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Main haplotypes and mutational analysis of vitamin K epoxide reductase (VKORC1) in a Swedish population: A retrospective analysis of case records
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry.
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Health Sciences, Internal Medicine . Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0003-1068-6168
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
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2006 (English)In: Journal of Thrombosis and Haemostasis, ISSN 1538-7933, Vol. 4, no 8, 1723-1729 p.Article in journal (Refereed) Published
Abstract [en]

Background: Vitamin K epoxide reductase (VKORC1) is the site of inhibition by coumarins. Several reports have shown that mutations in the gene encoding VKORC1 affect the sensitivity of the enzyme for warfarin. Recently, three main haplotypes of VKORC1; *2, *3 and *4 have been observed, that explain most of the genetic variability in warfarin dose among Caucasians.

Objectives: We have investigated the main haplotypes of the VKORC1 gene in a Swedish population. Additional objective was to screen the studied population for mutations in the coding region of VKORC1 gene.

Patients/methods: Warfarin doses and plasma S- and R-warfarin of 98 patients [with a target International Normalized Ratio (INR) of 2.0–3.0] have been correlated to VKORC1 haplotypes. Controls of 180 healthy individuals have also been haplotyped. Furthermore, a retrospective analysis of case records was performed to find any evidence indicating influence of VKORC1 haplotypes on warfarin response in the first 4 weeks (initiation phase) and the latest 12 months of warfarin treatment.

Results and conclusions: Our result shows that VKORC1*2 is the most important haplotype for warfarin dosage. Patients with VKORC1*2 haplotype had more frequent visits than patients with VKORC1*3 or *4 haplotypes, higher coefficient of variation (CV) of prothrombin time-INR and higher percentage of INR values outside the therapeutic interval (i.e. 2.0–3.0) than patients with VKORC1*3 or *4 haplotypes. Also, there was a statistically significant difference in warfarin dose (P < 0.001) and R-warfarin plasma levels (P < 0.01) between VKORC1*2 and VKORC1*3 or 4 haplotypes. Patients with VKORC1*2 haplotype seem to require much lower warfarin doses than other patients.

Place, publisher, year, edition, pages
2006. Vol. 4, no 8, 1723-1729 p.
Keyword [en]
INR, VKORC1, warfarin
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-14488DOI: 10.1111/j.1538-7836.2006.02039.xOAI: oai:DiVA.org:liu-14488DiVA: diva2:23583
Available from: 2007-05-21 Created: 2007-05-21 Last updated: 2013-09-03
In thesis
1. Studies on warfarin treatment with emphasis on inter-individual variations and drug monitoring
Open this publication in new window or tab >>Studies on warfarin treatment with emphasis on inter-individual variations and drug monitoring
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [sv]

Waran används sedan 60 år som blodförtunnande läkemedel för att förebygga eller förhindra progress av blodproppssjukdom. I Sverige behandlas årligen cirka 1 % av befolkningen med waran. I Östergötland uppskattas antal waranpatienter till cirka 3000. Waran hämmar enzymet VKORC1 som ansvarar för vitamin K omsättningen i kroppen. Vitamin K behövs som kofaktor för flera koagulationsfaktorer.

Behandling med waran är förenad med en svår balansgång och kräver en noggrann dosering. Stora skillnader i dosbehov mellan olika individer, beroende på ärftliga och miljöfaktorer, gör waran till ett svårhanterligt läkemedel. För låg dos medför otillräcklig effekt och därmed risk för minskat skydd mot blodproppssjukdom. För hög dos leder till allvarliga blödningskomplikationer. Uppskattningsvis 1 – 3 % livshotande blödningsfall registreras årligen efter waranbehandling. Därför måste behandlingen kontrolleras noga med analys av protrombinkomplex (PK) och dosjusteringar göras med ledning av resultaten. Två olika metoder finns att använda för mätning av PK. I Norden och i Japan används Owrens metod (utvecklat i Norge under 40- och 50-talet av Paul Owren). I de flesta andra länder används Quickmetoden (utvecklat i USA under 30-talet av Armand Quick). Den senare metoden är förenad med stora variationer mellan olika analyslaboratorier. I Norden, däremot, där Owrens metod används finns det ofta bra överensstämmelse mellan olika laboratorier i PK-resultat. Beroende på vilken PK-metod som används, kan samma patient få olika warandoser vilket ökar risker för under- eller överbehandling.

Vi har i samarbete med flera sjukhus och antikoagulationsmottagningar (AK-mottagningar) i sydöstra Sverige studerat dels mekanismerna bakom skillnader i warandos mellan olika patienter, och dels tittat varför de olika PK-metoder skiljer sig så mycket som de gör.

I studien har vi identifierat genetiska varianter av enzymet VKORC1. Av de undersökta patienter som gick på waran under längre tid, har vi identifierat en grupp som markant skiljde sig från de övriga. Denna grupp hade warandoser som var betydligt lägre än de övriga. När vi kartlade deras arvsmassa, fann vi att lågdospatienterna hade genvarianten VKORC1*2. Dessutom hade patienter med denna variant svårigheter att få stabila PK-värden. De gjorde också fler besök på AK-mottagningar än andra patienter. Vi har därför konstaterat att en del av de problem som är förenade med waranbehnadlingen kan förklaras av VKORC1*2 varianten. Vetskap om denna variant skulle troligen underlätta behandlingen framför allt under inledningsfasen då patienter med VKORC1*2 riskerar blödningar på grund av överdosering.

Vi har identifierat att provförspädning enligt Owrens metod är nödvändig för harmonisering av PK-resultatet mellan olika länder. Quickmetoden använder inte förspädning av patientprov till skillnad från Owrens metod. När vi modifierade en Quickmetod genom att förspäda prover enligt Owrens metod noterade vi en bra överensstämmelse mellan de två olika metoderna. Däremot var resultatet sämre utan provförspädning. Vi anser att Quickmetoder kan uppnå lika bra resultat som Owrens metod om prover förspäds som i Owrens metod. Det skulle gynna patienter som reser mellan olika regioner eller länder och leda till en bättre övervakning av waranbehandling internationellt.

I studien har dessutom en metod för mätning av waran i blodet utvecklats. Metoden som är den enda i sitt slag i Norden ger möjlighet att studera hur läkemedlet beter sig i kroppen. Vi har med denna metod kunnat upptäcka patienter som har onormala nedbrytningar av waran.

Abstract [en]

Warfarin was introduced more than 60 years ago and is used worldwide for the prophylaxis of arterial and venous thromboembolism in primary and secondary prevention. The drug is orally administered as a racemic mixture of (R)- and (S)-enantiomers. The (S)-form is mainly responsible for the anticoagulant effect and is metabolised by CYP2C9 enzyme in the liver microsomes. Warfarin exerts its pharmacological action by inhibiting the key enzyme (VKORC1) that regenerates vitamin K from an oxidised state to a reduced form. The latter is a cofactor for the post-translational modification of a number of proteins including coagulation factors II, VII, IX and X. The vitamin K-dependent modification provides these factors with the calcium-binding ability they require for the interaction with cell membranes of their target cells such as platelets.

Warfarin is monitored by measuring prothrombin time (PT) expressed as INR. Two main methods exist for PT analysis. The Owren method is used mainly in the Nordic and Baltic countries, in Japan, whereas the Quick method is employed in most other countries. Warfarin management is associated with some complications. Unlike many other drugs the dose for a given patient cannot be estimated beforehand, dose-response relationship is not predictable, and the prevention of thrombosis must be balanced against the risk of bleeding. Furthermore, the different PT methods used to monitor the drug are sometimes not in agreement and show significant discrepancies in results.

In an attempt to clarify the mechanisms influencing the inter-individual variations in warfarin therapy and to detect the factors that contribute to differences between PT methods, studies were conducted in collaboration with hospitals and anticoagulation clinics in the south-eastern region of Sweden. First, a stereo-specific HPLC method for measurement of warfarin enantiomers was developed and validated. With this method, the levels of plasma warfarin following its oral administration can be studied and evaluated. Abnormal clearance in some patients can be detected, and patient compliance can be verified. Furthermore, differing ratios of (S)- and (R)-isomers can be identified.

The impact of common VKORC1 polymorphisms on warfarin therapy was investigated. This study has shown that the VKORC1*2 haplotype is an important genetic determinant for warfarin dosage and is associated with difficulties in attaining and retaining therapeutic PT-INR. Further, significant differences in warfarin S/R-ratio was detected between patients with VKORC1*2 and VKORC1*3 or VKORC1*4 variants. This difference was not coupled with CYP2C9 genotype.

The effects of predilution of patient plasma samples, sources of thromboplastin and deficient plasma on between PT methods agreement were studied. This study has revealed that sample predilution according to the Owren method is to be preferred for the harmonisation of PT results. Undiluted samples, in contrast, according to the Quick method have shown reduced correlation between two different thromboplastin reagents. Sources of thromboplastin and deficient plasma were only of minor importance.

Place, publisher, year, edition, pages
Institutionen för biomedicin och kirurgi, 2007. 68 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1000
Keyword
Warfarin, VKORC1, Haplotype, Prothrombin time, INR
National Category
Other Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-8874 (URN)978-91-85715-45-9 (ISBN)
Public defence
2007-06-01, Berzeliussalen, Campus HU, Universitetssjukhuset, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2007-05-21 Created: 2007-05-21 Last updated: 2009-08-22

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Osman, AbdimajidEnström, CamillaArbring, KerstinSöderkvist, PeterLindahl, Tomas

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