The interplay of differentially γ-carboxylated vitamin K dependent proteins constitute variable VKCFD1 phenotypes

Abstract

Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in &gamma;-Glutamyl carboxylase (GGCX). VKCFD1 patients are treated with high dose of vitamin K, which corrects the haemostatic phenotype in most cases. In addition, these patients often exhibit non-haemostatic phenotypes such as skin hyperlaxity, skeletal and cardiac abnormalities. These phenotypes are most likely developed due to the undercarboxylation of one or more vitamin K dependent proteins. However, it remained elusive how GGCX mutations affect &gamma;-carboxylation of VKD proteins and which undercarboxylated protein leads to the development of a specific phenotype. <br /> In this PhD thesis, I have used CRISPR/Cas9 engineered GGCX knockout HEK293T cells to evaluate the effect of 22 GGCX missense mutations on &gamma;-carboxylation of 9 different vitamin K dependent proteins with respect to various concentrations of vitamin K. For this evaluation a specific ELISA assay was established, where only functional, &gamma;-carboxylated VKD proteins were detected. Furthermore, a GGCX in silico model was generated, where molecular docking was performed to identify the vitamin K hydroquinone binding site. <br /> All GGCX mutations were categorized into responder and low responder mutations, thereby determining the efficiency of vitamin K supplementation. It was observed that all VKCFD1 patients have at least one vitamin K responsive GGCX allele that is able to &gamma;-carboxylate clotting factors sufficient for a viable phenotype. For non-haemostatic phenotypes, the findings highlight that the deficiency to &gamma;-carboxylate Gla-rich protein leads to skin hyper-laxity whereas facial dysmorphologies are caused by reduced &gamma;-carboxylated MGP. Moreover, the vitamin K hydroquinone binding site in GGCX was identified, where mutations within this site severely affect &gamma;-carboxylation efficiency. <br /> With these new structural and functional data, the clinical outcome of each VKCFD1 genotype can be predicted thus enabling optimized treatment strategies.