Introduction: Vitamin K Dependent Coagulation Factor Deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in ɣ-glutamyl carboxylase (GGCX) which is often characterized by additional non-bleeding phenotypes including skin hyper-laxity or pseudoxanthoma elasticum (PXE) like phenotype. GGCX ɣ-carboxylates vitamin K dependent (VKD) proteins including blood clotting factors (e.g., FII) and non-coagulation proteins (e.g., BGLAP). Until now there are 26 GGCX missense mutations reported to cause VKCFD1. VKCFD1 patients are treated with vitamin K which reverses the coagulation defect in most cases.
The aim of this study is to characterize the effect of these GGCX mutations on VKD proteins to evaluate effective dose of vitamin K (K) needed for treatment.
Methods: A GGCX knockout HEK293T cell line was generated by CRISPR/Cas9 technology. The cDNAs of GGCX together with F2, F10, Growth Arrest Specific-6 (GAS6) or Osteocalcin (BGLAP) were cloned into a bicistronic vector. Cells were transfected with GGCX wt and mutants and treated with different K concentrations to determine ɣ-carboxylation by ELISA. Statistical analyses were performed using GraphPad Prism 7 software to impose dose–response curves to identify half maximal effective concentrations (EC50).
Results: Elevated K concentration increases ɣ-carboxylation of F2, F10, BGLAP, and GAS6 for R204C, V255M, S284P, or W493S as wt levels. Other mutations as R83P, L394R, or H404P revealed increased ɣ-carboxylation of the coagulation factors but not as high as wt, whereas mutant M174R, F299S, S300F show no recovery. Certain mutants show differential effect on ɣ-carboxylation for different VKD proteins as H404P and T591K can restore ɣ-carboxylation for F2 but not for BGLAP and GAS6, which is vice versa for R485P.
Conclusions: Our data suggests that patients harboring R204C, V255M, S284P or W493S will show reversible coagulation (and non-coagulation) phenotypes where therapy with K will lead to normal coagulation. Residual clotting factor activities can be achieved for R83P, L394R, H404P, W493S, T591K. Patients with mutation M174R, F299S, S300F will never reach physiological coagulation and ɣ-carboxylation of other VKD proteins indicating that catalytic activity is abolished. Non-coagulation phenotypes as observed for H404P and T591K can be potentially explained by reduced ɣ-carboxylation of BGLAP or GAS6.
