GGCX variants leading to biallelic deficiency to γ-carboxylate GRP cause skin laxity in VKCFD1 patients.

γ-Glutamyl carboxylase (GGCX) catalyzes the γ-carboxylation of 15 different vitamin K dependent (VKD) proteins. Pathogenic variants in GGCX cause a rare hereditary bleeding disorder called Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1). In addition to bleedings, some VKCFD1 patients develop skin laxity and skeletal dysmorphologies. However, the pathophysiological mechanisms underlying these non-hemorrhagic phenotypes remain elusive. Therefore, we have analyzed 20 pathogenic GGCX variants on their ability to γ-carboxylate six non-hemostatic VKD proteins in an in vitro assay, where GGCX variants were expressed in GGCX-/- cells and levels of γ-carboxylated co-expressed VKD proteins were detected by a functional ELISA. We observed that GGCX variants causing markedly reduced γ-carboxylation of Gla rich protein (GRP) in vitro were reported in patients with skin laxity. Reduced levels of γ-carboxylated Matrix gla protein (MGP) are not exclusive for causing skeletal dysmorphologies in VKCFD1 patients. In silico docking of vitamin K hydroquinone on a GGCX model revealed a binding site, which was validated by in vitro assays. GGCX variants affecting this site result in disability to γ-carboxylate VKD proteins and hence are involved in the most severe phenotypes. This genotype-phenotype analysis will help to understand the development of non-hemorrhagic phenotypes and hence improve treatment in VKCFD1 patients.

GGCX mutations show different responses to vitamin K thereby determining the severity of the hemorrhagic phenotype in VKCFD1 patients.

BACKGROUND: Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in γ-glutamyl carboxylase (GGCX). VKCFD1 patients are treated life-long with high doses of vitamin K in order to correct the bleeding phenotype. However, normalization of clotting factor activities cannot be achieved for all VKCFD1 patients. OBJECTIVE: The current study aims to investigate the responsiveness to vitamin K for all reported GGCX mutations with respect to clotting factors in order to optimize treatment. METHODS: This study developed an assay using genetically engineered GGCX-/- cells, in which GGCX mutations were analyzed with respect to their ability to γ-carboxylate vitamin K dependent pro-coagulatory and anti-coagulatory clotting factors by ELISA. Additionally, factor VII activity was measured in order to proof protein functionality. For specific GGCX mutations immunofluorescent staining was performed to assess the intracellular localization of clotting factors with respect to GGCX wild-type and mutations. RESULTS: All GGCX mutations were categorized into responder and low responder mutations, thereby determining the efficiency of vitamin K supplementation. Most VKCFD1 patients have at least one vitamin K responsive GGCX allele that is able to γ-carboxylate clotting factors. In few patients, the hemorrhagic phenotype cannot be reversed by vitamin K administration because GGCX mutations on both alleles affect either structural or catalytically important sites thereby resulting in residual ability to γ-carboxylate clotting factors. CONCLUSION: With these new functional data we can predict the hemorrhagic outcome of each VKCFD1 genotype, thus recommending treatments with either vitamin K or prothrombin complex concentrate.

Menaquinone-7 Supplementation Improves Osteogenesis in Pluripotent Stem Cell Derived Mesenchymal Stem Cells.

Development of clinical stem cell interventions are hampered by immature cell progeny under current protocols. Human mesenchymal stem cells (hMSCs) are characterized by their ability to self-renew and differentiate into multiple lineages. Generating hMSCs from pluripotent stem cells (iPSCs) is an attractive avenue for cost-efficient and scalable production of cellular material. In this study we generate mature osteoblasts from iPSCs using a stable expandable MSC intermediate, refining established protocols. We investigated the timeframe and phenotype of cells under osteogenic conditions as well as the effect of menaquinone-7 (MK-7) on differentiation. From day 2 we noted a significant increase in RUNX2 expression under osteogenic conditions with MK-7, as well as decreases in ROS species production, increased cellular migration and changes to dynamics of collagen deposition when compared to differentiated cells that were not treated with MK-7. At day 21 OsteoMK-7 increased alkaline phosphatase activity and collagen deposition, as well as downregulated RUNX2 expression, suggesting to a mature cellular phenotype. Throughout we note no changes to expression of osteocalcin suggesting a non-canonical function of MK-7 in osteoblast differentiation. Together our data provide further mechanistic insight between basic and clinical studies on extrahepatic activity of MK-7. Our findings show that MK-7 promotes osteoblast maturation thereby increasing osteogenic differentiation.