A multiplex assay to detect variations in the CYP2C9, VKORC1, CYP4F2 and APOE genes involved in acenocoumarol metabolism
Graphical abstract
Highlights
► We have developed a PCR multiplex SNaPshot reaction for CYP2C9, CYP4F2, VKORC1 and APOE. ► Polymorphism systems are related with appropriate dosage for acenocoumarol treatment. ► This SNaPshot assay is sensitive, simple, rapid and low cost. ► It can be included in the clinical routine prior to treatment with anticoagulants. ► This assay has been validated with other methodologies. (KASPar and TaqMan).
Introduction
Acenocoumarol is the most commonly used oral anticoagulant in European and Latin American countries. As a vitamin K antagonist, acenocoumarol is a highly effective antithrombotic drug for the treatment of thromboembolic disease, atrial fibrillation and artificial heart valves [1]. However, the appropriate dose of acenocoumarol is difficult to establish due to the numerous genetic and non-genetic factors associated with high interindividual variability in dose requirements, including age, gender, weight, height, drug interactions and genetic variations in the VKORC1 and CYP2C9 genes [2], [3] and other genes identified with controversial roles, such as CYP4F2 and APOE [4], [5], [6].
Our research group has developed and published [7] the first algorithm for individualization of the acenocoumarol dose in patients with venous thromboembolism that includes information about these four genes as well as clinical-demographic variables (age, body mass index and use of amiodarone and enzyme inducers). Our data suggest that inclusion of the CYP4F2 and APOE genotypes in the algorithm improves prediction for patients needing high doses, because these genotypes are associated with a higher dose of acenocoumarol (and probably warfarin). Based on this algorithm, we can explain 60.6% of the variability in the dose required to achieve a stable international normalized ratio (INR).
We therefore aimed to develop a method for simultaneously genotyping these four genes. The minisequencing technique has been shown to be a sensitive, reproducible, simple, flexible, and robust method as well as time- and cost-efficient when analyzing between two and ten SNPs [for example [8], [9]]. Therefore, we validated a SNaPshot system with four genes (CYP2C9, VKORC1, CYP4F2 and APOE) that are related to optimal acenocoumarol dosages and included the system in the pharmacogenetic algorithm developed by our group [7].
Section snippets
Variant alleles studied
The most important variants of CYP2C9 are CYP2C9*2 and CYP2C9*3. CYP2C9*2 has a point mutation in exon 3 that is responsible for the amino acid change Arg144Cys (rs1799853; 430C > T), and CYP2C9*3 has a single base substitution in exon 7 causing an Ile359Leu substitution (rs1057910; 1075A > C). The CYP4F2 gene has a point mutation in exon 11 causing a V433M change (rs2108622; 1347G > A). In terms of the VKORC1 gene, we studied VKORC1*2 (rs9934438), which is an SNP in the first intron of this gene
Results and discussion
High-throughput techniques have recently been developed to detect human mutations related to human disease. When analyzing a small number of SNPs, the most commonly employed genotyping systems are TaqMan® assays and minisequencing. The minisequencing technique allows the analysis of up to 10 SNPs in a single reaction. In the TaqMan® assay, the genotype of a sample is determined by measuring the signal intensity of two different dyes. Given that the real-time PCR instrument normally used in this
Acknowledgments
This study was supported by a research grant from Instituto de Salud Carlos III (FIS PI070710).
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Effect of CYP2C9 and VKORC1 genetic polymorphisms on mean daily maintenance dose of acenocoumarol in South Indian patients
2013, Thrombosis ResearchCitation Excerpt :This variability depends on numerous environmental factors. Further, genetic factors also play a significant role in the variability of dose requirement and acenocoumarol metabolism [7,8]. Oral anticoagulants are primarily metabolized by cytochrome P450 2C9 enzyme (CYP2C9) [9], and the target enzyme for these drugs was found to be vitamin K epoxide reductase complex subunit 1(VKORC1) [10].
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