Elsevier

Thrombosis Research

Volume 132, Issue 1, July 2013, Pages 123-126
Thrombosis Research

Regular Article
Resequencing of VKORC1, CYP2C9 and CYP4F2 genes in Italian patients requiring extreme low and high warfarin doses

https://doi.org/10.1016/j.thromres.2013.05.002Get rights and content

Abstract

Purpose

The aim of the present study was to investigate the genetic variability of VKORC1, CYP2C9 and CYP4F2 genes in patients who required a very low and high warfarin dose, in order to identify novel variants that could help to explain the particular extreme dose requirements.

Methods

Among patients followed and treated with warfarin at the Center of Haemostasis and Thrombosis of the PTV, we selected twelve patients showing a high divergence from warfarin standard doses required to achieve the therapeutic effect.

All VKORC1, CYP2C9 and CYP4F2 coding regions, 3’ and 5’ UTR and exon/intron boundaries were analyzed by direct sequencing.

Results

The 1173T and -1639A allele variants in VKORC1 gene, associated with warfarin sensitivity, were present, as expected, mostly in low dose patients while 3730A allele, linked to warfarin resistance, has been found only in high dose patients. Interestingly, we found that three out of six low dose subjects presented CYP2C9*3/*3 homozygous genotype, very rare in Caucasians.

Besides these common polymorphisms, we identified 5 SNPs in CYP2C9 gene and 19 SNPs in CYP4F2 gene. Among these, all polymorphisms identified in CYP2C9 gene were present only in low dose patients and three of them resulted in linkage with CYP2C9*2 and CYP2C9*3. Regarding CYP4F2 SNPs, we did not observe differences between the high and low dose patients. At the end, the whole sequencing did not reveal any novel polymorphism/mutation.

Conclusion

Further studies are required to identify other genetic factors contributing to extreme warfarin requirement.

Introduction

Warfarin (Coumadin) is a common oral anticoagulant used to prevention and long-term treatment of thromboembolic events in patients with atrial fibrillation, deep vein thrombosis, pulmonary embolism, heart valve prosthesis and other serious coronary malfunctions [1].

The correct dosing is necessary to avoid bleeding or risk of thrombotic events in the case of an excessive or a too low dose, respectively [2], [3].

The inter-individual variability in dose requirement is a multifactorial character and both environmental and genetic factors influence the drug metabolism and consequently the dose necessary for the therapeutic effect. Many pharmacogenetics studies have demonstrated that DNA variants in some genes play a key role: in particular the gene of VKORC1, encoding vitamin K epoxide reductase complex subunit 1, the warfarin drug target, and the gene of cytochrome CYP2C9, the primary enzyme involved in metabolism and subsequent inactivation of S-warfarin [4], [5], [6].

Polymorphisms in VKORC1 (-1639G > A, rs9923231; and 3730G > A, rs7294) and CYP2C9 (CYP2C9*2, rs1799853; and CYP2C9*3, rs1057910) genes have been found associated with required warfarin dose [6], [7], [8], [9], [10].

Many algorithms have been developed in the last years to predict the actual warfarin dose for each patients: when combined together CYP2C9 and VKORC1 polymorphisms and other clinical and environmental variables (e.g. age, weight, smoke, concomitant medication), they can explain about 47–56% of inter-individual variability in Caucasians [11], [12], [13].

In 2010 the US Food and Drug Administration approved revision of the warfarin label to include dosing recommendations (ranging from 0.5 to 7 mg/day) based on the different combinations of CYP2C9*2, CYP2C9*3, and VKORC1 -1639G > A variants [14].

Although these represent the most important genetic factors contributing to warfarin variability dose requirement, in 2008 another polymorphism in CYP4F2 gene (rs2108622) has been identified as involved in warfarin dosing [15] and confirmed as associated in different racial groups [16], [17], [18], [19].

A recent review/meta-analysis better defined the impact of CYP4F2 SNP on coumarin dose requirement [20].

However, the common variants in CYP2C9, VKORC1 and in CYP4F2 genes cannot explain the warfarin requirements in individuals with very high warfarin sensitivity or resistance. In our previous study, [21] we have identified a novel rare genetic variant in the CYP2C9 gene in a warfarin hyper-sensitive Italian patient who required only 2.25 mg/week of drug to achieve therapeutic anticoagulation effect; electrostatic calculations indicated an alteration on the enzyme surface that leads to a decrease of the recognition between the cytochrome and its redox partner NADPH P450 reductase.

In the present study we have investigated the genetic variability of VKORC1, CYP2C9 and CYP4F2 genes in six patients who required a very low weekly warfarin dose and six patients who required a very high weekly warfarin dose to achieve the therapeutic effect, in order to identify novel undescribed variations that could help to explain the particular extreme dose requirement.

Section snippets

Patients Recruitment and Monitoring of Anticoagulation

Twelve Italian patients with extreme warfarin doses requirements were selected, among about 500 treated patients present in our database and followed at the Center of Haemostasis and Thrombosis of the PTV (Policlinico Tor Vergata) Rome, Italy. In these patients the weekly therapeutic dose had showed a high divergence from warfarin standard dose. Patients were enrolled, on the basis of their required dosage, and divided into two groups: the low dosage group that includes six patients requiring a

Common Polymorphisms Linked to Warfarin Resistance and Sensitivity

Table 1 shows the distribution of common polymorphisms in VKORC1, CYP2C9 and CYP4F2 genes, associated with the variability of warfarin requirements, in the high and low dosage patients groups. Also the clinical diagnosis of the patients and their stable dose of warfarin have been reported. The 1173C > T (rs9934438) and -1639G > A (rs9923231) SNPs in VKORC1 gene associated with warfarin sensitivity, as expected, are in perfect linkage disequilibrium (LD) and show different frequencies among low and

Discussion

Previous studies on the pharmacogenetics of warfarin have proved that common polymorphisms in VKORC1, CYP2C9 and CYP4F2 genes, together with clinical and environmental factors explained just more than half of the variation in warfarin dose [11], [12], [13].

In this study we genotyped common polymorphisms in VKORC1, CYP2C9 and CYP4F2 genes in twelve Italian patients steadily anticoagulated with extreme warfarin doses. The presence at homozygous status of 1173T and CYP2C9*3 alleles, in 3 out of 6

Conflict of Interest Statement

The authors have no conflict of interest to declare.

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