Introduction
Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1; OMIM #277450) is a rare hereditary bleeding disorder caused by pathogenic variations in γ-Glutamyl carboxylase (GGCX) gene (Watzka et al., 2014). GGCX is an enzyme located in the ER that converts specific glutamic acid residues (Glu) into γ-carboxyglutamic acid residues (Gla) in vitamin K dependent (VKD) proteins (Rishavy & Berkner, 2012; Vermeer, 1990). This post-translational modification is essential and renders VKD proteins to their active form. VKCFD1 patients are diagnosed with bleeding symptoms caused by decreased activity of VKD clotting factors. Seven out of 15 VKD proteins have functions in the coagulation cascade including coagulation factor II (FII), VII (FVII), IX (FIX), X (FX), Proteins C (PC), S and Z (Kurachi & Davie, 1982; Leytus, Foster, Kurachi, & Davie, 1986; Nelsestuen, 1999; Stenflo, Fernlund, Egan, & Roepstorff, 1974). In addition to the haemorrhagic phenotype (Brenner et al., 1998; Darghouth et al., 2006; Spronk, Farah, Buchanan, Vermeer, & Soute, 2000), several VKCFD1 patients were reported to develop additional non-haemorrhagic phenotypes such as Pseudoxanthoma elasticum (PXE)-like syndrome causing skin hyper-laxity and folds, cardiac abnormalities, and osseous phenotypes such as midfacial hypoplasia or Keutel-like syndrome which is characterized by brachytelephalangy of the fingers and facial dysmorphologies (De Vilder, Eva Y G, Debacker, & Vanakker, 2017 Jan 25). These additional phenotypes are most likely developed due to the under-carboxylation of the other non-haemostatic VKD proteins which include matrix Gla protein (MGP), upper zone of growth plate and cartilage matrix associated protein (UCMA/GRP), osteocalcin (BGLAP), proline-rich Gla proteins (PRGPs) 1 and 2, and transmembrane Gla proteins (TMGs) 3 and 4, and growth arrest specific 6 (GAS6) (Kulman, Harris, Xie, & Davie, 2001; Manfioletti, Brancolini, Avanzi, & Schneider, 1993; Poser, Esch, Ling, & Price, 1980; Price, Fraser, & Metz-Virca, 1987; Viegas et al., 2009; Viegas et al., 2015). These proteins have diverse functions where BGLAP, MGP, and GRP have role in the regulation of physiological calcification in bone or soft tissues (Lacombe & Ferron, 2015; Willems, Vermeer, Reutelingsperger, & Schurgers, 2014) . GAS6 is a ligand of TAM receptor (Tyro, Axl, and Mer) and has function in cell signaling f.e. proliferation and platelet aggregation (Berkner & Runge, 2004). The function of the four VKD transmembrane proteins PRGP1 and 2, TMG3 and 4 is not yet clear.
The mechanism of GGCX variants leading to the non-haemorrhagic phenotypes is unknown. Previous studies have evaluated the effect of few selected GGCX variants on γ-carboxylation of either MGP or BGLAP among all non-haemostatic proteins (Tie et al., 2016; Watzka et al., 2014). However, whether the under-carboxylation of other known VKD proteins contributes to the non-haemorrhagic phenotypes remains elusive. VKCFD1 patients are mainly treated with high dose of vitamin K for correcting the clotting factor activities, but it is unclear whether life-long treatment with vitamin K is sufficient to rescue non-haemorrhagic phenotypes. Therefore, to devise new treatment strategies in the future, first we have to determine which undercarboxylated VKD protein/s are leading to specific phenotypes.
In the present study, an extensive genotype-phenotype analysis was performed by evaluating the effect of 22 reported GGCX variants on γ-carboxylation of six non-haemostatic VKD proteins in aGGCX-/- HEK293T cell line. We identified that undercarboxylated GRP causes the PXE-like phenotype by correlating the γ-carboxylation status from our analysis with the existing patient data. The knowledge of causative VKD protein will help to treat VKCFD1 patients in the future with personalized medicines. Additionally, we performed a KH2 docking on a GGCX in silicomodel, on which we have identified the KH2 binding site. Pathogenic variants affecting this site showed either loss-of-function or extreme reduced ability to γ-carboxylate VKD proteins.