Bleeding and non-bleeding phenotypes in patients with GGCX gene mutations

Watzka, Matthias; Geisen, Christof; Scheer, Monika; Wieland, Regina; Wiegering, Verena; Dörner, Thomas; Laws, Hans‐Jürgen; Gümrük, Fatma; Hanalıoğlu, Şahin; Ünal, Şule; Albayrak, Davut; Oldenburg, Johannes

doi:10.1016/j.thromres.2014.07.004

Cited by 34 publications

(72 citation statements)

References 49 publications

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“…Genetic screening for GGCX variations in patients with vitamin K-related disorders has identified more than 30 naturally occurring mutations (Watzka et al, 2014). However, the correlation between these GGCX mutants and their clinical phenotypes is not clear.…”

Section: Functional Study Of Ggcx Using Crispr-cas9-mediated Gene mentioning

confidence: 99%

Functional Study of the Vitamin K Cycle Enzymes in Live Cells

Tie

Stafford

2017

Methods in Enzymology

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Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH2) is oxidized to vitamin K epoxide (KO). KO must be recycled back to KH2 by the enzymes vitamin K epoxide reductase and vitamin K reductase in a pathway known as the vitamin K cycle. Our current knowledge about the enzymes of the vitamin K cycle is mainly based on in vitro studies of each individual enzymes under artificial conditions, which are of limited usefulness in understanding how the complex carboxylation process is carried out in the physiological environment. In this chapter, we review the current in vitro activity assays for vitamin K cycle enzymes. We describe the rationale, establishment, and application of cell-based assays for the functional study of these enzymes in the native cellular milieu. In these cell-based assays, different vitamin K-dependent proteins were designed and stably expressed in mammalian cells as reporter proteins to accommodate the readily used enzyme-linked immunosorbent assay for carboxylation efficiency evaluation. Additionally, recently emerged genome-editing techniques TALENs and CRISPR-Cas9 were used to knock out the endogenous enzymes in the reporter cell lines to eliminate the background. These cell-based assays are easy to scale up for high-throughput screening of inhibitors of vitamin K cycle enzymes and have been successfully used to clarify the genotypes and their clinical phenotypes of enzymes of the vitamin K cycle.

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Section: Functional Study Of Ggcx Using Crispr-cas9-mediated Gene mentioning

confidence: 99%

Functional Study of the Vitamin K Cycle Enzymes in Live Cells

Tie

Stafford

2017

Methods in Enzymology

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“…The disease was coined VK-dependent clotting factor deficiency-1 (VKCFD1, OMIM#277450), an autosomal recessive disorder, characterized by a mild to severe bleeding tendency and a moderate predisposition to thrombotic events [13,20]. VKCFD1 was shown to be associated with skeletal (midfacial hypoplasia, reduced bone mass, chondrodysplasia punctata) or cardiac abnormalities (patent ductus arteriosus Botalli, septal closure defects) in some patients [13,21,22,23,24,25,26,27,28,29,30]. Next to VKCFD1, a second autosomal recessive coagulation factor deficiency exists, VKCFD2 (OMIM#607473), caused by VKORC1 (vitamin K epoxide reductase complex, subunit 1; OMIM*608547) mutations and is also characterized by a deficiency of all VK-dependent clotting factors.…”

Section: Introductionmentioning

confidence: 99%

GGCX-Associated Phenotypes: An Overview in Search of Genotype-Phenotype Correlations

Vilder

Debacker

Vanakker

2017

IJMS

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Gamma-carboxylation, performed by gamma-glutamyl carboxylase (GGCX), is an enzymatic process essential for activating vitamin K-dependent proteins (VKDP) with important functions in various biological processes. Mutations in the encoding GGCX gene are associated with multiple phenotypes, amongst which vitamin K-dependent coagulation factor deficiency (VKCFD1) is best known. Other patients have skin, eye, heart or bone manifestations. As genotype–phenotype correlations were never described, literature was systematically reviewed in search of patients with at least one GGCX mutation with a phenotypic description, resulting in a case series of 47 patients. Though this number was too low for statistically valid correlations—a frequent problem in orphan diseases—we demonstrate the crucial role of the horizontally transferred transmembrane domain in developing cardiac and bone manifestations. Moreover, natural history suggests ageing as the principal determinant to develop skin and eye symptoms. VKCFD1 symptoms seemed more severe in patients with both mutations in the same protein domain, though this could not be linked to a more perturbed coagulation factor function. Finally, distinct GGCX functional domains might be dedicated to carboxylation of very specific VKDP. In conclusion, this systematic review suggests that there indeed may be genotype–phenotype correlations for GGCX-related phenotypes, which can guide patient counseling and management.

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“…14,15 Genetic screening of patients with vitamin K-related disorders has found more than 30 naturally occurring mutations in GGCX. 16 It is not clear, however, why some mutations cause bleeding disorders while others cause nonbleeding syndromes. This lack of knowledge comes in part from the fact that our current understanding of GGCX's function was obtained from in vitro experimentation under artificial conditions, 17 which has limited usefulness in understanding the clinical consequences of GGCX mutations.…”

Section: Introductionmentioning

confidence: 99%

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

Tie

Carneiro

Jin

et al. 2016

Blood

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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...

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Bleeding and non-bleeding phenotypes in patients with GGCX gene mutations

Cited by 34 publications

References 49 publications

Functional Study of the Vitamin K Cycle Enzymes in Live Cells

Functional Study of the Vitamin K Cycle Enzymes in Live Cells

GGCX-Associated Phenotypes: An Overview in Search of Genotype-Phenotype Correlations

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

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