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A new VKORC1 allelic variant (p.Trp59Arg) in a patient with partial resistance to acenocoumarol and phenprocoumon

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A new VKORC1 allelic variant (p.Trp59Arg) in a patient with partial resistance to acenocoumarol and phenprocoumon
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  LETTERS TO THE EDITOR A new  VKORC1   allelic variant (p.Trp59Arg) in a patient withpartial resistance to acenocoumarol and phenprocoumon E. B. WILMS,* D. J. TOUW,* J. M. H. CONEMANS,   R. VELDKAMP   and M. HERMANS  * Central Hospital Pharmacy, The Hague;   Laboratory of Molecular Diagnostics, Jeroen Bosch Hospital,    s Hertogenbosch; and    Department of Cardiology, Medisch Centrum Haaglanden, The Hague, the Netherlands To cite this article:  Wilms EB, Touw DJ, Conemans JMH, Veldkamp R, Hermans M. A new  VKORC1  allelic variant (p.Trp59Arg) in a patient withpartial resistance to acenocoumarol and phenprocoumon.  J Thromb Haemost   2008;  6 : 1224–6. A 79-year-old man (Caucasian) was treated with coumarinsfollowingmyocardialinfarction.TheInternationalNormalizedRatio(INR),however,didnotincreasewithstandarddosesforacenocoumarol and phenprocoumon. Upon administration of 8 mg day ) 1 acenocoumarol and subsequent 9 mg day ) 1 phen-procoumon, the INR remained 1.1. At this dose, phenprocou-mon serum concentration, determined by non-stereospecificreversed-phase high performance liquid chromatography (RP-HPLC) and ultraviolet detection, was 11 mg L ) 1 , above therange 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, pravast-atin, furosemide, spironolacton, pantoprazol, and temazepam,concurrently.AslongasthedesiredINRwasnotachieved,thepatient was protected with aspirin.The relatively high phenprocoumon and low vitamin Klevels allowed us to exclude alternative causes for treatmentfailure (malabsorption, non-compliance, metabolic interac-tions, excessive vitamin K intake). Based on earlier reports oncoumarin resistance [4–6], we concluded that treatment failurewas most likely a result of partial or total coumarin resistance.The desired anticoagulation level (target INR 2.5–3.5) wasachieved 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 phenpro-coumon has been successfully applied for over two years [2].The clotting factors VII, IX, X, prothrombin, andproteinCare activated by carboxylation. Vitamin K is an essentialcofactor, which is oxidized to vitamin K epoxide in thecarboxylation reaction. The enzyme vitamin K epoxide reduc-tase (VKOR) catalyses the reduction of vitamin K epoxide tovitamin K, which then can serve as cofactor again [7]. VKORcan 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 identifiedin recent years and is called vitamin K epoxide reductasecomplex 1 ( VKORC1 ) [5]. It is located on chromosome 16. Arecent review describes the identification of   VKORC1 , theprotein structure, function, and its interaction with coumarins[7,8]. Elevated sensitivity for vitamin K and resistance towardsanticoagulation by coumarins have been related to poly-morphisms in  VKORC1 . In resistant individuals, higher thannormal coumarin levels are needed to inhibit VKOR activity.Known  VKORC1  sequence variants associated withcoumarinresistanceare:p.Val29Leu,p.Val45Ala,p.Arg58Gly,p.Leu128Arg, p.Val66Met, and p.Asp36Tyr [5,6,9,10]. Toinvestigate whether a genetic predisposition of coumarinresistance was present in our patient, the  VKORC1  codingsequence was analyzed. Reference sequence AY587020 [11]annotates the wild-type  VKORC1  genomic sequence. DNAfrom the patient appeared heterozygous for a  g.T6621C  substitution in exon 2, which was clear from both the forwardand reverse sequences (Fig. 1).  g.T6621C   leads to the replace-ment of the tryptophan at position 59 by an arginine(p.Trp59Arg). None of the previously reported sequencevariations leading to coumarin resistance [4,5,8,9] was detectedin the patient DNA, and no alterations were found in the5 ¢ 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 (formerlydescribedas 1137C>T  )[11]inintron1usingreal-time PCRina 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 CCCCGA CCT CCC; Sigma-Aldrich Chemie B.V., Zwijndrecht,the Netherlands) and MGB probes (ATC GAC TCT TGGACT AGG A-FAM to detect  g.6484T   variant; CGA CCCTTG GAC TAG GA to detect  g.6484C   variant; AppliedBiosystems) were designed using Primer Express TM software(AppliedBiosystemsversion2.0.0). The patient appearedtobeheterozygous for the  g.6484C>T   variant in intron 1. Correspondence: Erik B. Wilms, Central Hospital Pharmacy,Escamplaan 900, 2547 EX The Hague, the Netherlands.Tel.: +31 703217217; fax: +31 703217153.E-mail: e.wilms@ahz.nlDOI: 10.1111/j.1538-7836.2008.02975.xReceived 2 January 2008, accepted 31 March 2008 Journal of Thrombosis and Haemostasis ,  6 : 1224–1249   2008 International Society on Thrombosis and Haemostasis  IndividualscarryingaCTorTTgenotypeonposition  g.6484 in intron 1 of   VKORC1 , in general, require less phenprocou-mon or acenocoumarol than individuals carrying a CCgenotype at this position [12,13]. In CT genotype patients, amean daily phenprocoumon dose of 2.6 mg (95% confidenceinterval 2.1–3.1;  n  = 29) was required to maintain the targetINR [13].Genotyping for Cytochrome  P450 2C9  (CYP2C9) variantsrevealed the absence of p.Cys144Arg (2C9*2) and p.Ile359Leu(2C9*3) allelic variants. (S)- and (R)-phenprocoumon differ inpotency (S being 1.5–2.5 times more potent than R). Withnormal CYP2C9 activity, the elimination half-life of (S)- and(R)-phenprocoumonare ofthe same orderofmagnitude (110– 130 h) and the ratio (S)/(R)-phenprocoumon does not changeovertimeandthereforedoesnotcontributetophenprocoumonresistance [14,15].Thus, the putative increased sensitivity to anticoagulantsbecause of the  g.6484CT   genotype of the patient was counter-acted by other factor(s) that resulted in decreased sensitivityinstead. Although the data are limited to one patient andfunctionality of the p.Trp59Arg protein was not assessed, weconsider it likely that the p.Trp59Arg sequence variation wasresponsible for the observed resistance to acenocoumarol andphenprocoumon. In support of this notion is the conservationof 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 observationsuggests p.Trp59Arg is likely to be important for enzymefunction.In summary, we describe the putative association of a novel VKORC1 sequencevariation(  g.T6621C  )leadingtoaminoacidsubstitution p.Trp59Arg with partial coumarin resistance. Acknowledgements We thank J. Poodt for sequencing  VKORC1  and CYP2C9analysis and C. Ingham for valuable comments on themanuscript. Disclosure of Conflict of Interests The authors state that they have no conflict of interest. References 1 Petersen D, Barthels M, Schumann G, Bu ¨ttner J. Concentrations of phenprocoumon in serum and serum water determined by high-per-formance liquid chromatography in patients on oral anticoagulanttherapy.  J Haemostasis  1993;  23 : 83–90.2 WilmsEB,VeldkampRF,vanMeegenE,TouwDJ.Partialresistanceto acenocoumarol and phenprocoumon caused by enzyme polymor-phism.  Ned Tijdschr Geneeskd   2006;  150 : 2095–8. Dutch.3 van Haard PM, Engel R, Postma T. Routine clinical determination of carotene, vitamin E, vitamin A, 25-hydroxyvitamin D3 and trans-vitamin K1 in human serum by straight phase HPLC.  Biomed Chro-matogr  1987;  2 : 79–88.4 O  Reilly RA, Aggeler PM, Hoag MS, Leong LS, Kropatkin ML.Hereditary transmission of exceptional resistance to coumarin anti-coagulant drugs. The first reported kindred.  N Eng J Med   1964;  271 :809–15. PatientWild typeForward C C C C C CG260 270G G GT TA AC C C CC CG G GT G G G G G GTa CA C C C C C CG270 280G G GT TA AC C C C C C C GGGGGGG GG 20 3020 30T T T A Reverse Fig. 1.  VitaminKepoxidereductasecomplex1( VKORC1 )sequencevariationinapatientwithpartialresistancetoacenocoumarolandphenprocoumon.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 VKOR-C1_6937ex2_reverse (5 ¢ GGGCCCTTCAGCCTCTAACAG-3 ¢ ; Sigma-Aldrich, Zwijndrecht, the Netherlands) in an MJ Research PTC-200 ThermalCycler (BioRad, Veenendaal, the Netherlands) and sequenced with the same primers (Baseclear, Leiden, the Netherlands). Letters to the Editor  1225   2008 International Society on Thrombosis and Haemostasis  5 Rost S, Fregin A, Ivaskevicius V, Conzelmann E, Hortnagel K, PelzH-J, Lappegard K, Seifried E, Scharrer I, Tuddenham EG, Mu ¨llerCR, Strom TM, Oldenburg J. Mutations in VKORC1 cause warfarinresistance and multiple coagulation factor deficiency type 2.  Nature 2004;  427 : 537–41.6 Bodin L, Horellou MH, Flaujac C, Loriot MA, Samama MM. Avitamin K epoxide reductase complex subunit-1 (VKORC1) mutationin a patient with vitamin K antagonist resistance.  J Thromb Haemost 2005;  3 : 1533–5.7 Oldenburg JM, Watzka M, Rost S, Mu ¨ller CR. VKORC1: moleculartarget of coumarins.  J Thromb Haemost  2007;  5 (Suppl. 1): 1–6.8 Kohn MH, Pelz HJ. A gene-anchored map position of the rat war-farin-resistance locus, Rw, and its orthologs in mice and humans. Blood   2000;  96 : 1996–8.9 Rieder RJ, Reiner AP, Gage BF, Nickerson DA, Eby CS, McLeodHL, Blough DK, Thummel KE, Veenstra DL, Rettie AE. Effect of VKORC1haplotypesontranscriptionalregulationandwarfarindose. N Engl J Med   2005;  352 : 2285–93.10 Loebstein R, Dvoskin I, Halkin H, Vecsler M, Lubetsky A, RechaviG, Amariglio N, Cohen Y, Ken-Dror G, Almog S, Gak E. A codingVKORC1 Asp36Tyr polymorphism predisposes to warfarin resis-tance.  Blood   2007;  109 : 2477–80.11 SeattleSNPs. NHLBI HL66682 Program for Genomic Applications,UW-FHCRC, Seattle, WA. http://pga.gs.washington.edu. Accessed12 December 2007.12 D  Andrea G, D  Ambrosio RL, Di Perma P, Chetta M, Santacroce R,Brancaccio V, Grandone E, Margaglione M. A polymorphism in theVKORC1 gene is associated with an interindividual variability in thedose-anticoagulant effect of warfarin.  Blood   2005;  105 : 645–9.13 Reitsma PH, van der Heijden JF, Groot AP, Rosendaal FR, Bu ¨llerHR. A C1173T dimorphism in the VKORC1 gene determines cou-marin sensitivity and bleeding risk.  PLoS Med   2005;  2 : 996–8.14 KirchheinerJ,UferM,WalterEC,KammererB,KahlichR,MeiselC,Schwab M, Gleiter CH, Rane A, Roots I, Brockmo ¨ller J. Effects of CYP2C9 polymorphisms on the pharmacokinetics of R- and S-phen-procoumon in healthy volunteers.  Pharmacogenetics  2004;  14 : 19–26.15 Ufer M, Svensson JO, Krausz KW, Gelboin HV, Rane A, Tybing G.IdentificationofcytochromesP4502C9and3A4asthemajorcatalystsof phenprocoumonhydroxylation in vitro .  Eur J Clin Pharmacol   2004; 60 : 173–82. Vitamin K epoxide reductase complex subunit 1 ( VKORC1  )polymorphism influences the anticoagulation responsesubsequent to vitamin K intake: a pilot study E. A. SCONCE,*   P. J. AVERY,   H. A. WYNNE   and F. KAMALI*  *  School of Clinical & Laboratory Sciences;   Institute of Ageing and Health; and     Schools of Mathematics and Statistics, Newcastle University,Newcastle upon Tyne, UK  To cite this article:  Sconce EA, Avery PJ, Wynne HA, Kamali F. Vitamin K epoxide reductase complex subunit 1  (VKORC1)  polymorphisminfluences the anticoagulation response subsequent to vitamin K intake: a pilot study.  J Thromb Haemost   2008;  6 : 1226–8. We recently demonstrated that daily supplementation withoral vitamin K can improve the stability of anticoagulationcontrol in previously unstable patients receiving chronictherapy with warfarin [1]. Vitamin K supplementation alsoantagonized the pharmacologic activity of warfarin, but tovarying degrees in different patients. Vitamin K epoxidereductase (VKOR) is the pharmacologic target for warfarinand influences the warfarin dose requirement [2,3]. Because of its influence upon VKOR activity, we explored, in this pilotstudy, whether the  VKORC1  genotype has any impact uponthe extent to which vitamin K affects the anticoagulationresponse and warfarin dose requirements.Bloodsampleswereobtainedfromthe70Caucasianpatientstakingpartintheearlierpublishedvitamin Ksupplementationstudy [1] for  post hoc  analysis. All were receiving chronictherapy with warfarin for stroke prophylaxis and had unstablecontrolofanticoagulation.Unstablecontrolwasdefinedasthepatient having a standard deviation of International Normal-ized Ratio (INR) of > 0.5 (i.e. from among the most unstable20%ofouranticoagulatedpopulation)andhavinghadatleastthree dose changes during the previous 6 months [1]. In thisstudy,patientshadbeenrandomlyallocatedtotwogroupsinadouble-blinded fashion. One group received a once-daily oralsupplement of 150  l g of vitamin K, and the other matchingplacebo, with their warfarin evening dose for 6 months. Thecriteria for patient selection and monitoring are describedelsewhere [1]. The study had prior approval from the JointNewcastleUniversityandHealthAuthorityEthicsCommittee,and written informed consent was acquired from all partici-pants. An overnight-fasted blood sample was taken at studyentry (day 0) and 1 week after supplementation with vita-min K for determination of plasma vitamin K and vitamin K2,3-epoxide concentrations, as vitamin K concentrations showlarge temporal variations and are influenced by vitamin Kconsumed in a meal [4]. The same blood sample was also used Correspondence: Farhad Kamali, Wolfson Unit of ClinicalPharmacology, School of Clinical and Laboratory Sciences,Newcastle University, Newcastle upon Tyne NE2 4HH, UK.Tel.: +44 191 222 8043; fax: +44 191 222 5827.E-mail: farhad.kamali@ncl.ac.ukDOI: 10.1111/j.1538-7836.2008.03003.xReceived 14 April 2008, accepted 26 April 2008 1226  Letters to the Editor   2008 International Society on Thrombosis and Haemostasis
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