Background: No factor X (FX)-deficient patients have been reported with complete deletion of F10 genes, or harbouring mutations leading to the total absence of FX activity. Further, F10 gene knockout in mice is lethal. However, a few severe FX deficient infants with life-threatening symptoms have been reported, which might underlie subtle amounts of FX ensuring a minimal haemostasis. Here, we characterized the novel missense (p.L251P) and nonsense (p.W461X, TAG stop codon) mutations, found in a compound heterozygous proposita, affected at birth by major bleeding symptoms, with radiological evidence of cerebral microbleeds, and currently on prophylaxis with a plasma-derived FX concentrate. Methods: Expression of recombinant FX (rFX) variants, evaluation of rFX antigen (Ag) secreted in medium and thrombin generation (TG) assays in the presence of specific FX (Fondaparinux) and anti-coagulant (anti-TFPI aptamer) inhibitors. Results: Residual FX antigen and coagulant activity (PT and aPTT) levels in proposita’s plasma were below 1%. Both coagulation parameters in parents were decreased in accordance with their heterozygosity (mother, p.W461X; father, p.L251P). We expressed the corresponding recombinant variants in HEK293 cells. As compared to wild-type rFX (rFX-wt), we detected in conditioned media low Ag levels (rFX-251P, 0.6±0.2%; rFX-461X, 1.3±0.1%) and very low (rFX-251P, 21 min) or not detectable (rFX-461X) ability to shorten TG lag time in FX-deficient plasma (25 min). Moreover, the missense variants, potentially arising from the spontaneous suppression of the nonsense mutation p.W461X (TAG codon), do not support a functional impact of translational readthrough: the rFX-461Y (TAC) and rFX-461Q (CAG) variants showed very low secretion (Ag: 3.6±1.2% and 1.0±0.3%, respectively) and barely detectable TG activity (19 and 20 min lag times), as compared with 0.5% rFX-wt (14 min) and FX-deficient plasma (25 min). The residual TG capacity of the rFX-251P variant was FX-dependent, as indicated by Fondaparinux inhibition, which made TG undetectable, and by inhibition of TFPI (anti-TFPI aptamer), which shortened the TG lag time (from 26 to <24 min). Conclusions: Our experimental approaches, which contribute to the knowledge of the very severe FX deficiency forms, suggest that i) the p.W461X nonsense mutation, truncating the FX carboxyl-terminal region involved in interactions with prothrombin, produced very low levels of FX variants with negligible activity even after translational readthrough, and ii) the p.L251P missense mutation would contribute, during the foetal and perinatal life, to the trace FX levels and the associated minimal FX-dependent TG capacity. This residual function is candidate to shape the life-threatening, but non-perinatally lethal, phenotype of the proposita.
Mutation-specific contributions to trace factor X levels account for a life-threating phenotype in a compound heterozygous factor X deficient patient
Mattia Ferrarese;Marcello Baroni;Armando D’Angelo;Francesco Bernardi;Alessio Branchini
2018
Abstract
Background: No factor X (FX)-deficient patients have been reported with complete deletion of F10 genes, or harbouring mutations leading to the total absence of FX activity. Further, F10 gene knockout in mice is lethal. However, a few severe FX deficient infants with life-threatening symptoms have been reported, which might underlie subtle amounts of FX ensuring a minimal haemostasis. Here, we characterized the novel missense (p.L251P) and nonsense (p.W461X, TAG stop codon) mutations, found in a compound heterozygous proposita, affected at birth by major bleeding symptoms, with radiological evidence of cerebral microbleeds, and currently on prophylaxis with a plasma-derived FX concentrate. Methods: Expression of recombinant FX (rFX) variants, evaluation of rFX antigen (Ag) secreted in medium and thrombin generation (TG) assays in the presence of specific FX (Fondaparinux) and anti-coagulant (anti-TFPI aptamer) inhibitors. Results: Residual FX antigen and coagulant activity (PT and aPTT) levels in proposita’s plasma were below 1%. Both coagulation parameters in parents were decreased in accordance with their heterozygosity (mother, p.W461X; father, p.L251P). We expressed the corresponding recombinant variants in HEK293 cells. As compared to wild-type rFX (rFX-wt), we detected in conditioned media low Ag levels (rFX-251P, 0.6±0.2%; rFX-461X, 1.3±0.1%) and very low (rFX-251P, 21 min) or not detectable (rFX-461X) ability to shorten TG lag time in FX-deficient plasma (25 min). Moreover, the missense variants, potentially arising from the spontaneous suppression of the nonsense mutation p.W461X (TAG codon), do not support a functional impact of translational readthrough: the rFX-461Y (TAC) and rFX-461Q (CAG) variants showed very low secretion (Ag: 3.6±1.2% and 1.0±0.3%, respectively) and barely detectable TG activity (19 and 20 min lag times), as compared with 0.5% rFX-wt (14 min) and FX-deficient plasma (25 min). The residual TG capacity of the rFX-251P variant was FX-dependent, as indicated by Fondaparinux inhibition, which made TG undetectable, and by inhibition of TFPI (anti-TFPI aptamer), which shortened the TG lag time (from 26 to <24 min). Conclusions: Our experimental approaches, which contribute to the knowledge of the very severe FX deficiency forms, suggest that i) the p.W461X nonsense mutation, truncating the FX carboxyl-terminal region involved in interactions with prothrombin, produced very low levels of FX variants with negligible activity even after translational readthrough, and ii) the p.L251P missense mutation would contribute, during the foetal and perinatal life, to the trace FX levels and the associated minimal FX-dependent TG capacity. This residual function is candidate to shape the life-threatening, but non-perinatally lethal, phenotype of the proposita.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
