title: Characterization of vitamin K-dependent carboxylase mutations that cause bleeding and nonbleeding disorders.
abstract: Vitamin K-dependent coagulation factors deficiency is a bleeding disorder mainly associated with mutations in γ-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 γ-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.
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC;
authors.forename: Jian-Ke
authors.initials: JK
authors.lastname: Tie
authors.affiliation: Instituto da Criança, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil;
authors.forename: Jorge D A
authors.initials: JD
authors.lastname: Carneiro
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC;
authors.forename: Da-Yun
authors.initials: DY
authors.lastname: Jin
authors.affiliation: Chromosome Genomic Medicine, São Paulo, Brazil; and.
authors.forename: Ciro D
authors.initials: CD
authors.lastname: Martinhago
authors.affiliation: R&D Group VitaK, Maastricht University, Maastricht, The Netherlands.
authors.forename: Cees
authors.initials: C
authors.lastname: Vermeer
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC;
authors.forename: Darrel W
authors.initials: DW
authors.lastname: Stafford
mesh_terms: D000015:Abnormalities, Multiple / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001780:Blood Coagulation Tests; D064113:CRISPR-Cas Systems; D002114:Calcinosis / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics; D019735:Carbon-Carbon Ligases / Q000235:genetics*; D002357:Cartilage Diseases / Q000235:genetics; D004252:DNA Mutational Analysis; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005260:Female; D017930:Genes, Reporter; D056726:Genetic Association Studies; D005838:Genotype; D057809:HEK293 Cells; D006228:Hand Deformities, Congenital / Q000235:genetics; D006470:Hemorrhage; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011666:Pulmonary Valve Stenosis / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D006475:Vitamin K Deficiency Bleeding / Q000235:genetics*; D000097568:Matrix Gla Protein
chemical_list: D002135:Calcium-Binding Proteins; D019735:Carbon-Carbon Ligases; D016326:Extracellular Matrix Proteins; D000097568:Matrix Gla Protein; C020660:glutamyl carboxylase