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Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4554-4559
RAPID COMMUNICATION
A Missense Mutation in  -Glutamyl Carboxylase Gene Causes Combined
Deficiency of All Vitamin K-Dependent Blood Coagulation Factors
By
Benjamin Brenner,
Beatriz Sánchez-Vega,
Sheue-Mei Wu,
Naomi Lanir,
Darrel W. Stafford, and
Jesus Solera
From the Thrombosis and Hemostasis Unit, Institute of Hematology,
Rambam Medical Center, and Bruce Rappaport Faculty of Medicine,
Technion, Haifa, Israel; the Unit of Molecular Genetics, S
Biochemistry, Hospital La Paz, Madrid, Spain; and the Department of
Biology, University of North Carolina-Chapel Hill, Chapel Hill, NC.
 |
ABSTRACT |
To identify potential mutations in the  -glutamyl carboxylase
gene, the sequence of all exons and intron/exon borders was determined
in 4 patients from a consanguineous kindred with combined deficiency of
all vitamin K-dependent procoagulants and anticoagulants and
results were compared with normal genomic sequence. All 4 patients were
homozygous for a point mutation in exon 9 that resulted in the
conversion of an arginine codon (CTG) to leucine codon (CGG) at residue
394. Screening of this mutation based on introduction of Alu I
site in amplified fragment from normal allele but not from the mutated
allele showed that 13 asymptomatic members of the kindred were
heterozygous for the mutation. The mutation was not found in 340 unrelated normal chromosomes. The segregation pattern of the mutation
which is the first reported in the -glutamyl carboxylase gene fits
perfectly with phenotype of the disorder and confirms the suggested
autosomal recessive pattern of inheritance of combined deficiency of
all vitamin K-dependent procoagulants and anticoagulants in
this kindred. The mutated carboxylase protein expressed in Drosophila
cells was stable but demonstrated threefold reduced activity compared
with WT carboxylase, confirming that the L394R mutation results in a
defective carboxylase.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
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
-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 -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 -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 -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 -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 -glutamyl carboxylase gene.
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MATERIALS AND METHODS |
Blood collection.
After approval of informed consent, citrated blood samples were
obtained for coagulation assays and EDTA samples were obtained for DNA
analysis.
Coagulation assays.
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.
Case reports.
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
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| Fig 1.
Pedigree of G family showing the segregation of the
mutation. Dashed lines indicate undefined number of generations.
Haplotypes are built with L394R mutation and intragenic polymorphisms.
Only haplotypes of members of the family available for the study are
shown. Affected subjects, carrier subjects, and unaffected subjects are
indicated by solid symbols, half-solid symbols, and open symbols,
respectively. The member indicated with a hatched symbol died of
bleeding.
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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 samples.
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).
Haplotype analysis.
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
Polymerase chain reaction (PCR) amplification and direct sequencing
of exons.
The 15 exons and intron/exon flanking sequences of the -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.
Analysis of L394R mutation.
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 an
Alu 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.
Expression studies.
Normal and mutant HGC were expressed in Drosophila cells using the
metallothionine promoter. DNA containing the human -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 CO2
and 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.
| |
RESULTS |
Identification of the mutation.
To identify potential mutations in the -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 -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.
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| Fig 2.
Identification of L394R mutation. (A) Schematic
representation of human -glutamyl carboxylase gene structure showing
the situation of exons. In detail is a fragment of exon 9 sequence
containing the nucleotide substitution at codon 394. The transversion T
to G (underlined) at that position causes a Leucine to Arginine
replacement in the protein. (B) Direct sequencing of the genomic DNA
from one patient depicts homozygosity for the mutation. (C) Analysis of
L394R mutation by PCR. Electrophoresis of amplified DNA using the
mutated oligonucleotide designed to introduce an Alu I
restriction site in normal allele but not in mutant allele. Lane 1, normal control; lane 2, patient's DNA homozygous for the mutant
allele; lane 3, heterozygous pattern.
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Expression studies.
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 3
demonstrates 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.
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| Fig 3.
Ten microliters (an amount equal to that for enzymatic
assays) of extracts from WT and L394R -glutamyl carboxylase was
fractionated by reducing sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. After Western blotting, the proteins
were identified by luminescence from antibodies directed against the
histidine tag.
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DISCUSSION |
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 -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 -glutamyl
carboxylase expression.
Weekly subcutaneous administration of 10 mg vitamin K resulted in an
increase of procoagulants levels in all 4 subjects with -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
-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 -glutamyl carboxylase.19-21 Site-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 -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 -glutamyl carboxylase. However,
-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 -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 -glutamyl carboxylase deficiency. This will
enable genetic counseling for a severe heritable bleeding disorder in
this kindred with a high consanguinity rate.
| |
ACKNOWLEDGMENT |
The authors are grateful to Rosalia Lavado and Cochava Mahler for
technical help.
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FOOTNOTES |
Submitted July 2, 1998;
accepted October 6, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Benjamin Brenner, MD, Thrombosis and
Hemostasis Unit, Institute of Hematology, Rambam Medical Center, Haifa,
31096, Israel.
| |
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H. M. H. Spronk, R. A. Farah, G. R. Buchanan, C. Vermeer, and B. A. M. Soute
Novel mutation in the gamma -glutamyl carboxylase gene resulting in congenital combined deficiency of all vitamin K-dependent blood coagulation factors
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G. S. Begley, B. C. Furie, E. Czerwiec, K. L. Taylor, G. L. Furie, L. Bronstein, J. Stenflo, and B. Furie
A Conserved Motif within the Vitamin K-dependent Carboxylase Gene Is Widely Distributed across Animal Phyla
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V. P. Mutucumarana, D. W. Stafford, T. B. Stanley, D.-Y. Jin, J. Solera, B. Brenner, R. Azerad, and S.-M. Wu
Expression and Characterization of the Naturally Occurring Mutation L394R in Human gamma -Glutamyl Carboxylase
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C. S. Walker, R. P. Shetty, K. Clark, S. G. Kazuko, A. Letsou, B. M. Olivera, and P. K. Bandyopadhyay
On a Potential Global Role for Vitamin K-dependent gamma -Carboxylation in Animal Systems. EVIDENCE FOR A gamma -GLUTAMYL CARBOXYLASE IN DROSOPHILA
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P. K. Bandyopadhyay, J. E. Garrett, R. P. Shetty, T. Keate, C. S. Walker, and B. M. Olivera
From the Cover: gamma -Glutamyl carboxylation: An extracellular posttranslational modification that antedates the divergence of molluscs, arthropods, and chordates
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Top
Abstract
Introduction