The 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 the VKORC1 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 (> or =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 K1 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 thatVKORC1 sequence is an important determinant of the warfarin dose response.

Summary. Background: Vitamin K epoxide reductase subunit 1 (VKORC1) is the molecular target of coumarin anticoagulants and mutations in VKORC1 have been identified previously in individuals who required high warfarin doses. Objective: Detailed characterization of the relationship between variation in VKORC1 and the warfarin resistance phenotype. Patients and methods: Serum warfarin concentration and coagulation parameters were determined in 289 subjects who required warfarin doses >20 mg day )1. The VKORC1 sequence was studied in selected study subjects. Results: Twenty-eight out of 289 (10%) subjects had serum warfarin >2.3 mg L )1 during stable therapeutic anticoagulation indicating pharmacodynamic warfarin resistance. Detailed analysis of 15 subjects from this group showed that eight out of 15 (53%) had nucleotide substitutions in VKORC1 predictive of p.V66M, p.L128R, p.V54L or p.D36Y. VKORC1 was normal in the remaining seven out of 15 (47%) subjects and in nine out of nine (100%) subjects with high warfarin dose requirement not caused by pharmacodynamic resistance. At referral, subjects with VKORC1 mutations received a median warfarin dose of 32 mg day )1(range 22-55) and had a median serum warfarin concentration of 4.6 mg L )1 (range 2.6-9.0). VKORC1 substitutions were associated with a requirement for high warfarin doses but not with adverse clinical events. Family members with VKORC1 nucleotide substitutions and not receiving warfarin had undetectable PIVKA-II and K 1 epoxide (K 1 O). Conclusions: Nucleotide variations in VKORC1 are a common cause of pharmacodynamic warfarin resistance but are not associated with adverse outcome during anticoagulation. Mutations associated with warfarin resistance do not cause a discernible defect in VKORC1 reductase function.

We demonstrate that coagulometric assays are not equally sensitive to clinically important functional defects of PC and that multiple assays may be required to identify all variants.

Summary We report a kindred with heritable protein C (PC) deficiency in which two siblings with severe thrombosis showed a composite type I and IIb PC deficiency phenotype, identified using commercial PC assays (proband: PC antigen 42 u/dl, amidolytic activity 40 u/dl, anticoagulant activity 9 u/dl). The independent PROC nucleotide variations c.669C>A (predictive of Ser181Arg) and c.131C>T (predictive of Asn2Ile) segregated with the type I and type IIb PC deficiency phenotypes respectively, but co‐segregated in the siblings with severe thrombosis. Soluble thrombomodulin (sTM)‐mediated inhibition of plasma thrombin generation from an individual with PC‐Asn2Ile was lower (endogenous thrombin potential (ETP) 56 ± 1% that of ETP determined without sTM) than control plasma (ETP 15 ± 2%) indicating reduced PC anticoagulant activity. Recombinant APC‐Asn2Ile exhibited normal amidolytic activity but impaired anticoagulant activity. Protein S (PS)‐dependent anticoagulant activity of recombinant APC‐Asn2Ile and binding of recombinant APC‐Asn2Ile to endothelial protein C receptor (EPCR) were reduced compared to recombinant wild‐type APC. Asn2 lies within the ω‐loop of the PC/APC Gla domain and this region is critical for calcium‐induced folding and subsequent interactions with anionic phospholipids, EPCR and PS. The disruption of these interactions in this naturally‐occurring PC variant highlights their collective importance in mediating APC anticoagulant activity in vivo.

Haemophilia A and B in one individual may arise from co-incident inheritance of independent mutations in the F8 and F9 genes. However, this association is rare and has been studied poorly at a genetic level. We report a male patient with abnormal bleeding and reduced factor VIII:C (26 IU dL(-1)) and factor IX:C (35 IU dL(-1)). This index case harboured a F8 c.979C>G transversion (predictive of p.Leu327Val) and a F9 c.845A>G transition (predictive of p.His282Arg) which have been previously associated with mild haemophilia A and B, respectively. Identical F8 and F9 mutations were identified in the mother and maternal grandmother. However, an affected maternal uncle showed only the F8 c.979C>G mutation, indicating haemophilia A alone. The sister of the index case was heterozygous only for F9 c.845A>G, indicating carriership of haemophilia B alone. The non-Mendelian inheritance of F8 c.979C>G and F9 c.845A>G in this kindred is consistent with recombination between F8 and F9 and illustrates the large recombination distance between these loci. Recognition of this phenomenon was essential for accurate genetic counselling in this kindred.