Simone Rost

Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2

Coumarin derivatives such as warfarin represent the therapy of choice for the long-term treatment and prevention of thromboembolic events. Coumarins target blood coagulation by inhibiting the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, a cofactor that is essential for the post-translational γ-carboxylation of several blood coagulation factors. Despite extensive efforts, the components of the VKOR complex have not been identified. The complex has been proposed to be involved in two heritable human diseases: combined deficiency of vitamin-K-dependent clotting factors type 2 (VKCFD2; Online Mendelian Inheritance in Man (OMIM) 607473), and resistance to coumarin-type anticoagulant drugs (warfarin resistance, WR; OMIM 122700). Here we identify, by using linkage information from three species, the gene vitamin K epoxide reductase complex subunit 1 (VKORC1), which encodes a small transmembrane protein of the endoplasmic reticulum. VKORC1 contains missense mutations in both human disorders and in a warfarin-resistant rat strain. Overexpression of wild-type VKORC1, but not VKORC1 carrying the VKCFD2 mutation, leads to a marked increase in VKOR activity, which is sensitive to warfarin inhibition.

Interethnic variability of warfarin maintenance requirement is explained by VKORC1 genotype in an Asian population

Chinese and Malay subjects have been reported to require less maintenance warfarin than Indians that could not be accounted for by cytochrome P450 (CYP) 2C9 variants. Vitamin K epoxide reductase complex 1 (VKORC1) is the target enzyme of warfarin, and VKORC1 intronic variants and haplotypes have recently been shown to influence VKORC1 activity and warfarin requirements.

VKORC1: molecular target of coumarins

Summary.  The genetic diagnosis of a single family with combined vitamin K‐dependent clotting factor deficiency (VKCFD2, OMIM #607473) finally led to the identification and molecular characterization of vitamin K epoxide reductase (VKORC1). VKORC1 is the key enzyme of the vitamin K cycle and the molecular target of coumarins, which represent the most commonly prescribed drugs for therapy and prevention of thromboembolic conditions. However, coumarins are known to have a narrow therapeutic window and a considerable risk of bleeding complications caused by a broad variation of intra‐ and inter‐individual drug requirement. Now, 3 years after its identification, VKORC1 has greatly improved our understanding of the vitamin K cycle and has led to the translation of basic research into clinical practise in at least three directions: (i) Mutations within VKORC1 have been shown to cause a coumarin‐resistant phenotype and a single SNP (rs9923231) within the VKORC1 promoter region has been identified as the major pharmacodynamic determinant of coumarin dose. Together with the previously described CYP2C9 variants and other dose‐influencing factors, such as age, gender and weight, individualized dosing algorithms have become available. (ii) Preliminary studies indicate that concomitant application of low‐dose vitamin K (80–100 μg day−1) and warfarin significantly improves INR stability and time of INR within the therapeutic range. (iii) Co‐expression studies of FIX and FX with VKORC1 have shown that VKOR activity is the rate‐limiting step in the synthesis of biologically active vitamin K‐dependent factors. Thus, co‐expression of VKORC1 leads to a more efficient production of recombinant vitamin K‐dependent coagulation factors such as FIX and FVII. This could improve production of recombinant factor concentrates in the future.

Evaluation of DHPLC in the analysis of hemophilia A

The manifestation of hemophilia A, a common hereditary bleeding disorder in humans, is caused by abnormalities in the factor VIII (FVIII) gene. A wide range of different mutations has been identified and provides the genetic basis for the extensive variability observed in the clinical phenotype. The knowledge of a specific mutation is of great interest as this may facilitate genetic counseling and prediction of the risk of anti-FVIII antibody development, the most serious complication in hemophilia A treatment to date. Due to its considerable size (7.2 kb of the coding sequence, represented by 26 exons), mutation detection in this gene represents a challenge that is only partially met by conventional screening methods such as denaturing gradient gel electrophoresis (DGGE) or single stranded conformational polymorphism (SSCP). These techniques are time consuming, require specific expertise and are limited to detection rates of 70-85%. In contrast, the recently introduced denaturing high performance liquid chromatography (dHPLC) offers a promising new method for a fast and sensitive analysis of PCR-amplified DNA fragments. To test the applicability of dHPLC in the molecular diagnosis of hemophilia A, we first assessed a cohort of 156 patients with previously identified mutations in the FVIII gene. Applying empirically determined exon-specific melting profiles, a total of 150 mutations (96.2%) were readily detected. Five mutations (3.2%) could be identified after temperatures were optimized for the specific nucleotide change. One mutation (0.6%) failed to produce a detectable heteroduplex signal. In a second series, we analyzed 27 hemophiliacs in whom the mutation was not identified after extensive DGGE and chemical mismatch cleavage (CMC) analysis. In 19 of these patients (70.4%), dHPLC facilitated the detection of the disease-associated nucleotide alterations. From these findings we conclude that the dHPLC technology is a highly sensitive method well suited to the molecular analysis of hemophilia A.

Site-directed mutagenesis of coumarin-type anticoagulant-sensitive VKORC1: evidence that highly conserved amino acids define structural requirements for enzymatic activity and inhibition by warfarin.

Coumarin and homologous compounds are the most widely used anticoagulant drugs worldwide. They function as antagonists of vitamin K, an essential cofactor for the posttranslational gamma-glutamyl carboxylation of the so-called vitamin K-dependent proteins. As vitamin K hydroquinone is converted to vitamin K epoxide (VKO) in every carboxylation step, the epoxide has to be recycled to the reduced form by the vitamin K epoxide reductase complex (VKOR). Recently, a single coumarin-sensitive protein of the putative VKOR enzyme complex was identified in humans (vitamin K epoxide reductase complex subunit 1, VKORC1). Mutations in VKORC1 result in two different phenotypes: warfarin resistance (WR) and multiple coagulation factor deficiency type 2 (VKCFD2). Here,we report on the expression of site-directed VKORC1 mutants, addressing possible structural and functional roles of all seven cysteine residues (Cys16, Cys43, Cys51, Cys85, Cys96, Cys132, Cys135), the highly conserved residue Ser/Thr57, and Arg98, known to cause VKCFD2 in humans. Our results support the hypothesis that the C132-X-X-C135 motif in VKORC1 comprises part of the redox active site that catalyzes VKO reduction and also suggest a crucial role for the hydrophobic Thr-Tyr-Ala motif in coumarin binding. Furthermore, our results support the concept that different structural components of VKORC1 define the binding sites for vitamin K epoxide and coumarin.

Novel mutations in the VKORC1 gene of wild rats and mice – a response to 50 years of selection pressure by warfarin?

BackgroundCoumarin derivatives have been in world-wide use for rodent pest control for more than 50 years. Due to their retarded action as inhibitors of blood coagulation by repression of the vitamin K reductase (VKOR) activity, they are the rodenticides of choice against several species. Resistance to these compounds has been reported for rodent populations from many countries around the world and poses a considerable problem for efficacy of pest control.ResultsIn the present study, we have sequenced the VKORC1 genes of more than 250 rats and mice trapped in anticoagulant-exposed areas from four continents, and identified 18 novel and five published missense mutations, as well as eight neutral sequence variants, in a total of 178 animals. Mutagenesis in VKORC1 cDNA constructs and their recombinant expression revealed that these mutations reduced VKOR activities as compared to the wild-type protein. However, the in vitro enzyme assay used was not suited to convincingly demonstrate the warfarin resistance of all mutant proteinsConclusionOur results corroborate the VKORC1 gene as the main target for spontaneous mutations conferring warfarin resistance. The mechanism(s) of how mutations in the VKORC1 gene mediate insensitivity to coumarins in vivo has still to be elucidated.

Compound heterozygous mutations in the γ-glutamyl carboxylase gene cause combined deficiency of all vitamin K-dependent blood coagulation factors

Hereditary combined deficiency of the vitamin K‐dependent coagulation factors II, VII, IX, X, protein C, S and protein Z (VKCFD) is a very rare autosomal recessive inherited bleeding disorder. The phenotype may result from functional deficiency of either the γ‐glutamyl carboxylase (GGCX) or the vitamin K epoxide reductase (VKOR) complex. We report on the third case of VKCFD1 with mutations in the γ‐glutamyl carboxylase gene, which is remarkable because of compound heterozygosity. Two mutations were identified: a splice site mutation of exon 3 and a point mutation in exon 11, resulting in the replacement of arginine 485 by proline. Screening of 100 unrelated normal chromosomes by restriction fragment length polymorphism and denaturing high‐performance liquid chromatography analysis excluded either mutation as a frequent polymorphism. Substitution of vitamin K could only partially normalize the levels of coagulation factors. It is suggested that the missense mutation affects either the propeptide binding site or the vitamin K binding site of GGCX.

Thirteen novel VKORC1 mutations associated with oral anticoagulant resistance: insights into improved patient diagnosis and treatment

Summary.  Background: Vitamin K 2,3‐epoxide reductase complex subunit 1 (VKORC1) is the molecular target of oral anticoagulants. Mutations in VKORC1 cause partial or total coumarin resistance. Objectives: To identify new VKORC1 oral anticoagulant (OAC) resistance (OACR) mutations and compare the severity of patient phenotypes across different mutations and prescribed OAC drugs. Patients/Methods: Six hundred and twenty‐six individuals exhibiting partial or complete coumarin resistance were analyzed by VKORC1 gene sequencing and CYP2C9 haplotyping. Results: We identified 13 patients, each with a different, novel human VKORC1 heterozygous mutation associated with an OACR phenotype. These mutations result in amino acid substitutions: Ala26→Thr, His28→Gln, Asp36→Gly, Ser52→Trp, Ser56→Phe, Trp59→Leu, Trp59→Cys, Val66→Gly, Gly71→Ala, Asn77→Ser, Asn77→Tyr, Ile123→Asn, and Tyr139→His. Ten additional patients each had one of three previously reported VKORC1 mutations (Val29→Leu, Asp36→Tyr, and Val66→Met). Genotyping of frequent VKORC1 and CYP2C9 polymorphisms in these patients revealed a predominant association with combined non‐VKORC1*2 and wild‐type CYP2C9 haplotypes. Additionally, data for OAC dosage and the associated measured International Normalized Ratio (INR) demonstrate that OAC therapy is often discontinued by physicians, although stable therapeutic INR levels may be reached at higher OAC dosages. Bioinformatic analysis of VKORC1 homologous protein sequences indicated that most mutations cluster into protein sequence segments predicted to be localized in the lumenal loop or at the endoplasmic reticulum membrane–lumen interface. Conclusions: OACR mutations of VKORC1 predispose afflicted patients to high OAC dosage requirements, for which stable, therapeutic INRs can sometimes be attained.

Evaluating multiplexed next-generation sequencing as a method in palynology for mixed pollen samples

The identification of pollen plays an important role in ecology, palaeo-climatology, honey quality control and other areas. Currently, expert knowledge and reference collections are essential to identify pollen origin through light microscopy. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach, but the assessment of mixed pollen samples originating from multiple plant species is still a tedious and error-prone task. Next-generation sequencing has been proposed to avoid this hindrance. In this study we assessed mixed pollen probes through next-generation sequencing of amplicons from the highly variable, species-specific internal transcribed spacer 2 region of nuclear ribosomal DNA. Further, we developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. We assessed in total 16 mixed pollen samples, 14 originated from honeybee colonies and two from solitary bee nests. The sequencing technique resulted in higher taxon richness (deeper assignments and more identified taxa) compared to light microscopy. Abundance estimations from sequencing data were significantly correlated with counted abundances through light microscopy. Simulation analyses of taxon specificity and sensitivity indicate that 96% of taxa present in the database are correctly identifiable at the genus level and 70% at the species level. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialised palynological expert knowledge.

Detection of large duplications within the factor VIII gene by MLPA1

Hemophilia A (OMIM +306700) is the most common inherited bleeding disorder, with an incidence of about 1:5000 male births. It is caused by mutations in the F8 gene, which comprises 26 exons and encodes the procoagulation factor (F) VIII – an essential cofactor in the intrinsic pathway of the coagulation cascade. The most prevalent mutation in the F8 gene is a genomic rearrangement, the intron-22-inversion, which is present in more than 40% of severe hemophilia A patients [1,2]. In addition, a wide spectrum of other causative mutations in the F8 gene has been published: missense and nonsense mutations, splice site mutations, small and large deletions, as well as insertions [3–8]. So far, only one duplication of a whole exon of the F8 gene, exon 13, has been described [9,10]. This may be an ascertainment bias because whole-exon duplications are difficult to detect by PCR-based methods, which are usually applied for mutation screening. Recently developed techniques for screening of quantitative genomic changes (copy number variations) significantly facilitate the detection of such rearrangements. Multiplex ligationdependent probe amplification (MLPA; [11]) has rapidly found wide-spread application and has, for example, identified a large number of different duplications in the DMD gene causing Duchenne and Becker muscular dystrophies [12]. Over the past 15 years, a cohort of about 2000 patients suffering from mild, moderate or severe hemophilia A was referred to our laboratory for genetic diagnosis. DNA was isolated from peripheral blood samples using standard procedures. All patients were screened for mutations in the F8 gene by (i) long-range PCR for intron-22-inversions and intron-1inversions, (ii) DGGE (denaturing gradient gel electrophoresis) for sequence alterations, and (iii) direct sequencing of all 26 exons and flanking intronic regions of the F8 gene. We could identify the causative mutations in all but 80 patients (about 96% detection rate). These 80 hemophiliacs comprise severe, moderate and mild cases and form the present study cohort. DNA samples of the 80 hemophilia A patients were analyzed by MLPA (Kit P178, MRC Holland, Amsterdam, The Netherlands) for quantitative genomic changes in the F8 gene according to the manufacturer s instructions. In brief, the F8 MLPA kit uses a mixture of 44 sequence-specific probes, which are hybridized to different target sequences within theF8 exons. Each MLPA probe consists of two adjacent oligonucleotides, which are ligated to each other after hybridization to their target sequence. Only ligated probes can be amplified in a single-tube PCR reaction using a universal primer pair that anneals to adaptors attached to the probe oligonucleotides. This results in the amplification of a set of exon-specific products between 130 and 476 bp in length. MLPA PCR products were separated on an ABI 310 automatic sequencer, preanalyzed by Genemapper V4.0 and evaluated by the Excelbased software Coffalyser V5.2, which normalizes the data in relation to control peaks from other genes and converts the peak areas into bar graphs. With a normal gene dosage, the ratio of control and test bars lies at 1.0 (variance: 0.8–1.2), in the case of a hemizygous duplication of an exon the corresponding peak is doubled, and in the case of a heterozygous duplication the peak should have a ratio of approx. 1.5. Among the 80 patients screened by MLPA analysis for quantitative genomic changes, we could detect large duplications in nine patients (11%). Patients A and B showed duplications of single exons: exon 13 and exon 14, respectively. The other duplications affected more than one exon: exons 1 to 5 were duplicated in patient C, exons 5 to 25 in patient D, exons 23 to 25 in the unrelated patients E and F, exons 2 to 25 in patient G, exons 14 to 21 in patient H, while in patient I exons 7 to 11 were found to be duplicated (Fig. 1A). All duplications could be confirmed by quantitative DHPLC analysis (Fig. 1B). Therefore, a semiquantitative multiplex PCR was performed by 25 cycles at touch-down PCR conditions (details on request), with each reaction comprising one or more exons expected to be duplicated, a human growth hormone (HGH) gene fragment as internal standard and one or more exons not involved in the duplication as PCR controls. Separation was done on an Correspondence: Johannes Oldenburg, Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Sigmund-Freud Str. 25, 53127 Bonn, Germany. Tel.: +49 228 287 5175/5176; fax: +49 228 287 4320. E-mail: johannes.oldenburg@ukb.uni-bonn.de