Evaluation of warfarin resistance using transcription activator-like effector nucleases-mediated vitamin K epoxide reductase knockout HEK293 cells

SummaryThe gene encoding vitamin K epoxide reductase complex subunit 1 (VKORC1), a component of the enzyme that is the therapeutic target site for warfarin, has recently been identified. In order to investigate the relationship betweenVKORC1 and warfarin dose response, we studied theVKORC1 gene (VKORC1) in patients with warfarin resistance. From a study group of 820 patients, we identified 4 individuals who required more than 25 mg of warfarin daily for therapeutic anticoagulation.Three of these had serum warfarin concentrations within the therapeutic range of 0.7–2.3 mg/l and showed wild-type VKORC1 sequence. The fourth warfarin resistant individual had consistently high ( ≥ 5.7 mg/l) serum warfarin concentrations, yet had no clinically discernible cause for warfarin resistance. VKORC1 showed a heterozygous 196G→ A transition that predicted aVal66Met substitution in the VKORC1 polypeptide. This transition was also identified in 2 asymptomatic family members who had never received warfarin.These individuals had normal vitamin-K dependent coagulation factor activities and undetectable serum PIVKAII and vitamin K 1 2,3 epoxide suggesting that their basal vitamin K epoxide reductase activity was not adversely affected by the VKORC1 Val66Met substitution.The association between a nucleotide transition in VKORC1 and pharmacodynamic warfarin resistance supports the hypothesis that VKORC1 is the site of action of warfarin and indicates that VKORC1 sequence is an important determinant of the warfarin dose response.

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A vitamin K epoxide reductase complex subunit 1 mutation in an Irish patient with warfarin resistance

Irish Journal of Medical Science (1971 -) ◽

10.1007/s11845-008-0126-2 ◽

2008 ◽

Vol 177 (2) ◽

pp. 159-161 ◽

Cited By ~ 8

Author(s):

F. N. Áinle ◽

A. Mumford ◽

E. Tallon ◽

D. McCarthy ◽

K. Murphy

Keyword(s):

Vitamin K ◽

Vitamin K Epoxide Reductase ◽

Epoxide Reductase ◽

Warfarin Resistance

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Overexpression of protein disulfide isomerase enhances vitamin K epoxide reductase activity

Biochemistry and Cell Biology ◽

10.1139/bcb-2021-0441 ◽

2022 ◽

Author(s):

Thomas Chetot ◽

Etienne Benoit ◽

Véronique Lambert ◽

Virginie Lattard

Keyword(s):

Vitamin K ◽

Protein Disulfide Isomerase ◽

Reductase Activity ◽

Functional Characterization ◽

Hek293 Cells ◽

Vitamin K Epoxide Reductase ◽

Disulfide Isomerase ◽

Vitamin K Cycle ◽

Epoxide Reductase ◽

Protein Disulfide

Vitamin K epoxide reductase (VKOR) activity is catalyzed by the VKORC1 enzyme. It is the target of vitamin K antagonists (VKA). Numerous mutations of VKORC1 have been reported and have been suspected to confer resistance to VKA and/or affect its velocity. Nevertheless, the results between studies have been conflicting, the functional characterization of these mutations in a cell system being complex due to the interweaving of VKOR activity in the vitamin K cycle. In this study, a new cellular approach was implemented to globally evaluate the vitamin K cycle in the HEK293 cells. This global approach was based on the vitamin K quinone/vitamin K epoxide (K/KO) balance. In the presence of VKA or when the VKORC1/VKORC1L1 were knocked out, the K/KO balance decreased significantly due to an accumulation of vitamin KO. On the contrary, when VKORC1 was overexpressed, the balance remained unchanged, demonstrating a limitation of the VKOR activity. This limitation was shown to be due to an insufficient expression of the activation partner of VKORC1, as overexpressing the protein disulfide isomerase (PDI) overcomes the limitation. This study is the first to demonstrate a functional interaction between VKORC1 and the PDI enzyme.

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Formation of 3-Hydroxy-2,3-dihydrovitamin K1 In Vivo: Relationship to Vitamin K Epoxide Reductase and Warfarin Resistance

Journal of Nutrition ◽

10.1093/jn/114.5.902 ◽

1984 ◽

Vol 114 (5) ◽

pp. 902-910 ◽

Cited By ~ 9

Author(s):

Peter C. Preusch ◽

John W. Suttie

Keyword(s):

Vitamin K ◽

Vitamin K Epoxide Reductase ◽

Epoxide Reductase ◽

Warfarin Resistance

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Warfarin Resistance Due to a Val29Leu Mutation in the VKORC1 Protein.

Blood ◽

10.1182/blood.v106.11.4146.4146 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4146-4146

Author(s):

Jason D. Huff ◽

Heather L. Lawson ◽

V. C. Harish ◽

Lin Zhang ◽

John Owen

Keyword(s):

Vitamin K ◽

Human Subjects ◽

Clinical Problem ◽

Pcr Primers ◽

International Normalized Ratio ◽

Valuable Insight ◽

Vitamin K Epoxide Reductase ◽

Epoxide Reductase ◽

Exon 1 ◽

Warfarin Resistance

Abstract Warfarin resistance is a common clinical problem encountered by physicians of all subspecialities. Medical noncompliance and dietary indiscretions are felt to be the usual causes of a subject’s “resistance” to anticoagulation with coumarin derivatives. Warfarin’s target is the vitamin K epoxide reductase complex (VKOR). This complex functions within the vitamin K dependent γ-carboxylase system to recycle oxidized vitamin K. Until recently, the genetic sequence of components of this complex remained a mystery (Li et al., Nature 2004). Mutations in a subunit of this complex named vitamin K epoxide reductase complex subunit 1 (VKORC1) were isolated in human subjects with warfarin resistance (Rost et al., Nature 2004). Knowledge of this coding sequence has enabled us to study individual subjects suspected to have a genetic source for their warfarin resistance. Our study subject is a 24-year-old Caucasian female who received warfarin after developing bilateral pulmonary emboli. She required 30 mg of warfarin daily to achieve an international normalized ratio greater than 2.0. After institutional review board approval and with the patient’s informed consent, genomic DNA was extracted from the subject’s peripheral blood. The three exons comprising the VKORC1 gene were amplified by polymerase chain reaction (PCR). PCR primers were designed using the published sequence and online Primer3 software. Prolonged extension times were required to obtain good amplification of all three exons. After purification, the PCR products were sequenced and analyzed. The sequence analysis demonstrated a heterozygous G→T mutation in exon 1 at base pair 85. This results in a Val29Leu substitution, which has been described in a warfarin resistant patient by Rost et al. The second and third exons matched completely with the published sequence. The gene frequency of the mutation was then determined by examining 400 random, discarded DNA samples. The HypCH4 IV restriction enzyme cleaves at 2 positions in exon 1 in the wild-type genotype, but at just one position if our known mutation is present. The mutation was not identified in any sample. In conclusion, we have identified a second patient with a Val29Leu substitution in vitamin K epoxide reductase resulting in warfarin resistance. Our analysis of 400 random samples has not identified any other example of this mutation, indicating that this is a rare occurrence. Genetic analysis of patients requiring more than 20 mg of warfarin daily would likely reveal more mutations in the VKORC1 protein. The identification of such mutations would provide valuable insight into the structure-function relationships of this protein complex.

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Presence of a T383G Mutation in the Vitamin K Epoxide Reductase Gene (VKORC1) in a Patient Resistant to Four Different Vitamin K Antagonists.

Blood ◽

10.1182/blood.v104.11.4068.4068 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4068-4068

Author(s):

Michel-Meyer M.M. Samama ◽

Laurent L. Bodin ◽

Marie Helene M.H. Horellou ◽

Florence F. Parent ◽

Anne A. Kereveur ◽

...

Keyword(s):

Molecular Weight ◽

Vitamin K ◽

Low Molecular Weight Heparin ◽

Vitamin K Antagonists ◽

Low Molecular Weight ◽

Clotting Factors ◽

Vitamin K Epoxide Reductase ◽

Vitamin K Cycle ◽

Epoxide Reductase ◽

Warfarin Resistance

Abstract Resistance to vitamin K antagonists is a rare disorder which until recently has not been associated to a genetic factor. The existence of inherited warfarin resistant rat strains has been related to mutations of the genes involved in the vitamin K cycle. Vitamin K dependent carboxylase was cloned in 19911, while human vitamin K epoxide reductase complex 1 (VKORC1), another enzyme implicated in vitamin K cycle, has been cloned more recently2,3. Mutations in this gene are responsible for both human and rat warfarin resistance 3. In 1997, we reported 4 a 63-year old patient with recurrent pulmonary embolism and deep vein thrombosis without known hereditary or acquired thrombophilia who was found resistant to warfarin (up to 45mg/day), fluindione (up to 80mg/day), acenocoumarol (up to 12 mg/day) and phenprocoumon (up to 30 mg/day). With phenprocoumon, long-acting vitamin K antagonist, drug concentration reached 85 mg/L (usual range 1–5 mg/L) but the INR remained around 1. Daily low molecular weight heparin (LMWH) was continued 3 months. Treatment was discontinued on 2 occasions and a recurrent thrombotic episode was observed. The patient is now receiving a single daily dose of low molecular weight heparin (80 mg Enoxaparin, body weight 120 kg, 67 IU/kg once a day) for the past 10 years without thrombotic or bleeding episodes. Osteodensitometry remains normal after 9 years of treatment. Careful biochemichal investigation had demonstrated a deficiency in vitamin K dependent carboxylation and absence of blockade of vitamin K epoxide reductase by phenprocoumon. This year, we sequenced the three exons of VKORC1 in this patient and detected a heterozygous T383G transversion resulting in a leucine to arginine substitution (L128R). This mutation has been recently identified by Rost3 et al. in an individual with warfarin resistance. It was not found in 259 control subjects that we had tested. Interestingly, another gene mutation of the VKORC1 can be responsible for a combined deficiency of vitamin K dependent clotting factors 3. In conclusion, a resistance to all vitamin K antagonists, including warfarin up to 45 mg/day is extremely rare and the mutation T383G of the VKORC gene has been reported in only one patient before the case reported here. Testing for mutation in VKORC1 will help in explaining some cases of anti vitamin K resistance.

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