A multiplex assay to detect variations in the CYP2C9, VKORC1, CYP4F2 and APOE genes involved in acenocoumarol metabolism

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Abstract

Objectives

We have developed a genotyping system to determine the alleles of genes related to interindividual variability in acenocoumarol dosage requirements. This genotyping system is intended for routine clinical use and therefore it is essential that it be simple, fast and inexpensive.

Design and methods

We developed a PCR multiplex SNaPshot reaction that targets 6 SNPs (single nucleotide polymorphisms) in CYP2C9, CYP4F2, VKORC1 and APOE genes, which are associated with acenocoumarol dose requirements.

Results

We tested the multiplex in 152 samples and found it to be 100% concordant with the results of other methods.

Conclusions

We successfully produced a reliable multiplex system for simultaneously typing 6 SNPs. This system may be used as a model for accurate, simple and inexpensive genotyping of SNPs related to dose requirements. This information allows the prediction of drug efficiency in patients prior to treatment with acenocoumarol and the prevention of adverse drug reactions.

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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