A Missense Mutation in γ-Glutamyl Carboxylase Gene Causes Combined Deficiency of All Vitamin K-Dependent Blood Coagulation Factors

VITAMIN K IS A NECESSARY cofactor for the hepatic carboxylation of glutamic acid residues in a number of proteins, including the procoagulants factors II, VII, IX, and X; the anticoagulants protein C and protein S; and other proteins such as osteocalcin and matrix Gla protein. This carboxylation is required for normal hemostasis, because it enables calcium binding and attachment of the procoagulants and anticoagulants to phospholipids.1 2 

γ-Glutamyl carboxylase is an integral membrane microsomal enzyme located in the rough endoplasmic reticulum. It carboxylates glutamate residues located in the Gla domain of vitamin K-dependent coagulation factors.3,4 The carboxylation reaction is dependent on reduced vitamin K (KH₂), which is converted to vitamin K epoxide during carboxylation, and must be regenerated by the vitamin K epoxide reductase for carboxylation to continue.5 

Hereditary combined deficiency of vitamin K-dependent procoagulants is a rare bleeding disorder that has been reported in only a few patients.6-13 Deficiency of the anticoagulants protein C and protein S has been reported in some of these patients.12 13 Theoretically, this disorder may stem from functional deficiency of either the γ-glutamyl carboxylase or the vitamin K epoxide reductase.

We have previously reported on an offspring of consanguinous marriage in a kindred with hereditary deficiency of all vitamin K-dependent procoagulants and anticoagulants.12 Normal epoxide reductase function was demonstrated by undetectable vitamin K epoxide serum levels. Impairment of Gla-dependent calcium binding was suggested by cross-immunoelectrophoresis studies of prothrombin. Therefore, we suggested at that time that the abnormality in the kindred resulted from γ-glutamyl carboxylase deficiency and speculated the inheritance to be autosomal recessive. Over the past 7 years we have identified 3 additional siblings in this kindred with the same deficiency.

Human γ-glutamyl carboxylase cDNA has recently been isolated and sequenced.14 It contains an open reading frame of 2274 nucleotides encoding a 758 amino acid polypeptide chain. The gene is located at 2p1.2,15 spans about 13 kb, and contains 15 exons.16 

We report here the identification of a T to G transversion at codon 394 of the γ-glutamyl carboxylase gene that results in the substitution of arginine for leucine. The mutation was identified in all 4 siblings with clinical and analytical findings of hereditary deficiency of all vitamin K-dependent coagulation factors. This is the first reported mutation in the γ-glutamyl carboxylase gene.

After approval of informed consent, citrated blood samples were obtained for coagulation assays and EDTA samples were obtained for DNA analysis.

Factors II, VII, IX, and X activities were assayed by a one-stage coagulation assay.12 Protein S:Ag (PS:Ag) was analyzed by electroimmunoassy, using the following antibody solutions. Tris Tricine (0.08 mol/L Tris and 0.02 mol/L Tricine) containing 0.2% goat anti-protein S antibodies that recognize total protein S. A 1% agarose (Seakem; FMC Bioproducts, Rockland, ME) was used in all electroimmunoassays and gels were run at room temperature. Polyclonal antibodies used were commercial (Stago, Asnieres, France). Protein C activity was assayed by chromogenic substrate (Stachrom-protein C; Stago). Normal range for each assay was determined by studying 30 normal individuals.

Patient no. 20, the 10th female offspring of consanguinous asymptomatic parents of an Arab origin, presented in 1982 shortly after birth with multiple ecchymoses and bleeding from puncture sites (Fig 1). An older sibling (hatched symbol) had died in infancy from uncontrolable umbilical bleeding. The prothrombin time (PT) of patient no. 20 was longer than 120 seconds, and the activated partial thromboplastin time (APTT) was longer than 180 seconds. No response to 1 mg of vitamin K was observed and symptoms subsided after plasma transfusion.12 Coagulation workup showed factor II:C to be 2 U/dL, factor VII:C to be 3 U/dL, factor IX:C to be 8 U/dL, and factor X:C to be 2 U/dL. At 6 weeks, she presented with intracerebral bleeding. After diagnosis of deficiency of all vitamin K-dependent procoagulants, therapy with subcutaneous vitamin K (10 mg) resulted in partial increase of factors II, VII, IX, and X plasma activity levels (Table 1). Study of natural anticoagulants showed that protein C activity was 45 U/dL and protein S:Ag was 34 U/dL. Weekly subcutaneous vitamin K administrated at a dose of 10 mg during the past 14 years was successful in preventing bleeding, except for one episode of hemarthrosis and another episode of epistaxis after dilantin therapy.12 

Patient no. 21, the first female offspring of consanguinous asymptomatic parents, presented at 5 months with knee hemarthrosis and prolonged APTT and PT, factor II:C of 24 U/dL, factor VII:C of 23 U/dL, factor IX:C of 8 U/dL, and factor X:C of 20 U/dL, PC activity of 42 U/dL, and PS:Ag of 35 U/dL. After diagnosis of deficiency of all vitamin K-dependent procoagulants and anticoagulants, weekly therapy with vitamin K (10 mg subcutaneously) was initiated, resulting in an increase in coagulation factor levels (Table 1), keeping an international normalized ratio (INR) of 2.0 to 3.5 without further significant bleeding during the 7 years of follow-up.

Patient no. 22, a male neonate, and patient no. 23, a female newborn, were examined shortly after birth and were found to have deficiency of all vitamin K-dependent procoagulants and anticoagulants. Therapy with 10 mg of vitamin K administered weekly subcutaneously was successful in preventing bleeding episodes during a follow-up of 5 and 4 years, respectively. Thus, chronic weekly administration of vitamin K resulted in a stable increase of procoagulant and anticoagulant levels (Table 1) and successfully prevented bleeding during a follow-up period of 30 patient years.

None of the patients had an increase in serum liver enzymes, malabsorption, or any other clinical findings suggestive of vitamin K deficiency. In addition, none of the 4 siblings had skeletal abnormalities by x-ray imaging.

Genomic DNA was isolated from the peripheral blood of 23 members of the kindred and 170 unrelated controls (135 from the Spanish population and 35 from the Israeli population).

For the construction of haplotypes, L394R mutation and the following intragenic polymorphisms were included: microsatellite of intron 6,EcoRI polymorphism, coding sequence polymorphism at nucleotide 8779 (exon 8), and silent polymorphism in exon 9. Protocols to perform analysis of intragenic polymorphisms were as described.16 

The 15 exons and intron/exon flanking sequences of the γ-glutamyl carboxylase gene were screened for mutations. All functionally important fragments of the gene were included and both strands were sequenced. The screening consisted of PCR amplification and further direct sequencing of amplified products using a commercial kit, according to the manufacturer’s instructions (Amersham, Arlington Heights, IL). The primers and PCR conditions for each exon are collected in Table 2.

We designed a specific PCR approach for the analysis of L394R mutation. First, we amplified a 810-bp fragment with primers E-IX-5 and E-X-3 (Table 2), including exons 9 and 10. The reaction mixture contained in a volume of 25 μL the following: 50 ng of genomic DNA, 400 ng of each primer, 200 μmol/L dNTPs, 1.5 mmol/L MgCl₂, 2.5 μL of 10× buffer, and 1 U of Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany). The mixtures were overlaid with 20 μL of mineral oil. We performed 30 cycles of amplification with the following temperature profile: denaturation at 94°C for 45 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 1 minute.

The amplified fragment, diluted 1/10, was used as template for a second PCR. The second fragment (129 bp) was amplified with the primers IX-mut (5′-TAT AAC AAC TGG ACA AAT GAG C-3′) and 1370C (5′-CCC AGG GTT AAG GTA GCC-3′). The primer IX-mut includes a nucleotide modification (underlined) over the genomic sequence of the gene. The A instead of G at that specific site of the primer, along with the normal sequence of the gene, leads to the introduction of anAlu I site at PCR product from the normal allele (AGCT), but not from the mutant allele (AGCG). The 25 μL mixture reaction contained the following: 0.1 μL from the first PCR product, 400 ng of each primer, 200 μmol/L dNTPs, 1.5 mmol/L MgCl₂, 5% dimethyl sulfoxide (DMSO), 2.5 μL of 10× buffer, and 1 U of Taq DNA polymerase (Boehringer Mannheim). Amplification was performed with 30 cycles of the following profile: denaturation at 94°C for 1 minute, annealing at 55°C for 45 seconds, and extension at 72°C for 5 seconds.

The PCR product was digested with Alu I and subjected to electrophoresis in 4% agarose.

Normal and mutant HGC were expressed in Drosophila cells using the metallothionine promoter. DNA containing the human γ-glutamyl carboxylase cDNA and the hygromycin-resistant gene were cotransfected into S2 Drosophila cells with calcium chloride. Positive clones were selected with hygromycin at 150 μg/mL. For expression, the metallothionine promoter was induced with 500 μmol/L cupper sulphate when the cell density had reached about 5 million cells/mL. Twenty-four hours after induction, the cells were harvested, concentrated and mixed with a cocktail of protease inhibitors.17 For carboxylase assays, 3 million cells in 35 μL were lysed with 1.4% CHAPS/phosphatidyl choline at 10 mmol/L MOPS, pH 7.5, and 700 mmol/L NaCl on ice for 20 minutes. Reaction was performed at a total volume of 125 μL with 1.2 mmol/L FLEEL, 16 μmol/L proFIX, 820 mmol/L ammonium sulfate, 222 μmol/L reduced vitamin K, and 1.4 mmol/L CO₂and incubated at 25°C for 30 minutes. The samples were processed as previously described.17 For estimation of the relative amounts of normal and mutant carboxylase, samples were Western blotted and probed with an antibody. The first, RGS.His (Qiagen, Valencia, CA) was followed by a peroxidase-conjugated goat antimouse antibody (Jackson Laboratories, West Grove, PA), and the bands were visualized with Amersham’s ECL reagent.

To identify potential mutations in the γ-glutamyl carboxylase gene, the sequence of all exons and intron/exon borders was determined and the results were compared with the normal genomic sequence.16 The results of this analysis that were preliminarily reported at the ISTH Florence meeting18 showed that all patients were homozygous for a point mutation in exon 9, which resulted in the conversion of an arginine codon (CTG) to the leucine codon (CGG) at residue 394 (Fig 2A and B). There were no other nucleotide changes that would lead to an amino acid substitution. Exon 9 codes for a carboxylase domain showing some sequence similarity to cytochrome b and is completely conserved in human and bovine carboxylase. We have designed a PCR strategy for the screening of this mutation based on the introduction of an Alu I site in the amplified fragment from the normal allele but not from the mutated allele (Fig 2C). With this approach, 340 unrelated normal chromosomes were analyzed and the mutation was not found in any case. This PCR strategy along with haplotype analysis of the 4 known intragenic polymorphisms in the γ-glutamyl carboxylase gene was used in every member of the pedigree and confirmed 6 normal siblings, 13 heterozygotes, and the 4 homozygotes for the L394R mutation in the kindred (Fig 1). Carriers of the mutation showed no clinical or analytical alteration.

To address the question of whether the expressed, mutated protein was stable and was present in our assays in amounts similar to normal carboxylase, normal and mutant carboxylase were expressed in Drosophila cells.18 Both constructs had a histidine tag at their amino terminus, which had no effect on carboxylase activity. Drosophila cells are free of endogenous carboxylase activity and are therefore appropriate for comparison of carboxylase activity. Figure 3 shows that approximately equivalent amounts of carboxylase were present in extracts from normal and L394R carboxylase preparations. Table 3demonstrates that the mutation of leucine 394 to arginine results in an at least threefold reduction in carboxylase activity and demonstrates that the cause of the defect is truly carboxylase related.

The segregation pattern of the L394R mutation fits perfectly with the phenotype of the disease and confirms the suggested autosomal recessive pattern of inheritance for combined deficiency of the vitamin K-dependent coagulation factors in this family. Haplotype analysis provided further proof for the common origin of both alleles of the L394R mutation in affected patients.

Reported cases of mild or moderate combined deficiency of vitamin K-dependent procoagulants were usually diagnosed at an older age, when the patients presented with mucucutaneous or postsurgical bleeding.8-10 Most reports concern isolated cases with deficiency of all vitamin K-dependent coagulation factors.

The markedly low levels of vitamin K-dependent coagulation factors in patient no. 20 who presented as a neonate could partly result from the added effect of immaturity of neonatal liver.

Expression studies demonstrated at least threefold reduced activity of the L394R γ-glutamyl carboxylase. This fits nicely with the detectable plasma procoagulants levels at diagnosis and may explain why the L394R mutation that results in moderate to severe reduction of vitamin K-dependent coagulation factors levels is viable, in contrast to mutations that result in total abrogation of γ-glutamyl carboxylase expression.

Weekly subcutaneous administration of 10 mg vitamin K resulted in an increase of procoagulants levels in all 4 subjects with γ-glutamyl carboxylase L394R mutation. Although the increase was more pronounced in factor IX:C levels and less in factor X:C levels, it was sufficient for achieving hemostatic levels. In fact, during 30 patient years on vitamin K therapy, no major bleeding and only rare minor bleeding episodes were observed. Interestingly, a previously reported 2 siblings responded with total correction of plasma procoagulant levels after parenteral administration of vitamin K.9 

Recognition of the vitamin K-dependent coagulation factors by γ-glutamyl carboxylase is dependent on 18 amino acid propeptide at the N-terminal of the coagulation factor, which serves as a docking site for interaction with γ-glutamyl carboxylase.19-21Site-directed mutagenesis studies suggest that regions around residues 234, 406, and 513 define in part the propeptide binding site.22 The L394R mutation is in proximity to a propeptide binding site on γ-glutamyl carboxylase,23 suggesting the possibility of reduced propeptide binding. Theoretically, the observed increase in coagulation factor levels after high-dose vitamin K administration may be explained by an increased affinity or by an overcome of a normal or reduced affinity of vitamin KH₂ to the L394R γ-glutamyl carboxylase. However, γ-glutamyl carboxylase is a complicated enzyme with several substrates, and further experiments are required to elucidate the role of the L394R mutation on different aspects of carboxylation.

The enzymatic aspects of carboxylation have been characterized in Devon Rex cats and congenital deficiency of glutamyl carboxylase.24 Phenotypical expression of defective glutamyl carboxylase in affected cats is similar to the clinical phenotype of the L394R mutation. In that study, kinetic parameters showed a potential impaired recognition of the propeptide sequence in nascent vitamin K coagulation polypeptides.24 

The L394R mutation is the first reported naturally occurring mutation in the human γ-glutamyl carboxylase gene that is responsible for a combined deficiency of vitamin K-dependent coagulation factors. Identification of L394R mutation will allow future direct diagnosis of potential carriers of γ-glutamyl carboxylase deficiency. This will enable genetic counseling for a severe heritable bleeding disorder in this kindred with a high consanguinity rate.

The authors are grateful to Rosalia Lavado and Cochava Mahler for technical help.