Effect Of VKORC1 And CYP2C9 Genotype On Inter-individual Warfarin Dose: A Prospective Study In Chinese Patients

Context and objectivesAs a derivant of bishydroxycoumarin,warfarin is the most commomly prescribed oral anticogulant in the world from 40s 20th centry when it was invented.For the narrow therapeutic index,it is easy to lead to adverse effects,such as thrombogenesis when the dose is insufficient or bleeding when the dose is excessive.The risk of bleeding is increased several times,and even cause death when use warfarin as anticogulant,especially in the first weeks to months during the early stages of treatment.Another characteristic of warfarin therapy is the large different stable dose between different races as well as interindividuals,with more than 20-fold of the stable dose range.As such,it is difficult for for clinicians to safely and efficiently prescribe warfarin for diffenent patients.Multiple factors affect the interindividual variation of warfarin dose,which can be sorteded into genetic factors and non-genetic factors.Non-genetic factors include age, gender,body surface area,drug interactions,dietary and morbid state.However, because the influence of non-genetic factors is limited,they are not the main reason of the variation of warfarin dose.Recently,with the rapid development of pharmacogenomics and elucidation of molecular mechanism of warfarin pharmacologic action,the effect of genetic factors on the variation of warfarin dose is more and more important.At present,there are more than 30 genes related to the pharmacodynamics and pharmacokinetics of warfarin,and CYP2C9 and VKORC1 are the two most important genetic factors.CYP2C9 is the major metabolic enzyme of warfarin.Up to now,there are 30 alleles of CYP2C9 be found,the common alleles are ~*1(wild type),~*2(Arg144Cys),~*3 (Ile359Leu).Patients carrying variant alleles have obviously lower enzymatic active and 2～3 times of bleeding risk than those carrying wild type alleles.VKORC is the target protein of warfarin.Warfarin exert its anticoagulation by inhibit the activity of VKORC,which function is reduces vitamin K 2,3-epoxide to the biologically active vitamin K hydroquinone.Several SNPs in VKORC1 gene were found to be related to the variation of warfarin dose,the most common SNPs were 1173C＞T in intron 1 and 3730G＞A in 3' UTR.These SNPs were in strong Linkage Disequilibrium and the Haplotypes divided into two groups,A(H1 and H2) and B(H7,H8 and H9), associated with lower and higher warfarin dosage requirements respectively.The contributions of the two genes on the variation of warfarin dose were studied by many researchers,these studies showed CYP2C9 and VKORC1 could account 5-22%and 6-37%interindividual variation of warfarin dose respectively.In the recent years,several predictive warfarin stable dose algorithms have been founded based on genetic factors and non-genetic factors in same given populations.Before widely used in clinic,it is necessary for these algorithms to be validated for their safe and effectiveness by prospective random case-control study.At present,there are few prospective studies to validate these algorithms,which had insufficient persuasion for the defect of design or shortage of cases.Although there were warfarin stable dose algorithms for Chinese population,they are never be validated prospectively.The objectives of this study were to:1) Determine the relation of interindividual variation of warfarin dose and polymorphisms of VKORC1 and CYP2C9 in Chinese population;2)Develop a warfarin stable dose predictive algorithm incorporated genetic factors and non-genetic factors by a retrospective study;3) Validate the practicality and feasibility of this algorithm by a prospective random case-control study and search for a suitable personalized medicine model of warfarin in Chinese population;4) Develop a SYBR Green-based real-time PCR assay for SNP genotyping,which is simple,rapid,accurate,inexpensive and suitable for VKORC1 and CYP2C9 genotyping in clinical laboratory.Design and methodsThis study contains three sections:1) choose candidate genes and target SNPs, develope and evaluate genotyping method;2)retrospective study of warfarin personalized medicine and foundation of warfarin stable predictive algorithm;3) Validated the feasibility of this algorithm by a prospective study.We set up two mehtods,PCR/DHPLC and SYBR Green-Ⅰbased real-time PCR for genotyping VKORC1 T6484C and CYP2C9 A1075C.With direct DNA sequencing as gold standard,the sensitivity and specificity,time and cost of the two methods were compared,the one with more rapid,accurate and cost-effective was choosed for genotyping in this study.In the retrospective study,266 patients required warfarin stable dose were enrolled. We collected data on age,gender,height,weight,smoking,drinking,coadministration drugs and eating habit.A 5-ml blood sample of each patient was taken for genotype analysis.Correlations of stable warfarin dosage and each factor were analysised withα=0.05 as the size of a test.Factors that were not statistically significant were rejected.A multiple regression analysis was performed using factors with statistical significance to determin coefficient of determination(R~2),which means the proporation of the reason of interindividual warfarin dose could be explained by all the factors used.A warfarin stable dose predective algorithm was founded based on the multiple regression equation.In the prospective study,112 patients accorded with the selected standard and were first-time warfarin users were enrolled,divided into study group and control group randomly.In the study group,patients were genotyped for VKORC1 and CYP2C9 polymorphisms before taking warfarin,and the predicted doses were calculated by using warfarin stable dose predictive algorithm.The first three dosage of warfarin were taken according to the predicted doses,and the doses were adjusted based on the variation of INR values.In the control group,patients' warfarin therapy were performed by tradictional model,and the warfarin doses were adjusted to stable dosage gradually.The frequency of INR monitoring was one time every from initial warfarin therapy to leave hospital,one time every week after discharge,and one time every month after stable dose acquired.Recored the time to stable dose and the time to adverise outcome,survival analyses were used to analysis experimental data.The endpoints were 1) dyas until an adverse outcome(defined as:any INR＞3.5,bleeding or venous thrombosis),and 2) days until stable dose acquired.The log-rank test compared the difference of time-to-event between study group and control group.A Cox proportional hazards-regression model generated HR(hazard ration) for study group and control group on the time to stable dose,and analysised the influence of each factors on endpoints.Results and discussionBoth the two SNP genotyping methods for VKORC1 and CYP2C9,DHPLC and real-time PCR assays were very accurate.The results showed complent concordance between DNA sequencing results and real-time PCR genotyping,and 99% concordance between DHPLC assay and DNA sequencing results.Compared with DHPLC,real-time PCR assay is convenient,time savings and inexpensive with relative throughput.It is suitable for VKORC1 and CYP2C9 genotyping in prospective application of warfarin therapyAmong the 266 patients in retrospective study,warfarin stable dosage ranged from 0.625 to 8.125mg/d.The mean of stable dose was 2.95±1.18mg/d and the coefficient of variance(CV) was 40%.The maximum dose exceeded the minimun dose by 13 times.Such further confirmed the existance of huge difference of warfarin among population.In the non-genetic factors,we selected eight factors,including gender,age, BSA,smoking,drinking,diabetes,indications and mean INR,to analysis their influence on interindividual warfarin stable dosage.The correlation analysis for the 8 factors and warfarin stable dosage(natural logarithmic transformation) shown gender, smoking,drinking,diabetes and indication had no relation to warfarin stable dosage, while age,BSA and mean INR had significant linear correlation to warfarin stable dosage.We found that the dose requirements fell with age,decreasing by approximately 0.2 mg per decade between the ages of 10 to 90 years irrsepective of other factors,and age accounted for 7.1%of interindividual variation of warfarin dosage.Warfarin dose requirement increased by 1.54mg per square meter of BSA irrsepective of other factors,and BSA accounted for 4.9%of interindividual variation of warfarin dosage.Correlation analysis showed that VKORC1 and CYP2C9 polymorphism had significant linear correlation to warfarin stable dosage.Irrsepective of other factors,VKORC1 and CYP2C9 accounted for 26.5%and 21%of interindividual variation of warfarin dosage respectively.Stepwise regression analysis was used to re-analysis the factors significantly related warfarin stable dose.The multiple regression shown age,BSA,VKORC1 and CYP2C9 were significant related to interindividual variation of warfarin dosage.The multiple regression equation of the optimized regression model was regarded as warfarin stable dose predictive algorithm,and this model accounted for 54.1%of interindividual variation of warfarin dosage.A total of 112 patients enrolled in prospective study.Of the total 95 patients completed the whole follow-up process,49 were in study group,and 46 were in control group.The mean time to stable dose of study group and control group were 27.4±1.8 days and 34.8±1.9 days respectively,and the median time were 24.0±1.6 days and 33.0±4.4 days respectively.The times to stable dose of the two groups had significant difference(P=0.011),which indicated that the time of stable warfarin dose adjustment could be shorten efficiently when patients taken warfarin according to the dose calculated by the predictive algorithm.Cox proportional hazards-regression analysis for group,gender,age,BSA,VKORC1 and CYP2C9 genotypes showen that only two factors,group and age had significant influence on time of stable warfarin dose adjustment(P=0.019,P=0.037).In the follow-up phase,the number of patients acquired stable dose in study group was 1.764(HR:1.764,95%CI:1.084-2.869, P=0.022) times than that in control group on the condition that the two groups had same total number of patients.There was a tendency that the time to stable dose prolonged with increasing age.In the four age groups,the time to stable dose of patients in the group of≥50 years was significantly increased than those of the other three groups.Of the 4 secondary indexes of prospective study,only the proporations of patients acquired stable dose had significant difference between study group and control group,the proporations were 81.6%and 63%respectively.The other three indexes,time of adjustment in hospital,incidence of adverse outcome and time to adverse outcome had no significant difference between the two groups.A total of 69 patients in the two groups acquired stable dose during follow-up phase.The mean predicted dose and mean actual dose of the 69 patients were 2.89±0.66 mg/d and 2.82±1.09mg/d respectively,with a mean overestimation of 0.07±0.81mg/d. predicted dose and actual dose had a significant linear correlation(r=0.676,P＜0.001). 66.7%patients' predicted doses were within acceptable range.The results of prospective study indicated stable warfarin dose predictive algorithm incorporated genetic factors and non-genetic factors had high predictive effectiveness.ConclusionThe results of retrospective study indicated that age,BSA,VKORC1 and CYP2C9 polymorphisms had significant influence on interindividual variation of warfarin dose, and genetic factors play a key role in this variation.Stable warfarin dose predictive algorithm incorporated genetic factors and non-genetic factors accounted for 54.1%of the interindividual variation of warfarin dose.The results of prospective randomized case-control study indicated that the time of stable warfarin dose adjustment of the patients taken warfarin based on pharmacogenetics was apparently decreased than that of the patients taken warfarin traditionally.Predicted dose and actual dose of patients had a significant linear correlation.Our study confirmed that the warfarin dose predictive algorithm had a high clinical value.It is useful to improve the safety and effectivity of warfarin therapy.