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Keywords:

  • oral anticoagulant;
  • VKORC1;
  • pharmacogenomic

Oral anticoagulants (OAC) exert their effects by specifically inhibiting the vitamin K epoxide reductase multiprotein complex (VKOR1). These molecules are used in the treatment and prevention of thrombotic diseases. Warfarin, a coumarin compound, is the most used worldwide. In France, an indanedione derivative, fluindione, is traditionally prescribed in more than 80% of patients. There is a wide intersubject variation of OAC efficacy, due to environmental (including co-treatments, age, diet and ethnic origin) and genetic factors. Among genetic factors, two have been clearly identified: polymorphisms in a cytochrome isoform, CYP2C9, involved in OAC metabolism, and polymorphisms and mutations in the gene encoding the vitamin K epoxide reductase complex subunit 1 (VKORC1; OMIM ID 608547) (Yin & Miyata, 2007), a component of VKOR1. VKORC1 was recently localized on chromosome 16 (16p12-q21). The protein metabolizes vitamin K 2,3-epoxide in vitamin K hydroquinone, a cofactor essential for the post-translational γ-carboxylation of several blood coagulation factors. The expressed enzyme is 163 amino acids long with three transmembrane domains within the endoplasmic reticulum. A shorter isoform (named isoform 2) of 156 amino acids, produced by an alternative splicing has also been described. Mutations in the coding sequence of VKORC1 have been associated with OAC resistance (Harrington et al, 2005; Rieder et al, 2005; Wilms et al, 2008), whereas several single nucleotide polymorphisms in the non-coding region have been shown to influence warfarin dose requirement (Yin & Miyata, 2007).

We describe a new mutation of VKORC1 in a patient who presented a selective resistance to fluindione but was sensitive to warfarin. This 41-year-old male patient of Bengali origin was referred to the anticoagulation clinic (CREATIF) at Lariboisière Hospital, because his International Normalized ratio (INR) was low and fluctuated with a high daily dose of fluindione (60 mg, i.e. three times the usual dose). This patient was treated by OAC for a mitral valve prosthesis St Jude no 31. The prosthetic heart valve was necessary to treat a severe rheumatic heart disease complicated by a grade II dyspneoa (New York Heart Association) and permanent atrial fibrillation. After surgery, the patient did not receive any antibiotics; he had no history of thyroid disease, no vitamin K treatment and a regular equilibrated diet. Current medications were pravastatin and esomeprazole. The patient was initially treated by the CREATIF team with a high dose of Warfarin 12 mg/d, leading to a significant overcoagulation (INR:7). He was finally equilibrated with 7 mg/d warfarin to achieve the therapeutic INR target (3–4·5). After elimination of all external explanations for OAC resistance, he was investigated at the genetic level. After obtaining written informed consent, DNA was extracted from peripheral blood leucocytes according to standard protocols. The cytochrome gene CYP2C9 was genotyped for *2 (c.C416T, p.Arg144Cys, exon 3; rs1799853) and *3 (c.A1061C, p.Ile359Leu, exon 7; rs1057910) by polymerase chain reaction-restriction fragment length polymorphism using restriction enzymes AvaII and NsiI respectively. The coding sequence of VKORC1 was directly sequenced on both strands using the following primers: Ex1 Forward primer: 5′-ctccgtggctggttttctc-3′ and Reverse primer: 5′-ccgatcccagactccagaat-3′; Ex2 Forward primer: 5′-tgacatggaatcctgacgtg-3′ and Reverse primer 5′-cctgttagttacctccccaca-3′; Ex3 Forward primer: 5′-agtgcctgaagcccacac-3′ and Reverse primer 5′-gcacatttggtccattgtca-3′.

The patient was heterozygous for the CYP2C9*3 allele, which usually leads to a poor metabolism of warfarin (Bodin et al, 2005a). This polymorphism, observed in 10% of Caucasians, is associated with an unstable decreased activity of the CYP2C9 isoform, leading to an increased risk of overanticoagulation and bleeding. Patients heterozygous or a fortiori homozygous for CYP2C9*3 require more time to achieve stable dosing (Bodin et al, 2005a).

In addition, the patient presented a new missense mutation in the exon 1 of VKORC1:p.Leu27Val or NM_024006·4:c.79C > G. Pathogenicity is suggested by both nucleotide and amino acid extreme conservation among species (back to the Drosophila fly for Amino acid). On the other hand, the physico-chemical difference between Leucine and Valine is low (Graham score:32). The substitution does not modify any splicing sites as predicted by ‘Gene splicer’ and ‘Splice site finder’ software. The mutation is located within the coding sequence of the protein, and is predicted to be localized within the putative first transmembrane domain. No nucleotidic variation was found in the other exons of VKORC1. In 40 healthy Caucasian subjects (80 alleles), screening for this C-to-G substitution failed to identify this unpreviously published nucleotidic variation. However, as this patient is of Bengali origin, it remains possible that this variation is a rare, undescribed polymorphism. At the present time, VKORC1 has been little studied in this ethnical group; a very limited study on a small cohort of Indian subjects showed they required statistically higher doses of warfarin (Gan et al, 2003).

The described mutation is localized near p.Vall28Leu, which has been described to cause very severe warfarin resistance in an experimental cellular model (Rost et al, 2004).

The response to OAC is influenced by both environmental and genetic factors, with two genes playing a major role, CYP2C9 and VKORC1. To date, <10 different mutations of VKORC1 associated with OAC resistance have been published (For review see Table I; p.Val29Leu, p.Val45Ala, p.Trp59Arg, p.Val66Met p.Arg58Gly, p.Arg98Trp, p.Leu128Arg; Harrington et al, 2005; Wilms et al, 2008; Rost et al, 2004; Bodin et al, 2005b). These mutations are located either in the first and third transmembrane domains, or in the intraluminal loop of the protein (Li et al, 2004). The p.His68Tyr mutation was associated with an OAC resistance in a French female patient (K. Peoc'h, unpublished observations). The p.Asp36Tyr substitution appeared to be a common polymorphism within both Jewish and Ethiopian populations (Aklillu et al, 2008).

Table I.   Comparison of the different VKORC1 mutations.
Mutationp.Leu27Valp.Val29Leup.Val45Argp.Arg58 Glyp.Trp59Argp.Val66Metp.His68Tyrp.Leu128Arg
  1. WR, warfarin resistance (>20 mg/d); FR, fluindione resistance (>80 mg/d); F, fluindione; Hu, human; Mo, mouse; ND, not determined; TM, transmembrane region; CL, cytosolic loop.

Resistant to OACFRWRWRWRAcenocoumarol phenprocoumonFR, WRFR, WRWR
Level of resistanceHighHighLowLowLowHighNDHigh
DescriptionClinical settingCell modelCell modelCell modelClinical settingClinical settingClinical settingCell model
LocalizationFirst TMFirst TMFirst CLFirst CLFirst CLFirst CLFirst CLThird TM
Conservation of amino acids among species++++++Hu, Mo, Rat++++++Hu, Mo, RatHu, Mo, Rat+++
ReferencePresent studyRost et al (2004)Rost et al (2004)Rost et al (2004)Wilms et al (2008)Harrington et al (2005); Bodin et al (2005b)Osman et al (2006); K. Peoc'h, unpublished resultsRost et al (2004)

To our knowledge, this is the first description of an isolated fluindione resistance associated with a VKORC1 mutation. The previously published p.Val66Met was shown to be associated with fluindione resistance, but was also associated with resistance to warfarin, acenocoumarol and phenprocoumon (Bodin et al, 2005b).

The present description increases the range of OAC resistance associated with VKORC1 mutations, and underlines the clinical interest of VKORC1 sequencing in the management of patients with recurrent venous thromboembolism. Additionally, it demonstrated that the effect of CYP2C9 polymorphisms was weaker than those of VKORC1 mutations on OAC treatment equilibrations in the clinical setting. Physicians need to first consider the presence of VKORC1 mutations as a risk of resistance to OAC, before taking into account the CYP2C9 genotype risk of overdose and bleeds. Finally, this study showed that resistance to OAC is not always crossed between coumarin and indanedione compounds, which indicates that switching between these two classes of anticoagulants may be useful for establishing anticoagulation when resistance to one of these molecules occurs.

References

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  2. References
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