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A 79-year-old man (Caucasian) was treated with coumarins following myocardial infarction. The International Normalized Ratio (INR), however, did not increase with standard doses for acenocoumarol and phenprocoumon. Upon administration of 8 mg day−1 acenocoumarol and subsequent 9 mg day−1 phenprocoumon, the INR remained 1.1. At this dose, phenprocoumon serum concentration, determined by non-stereospecific reversed-phase high performance liquid chromatography (RP-HPLC) and ultraviolet detection, was 11 mg L−1, above the range associated with an elevated INR (0.6–5 mg L−1) [1]. Vitamin K serum concentration was low (0.6 nmol L−1, reference range 0.8–5.3 nmol L−1) [2,3].

The patient was treated with irbesartan, carvedilol, pravastatin, furosemide, spironolacton, pantoprazol, and temazepam, concurrently. As long as the desired INR was not achieved, the patient was protected with aspirin.

The relatively high phenprocoumon and low vitamin K levels allowed us to exclude alternative causes for treatment failure (malabsorption, non-compliance, metabolic interactions, excessive vitamin K intake). Based on earlier reports on coumarin resistance [4–6], we concluded that treatment failure was most likely a result of partial or total coumarin resistance.

The desired anticoagulation level (target INR 2.5–3.5) was achieved with 18–21 mg phenprocoumon daily (6–7 tablets) with a serum concentration of 28 mg L−1. For this patient, anticoagulation treatment with this high dose of phenprocoumon has been successfully applied for over two years [2].

The clotting factors VII, IX, X, prothrombin, and protein C are activated by carboxylation. Vitamin K is an essential cofactor, which is oxidized to vitamin K epoxide in the carboxylation reaction. The enzyme vitamin K epoxide reductase (VKOR) catalyses the reduction of vitamin K epoxide to vitamin K, which then can serve as cofactor again [7]. VKOR can be inhibited by coumarins, resulting in a lower rate of vitamin K formation and thereby a lower rate of activation of clotting factors [4,7].

The gene coding for subunit 1 of VKOR has been identified in recent years and is called vitamin K epoxide reductase complex 1 (VKORC1) [5]. It is located on chromosome 16. A recent review describes the identification of VKORC1, the protein structure, function, and its interaction with coumarins [7,8]. Elevated sensitivity for vitamin K and resistance towards anticoagulation by coumarins have been related to polymorphisms in VKORC1. In resistant individuals, higher than normal coumarin levels are needed to inhibit VKOR activity.

Known VKORC1 sequence variants associated with coumarin resistance are: p.Val29Leu, p.Val45Ala, p.Arg58Gly, p.Leu128Arg, p.Val66Met, and p.Asp36Tyr [5,6,9,10]. To investigate whether a genetic predisposition of coumarin resistance was present in our patient, the VKORC1 coding sequence was analyzed. Reference sequence AY587020 [11] annotates the wild-type VKORC1 genomic sequence. DNA from the patient appeared heterozygous for a g.T6621C substitution in exon 2, which was clear from both the forward and reverse sequences (Fig. 1). g.T6621C leads to the replacement of the tryptophan at position 59 by an arginine (p.Trp59Arg). None of the previously reported sequence variations leading to coumarin resistance [4,5,8,9] was detected in the patient DNA, and no alterations were found in the 5′UTR (positions g.5086-5311 analyzed), in exon 1 (positions g.5312-5484) and exon 3 (positions g.8699-8907). In addition, we analyzed the presence of g.6484C>T variant (formerly described as 1137C>T) [11] in intron 1 using real-time PCR in a 7500 Fast Real-Time PCR System (Applied Biosystems, Nieuwerkerk a/d. lJssel, the Netherlands). Primers (forward, TGG AAT CCT GAC GTG GCC; reverse, TCT GTT CCC CGA CCT CCC; Sigma-Aldrich Chemie B.V., Zwijndrecht, the Netherlands) and MGB probes (ATC GAC TCT TGG ACT AGG A-FAM to detect g.6484T variant; CGA CCC TTG GAC TAG GA to detect g.6484C variant; Applied Biosystems) were designed using Primer Express™ software (Applied Biosystems version 2.0.0). The patient appeared to be heterozygous for the g.6484C>T variant in intron 1.

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Figure 1.  Vitamin K epoxide reductase complex 1 (VKORC1) sequence variation in a patient with partial resistance to acenocoumarol and phenprocoumon. Electropherogram of the patient DNA showing heterozygosity for the VKORC1 g. 6621T>C sequence variant. DNA was isolated from whole blood. Exon 2 of the VKORC1 gene was amplified using primers VKORC1_6546ex2_forward (5′-CTTTCTCGGGCAGGGTCCAAG-3′) and VKORC1_6937ex2_reverse (5′GGGCCCTTCAGCCTCTAACAG-3′; Sigma-Aldrich, Zwijndrecht, the Netherlands) in an MJ Research PTC-200 Thermal Cycler (BioRad, Veenendaal, the Netherlands) and sequenced with the same primers (Baseclear, Leiden, the Netherlands).

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Individuals carrying a CT or TT genotype on position g.6484 in intron 1 of VKORC1, in general, require less phenprocoumon or acenocoumarol than individuals carrying a CC genotype at this position [12,13]. In CT genotype patients, a mean daily phenprocoumon dose of 2.6 mg (95% confidence interval 2.1–3.1; n = 29) was required to maintain the target INR [13].

Genotyping for Cytochrome P450 2C9 (CYP2C9) variants revealed the absence of p.Cys144Arg (2C9*2) and p.Ile359Leu (2C9*3) allelic variants. (S)- and (R)-phenprocoumon differ in potency (S being 1.5–2.5 times more potent than R). With normal CYP2C9 activity, the elimination half-life of (S)- and (R)-phenprocoumon are of the same order of magnitude (110–130 h) and the ratio (S)/(R)-phenprocoumon does not change over time and therefore does not contribute to phenprocoumon resistance [14,15].

Thus, the putative increased sensitivity to anticoagulants because of the g.6484CT genotype of the patient was counteracted by other factor(s) that resulted in decreased sensitivity instead. Although the data are limited to one patient and functionality of the p.Trp59Arg protein was not assessed, we consider it likely that the p.Trp59Arg sequence variation was responsible for the observed resistance to acenocoumarol and phenprocoumon. In support of this notion is the conservation of the tryptophan at position 59 of VKOR among the species Homo sapiens, Mus musculus, Rattus norvegicus, Fugu rubripes, Xenopus laevis; but not in Anopheles spp. [5]. This observation suggests p.Trp59Arg is likely to be important for enzyme function.

In summary, we describe the putative association of a novel VKORC1 sequence variation (g.T6621C) leading to amino acid substitution p.Trp59Arg with partial coumarin resistance.

Acknowledgements

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References

We thank J. Poodt for sequencing VKORC1 and CYP2C9 analysis and C. Ingham for valuable comments on the manuscript.

Disclosure of Conflict of Interests

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References

The authors state that they have no conflict of interest.

References

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References
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