Characterization of vitamin K–dependent carboxylase mutations that cause bleeding and nonbleeding disorders

Abstract

Key Points• CRISPR-Cas9-mediated GGCX knockout cell-based assay clarifies the correlation between GGCX genotypes and their clinical phenotypes.• A GGCX mutation decreases clotting factor carboxylation and abolishes MGP carboxylation, causing 2 distinct clinical phenotypes.Vitamin K-dependent coagulation factors deficiency is a bleeding disorder mainly associated with mutations in g-glutamyl carboxylase (GGCX) that often has fatal outcomes. Some patients with nonbleeding syndromes linked to GGCX mutations, however, show no coagulation abnormalities. The correlation between GGCX genotypes and their clinical phenotypes has been previously unknown. Here we report the identification and characterization of novel GGCX mutations in a patient with both severe cerebral bleeding disorder and comorbid Keutel syndrome, a nonbleeding malady caused by functional defects of matrix g-carboxyglutamate protein (MGP). To characterize GGCX mutants in a cellular milieu, we established a cell-based assay by stably expressing 2 reporter proteins (a chimeric coagulation factor and MGP) in HEK293 cells. The endogenous GGCX gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. Our results show that, compared with wild-type GGCX, the patient's GGCX D153G mutant significantly decreased coagulation factor carboxylation and abolished MGP carboxylation at the physiological concentration of vitamin K. Higher vitamin K concentrations can restore up to 60% of coagulation factor carboxylation but do not ameliorate MGP carboxylation. These results are consistent with the clinical results obtained from the patient treated with vitamin K, suggesting that the D153G alteration in GGCX is the causative mutation for both the bleeding and nonbleeding disorders in our patient. These findings provide the first evidence of a GGCX mutation resulting in 2 distinct clinical phenotypes; the established cell-based assay provides a powerful tool for studying the clinical consequences of naturally occurring GGCX mutations in vivo. (Blood. 2016;127(15):1847-1855 Introduction Vitamin K-dependent carboxylation is a posttranslational modification that converts specific glutamate (Glu) residues to g-carboxyglutamate (Gla) residues in vitamin K-dependent proteins. It is required for the functioning of numerous vitamin K-dependent proteins involved in a broad range of biological functions, including blood coagulation. Carboxylation is catalyzed by the enzyme g-glutamyl carboxylase (GGCX), which uses a reduced form of vitamin K (KH 2 ) as a cofactor. Concomitant with each glutamate modification, KH 2 is oxidized to vitamin K epoxide (KO). Because humans cannot synthesize vitamin K, KO must be converted back to KH 2 by the enzymes KO reductase and the as-yet unknown vitamin K reductase in a pathway known as the vitamin K cycle. 1Defects of vitamin K-dependent carboxylation have long been known to cause bleeding disorders, referred to as combined vitamin Kdependent coagulation factors deficiency (VKCFD). Patients with VKCFD have decreased levels of mul...

Functional Study of the Vitamin K Cycle Enzymes in Live Cells

“…The cDNA of GGCX or its mutants was cloned into mammalian expression vector pBudCE4.1-Met.Luc and transiently expressed in the GGCX-deficient dual-reporter cells as described in Section 3.3. Four hours posttransfection, the transfection medium was replaced by complete growth medium containing 11 μ M vitamin K and incubated for 48 h. Carboxylation activity was determined by measuring the level of carboxylated reporter proteins in the cell culture medium using sandwich-based ELISA (Tie et al, 2016). …”

“…Using this cell-based assay, we first studied GGCX mutations found in a child with severe cerebral bleeding and comorbid Keutel syndrome, a nonbleeding malady caused by functional defects of MGP (Tie et al, 2016). Clinical data showed that high doses of vitamin K administration improved the bleeding diathesis of the patient, but not the functional defect of MGP.…”

“…Menaquinone supplementation has been shown to improve extrahepatic vitamin K status, but to have no effect on thrombin generation in healthy subjects (Theuwissen et al, 2012). However, results from the GGCX-deficient cell-based assay show that both vitamin K and menaquinones can efficiently support the carboxylation of both coagulation factors and MGP by wild-type GGCX (Tie et al, 2016). The different contributions of phylloquinone and menaquinones to hepatic and extrahepatic carboxylation may therefore be caused by the way different tissue absorbs and utilizes these K vitamins.…”

Splice-Site Mutation of Exon 3 Deletion in the Gamma-Glutamyl Carboxylase Gene Causes Inactivation of the Enzyme

“…It is also possible that this splice-site mutation could result in several variant mRNA transcripts of GGCX, as has previously described for other genes (Gallinaro et al , 2006; Kallabi et al , 2015). The affected patients reported by Kariminejad et al (2014) have normal coagulation factor carboxylation, but defects in their MGP carboxylation; it is therefore possible that some of the mRNA transcript may produce a GGCX mutant protein that causes MGP-carboxylation defects but has a smaller effect on coagulation factor carboxylation, as we have previously observed in a VKCFD patient (Tie et al , 2016). …”

“…This approach, unlike the traditional in vitro GGCX activity assay, allows us to assess the functionality of GGCX using its natural protein substrates in a cellular milieu that requires the enzyme to interact with its physiologic components for VKD carboxylation (Tie et al , 2016). Our result shows that the GGCX-Δex3 mutant abolished carboxylation activity for both reporter proteins (Figure 1a).…”