Characterization of GGCX mutations identified in VKCFD1 patients

S. Ghosh1, K. J. Czogalla1, K. Liphardt1, J. Müller1, K. Höning1, V. Hornung1,2, M. Watzka1, J. Oldenburg1 (1Bonn, Germany, 2Munich, Germany)

Bleeding Disorders - Clinical Studies
Date: 18.02.2017,
Time: 08:30 - 09:45

Objective: Vitamin K Dependent Coagulation Factor Deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in gamma-glutamyl carboxylase (GGCX) gene. GGCX carboxylates glutamate residues of vitamin K dependent (VKD) proteins, including blood clotting factors (e.g. FVII, Protein C). This modification is essential for the activity of VKD coagulation factors and thus for maintaining hemostasis. Until now there are 30 GGCX mutations reported to cause VKCFD1. These patients are treated with high dose of vitamin K which reverses the coagulation defect in most cases. The aim of this study is to characterize the effect of GGCX mutations on VKD coagulation factors and thus to evaluate the effective dose of vitamin K needed for appropriate patient treatment.

Methods: A GGCX knockout (KO) HEK 293T cell line was generated by CRISPR/Cas9 technology. The cDNAs of GGCX together with F7 or protein C were cloned into a bicistronic vector. GGCX mutations were introduced by site directed mutagenesis. KO cells were transfected with wild-type (wt) and mutant GGCX variants and treated with different vitamin K concentrations in order to determine half maximal effective concentrations (EC50). After 72 hours, cell culture supernatants were collected and activity of FVII was measured by an aPTT assay. Protein C activity was measured by thrombin generation assay. Finally, GGCX activity was determined by normalizing the FVII or protein C activity with GGCX antigen levels.

Results: CRISPR/Cas9 gene editing resulted in GGCX deficient cells with a deletion of 46 nucleotides in exon 8. Transfection of wt GGCX together with FVII or protein C led to sufficient activity of these reporter proteins dependent on the vitamin K level. GGCX variants harboring mutations W157R, S300F, R325Q, I532T have reduced FVII activity of 16%, 12%, 57%, and 2%, respectively compared to wt GGCX. Elevated concentrations of vitamin K led to increased FVII activity for the mutant R325Q similar to wt GGCX. However, this recovery was not seen for S300F and I532T. Protein C activities for co-transfected GGCX variants showed similar EC50 values as detected for FVII.

Conclusion: Our data are in accordance with reported patient phenotype. Hence, this approach will be helpful to deduce the molecular mechanism in VKCFD1 patients and to determine specific K doses to rescue the coagulation defect.