Compound heterozygosity of novel missense mutations in the gamma-glutamyl-carboxylase gene causes hereditary combined vitamin K–dependent coagulation factor deficiency

Abstract

Hereditary combined vitamin K-dependent (VKD) coagulation factor deficiency is an autosomal recessive bleeding disorder associated with defects in either the ␥-carboxylase, which carboxylates VKD proteins to render them active, or the vitamin K epoxide reductase (VKORC1), which supplies the reduced vitamin K cofactor required for carboxylation. Such deficiencies are rare, and we report the fourth case resulting from mutations in the carboxylase gene, identified in a Tunisian girl who exhibited impaired function in hemostatic VKD factors that was not restored by vitamin K administration. Sequence analysis of the proposita did not identify any mutations in the VKORC1 gene but, remarkably, revealed 3 heterozygous mutations in the carboxylase gene that caused the substitutions Asp31Asn, Trp157Arg, and Thr591Lys. None of these mutations have previously been reported. Family analysis showed that Asp31Asn and Thr591Lys were coallelic and maternally transmitted while Trp157Arg was transmitted by the father, and a genomic screen of 100 healthy individuals ruled out frequent polymorphisms. Mutational analysis indicated wild-type activity for the Asp31Asn carboxylase. In contrast, the respective Trp157Arg and Thr591Lys activities were 8% and 0% that of wildtype carboxylase, and their compound heterozygosity can therefore account for functional VKD factor deficiency. The implications for carboxylase mechanism are discussed. IntroductionHereditary combined vitamin K-dependent (VKD) factor deficiency is a bleeding disorder characterized by the reduced activities of the procoagulant factors II, VII, IX, and X and anticoagulant proteins C, S, and Z. [1][2][3][4][5][6][7][8] The inheritance of the disease is autosomal recessive and is due to mutations in the genes for either the ␥-carboxylase 9-12 or the vitamin K epoxide reductase (VKORC1). 13 The carboxylase converts clusters of Glus to ␥-carboxylated Glus (Glas) in the Gla domains of VKD proteins, which renders them active by generating a calcium-binding module that binds either to anionic phospholipids that become exposed on cell surfaces or to hydroxyapatite in the extracellular matrix. 14,15 The carboxylase uses reduced vitamin K (KH 2 ) as a cofactor to drive Glu carboxylation, and the KH 2 becomes oxygenated to a vitamin K epoxide (KO) product that must be recycled for continuous carboxylation. Recycling is accomplished by VKORC1, which is the target of anticoagulant therapy with coumarin derivatives like warfarin that block KH 2 regeneration and consequently inhibit VKD protein carboxylation. Both VKORC1 and the carboxylase are integral membrane enzymes that reside in the endoplasmic reticulum (ER), where the VKD hemostatic factors are modified during their secretion from the cell. The concerted action of these 2 enzymes can therefore explain why congenital defects in either the carboxylase or VKORC1 lead to combined functional deficiency of the VKD factors.The interactions between VKD proteins and the carboxylase are complex and not well understood. 16,17 All VKD prot...

Fatal hemorrhage in mice lacking γ-glutamyl carboxylase

“…Two compound heterozygous individuals were recently reported to exhibit skeletal and cardiac developmental abnormalities potentially related to ␥-carboxylase deficiency. 32 These observations suggest that partial activity of ␥-carboxylase is sufficient for embryonic developmental signaling. Of note, lowlevel expression of a prothrombin or factor V transgene rescues the prenatal as well as the midembryonic lethal phenotypes of prothrombin-and factor V-deficient mice, respectively, 10,50,51 consistent with the notion that a low level of thrombin generation is sufficient for normal development.…”

“…Partial deficiency of ␥-carboxylase has been reported in several human patients with VKCFD, [30][31][32] with the decrease in ␥-carboxylase activity resulting in combined deficiency of all of the vitamin K-dependent coagulation factors, though without any apparent fetal or embryonic loss. Two compound heterozygous individuals were recently reported to exhibit skeletal and cardiac developmental abnormalities potentially related to ␥-carboxylase deficiency.…”

“…26 Although patients with VKCFD exhibit variable levels of bleeding, abnormalities related to other ␥-carboxylase substrates are not generally observed. However, in a recent report, 32 2 affected patients were noted to have skeletal and cardiac developmental abnormalities possibly related to ␥-carboxylase deficiency. Of note, all the reported human mutations appear to be hypomorphic, with no individuals yet identified with complete ␥-carboxylase deficiency.…”

Co-Existent Pseudoxanthoma Elasticum and Vitamin K-Dependent Coagulation Factor Deficiency

“…The VKORC1 coding region was analyzed using primers described previously. 13 The entire coding region and intron/exon boundaries of GGCX, ABCC6, and VKORC1 were analyzed via direct sequencing using an Applied Biosystems 3730 Sequencer (Applied Biosystems, Foster City, CA). For GGCX sequences the nucleotide numbers are derived from cDNA (GenBank accession no.…”

Novel Insight into the Mechanism of the Vitamin K Oxidoreductase (VKOR)

“…The vitamin K oxidoreductase (VKOR) 3 supports carboxylation and consequent activation of vitamin K-dependent (VKD) proteins. Carboxylation occurs in the endoplasmic reticulum, where the ␥-glutamyl carboxylase converts clusters of Glu to carboxylated Glu (Gla), transforming the Gla domain of VKD proteins into a calcium-binding module that functions either at cell surfaces or in the extracellular matrix (1,2).…”