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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Compound heterozygosity of novel missense mutations in the
gamma-glutamyl-carboxylase gene causes hereditary combined
vitamin K–dependent coagulation factor deficiency
Dhouha Darghouth, Kevin W. Hallgren, Rebecca L. Shtofman, Amel Mrad, Youssef Gharbi, Ahmed Maherzi,
Radhia Kastally, Sophie LeRicousse, Kathleen L. Berkner, and Jean-Philippe Rosa
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. (Blood. 2006;108:1925-1931)
© 2006 by The American Society of Hematology
Introduction
Hereditary 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.1-8 The inheritance of the disease is autosomal
recessive and is due to mutations in the genes for either the
␥-carboxylase9-12 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 (KH2) as a cofactor to drive Glu carboxylation, and the KH2 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 KH2 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 proteins have a
domain, usually a propeptide, that confers high-affinity binding to
the carboxylase and consequent conversion of the multiple Glus to
Glas by a processive mechanism.18,19 Allosteric changes caused by
VKD substrate binding regulate carboxylation: Binding of the
propeptide and Glu residues to the carboxylase is reciprocally
modulated,20,21 and binding of both domains increases carboxylase
affinity for KH2.22 Consequently, VKD factor association with
carboxylase results in more efficient vitamin K utilization, which is
significant because the availability of KH2 appears to regulate
carboxylation.23-26 In the absence of VKD substrate binding,
KH2 epoxidation to KO does not occur,27 and this regulation
prevents the unfettered formation of highly reactive and undesirable
From the Laboratory of Hemostasis and Thrombosis, U689 Institut National de
la Santé et de la Recherche Médicale, Institut Fédératif de Recherche 139,
University Paris 7, Hôpital Lariboisière, France; Departments of Molecular
Cardiology and Molecular Genetics, Lerner Research Institute, Cleveland
Clinic Foundation, OH; Laboratory of Hematology, Hôpital Habib Thameur,
Tunis, Tunisia; and Department of Pediatrics, Hôpital Mongi Slim, La
Marsa, Tunisia.
data; and J.-P.R. and K.L.B. designed the research and cowrote the
manuscript.
Submitted December 28, 2005; accepted April 6, 2006. Prepublished online as
Blood First Edition Paper, May 18, 2006; DOI 10.1182/blood-2005-12-010660.
Reprints: Jean-Philippe Rosa, Laboratory of Hemostasis and Thrombosis,
U689 Institut National de la Santé Et de la Recherche Médicale, Hôpital
Lariboisière, 41 boulevard de la Chapelle, 75475 Paris Cedex 10, France;
e-mail: [email protected].
Supported by grants from the Tunisian Ministry of Superior Education, the
Groupe d’Etude de l’Hémostase et de la Thrombose and Société Française
d’Hématologie (D.D.), and the National Institutes of Health (HLB 55666)
(K.L.B.).
D.D., K.W.H., R.L.S., and S.L.R. performed the research; A. Mrad, Y.G., and
R.K. provided biologic assessment of the patient; A. Maherzi collected clinical
BLOOD, 15 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 6
K.L.B. and J.-P.R. contributed equally to this study.
The online version of this article contains a data supplement.
An Inside Blood analysis of this article appears at the front of this issue.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2006 by The American Society of Hematology
1925
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1926
BLOOD, 15 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 6
DARGHOUTH et al
vitamin K intermediates. At present, the residues that make up the
carboxylase active site to facilitate the reaction are largely unknown, due in part to the lack of a crystal structure or homology
with other proteins that might indicate functional residues. Structurefunction relationships for VKORC1 are even less well defined,
because the gene for this enzyme has only recently been
identified.13,28
Mutations associated with hereditary combined VKD coagulation factor deficiency are rare. The carboxylase is encoded by a
single gene,29 and only 3 naturally occurring missense mutations
have previously been identified: Leu394Arg,10 which is impaired in
both Glu binding and propeptide binding,30 Trp501Ser,9,11 which
shows decreased propeptide binding,31 and Arg485Pro,12 whose
functional analysis has not yet been reported. In all 3 cases, vitamin
K supplementation resulted in at least partial restoration of VKD
factor function, consistent with the effect of VKD protein binding
on carboxylase affinity for vitamin K. Two carboxylase gene
mutations in noncoding regions are also known that include a splice
site mutation in intron 212 and a short deletion affecting a putative
cis-acting element in the promoter of the gene.32 In the case of
VKORC1, only the missense mutation Arg98Trp has been identified,13
which appears to cause a large decrease in VKORC1 activity.
We report a case of combined deficiency in VKD hemostatic
factors that is associated with compound heterozygosity in the
carboxylase gene, which was identified in a Tunisian family. No
mutations were found in the VKORC1 gene. Sequence analysis
revealed 3 new heterozygous missense mutations, Asp31Asn,
Trp157Arg, and Thr591Lys, and functional analysis indicated that
the VKD factor deficiency phenotype is due to compound heterozygosity in Trp157Arg and Thr591Lys. The relevance of these
residues to carboxylase function is discussed.
Patient, materials, and methods
Coagulation assay
Blood samples collected by venipuncture into 0.4% sodium citrate, at a
ratio of 9:1, were centrifuged at 3000g for 20 minutes at 4°C before freezing
at ⫺20°C. The activities of factors II, V, VII, IX, and X were measured in
one-stage clotting assays using Diagnostica Stago (Parsippany, NJ) reagents. Antigen levels of factors II and IX were determined by an
enzyme-linked immunosorbent assay (ELISA). Protein C and S activities
were assayed by the chronometric method from Diagnostica Stago. The
prothrombin time (PT) and kaolin-activated partial thromboplastin time
(APTT) were performed using the Neoplastin CI5 or C.K. Prest kits (both
from Diagnostica Stago), respectively, on an STA instrument.
Case report
The proposita, the second female offspring of asymptomatic parents of
Tunisian origin, was admitted at 2 years of age with acute infection,
ecchymosis, severe epistaxis, and gingivorrhagia that required blood
transfusion. The patient presented with developmental retardation and
stunted growth (⫺3 or ⫺2 standard deviations for weight and size,
respectively) and exhibited facial dysmorphia that was likely due to skeletal
anomalies. Malabsorption was ruled out by a normal duodenal biopsy. Both
the PT and APTT were prolonged, and coagulation testing revealed
defective activities of the VKD coagulation factors but normal activity of
factor V (Table 1). Intramuscular injections of high doses of vitamin K (ie,
10 mg/d phylloquinone for 2 weeks) did not modify either the PT or APTT
of the proposita (Table 1). The coagulation values of her parents and a sister
were within the normal range; her brother appeared affected, consistent
with a severe bleeding episode from his lip that had required infusion of
PPSB (a plasma fraction enriched in the VKD factors II, VII, IX, and X)
when he was 1 year old. The brother was subsequently diagnosed for
Table 1. Coagulation factor values in the proposita
and family members
Factors
Patient
Brother
Mother
Father
Sister
II:C, %
9
14
119
97
86
V:C, %
87
47
74
85
97
VII:C, %
6
7
100
68
84
IX:C, %
7
—
92
85
—
116
X:C, %
5
7
82
109
II Ag, %
60
—
110
80
80
IX Ag, %
73
70
—
—
—
PC, %
⬍1
⬍1
89
112
88
PS, %
⬍1
⬍1
44
40
87
⬎ 60/11.5
12.7/11.5
13.7/11.5
13.7/11.5
PT, s
49.5/11.5
PT, %
10
PT, s*
48/11.5
⬍ 10
80
70
70
—
—
—
—
PT, %*
9
—
—
—
—
APTT, s
60/30
56/30
36/30
38/30
33/30
APTT, s*
60/30
—
—
—
—
AT, %
102
86
84
103
102
Coagulation (C) and antigen (Ag) levels are indicated as the percent of normal
values.
PC indicates protein C; PS, protein S; AT, antithrombin activity; and —, not done.
*The PT and APTT times were also measured after intramuscular phylloquinone
administration (2 weeks at 10 mg/d).
combined deficiency in VKD hemostatic factors (Table 1). At age 11, the
proposita developed heart failure due to incomplete closure of the
ventricular septum, and incomplete septal closure was also observed in the
affected brother (at 11⁄2 years) during a medical examination. An older
sibling died at 4 days old without any diagnosis.
Sequence analysis of the carboxylase and VKORC1 genes
The genomic DNA of the proposita and 4 members of her family was
isolated from frozen peripheral blood, as described.33 Blood collection was
achieved with informed written consent from all donors in accordance with
the Helsinki protocol. The study protocol was approved by the Ethics
Committee of Hôpital Habib Thamenr, Tunis, Tunisia. The 13-kb gene
encoding the carboxylase and 5-kb gene encoding VKORC1 were entirely
sequenced using the polymerase chain reaction (PCR) and subsequent
direct sequencing of the PCR products. The primers and PCR conditions for
the carboxylase and VKORC1 genes are summarized, respectively, in
Tables 2 and S1 (Table S1 is available on the Blood website; see the
Supplemental Table link at the top of the online article).
mRNA analysis
Reverse transcriptase–PCR was used to analyze the size and amount of
carboxylase mRNA in the proposita. Total RNA was isolated from platelets
and used as the template (20 ng) for first strand–specific cDNA synthesis in
a 2.5-␮L reaction mixture that contained PCR buffer (10 mM Tris [pH 8.3],
50 mM KCl), 5 mM MgCl2, 2.5 ␮M oligo(dT)16, 1 mM of each dNTP, 10
units of RNAse inhibitor, and 25 units of MuLV reverse transcriptase
(Applied Biosystems, Weiterstadt, Germany). The reaction mixture was
successively incubated for 10 minutes at room temperature, 30 minutes at
42°C, 10 minutes at 95°C, and 10 minutes at 4°C. The reaction product then
served as the template for PCR in a 12.5 ␮L reaction mixture containing
PCR buffer, 2 mM MgCl2, 0.15 ␮M of the forward and reverse primers
(Proligo, St Louis, MO), and 1.25 units of AmpliTaq gold DNA polymerase
(Applied Biosystems). PCR was conducted for 41 cycles (1 minute at 94°C,
1 minute at 66°C, and 1 minute at 72°C) followed by an extension step for
10 minutes at 72°C. Amplification products were then electrophoresed on
1.5% (wt/vol) agarose gels stained by ethidium bromide and were also
directly sequenced.
Screen for polymorphisms
The genomic DNA of 100 unrelated controls (50 each from the Tunisian or
French population) was isolated from frozen blood (Nucleospin Blood
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BLOOD, 15 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 6
NOVEL COMPOUND HETEROZYGOUS CARBOXYLASE MUTATIONS
1927
Table 2. Primers and PCR conditions used to sequence the carboxylase gene
Gene
region
Exon 1
Exon 2
Exon3/4/5
Exon 6
Exon 7
Exon 8
Exon 9/10
Exon 11
Exon 12
Exon 13
Exon 14
Exon 15
PCR conditions
94°C/1 min, TD 70/65°C/30 s, 2°Cn, 72°C/1 min (2% DMSO)
94°C/1 min, TD 61/55°C/30 s, 2°Cn, 72°C/1 min
94°C/1 min, 55°C/30 s, 72°C/1 min
94°C/1 min, 55°C/30 s, 72°C/1 min
94°C/1 min, TD 65/55°C/30 s, 2°Cn, 72°C/1 min
94°C/1 min, TD 65/55°C/30 s, 2°Cn, 72°C/1 min
94°C/1 min, TD 65/55°C/30 s, 2°Cn, 72°C/1 min
94°C/1 min, 55°C/30 s, 72°C/1 min
94°C/1 min, TD 52/48°C/30 s, 2°Cn, 72°C/1 min
94°C/1 min, TD 52/48°C/30 s, 2°Cn, 72°C/1 min
94°C/1 min, TD 52/48°C/30 s, 2°Cn, 72°C/1 min
94°C/1 min, TD 65/55°C/30 s, 2°Cn, 72°C/1 min
Primer
names
Primer sequences
E-1-U
GCGTCCTCAACTCGGCGTCACTC
E-1-L
CTCCACCTCAAATCAAAGAAATC
E-2-U
GAGCTGTTGGTGCAGTGATTTCT
E-2-L
AGAGATTGTCATTCTCCACTCT
E-3-U
CCAATGACCAACTCCCCTAT
E-5-L
TCCTCCCTCTGTCCTAAAAT
E-6-U
TGTAACTCAGGAGCATGGATTC
E-6-L
CATTACTGAGAGAGATGAGTCACCT
E-7-U
GCTGTGAATGTGCTTTGATGTG
E-7-L
AAGCCCCAGTCCTCTTATC
E-8-U
AGGCCCAGCCAAACTCCT
E-8-L
CTCACACTGACCCCATCC
E-9-U
GCTGATTCCCCTCTGTGCTG
E-10-L
AACCAGCTATGCCCACAAC
E-11-U
GGTGGCTGTGATGTCCTTAGAA
E-11-L
CCCCATGGCAGAGTGAAC
E-12-U
GCCATGGGGTGGGATGATGAAC
E-12-L
CAGGCAACTGACAAGGGA
E-13-U
AGAAAGAAGCCAAGAGTCAT
E-13-L
GGCTAGAACATCATTCATAACC
E-14-U
CTAGCTGGCAGAAGAGGAGTT
E-14-L
AGAATGGCAGGAAAAGATACC
E-15-U
GGCTGTTCCTACCCTATCC
E-15-L
ACCTCCCCTTCTGCTCACC
GGGCTCAAGCGAACCTC
P1
94°C/30 s, TD 65/55°C/30 s, 1°Cn, 72°C/30 s (1.5 mM MgCl2)
1U
379L
CTCCCCGACCCCATTAGT
P2
94°C/15 s, TD 65/55°C/30 s, 2°Cn, 72°C/1 min
284U
GCGTCCTCAACTCGGCGTCACTC
1124L
GGGCTCCACCTCAAATCAAAGAAATC
In1
94°C/15 s, 55°C/30 s, 72°C/1 min
969U
ACTGTAGTCTGAGGGGTTCTGG
2261L
TAGTCACATTTTGGGCTGGTTA
724U
GCAGAGCAATGGCGGTGTC
6 kb
94°C/15 s, TD 70/62°C/30 s, 68°C/4 min* (3 mM MgCl2)
6065L
AGGGAGGCAGCGGAGAGTGGT
AGATGTGCCCAGGATAGAT
ln5–6
94°C/30 s, 55°C/30 s, 72°C/30 s (1.5 mM MgCl2)
5812U
6511L
GGCAAGAATAAAATAAGGAG
4 kb
94°C/15 s, TD 66/58°C/30 s, 2°Cn, 68°C/3 min* (3 mM MgCl2)
6261U
TTCTTCCTTGGTGCCTGATACTGTC
10364L
AGCCTCTCCTCACTTTCCTCCATAC
3.5 kb
94°C/15 s, 60°C/30 s, 68°C/2.5 min (1 mM MgCl2)
10470U
GGGATGATGGTGGTAAAGGT
3⬘ NC
94°C/30 s, 55°C/30 s, 72°C/30 s (1.5 mM MgCl2)
13595L
GGAGGAGTGGGGGAGAGTAT
13321U
GAAGGGGAGGTAAAGTAAGAAT
13676L
AATCCTGGAGTAGACACAATCA
The gene regions correspond to the PCR fragments shown in Figure 1A. The positions within the gene are indicated by the oligonucleotide primer number (U, upper; L,
lower). PCR was performed for 30 cycles using the “touchdown” (TD) method: After a 94°C denaturation step, a hybridization step was performed (30 seconds, initial and final
temperatures are indicated) with 2°C temperature decrements (n) every 3 cycles. The last 15 cycles were performed at the final temperatures indicated. The products were
then extended at 72°C for 1 minute, except for the 3.5 kb, 4 kb, and 6 kb fragments, which were extended at 68°C for 2.5, 3, and 5 minutes, respectively.
NC indicates noncoding sequence.
*The extensions during the final 15 cycles of TD for the 4 kb and 6 kb fragments were 68°C for 5 and 4 minutes, respectively.
Quick Pure; Macherey-Nagel, Easton, PA) and screened for 3 carboxylase
mutations (Asp31Asn, Trp157Arg, and Thr591Lys) identified in the
proposita. Differential hybridization PCR was used to search for the
1235G⬎A mutation in exon 2 and the 11134C⬎A mutation in exon 13 that
corresponded to the amino acid mutations Asp31Asn and Thr591Lys,
respectively. Primer 5⬘ ACTCTCAACCAAATTGCTCCCA was used in
combination with primers 5⬘ TCAGGGCCCAGGCAGG or 5⬘ TCAGGGCCCAGGCAGA, which are specific for 1235G or A in exon 2. Amplification
generates a 179 bp fragment with the following conditions: 95°C for 1
minute, 62°C to 52°C for 30 seconds, and 72°C for 1 minute. The second set
of PCR reactions used primer 5⬘ GGGGATGATGGTGGTAAAGGTG with
either 5⬘ AAGAAGGGCTAGGTGATGTCGT or 5⬘ AAGAAGGGCTAGGTGATGTCTT, which are specific for 11134C or A in exon 13, to amplify a
686 bp fragment using the following conditions: 95°C for 1 minute, 72°C to
66°C for 30 seconds, and 72°C for 1 minute. The resulting fragments were
analyzed by 1.5% (wt/vol) agarose gel electrophoresis. The third mutation,
1811T⬎C in exon 4, which corresponds to the amino acid mutation
Trp157Arg, was screened in healthy individuals by PCR followed by
sequencing. The primers 5⬘ TGTGCCGCTTCCCCTTGCTG and 5⬘ GC-
CATTTTGCTTTGTGTCAT were used to generate a 753 bp PCR fragment
(94°C for 1 minute, 65°C to 55°C for 30 seconds, and 72°C for 1 minute),
which was then directly sequenced.
Construction and analysis of mutant carboxylase enzymes
Generation of carboxylase cDNAs with the individual mutations
Asp31Asn, Trp157Arg, or Thr591Lys was accomplished using the
Quick Change XL site-directed mutagenesis kit (Stratagene, La Jolla,
CA) according to the manufacturer’s instructions and using the following oligonucleotide primers: Asp31Asn: (upper) GATCTCAGGGCCCAGGCAGAACAGCCGAATAGGGAAACTCTTG and (lower) CAAGAGTTTCCCTATTCGGCTGTTCTGCCTGGGCCCTGAGATC; Trp157Arg:
(upper) CTCCTGGACAAGACATCACGGAACAACCACTCCTAT and (lower)
ATAGGAGTGGTTGTTCCGTGATGTCTTGTCCAGGAG; Thr591Lys: (upper) GTACCATAAGGTGTATAAGACATCACCTAGCCCTTCTTGC and
(lower) GCAAGAAGGGCTAGGTGATGTCTTATACACCTTATGGTAC.
Briefly, a full-length carboxylase cDNA34 was hybridized to the
indicated primers and subjected to Pfu polymerase (Stratagene) extension.
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1928
DARGHOUTH et al
After 20 cycles of amplification, parental wild-type strands were digested
with the methyl-sensitive restriction enzyme DpnI, and amplified mutant
strands were self-ligated and used to transform DH5␣ Escherichia coli. A
BamHI fragment containing the full-length cDNA was then isolated and
subcloned into the baculovirus expression vector pBacPak9 (Invitrogen),
and the carboxylase cDNA was entirely sequenced on both strands. The
plasmid was cotransfected into SF21 insect cells with Bsu36I-digested viral
DNA (BacPAK6; Clontech, Palo Alto, CA), and virus was isolated from
individual plaques and amplified. Virally infected SF21 cells were screened
for carboxylase by preparing lysates, as before,35 followed by Western
analysis using an antibody against the C terminus of the carboxylase.34 Two
independent viral isolates of each mutant were subsequently tested for
carboxylase activity by measuring [14C]-CO2 incorporation into the peptide
Boc-Glu-Glu-Leu-Ome 35 and for epoxidase activity by measuring conversion of KH2 to KO.36
Results
Combined deficiency in VKD coagulation factors in the
proposita is associated with compound heterozygosity of
mutations Asp31Asn, Trp157Arg, and Thr591Lys in the
carboxylase gene
Combined deficiency in VKD factors in a 2-year-old girl was
diagnosed based on frequent bleeding, prolonged PT and APTT,
and severe defects in the activities of both procoagulant and
anticoagulant VKD factors (Table 1). Factors II, VII, IX, and X and
proteins C and S showed activities ranging from less than 1% to
9%, while the activity of factor V, which is not a VKD protein, was
normal. The combined deficiency prompted us to analyze the genes
for both VKORC1 and the carboxylase. Sequence analysis of the
entire 5 kb VKORC1 gene did not detect any mutations in the
proposita (Table S1). In contrast, the 13 kb carboxylase gene
sequence (Figure 1A) revealed 3 heterozygous base substitutions: a
1235G⬎A transition (corresponding to 177G⬎A in the mRNA) in
exon 2, a 1811T⬎C transition (555T⬎C in the mRNA) in exon 4,
and a 11134C⬎A transversion (1858C⬎A in the mRNA) in exon
13 (Figure 1B-D). These same heterozygous substitutions were
found in carboxylase mRNA isolated from platelets and analyzed
by reverse transcriptase–PCR, indicating comparative expression
levels of both alleles. The substitutions predicted the 3 amino acid
BLOOD, 15 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 6
Figure 2. The Tunisian family pedigree shows cosegregation of the 3 carboxylase mutations with VKD coagulation factor deficiency. Affected and unaffected
subjects are indicated by solid or open symbols, respectively. The arrow points to the
proposita. The female indicated by the hatched circle was dead in early infancy for
unknown reasons. Mutated residues are in red, and the chromosomal colors indicate
maternal (blue) or paternal (black) inheritance.
mutations Asp31Asn, Trp157Arg, and Thr591Lys (Figure 1B-D),
none of which have been reported to date.
These 3 mutations were not found in the genomes of 100
healthy French or Tunisian individuals, thus ruling out frequent
polymorphisms. Two known polymorphisms in the proposita’s
carboxylase gene were identified that have previously been reported37: 9167T⬎C in exon 9, which is a silent polymorphism, and
8762G⬎A in exon 8, which is present in roughly half of the
population and results in a Gln325Arg substitution that does not
affect activity (K.W.H., R.L.S., and K.L.B., unpublished results, June
2005). In addition, a novel polymorphism at position 5338 in intron 4-5
was identified that corresponds to a homozygous G⬎A transition. To
determine whether this substitution affected either the size or amount of
carboxylase mRNA, reverse transcriptase–PCR products of platelet
RNA from the proposita were compared with that of healthy
subjects. This analysis did not reveal any differences, indicating
that this intron mutation did not contribute to the defect in VKD
factor function. The combined results, then, strongly indicate that
the VKD factor deficiency in the proposita is highly likely to be
associated with coding mutations in the carboxylase gene.
The combined deficiency in the function of VKD factors
cosegregates with mutations Asp31Asn, Trp157Arg,
and Thr591Lys
The analysis of family members (Figure 2) showed that the mother,
who was asymptomatic, was a compound heterozygote for the
mutations Asp31Asn and Thr591Lys like her affected daughter, the
Figure 1. PCR strategy and identification of carboxylase coding substitutions. (A) The entire carboxylase
gene was sequenced using PCR fragments generated
with the primers that are indicated in Table 2. The doubleheaded arrows indicate exonic (E1-E15) and other fragments (whose nucleotide positions at the 5⬘ and 3⬘ ends
are shown below the lines) whose names are the same as
those used in Table 2. (B-D) Electropherograms corresponding to the 3 new mutations identified in this work are
shown: (B) Asp31Asn in exon 2, (C) Trp157Arg in exon 4,
and (D) Thr591Lys in exon 13. The positions of the
mutations are indicated on top of the electropherograms
using the mRNA numbering for the carboxylase (GenBank
accession M81592). Wild-type and mutant nucleotides are
indicated on top of the corresponding peaks, and wild-type
(black) and mutant (red) nucleotide and amino acid sequences are shown below the electropherogram.
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BLOOD, 15 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 6
proposita. However, the mother was homozygous for the wild-type
Trp157 residue, suggesting that Asp31Asn and Thr591Lys were
carried by the same allele. This interpretation was confirmed by the
analysis of the father, who was unaffected as well and homozygous
for the wild-type residues Asp31 and Thr591 but heterozygous for
the mutant residue Trp157Arg. We thus conclude that the proposita is
a compound heterozygote, with Asp31Asn and Thr591Lys being
coallelic and of maternal origin and with Trp157Arg being derived from
the paternal allele. Analysis of the proposita’s siblings supported this
conclusion: The unaffected sister tested normal for both alleles while the
clinically affected brother was identical to the proposita, carrying all 3
mutations. Thus, the segregation pattern of the Asp31Asn, Thr591Lys,
and Trp157Arg mutations confirmed the autosomal recessive inheritance of combined deficiency of the VKD coagulation factors in this
family (Figure 2).
Functional analysis of the carboxylase mutants
Wild-type and mutant carboxylases were expressed in insect cells,
which do not contain endogenous activity but synthesize active
enzyme that is exogenously introduced.38 Similar levels of carboxylase protein were observed following infection of insect cells with
baculoviruses containing either wild-type or mutated carboxylases
(Figure 3), indicating that the mutations did not impair expression.
When tested for activity in a carboxylase assay that measures
[14C]-CO2 incorporation into a Glu-containing peptide substrate,
the Asp31Asn mutant showed wild-type activity (Table 3). In
contrast, the Trp157Arg mutant exhibited activity that was only 8%
to 10% that of wild-type, and the Thr591Lys mutant, as well as a
double mutant containing both the Thr591Lys and Asp31Asn
substitutions, did not show detectable activity. The carboxylase
drives Glu carboxylation through epoxidation of KH2 to KO. In the
wild-type enzyme these 2 activities are coupled (ie, showing an
approximate 1:1 stoichiometry).39 To determine if any of the
mutants showed differences in epoxidation versus carboxylation,
epoxidase activity was measured. The ratio of epoxidase to
carboxylase activity in Asp31Asn and Trp157Arg was similar to
that of wild-type enzyme, indicating that the coupling mechanism
was unaffected. The actual epoxidase value for Trp157Arg was
substantially less than that of wild-type enzyme, and neither
Thr591Lys nor Thr591Lys/Asp31Asn showed detectable activity (Table
3). The Trp157Arg and Thr591Lys mutations, then, can account for
the combined deficiency in the function of VKD factors.
Discussion
We report the identification of 3 novel missense mutations in the
carboxylase gene associated with combined deficiency of hemostatic VKD factors. The carboxylase mutations could not be
accounted for by frequent polymorphisms, and no mutations in the
Figure 3. Expression of carboxylase mutants in insect cells. Lysates from insect
cells mock-infected or infected with baculoviruses containing either wild-type or
mutant carboxylases were analyzed with Western analysis using anti–C-terminal
carboxylase antibody.
NOVEL COMPOUND HETEROZYGOUS CARBOXYLASE MUTATIONS
1929
Table 3. Trp157Arg and Thr591Lys show impaired carboxylase
and epoxidase activities
Sample
Epoxidase
activity,
pmol/h ⴛ 10ⴚ2, %
Carboxylase
activity,
pmol/h ⴛ 10ⴚ2
Epoxidationcarboxylation
ratio
Wild type
27.2
97
25.0
1.1
Wild type
28.1
100
24.7
1.1
Asp31Asn
29.1
104
26.2
1.1
Asp31Asn
29.1
104
27.0
1.1
Trp157Arg
2.2
8
1.8
1.2
Trp157Arg
2.7
10
1.8
1.5
Thr591Lys
ND
0
0.2
—
Thr591Lys
ND
0
0.2
—
Asp31Asn/Thr591Lys
ND
0
0.2
—
Asp31Asn/Thr591Lys
ND
0
0.2
—
Mock
ND
0
0.2
—
Mock
ND
0
0.2
—
Lysates containing equivalent amounts of wild-type or mutant carboxylase
protein (as determined by a quantitative Western analysis) were assayed for
conversion of KH2 to KO (epoxidase activity) or for incorporation of [14C]-CO2 into a
Glu substrate (carboxylase activity). Epoxidase activity was quantitated by reference
to a KO standard, and carboxylase activity was determined by using a specific activity
of 50 cpm/pmol for [14C]-CO2.
ND indicates not detected; —, not applicable.
gene for VKORC1 were detected. The proposita is unusual with
respect to the previously identified patients with carboxylase
missense mutations9-12 because of the compound heterozygosity in
the mutations. The mutations Trp157Arg and Thr591Lys, which were
derived from the father and mother, respectively (Figure 2), resulted in
impaired carboxylation in vitro (Table 3) that can account for the
combined VKD factor deficiency. The carboxylase also modifies VKD
proteins with nonhemostatic functions that include bone development,
apoptosis, and signal transduction.15 While the activities of these
nonhemostatic VKD proteins are not typically screened when combined
VKD factor deficiency is suspected, they are also very likely affected by
carboxylase defects. The proposita and her affected brother both
appeared to have skeletal abnormalities, which could be due at least in
part to functional deficiencies in VKD proteins like osteocalcin and
matrix Gla protein.40 Incomplete closure of the ventricular septum was
also observed in both siblings, which appears to be due to poor VKD
protein carboxylation, because anatomic abnormalities associated with
other syndromes that affect ventricular septal closure41 were not
observed.
The mutations in the proposita are informative in understanding
how carboxylase residues contribute to function, which currently is
not well understood. As shown in Figure 4A, all 3 of the newly
identified mutations are located away from the 3 previously known
missense mutations that cause combined VKD factor deficiency,
and 2 residues lie within regions of the carboxylase that have been
functionally implicated by cross-linking studies using Glu or
propeptide sequences. A third functionally important region, the
vitamin K binding site, has not yet been identified but must be
closely juxtaposed to the Glu binding site in the 3-dimensional
structure to allow the chemical reactivity between vitamin K and
Glu that ultimately leads to Glu carboxylation. The carboxylase
active site, then, is unusual in accommodating the binding of both a
hydrophilic substrate (Glu) and a hydrophobic cofactor (vitamin
K), and the structure of the carboxylase reflects this property: The
C-terminal half is hydrophilic and located in the lumen of the ER46
while most of the N-terminal half is hydrophobic. Membrane
association of N-terminal sequences has been confirmed by the
ability of short carboxylase-derived peptides to confer membrane
insertion in vitro.47 The carboxylase active site, then, may be
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1930
BLOOD, 15 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 6
DARGHOUTH et al
Figure 4. Trp157 and Thr591 are evolutionarily conserved residues. (A) Mutations implicated in combined VKD factor deficiency that were previously identified (small
font) or that are identified in this work (large font) are shown along with regions of the
carboxylase that are hydrophobic (gray bars) or that cross-link in vitro to propeptide or
Glu-containing peptide.42-44 (B-D) The alignment of carboxylases from evolutionarily distant
organisms45 is shown for sequences surrounding the residues (single-letter codes) whose
mutations were identified in the proposita: Asp31Asn (B), Trp157Arg (C), and Thr591Lys
(D). Residues that are identical or similar in all proteins are highlighted in black or gray,
respectively.
similar to that of other enzymes (reviewed by Bracey et al48) that
contain membrane-bound residues that interact with lumenal (or
cytoplasmic) residues to form the active site.
Trp157Arg, which had only 8% to 10% activity compared with
wild-type enzyme (Table 3), resides within a hydrophobic region
suggested by evolutionary comparison to be important to function.
Thus, carboxylases that depend upon vitamin K have been
identified in distantly related organisms that include Conus,
Drosophila, and fish,49-53 and the comparison of these orthologs to
the mammalian carboxylases has been valuable in evaluating
functional residues (Figure 4B-D). The sequences around Trp157Arg
are highly conserved, suggesting functional relevance, and the
hydrophobicity and number of aromatic amino acids in this region
raise the question of whether residue Trp157 is part of the vitamin
K binding site. The proposita did not respond to vitamin K
supplementation (Table 1), which contrasts with the previously
identified patients with carboxylase mutations.9-12 Those patients
had mutations (Leu394Arg and Trp501Ser) that were subsequently
shown to decrease VKD substrate binding.30,31 Because VKD
substrate binding regulates carboxylase affinity for vitamin K,22
this decrease impaired vitamin K binding (ie, causing an increase in
Km for vitamin K), which can explain why these patients responded
to vitamin K supplementation. The lack of response of the
proposita, then, suggests that the Trp157Arg mutation impairs
vitamin K catalysis rather than binding. In this regard, the change
in charge caused by the substitution of an Arg for Trp is notable:
Previous studies54 indicate that the carboxylase active site provides
an unusual environment for the deprotonation of the catalytic base
that reacts with vitamin K to initiate carboxylation, and this
environment would be disrupted by a charged residue. The fact that
the proposita did not respond to vitamin K supplementation also
suggests that the Trp157Arg carboxylase is normally saturated with
vitamin K, due either to the mutation causing a decrease in Km
(which has been observed with other carboxylase mutants55) or to
vitamin K normally being present at saturating levels. The later case
would be surprising, because the availability of reduced vitamin K has
been shown to regulate carboxylation, at least in cell lines.23-26
The Thr591Lys mutation, which resulted in loss of detectable
carboxylase and epoxidase activities (Table 3), lies within the ER
lumenal hydrophilic region of the carboxylase (Figure 4A). Sequences surrounding Thr591 do not show extensive evolutionary
conservation; however, this residue is invariant in all metazoan
carboxylases that have been identified51,52,56 (Figure 4D). This
region is completely absent in an ortholog of the VKD carboxylase
that is present in the bacterial pathogen Leptospira, which is
thought to have been acquired by horizontal transfer of sequences
from the mammalian host into the bacterium.45 Adaptation has
resulted in a Leptospira enzyme with altered properties from
mammalian carboxylase (ie, with no detectable carboxylase activity and unregulated epoxidase activity that occurs in the absence of
VKD substrate).45 Epoxidase activity in the Leptospira enzyme but
not in Thr591Lys suggests that the function of the Thr591 residue
either is in the coupling mechanism or is specific to the carboxylase
part of the reaction, with both epoxidation and carboxylation being
impaired because of the mechanism regulating their coupling.27
The Thr591Lys mutation is nonconservative and introduces a
charged residue, which may account for the dramatic loss of
activity that was observed, and studies that test whether more
conservative substitutions are as deleterious will be of interest.
The Asp31Asn mutation lies within a region that shows almost
no evolutionary conservation (Figure 4B), and the Asp31 residue
itself is not well conserved even among mammals (eg, bovine
carboxylase contains a Gly instead of an Asp57). Asp31Asn
exhibited wild-type activity (Table 3), but this mutation was not
observed in a screen of 100 healthy individuals, and so either this
substitution is a rare polymorphism or Asp31 is important to some
function not assessed in the activity assays that were used. The first
approximately 60 N-terminal carboxylase residues are thought to
be cytoplasmically exposed,47 which likely separates Asp31 from
the active site. Topologic separation therefore suggests that the
N-terminus is important for some function other than activity,
which may or may not involve Asp31. Thus, whether the Asp31Asn
mutation also contributes to the combined deficiency in VKD
factors remains to be established. Future studies that address the
functional consequence of all 3 mutations will clearly be of interest
in defining how molecular alterations in the carboxylase cause
this disease.
Acknowledgments
We are grateful to Raymonde Bredoux for helpful advice on the
PCR experiments, Odile Issertial for expert technical assistance in
polymorphism analysis, and Narges El Golli for help in the
mutagenesis experiments. We also thank Drs Kurt W. Runge and
Mark A. Rishavy for helpful comments during the preparation of
this manuscript.
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2006 108: 1925-1931
doi:10.1182/blood-2005-12-010660 originally published
online May 23, 2006
Compound heterozygosity of novel missense mutations in the
gamma-glutamyl-carboxylase gene causes hereditary combined vitamin
K −dependent coagulation factor deficiency
Dhouha Darghouth, Kevin W. Hallgren, Rebecca L. Shtofman, Amel Mrad, Youssef Gharbi, Ahmed
Maherzi, Radhia Kastally, Sophie LeRicousse, Kathleen L. Berkner and Jean-Philippe Rosa
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