Factor VII (FVII) is the serine protease triggering blood coagulation. The deficiency of FVII is associated to variable bleeding tendency and its complete absence is virtually lethal. The mutational pattern of FVII is heterogeneous and mainly characterized by missense changes. The few nonsense mutations so far described in homozygous state, C72Stop and K316Stop, are associated to moderate or life-threatening symptoms, respectively. We investigated the R402Stop nonsense mutation, identified in the homozygous condition in two asymptomatic patients. FVII antigen and coagulant levels in patients’ plasma were 0,8% and 5% of normal, respectively, thus suggesting the presence of a truncated molecule (FVII-R402Stop, wild type stop codon at the 407 position), poorly secreted but with improved procoagulant activity. Functional FXa and Thrombin generation assays confirmed these observations in plasma. The deleted residues are located in the carboxyl-terminal region, which has been shown to be crucial for secretion of the highly homologous coagulation serine proteases PC and FIX. To investigate this issue, we expressed the naturally truncated FVII-402Stop and the FVII variants 403-406Stop. Similarly to the 402Stop, the rFVII-403Stop and rFVII-402Stop variants were poorly secreted (~1%). We found an inverse relationship between secreted protein levels in medium and the extent of the deletion for the rFVII-406Stop (50-60% of WT), rFVII-405Stop (15-20%) and rFVII-404Stop (9-12%). FXa generation as well as PT-based assays revealed a normal specific activity for the rFVII-406Stop, rFVII-405Stop, rFVII-404Stop variants. Intriguingly, upon concentration of conditioned media, the specific activity of the rFVII-402Stop appeared to be 2-3 fold higher than that of rFVII-wt, thus contributing to explain its association to an asymptomatic phenotype. Altogether these findings demonstrate the importance of the carboxyl-terminal region for FVII biosynthesis/secretion, and also support its participation in FVII function.
Association of the homozygous nonsense mutation R402X in coagulation factor VII with asymptomatic phenotype
BRANCHINI, Alessio;RIZZOTTO, Lara;CANELLA, Alessandro;PINOTTI, Mirko;BERNARDI, Francesco
2010
Abstract
Factor VII (FVII) is the serine protease triggering blood coagulation. The deficiency of FVII is associated to variable bleeding tendency and its complete absence is virtually lethal. The mutational pattern of FVII is heterogeneous and mainly characterized by missense changes. The few nonsense mutations so far described in homozygous state, C72Stop and K316Stop, are associated to moderate or life-threatening symptoms, respectively. We investigated the R402Stop nonsense mutation, identified in the homozygous condition in two asymptomatic patients. FVII antigen and coagulant levels in patients’ plasma were 0,8% and 5% of normal, respectively, thus suggesting the presence of a truncated molecule (FVII-R402Stop, wild type stop codon at the 407 position), poorly secreted but with improved procoagulant activity. Functional FXa and Thrombin generation assays confirmed these observations in plasma. The deleted residues are located in the carboxyl-terminal region, which has been shown to be crucial for secretion of the highly homologous coagulation serine proteases PC and FIX. To investigate this issue, we expressed the naturally truncated FVII-402Stop and the FVII variants 403-406Stop. Similarly to the 402Stop, the rFVII-403Stop and rFVII-402Stop variants were poorly secreted (~1%). We found an inverse relationship between secreted protein levels in medium and the extent of the deletion for the rFVII-406Stop (50-60% of WT), rFVII-405Stop (15-20%) and rFVII-404Stop (9-12%). FXa generation as well as PT-based assays revealed a normal specific activity for the rFVII-406Stop, rFVII-405Stop, rFVII-404Stop variants. Intriguingly, upon concentration of conditioned media, the specific activity of the rFVII-402Stop appeared to be 2-3 fold higher than that of rFVII-wt, thus contributing to explain its association to an asymptomatic phenotype. Altogether these findings demonstrate the importance of the carboxyl-terminal region for FVII biosynthesis/secretion, and also support its participation in FVII function.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
