VKORC1 Gene Analysis in an Iranian Warfarin Resistant Patient
The vitamin K epoxide reductase subunit 1 (VKORC1) has
been identified recently. It is a component of the enzyme vitamin K epoxide
reductase that is the therapeutic target site of warfarin. In order to
investigate the relationship between VKORC1 gene and warfarin dose
response, we studied this gene in an Iranian warfarin resistant patient
who receive more than 100 mg warfarin per day. The results showed that
although warfarin concentration in his plasma was extremely higher than
therapeutic level (22.8 mg L-1) but no mutation(s) found in
the exons of VKORC1 gene. Other genes may be contributed in resistance
to warfarin in this patient.
Warfarin is the most commonly used oral anticoagulant drug for the
prevention and treatment of thromboembolic events (Landefeld and Beyth,
1993). The required dose of this drug is variable, in particular between
individuals but also in any patient and depends on several factors.
The therapeutic response to warfarin is influenced by pharmacokinetic,
pharmacodynamic, genetic variability (Loebstein et al., 2001; Meyer,
2000) so prediction of warfarin dose response in patients is difficult
and the bleeding risk could be significantly high. Physicians are very
interested in developing strategies in order to predict warfarin dose
Anticoagulant effect of warfarin is exerted by inhibiting the vitamin
k epoxide reductase enzyme complex (VKOR) which exists in vitamin K cycle
(Stafford, 2005). Before the year 2004, glutathione S-transfrase has been
identified as a component of VKOR that is responsible for resistance to
warfarin in animal models (Cain et al., 1998). However, this proposal
remains valid but requires additional experimental verification. Another
component of VKOR termed VKORC1 has now been identified and 6 heterozygous
missense mutations found in patients suffering from warfarin resistance,
shown that it is the most probable target site of warfarin both in rat
and human (Rost et al., 2004; Li et al., 2004; Harrington
et al., 2005; Bodin et al., 2005).
The objective of the present study was to evaluate the relationship between
VKORC1 genotype and warfarin dose requirement in an Iranian patient
who is resistant to warfarin.
MATERIALS AND METHODS
A 56 years old man with rheumatic heart disease and three prosthetic
valves was referred to us from the anticoagulation clinic of Imam Hospital
(Tehran, Iran). The patient was firstly operated on 1974 for replacement
of mitral and tricuspid valves. In the year 1992, he had re-do operation
because of tricuspid prosthetic valve malfunction and aortic valve disease
and these two valves replaced by prosthetic valves. The mitral prosthetic
valve had no problem and remained as before.
He had a borderline blood glucose level and positive HBs antigen but
otherwise nearly normal lab tests. He was taking the usual cardiac drugs
and no any special drug with significant effect on warfarin metabolism.
This patient has been receiving more than 100 mg warfarin per day for
several years (normal amount is 5-10 mg warfarin per day). The INR of
this patient was just hardly in the therapeutic range with this amount
DNA was extracted from peripheral blood leukocytes using DNGTM-plus
solution (fermentase). DNA sample was amplified by polymerase chain reaction
(PCR) in a final volume of 100 μL, consists of 0.5 μM from each
primer, 0.2 mM deoxynucleoside triphosphate (dNTP), 1 μg
||Primers used for amplification and sequencing
of the VKORC1 gene
genomic DNA, 2.5 U Taq polymerase (fermentas) in 1x PCR buffer. The PCR
were done in 35 cycles that in each one denaturation occurred 45 sec in
93 °C, annealing occurred 30 sec in 58 °C for exon one and two and
56 °C for exon three and elongation occurred 90 sec in 72 °C. Primer
sequences are shown in Table 1. Then amplified DNA fragments
of all three exons were sequenced to find any mutation(s).
In the parallel study assaying of warfarin concentration using HPLC method
in his plasma was performed (Sadrai et al., 2008) and showed a
very high concentration (22.8 mg L-1) of plasma warfarin level,
which is very higher than therapeutic warfarin level in Iranian patients
(1.19 ± 0.3 mg L-1) that is determined by Andalibi et
RESULTS AND DISCUSSION
The coagulation assay of this patient showed the following results:
Pt = 24 sec (normal 11-13), Aptt = 36 sec (normal 33-37), Factor II =
36%, Factor VII = 35%, Factor IX = 50%, Factor V and VIII = 100%, INR
= three, that are consistent with moderate decrease in function of vitamin
K dependent coagulation factors, excluding the possibility of non cooperation
in drug ingestion. Also very high concentration of warfarin plasma level
could exclude a significant impairment of bioavailability and intestinal
absorption of the drug. Therefore, this patient was warfarin resistant.
However, the results of DNA extraction and sequencing showed a predicted
normal sequence of all three exons and exclude these exons as the site
of mutation and warfarin resistance in our patient. Our result is in contradict
of the results of the some previous studies (Rost et al., 2004;
Li et al., 2004; Harrington et al., 2005; Bodin et al.,
2005) that reported VKORC1 as the site of mutation for warfarin resistance.
So other genes than VKORC1 may be responsible for warfarin resistance
in such patient that should be surveyed later.
VKORC1 gene is not the only gene that is responsible for
resistance to warfarin in individuals, because in some warfarin resistant
patients this gene is normal. More studies should be done about the other
genes that are important for pharmacodynamics and pharmacokinetics of
The authors wish to thank Imam Hospital and Alzahra University for
their grateful support of this research.
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