with CT and TT genotypes in GGCX gene rs259251 loci required for significantly higher warfarin dose than those with CC genotype; therefore, rs2592551
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polymorphism may one of the factors affecting the warfarin dose in patients with atrial fibrillation
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(Kamali et al., 2013).
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The study also found that CYP2C19 phenotypes exerted influences on different warfarin dose. The volume of warfarin maintenance dose in the extensive metabolizer was lowered than that in the
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intermediate metabolizers, which revealed that CYP2C19*2 mutation was the main reason for the dose of poor metabolizers. In addition, the CYP2C19 gene polymorphism accounted for inter-
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individual differences in warfarin dose requirements among different ethnicities (Sun et al., 2015). Mamiya et al introduced the CYP2C19 genetic polymorphism into population pharmacokinetics.
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Their research reported that the phenobarbital clearance rate in the poor metabolizers of CYP2C19
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was 18.8% lower than that in the extensive metabolizer (Mamiya et al., 2000).
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In conclusion, CYP2C19 genetic polymorphisms were highly associated with warfarin dose in patients with NVAF. Furthermore, CYP2C19 genotypes may be used as an indicator for clinically appropriate medication in patients with NVAF. However, this experiment still has the limitation
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which may be attributed to small genetic sample size, possibly leading to bias in our results. In addition, due to limitation in time and funding, other genes related to the metabolism and drug dose of warfarin in NVAF were not studied. Therefore, further study on the polymorphic CYP2C19 gene and other related genes, such as CYP2D6, CYP2C9, CYP2C19 and CYP1A2, as well as the improvement of genetic polymorphism mechanisms will promote clinical medicine rationalization of warfarin, which also plays a role in the prevention and treatment of patient with NVAF.
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ACCEPTED MANUSCRIPT Acknowledgements
Competing interests
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All authors in our study have no conflict of interest.
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The authors express their gratitude to the reviewers for their valuable advice.
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Jin, T., Zhang, X., Geng, T., Shi, X., Wang, L., Yuan, D. and Kang, L., 2016. Genotypephenotype analysis of CYP2C19 in the Tibetan population and its potential clinical implications in drug therapy. Mol Med Rep. Kamali, X., Wulasihan, M., Yang, Y.C., Lu, W.H., Liu, Z.Q. and He, P.Y., 2013. Association of GGCX gene polymorphism with warfarin dose in atrial fibrillation population in Xinjiang. Lipids Health Dis 12, 149. Kodani, E., Atarashi, H., Inoue, H., Okumura, K., Yamashita, T., Origasa, H. and Investigators, J.R.R., 2015. Secondary Prevention of Stroke with Warfarin in Patients with Nonvalvular Atrial Fibrillation: Subanalysis of the JRHYTHM Registry. J Stroke Cerebrovasc Dis. Lee, J.Y., Kim, N., Kim, M.S., Choi, Y.J., Lee, J.W., Yoon, H., Shin, C.M., Park, Y.S., Lee, D.H. and Jung, H.C., 2014. Factors affecting first-line triple therapy of Helicobacter pylori including CYP2C19 genotype and antibiotic resistance. Dig Dis Sci 59, 1235-43. Li, Y., Zhu, J. and Ding, J., 2015. VKORC1 -1639G/A and 1173 C/T Genetic Polymorphisms Influence Individual Differences in Warfarin Maintenance Dose. Genet Test Mol Biomarkers 19, 488-93. Mamiya, K., Hadama, A., Yukawa, E., Ieiri, I., Otsubo, K., Ninomiya, H., Tashiro, N. and Higuchi, S., 2000. CYP2C19 polymorphism effect on phenobarbitone. Pharmacokinetics in Japanese patients with epilepsy: analysis by population pharmacokinetics. Eur J Clin Pharmacol 55, 821-5. 13
ACCEPTED MANUSCRIPT Mandic, D., Bozina, N., Mandic, S., Samardzija, M., Milostic-Srb, A. and Rumora, L., 2015. VKORC1 gene polymorphisms and adverse events in Croatian patients on warfarin therapy. Int J Clin Pharmacol Ther 53, 905-13. Noai, M., Soraoka, H., Kajiwara, A., Tanamachi, Y., Oniki, K., Nakagawa, K., Ishitsu, T. and Saruwatari, J., 2016. Cytochrome P450 2C19 polymorphisms and valproic acid-induced weight gain. Acta Neurol Scand 133, 216-23. Peng, X.E., Chen, H.F., Hu, Z.J. and Shi, X.S., 2015. Independent and combined effects of environmental factors and
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Scordo, M.G., Pengo, V., Spina, E., Dahl, M.L., Gusella, M. and Padrini, R., 2002. Influence of CYP2C9 and CYP2C19 genetic polymorphisms on warfarin maintenance dose and metabolic clearance. Clin Pharmacol Ther 72, 702-10.
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effector nucleases-mediated vitamin K epoxide reductase knockout HEK293 cells. J Thromb Haemost 11, 1556-64. Verhoef, T.I., Redekop, W.K., Daly, A.K., van Schie, R.M., de Boer, A. and Maitland-van der Zee, A.H., 2014. Pharmacogenetic-guided dosing of coumarin anticoagulants: algorithms for warfarin, acenocoumarol and
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phenprocoumon. Br J Clin Pharmacol 77, 626-41. Yan, X., Yang, F., Zhou, H., Zhang, H., Liu, J., Ma, K., Li, Y., Zhu, J. and Ding, J., 2015. Effects of VKORC1 Genetic 3577-84.
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Polymorphisms on Warfarin Maintenance Dose Requirement in a Chinese Han Population. Med Sci Monit 21,
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ACCEPTED MANUSCRIPT Table 1. The Primer Sequences Length SNP 169 bp
CYP2C19*3
233 bp
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Primer sequences 5'-AATTACAACCAGAGCTTGGC-3' 5'-TATCACTTTCCATAAAAGCAAG-3' 5'-TATTATTATCTGTTAACTAATATGA-3' 5'-ACTTCAGGGCTTGGTCAATA-3'
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SNP, single nucleotide polymorphism.
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Table 2. Distribution Frequency of CYP2C19 Genotype Phenotypes Genotypes Cases/n Frequency/% Extensive metabolizer CYP2C19 * 1 * 1 21 11.93% Intermediate CYP2C19* 1* 2 43 24.43% metabolizer Intermediate CYP2C19* 1* 3 34 19.32% metabolizer Poor metabolizer CYP2C19 * 2 *2 51 28.98% Poor metabolizer CYP2C19* 2* 3 21 11.93% Poor metabolizer CYP2C19* 3* 3 6 3.41%
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ACCEPTED MANUSCRIPT Table 3. Distribution Frequency of CYP2C19 Allele Alleles Cases/n 119 166 67
33.81% 47.16% 19.03%
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CYP2C19* 1 CYP2C19* 2 CYP2C19* 3
Frequency/%
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ACCEPTED MANUSCRIPT *2*3 (n = 57.221) ± 14.5 21 (3/18) 162.5 ± 10.2 62.5 ± 5.9
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Table 4. Basic Indexes of Patients with Different Genotypes *1*1 (n = *1*2 (n = *1*3 (n = *2*2 (n = Indexes Age 59.521) ± 18.7 57.443) ± 16.3 56.334) ± 16.1 61.051) ± 19.8 21 (5/16) 43 (9/34) 34 (9/25) 51 (15/36) Gender (M/F) Height 163.3 ± 7.0 166.0 ± 7.8 164.7 ± 8.9 164.7 ± 7.9 60.7 ± 6.4 Weight 61.4 ± 5.9 62.2 ± 6.2 61.7 ± 7.2 BMI 22.8 ± 2.8 22.4 ± 2.4 23.5 ± 2.6 22.5 ± 2.5 BSA 0.77 ± 0.08 0.78 ± 0.08 0.81 ± 0.07 0.77 ± 0.08
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M, male; F, female; BMI, body mass index; BSA, body surface area.
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23.8 ± 2.8 0.79 ± 0.08
*3 *3 (n = 6)± 62.2 612.2 (1/5) 158.0 ± 10.1± 58.0 8.7 ± 23.3 3.4 ± 0.74 0.11
ACCEPTED MANUSCRIPT Table 5. Association between Different Basic Indexes and Warfarin Maintenance Doses Warfarin maintenance dose
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3.44 ± 0.78 3.55 ± 0.74
42 134
3.60 ± 0.83 3.47 ± 0.74
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> 0.05
> 0.05
3.57 ± 0.74 3.35 ± 0.80
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> 0.05
3.53 ± 0.79 3.46 ± 0.72
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Index Age (year) ≥60 < 60 Gender Male Female Height (cm) ≥ 60 < 160 Weight (kg) ≥ 60 < 60 BMI (kg/ m2) < 18.5 18.5~23.9 ≥. 24.0 BSA (m2) ≥ 0.77 < 0.77
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> 0.05
4.00 ± 0.94 3.46 ± 0.72 3.54 ± 0.82 > 0.05 3.54 ± 0.76 3.49 ± 0.76
ACCEPTED MANUSCRIPT Table 6. Warfarin Maintenance Dose of Patients with Different Genotypes INR value
Warfarin maintenance dose
*1 *1 (n = 21)
2.50 ± 0.18
4.46 ± 0.40
*1 *2 (n = 43)
2.50 ± 0.17
3.62 ± 0.67*
*1 *3 (n = 34)
2.47 ± 0.16
3.67 ± 0.78*
*2 *2 (n = 51)
2.54 ± 0.19
3.24 ± 0. 54*+
*2 *3 (n = 21)
2.45 ± 0.18
*3 *3 (n = 6)
2.37 ± 0.14
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3. 03 ± 0. 46*+ 2.14 ±0.06*+✿
, Compared with *1*1, P < 0.05; +, Compared with *1 *2 and *1 *3, P < 0.05; ✿, Compared
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ACCEPTED MANUSCRIPT Table 7. Warfarin Maintenance Doses of Patients with Different Phenotypes Genotypes INR value Warfarin maintenance dose Extensive metabolizer (n = 21) Intermediate metabolizer (n = 77) Poor metabolizer (n = 78)
2.50 ± 0.18 2.49 ± 0.17 2.50 ± 0.19
4.46 ± 0.40 3.64 ± 0.72* 3.10 ± 0.58+
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*, compared with extensive metabolizer, P < 0.05; +, compared with intermediate metabolizer, P < 0.05; all P values were adjusted for age, gender, and other confounding factors by covariance analysis; INR, international normalized ratio.
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ACCEPTED MANUSCRIPT Highlights 1. First report about CYP2C19*2 and *3 polymorphism and warfarin maintenance dose. 2. The patients with CYP2C19*1/*1 had the lowest warfarin maintenance dose.
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3. CYP2C19*1/*2 and *1/*3 had lower warfarin dose than CYP2C19*2/*2 and *2/*3.
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4. Warfarin maintenance dose: extensive < intermediate < poor metabolizer.
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5. CYP2C19 genotype may be used as an indicator for medication in NVAF patients.
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