Identification of a Novel Coagulation Factor X Compound Heterozygous Mutation Associated with Differential Initiating Clotting Pathway Function.

Abstract Abstract 1129 Introduction: Factor × (FX) deficiency is a rare form of haemophilia characterized by a decrease in circulating FX antigen and/or activity levels, which can result in a variable bleeding diathesis. In heterozygous patients, bleeding may be absent or mild while homozygous and doubly heterozygous patients have phenotypes that are often associated with moderate or severe bleeding. Patient History: In this study, a propositus now aged 75 with a moderate bleeding diathesis is described. Neither parent had a history of bleeding. The patient was originally diagnosed as FX-deficient based on clinical measurements of coagulation factors. With prostate surgery, he had unexpected bleeding, that could not be explained surgically, requiring large volumes of plasma and red cell concentrates. Other surgical challenges, including dental extractions, were not complicated by bleeding but were preceded by plasma infusion. Methods: Plasma FX antigen levels were assayed by western blot using FX-specific monoclonal antibodies. To follow activity, prothrombin time and activated partial thromboplastin time clotting tests were used to evaluate the extrinsic and intrinsic initiating branches of coagulation, respectively. The entire F10 gene (8 exons and flanking intronic sequences) was amplified using PCR and sequenced to identify mutations. Results: DNA sequence analysis identified two mutations, which were presumably on different alleles based on a lack of parental bleeding. The first was a previously reported mutation that disrupts the splice site between exons 1 and 2 (IVS1 +1 G>A) and was hypothesized to lead to premature degradation of FX mRNA transcripts (Wang WB et al, Haemophilia 2005). This explains a 50% loss of antigen in our heterozygous patient. The second was a novel mutation at nucleotide 28145 (C>T) which results in an Arg386 to Cys (Arg386Cys) substitution in the serine protease domain of FX. Quantification of plasma FX antigen by western blot analysis revealed 15% of normal, which correlated precisely with 15% extrinsic pathway activity. However, intrinsic pathway clotting activity was only 5% of normal. The fragmentation of FX antigen in plasma after initiation of coagulation was followed over time. When initiated through the extrinsic pathway, the patient's FX fragmentation profile was identical to normal plasma. However, when clotting was triggered through the intrinsic pathway, activation to FXa and appearance of other fragments was notably slower. This further confirms that the patient's novel FX defect predominantly affects the intrinsic pathway while maintaining normal function in the extrinsic pathway. Conclusions: Here we describe a compound heterozygous FX deficiency. The first mutation has been reported once before (IVS1 +1 G>A) and accounts for 50% loss of FX antigen. The second FX mutation we identified is novel and may result in alternate disulfide bond formation; in particular at the nearby sole covalent link between the heavy and light chains of FX. This may explain the 35% further reduction in FX plasma antigen to 15% for this patient. Interestingly, the differential effect of Arg386Cys on the extrinsic and intrinsic coagulation pathways suggests that Arg386 may be involved in the substrate recognition by the intrinsic FIXa/FVIIIa Xase complex. As this Xase functions to amplify coagulation, Arg386Cys may be predicted to most affect hemostasis under severe conditions such as surgery. Production of recombinant FX containing this mutation is currently underway. Disclosures: No relevant conflicts of interest to declare.

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