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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 920-926
Exclusion of the First EGF Domain of Factor VII by a Splice Site
Mutation Causes Lethal Factor VII Deficiency
By
John H. McVey,
Emma J. Boswell,
Osamu Takamiya,
Gabriel Tamagnini,
Victor Valente,
Teresa Fidalgo,
Mark Layton, and
Edward G.D. Tuddenham
From the Haemostasis Research Group, MRC Clinical Sciences Centre,
ICSM, London, UK; the School of Allied Medical Science, Shinshu
University, Matsumoto, Japan; Servico de Hematologia, Centro Hospitalar
de Coimbra, Coimbra, Portugal; and the Department of Haematological
Medicine, King's College Hospital, London, UK.
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ABSTRACT |
We have studied a family with homozygous lethal, blood coagulation
factor VII (FVII) deficiency. To identify the mutation responsible for
the deficiency, exons 2 to 8 and the intron-exon junctions of their
FVII genes were amplified from peripheral white blood cell DNA by
polymerase chain reaction and screened by single-strand conformational
polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation,
a homozygous G to A substitution at nucleotide position 6070, in the
invariant GT dinucleotide at the 5 splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction
endonuclease Mlu I. Analysis of the splicing pattern of ectopic
transcripts in lymphocytes in the parents revealed that this mutation
is associated with skipping of exon 4, which produces an mRNA encoding
FVII with an in-frame deletion of the first epidermal growth
factor-like domain (EGF 1). Transient transfection of COS-7 cells with
an expression vector containing the  EGF 1 FVII cDNA shows that this
mutant protein is not expressed. The identification of the molecular
basis of the FVII deficiency in this family allowed mutation-specific
prenatal diagnosis to be performed in a subsequent pregnancy. In this
family complete FVII deficiency is associated with a severe bleeding
diathesis but no developmental abnormalities, lending weight to the
hypothesis that fetal FVII is not required for the putative angiogenic
functions of tissue factor in humans.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
COAGULATION FACTOR VII (FVII) is a
vitamin K-dependent plasma glycoprotein that circulates in blood as a
single-chain zymogen composed of 406 amino acid residues
(Mr 50,000 kD).1 Upon vascular injury and in the presence of calcium, FVII forms a one-to-one stoichiometric complex with its cell surface receptor and cofactor tissue factor (TF). Once complexed to TF, FVII is rapidly cleaved to
its active form, FVIIa, and converts zymogen factor X and factor IX to
their active enzymes. The formation of an active complex between TF and
FVIIa is widely thought to represent the primary stimulus for blood
coagulation.
FVII zymogen is converted to its active form by proteolytic cleavage at
a single site (Arg 152-Ile 153), resulting in a two-chain molecule
composed of an N-terminal light chain, linked by a single disulfide
bridge to a C-terminal heavy chain. The light chain largely consists of
an amino terminal  -carboxyglutamic acid-rich domain, followed by
two epidermal growth factor (EGF)-like modules. The heavy
chain consists of the serine protease catalytic domain. The FVII gene
that spans 12.5 kb consists of nine exons; exons 1a and 1b encode the
5 untranslated region and most of the pre-pro leader sequence, whereas
exons 2 to 8 encode the mature protein.2
Hereditary FVII deficiency is a rare autosomal recessive bleeding
disorder with variable clinical expression.3 In cases of
congenital FVII deficiency there often seems to be little correlation between FVII activity measured in vitro and hemorrhagic symptoms; the
reasons for this discrepancy are not clearly understood. However, patients with less than 1% to 2% FVII activity often have clinical complications similar to those experienced by patients with severe hemophilia A, consistent with the key role of FVII in the initiation of
blood coagulation. In addition, an increased incidence of intracranial hemorrhage has been reported that occurs most often in infants and is
usually ascribed to birth trauma, which represents a major hemostatic
challenge.4
The recently reported targeted disruption of the mouse TF gene
indicates that TF plays an essential role in establishing
and/or maintaining vascular integrity in the developing embryo
because TF / embryos die in utero between days
8.5 and 10.5 due to defective yolk-sac vessel and vitello-embryonic
circulation.5-7 It is not clear whether TF exerts its role
in vessel development by generation of downstream coagulation
proteinases, intracellular signaling, or requires other unknown
ligands.
In the present study, we have identified a family with homozygous,
lethal FVII deficiency. We describe the molecular basis of the FVII
deficiency and prenatal, mutation-specific diagnosis in a subsequent
pregnancy. In this family homozygous lethal FVII deficiency is
associated with a severe bleeding diathesis but not with developmental
abnormalities, lending weight to the hypothesis suggesting that the
requirement for TF in blood vessel development is independent of fetal
FVII.
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MATERIALS AND METHODS |
Coagulation studies.
Blood was collected by venipuncture in  volume of 3.2%
sodium citrate after informed consent. The prothrombin time was
performed using rabbit brain thromboplastin. The coagulation activity
of factors II, V, VII, and X were assayed by one-stage method: the
activity of factors VIII and IX were studied with a one-stage method by
activated partial thromboplastin time using the respective deficient
plasma as substrates.
DNA amplification.
Exons 2 to 8 and the adjacent intronic sequences of the FVII genes of
the propositus (Fig 1, IV-2) were amplified
by polymerase chain reaction (PCR) using the oligonucleotides described
previously.8 Five hundred nanograms of genomic DNA, 0.5 µg of each oligonucleotide, and 1.5 U of Taq DNA polymerase
were added to 90 µL buffer containing 1.5 mmol/L MgCl2,
10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 0.01% gelatin, 200 µmol/L each deoxynucleotide triphosphate, and 92.5 kBq of
 -33P-dATP (74 TBq/mmoL, 370 MBq/mL; Du Pont-NEN,
Stevenage, UK). The reaction mixture was then amplified using the
conditions described previously.9
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| Fig 1.
The pedigree of the family. The propositus
is indicated by an arrow. ( ), IV.1 died at age 1 month with cerebral
hemorrhage and hydrocephaly. ( ), IV.2 died at age of 12 days with
hydrocephalus, intraventricular clots, and cerebral edema.
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Single-strand conformational polymorphism (SSCP)
analysis.
SSCP analysis was performed according to the method of
Hayashi.10 Of the PCR product, 3.5 µL were diluted
10-fold in 85% formamide, 20 mmol/L EDTA, 0.05% bromophenol blue, and
0.05% xylene cyanol. The samples were then heated at 80°C for 3 minutes to denature the DNA, and snap-cooled on ice for 5 minutes
before loading onto a 4.5% nondenaturing acrylamide gel (%T, 4.5%;
%C, 2.25%). Electrophoresis was performed at 40 W for 1.5 to 3 hours at 4°C. The gels were dried before autoradiography. Amplified products from exon 8 were digested with the restriction endonucleases NarI, PstI, and BstXI according to the
manufacturer's instructions (Promega Corp, Southampton, UK) before
SSCP analysis.
DNA cloning and sequencing.
The amplified DNA fragments were gel purified on a 2% agarose gel and
cloned at the EcoR V site of the plasmid vector pBSK (Stratagene, Cambridge, UK). The inserts were sequenced by the dideoxy
chain termination method using a T7 DNA sequencing kit (Pharmacia
Biotech, St Albans, UK). Ten independent clones were sequenced for each
fragment.
Reverse transcription (RT)-PCR.
Polyadenylated RNA was isolated from peripheral lymphocytes using a
commercial kit (Pharmacia Biotech) after purification of lymphocytes
from a 10-mL blood sample on a Ficoll-Paque gradient (Pharmacia Biotech). First-strand cDNA synthesis was performed using a
commercial kit (Pharmacia Biotech) with the addition of 200 ng of a
FVII specific antisense primer: 5-ACCTTGCCCCCCACAATTCG-3. The entire
reaction was used for PCR amplification using the oligonucleotide primers 5-GACACCCCGTCTGCCAGCAG-3 and 5-ACCCAGGAGGAAGCCCACGG-3 under the following conditions: initial denaturation at 94°C for 3 minutes, then 30 cycles of: denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, elongation at 72°C for 60 seconds, and a final elongation step at 72°C for 10 minutes. One
tenth of this PCR reaction was then used in a further PCR reaction
using nested oligonucleotide primers 5-TACCCCTCGTGGCACCGACA-3 and 5-CCAACGCGTTCCTGGAGGAG-3 under identical PCR conditions. The products
were purified by 2% agarose gel electrophoresis and cloned at the
EcoR V site of the plasmid vector pBSK (Stratagene). The inserts were sequenced by the dideoxy chain termination method using a
T7 DNA sequencing kit (Pharmacia Biotech).
Construction of expression vectors for wild-type (wt) and  EGF 1 FVII.
A cDNA for wtFVII was provided by Professor E. Davie (University of
Washington, Seattle, WA). The EGF 1 FVII was generated from the
wtFVII cDNA by a two-stage PCR.11 Oligonucleotide primer pairs 5-GCAGGGGCAGCACTGCAGA-3,
5-TCAGCTGGTCATCCTTGTGACTGTAAGAAATCCAGAACAGC-3 and
5-TGGGGTTTGCTGGCATTTCTTT-3, 5-GCTGTTCTGGATTTCTTACAGTCACAAGGATGACCAGCTGA-3 were used to generate fragments of the FVII
cDNA, spanning nucleotides 8-350 and 466-664 respectively, which
overlap by 41 bp corresponding to the desired join of exons 3 to 5. These fragments were gel purified and used in a subsequent PCR with two
further oligonucleotide primers 5-TTTCATCATGGTCTCCCAGGCC-3 and
5-TTTTTTCTAGAATAGGTATTTTTCCACATGGA-3. The PCR product was cloned and
the nucleotide sequence verified by the dideoxy chain termination
method using cycle sequencing kit (PE Applied Biosystems, Warrington,
UK). The fragment was then subcloned into the wtFVII cDNA as a
HindIII-XbaI fragment replacing nucleotides 29-639. Finally, the wtFVII and the EGF 1 FVII cDNAs were cloned into the
expression vector pcDNA3 (Invitrogen, Leek, The Netherlands).
Cell culture and expression of FVII.
COS-7 cells (European Collection of Animal Cell Cultures Catalogue
[ECACC] No. 87021302) were grown in Eagle's minimum
essential medium (MEM), 10% fetal calf serum, 5,000 IU/mL strep, and 5 µg/mL soluble vitamin K (menadione; Sigma, Poole, UK) in a 5%
CO2 atmosphere. Cells were transfected by electroporation
using the Gene Pulser apparatus (BioRad, Hemel Hempstead, UK) set at
250 V, 250 µFD. Plasmid (50 µg) was used to transfect 2 × 107 cells. One microgram of a  -galactosidase expression
vector pCH110 (Pharmacia Biotech) containing the SV40 promoter was used
as a cotransfectant to correct for variation in transfection
efficiency. The electroporated cells were transferred to a 10-mL volume
of MEM and grown for 24 hours. The cells were then washed twice with phosphate-buffered saline (PBS) and grown in 10 mL of protein-free medium (Hybrimax; Sigma), 5,000 IU/mL strep, and 5 µg/mL soluble vitamin K (menadione; Sigma) for a further 48 hours. The conditioned media were then collected.
Detection of FVII secreted from COS-7 cells.
The 10-mL conditioned medium was reduced to 1 mL by centrifuging the
solution through the filter of a Centricon-10 concentrator (10,000 kD cut-off) (Amicon, Stonehouse, UK). FVII antigen was assayed using a commercial enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Stago Asserachrom; Shield Diagnostics, Dundee, UK) and Western blot analysis using a
rabbit polyclonal antibody. FVII:Ag levels were referenced to a normal
pool plasma which was calibrated against the first
international standard for factors II, VII, IX, and X (National
Institute for Biological Standards and Control [NIBSC],
South Mimms, UK). FVII:Ag could be measured in the range 1.04 to 104 mU/mL. Equal amounts of protein of each sample were electrophoresed on
10% precast Nupage gels (Novex, Frankfurt, Germany) with
MOPS buffer (50 mmol/L 3-(N-morpholino) propane sulfonic
acid, 50 mmol/L Tris-HCl (pH 7.7), 3.5 mol/L sodium lantyl sulfate, 1 mmol/L EDTA) at constant voltage of 200 V according to the
manufacturer's instructions. The proteins were transferred from the
gel to a nitrocellulose membrane by electrophoretic transfer. FVII on
the nitrocellulose membrane was detected using the rabbit polyclonal
anti-human FVII detection antibody in the ELISA kit (Stago Asserachrom;
Shield Diagnostics). The rabbit polyclonal antibody was detected using a horseradish peroxidase conjugated goat anti-rabbit IgG antibody (BioRad) and ECL+ chemiluminescence detection reagents according to the
manufacturer's instructions (Amersham International, Little Chalfont,
UK).
Protein and -galactosidase assay.
Cells were obtained by brief exposure to trypsin/EDTA. They were then
washed in PBS, transferred to a 1.5-mL microfuge tube, and centrifuged
at 5,000g for 5 minutes. The pellet was resuspended in 500 µL
of 0.25 mol/L Tris HCl, pH 8.0, and the cells were disrupted by three
freeze/thaw cycles. The debris was eliminated by centrifugation at
12,000g for 5 minutes at 4°C. Soluble proteins in the cell lysate (supernatant) were quantified using a Coomassie blue protein staining assay (BioRad). -galactosidase activity was assayed as
previously described.12
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RESULTS |
Case history and coagulation studies.
A baby boy was referred to the Paediatric Hospital in Coimbra with
vomiting, prostration, and sudden onset twitching. He had been
delivered 10 days previously by forceps, after an uneventful pregnancy;
the Apgar score was 9/10/10. He had been discharged home on the third
day after birth and had apparently been healthy until that morning.
His parents are consanguineous and both enjoy good health. However, a
baby girl from a previous pregnancy had died at the age of 1 month with
cerebral hemorrhage and hydrocephaly (Fig 1, IV-1).
Physical examination showed a prostrated baby boy, slightly pale with
hypertonicity of the limbs, pausal respiration, and tense fontanelle. A
computed axial tomography (CAT) scan showed hydrocephalus with
compression of the IVth ventricle, numerous intraventricular clots, and
marked cerebral edema. Coagulation studies revealed a prolonged
prothrombin time, whereas the activated partial thromboplastin time was
within the normal range. The procoagulant activities of the propositus
were all within the normal range except for FVII activity, which was
less than 1% (Table 1). A diagnosis of
factor VII deficiency was made and fresh frozen plasma was
administered.
Lumbar puncture performed under cover of fresh frozen plasma showed
hemorrhagic fluid under tension. Emergency external drainage of the
hydrocephalus was performed by removing 85 mL of hemorrhagic fluid,
resulting in transient relief. A control CAT scan showed extensive
bilateral cerebral hemorrhage and he died 44 hours after admission.
Identification of the mutation in the FVII gene.
To identify the mutation responsible for the FVII deficiency in this
family, exons 2 to 8 and the adjacent intronic sequences of the FVII
genes of the propositus were amplified by PCR. SSCP analysis of PCR
products revealed no difference in mobilities between the propositus
and normal controls analyzed in parallel except with PCR fragments
which spanned exons 3 and 4 (Fig 2). Exons
3 and 4 were amplified and analyzed as a single fragment. To determine
the nucleotide substitution responsible for the altered electrophoretic
mobility detected by SSCP analysis, the PCR-amplified DNA fragment was
cloned and sequenced. A G to A point mutation was found at nucleotide
position 6070 (Fig 3). This mutation is in
the invariant GT dinucleotide at the 5 splice site of intron 4. All 10 clones sequenced corresponded to the mutant FVII sequence. The mutation
abolishes a MluI restriction site by changing the sequence from
ACGCGT to ACGCAT. PCR fragments of exons 3 and 4 from the propositus
and his parents were digested with MluI and analyzed by agarose
gel electrophoresis and Southern blot analysis with the human FVII cDNA
(Fig 4). The propositus had only a 310-bp band and was therefore homozygous for the loss of the MluI
site. His parents had both the 310- and 261-bp bands, indicating that they are heterozygous for the mutation.
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| Fig 2.
PCR-SSCP analysis of exons 3 and 4 of the factor VII
genes of: lane 1, normal control; lane 2, affected child (IV.2); lane 3, father (III.5); lane 4, mother (III.6); lane 5, double-stranded control. An arrow indicates extra band with altered migration, when
compared with normal control bands.
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| Fig 3.
Nucleotide sequence of mutant DNA spanning exon 4 and
intron 4 of the gene for FVII. The DNA was sequenced by the dideoxy chain termination method after subcloning into Bluescript
plasmid vector (Stratagene, Cambridge, UK). The antisense sequence is shown. An arrow indicates the mutated base.
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| Fig 4.
Restriction endonuclease digestion of PCR products. (A) A
fragment spanning exons 3 and 4 and the flanking introns was PCR amplified and digested with the restriction endonuclease Mlu I before electrophoresis on a 2% agarose gel, Southern blotted, and
probed with the FVII cDNA sequence. Lane 1, normal control; lane 2, affected child, IV.2; lane 3, father, III.5; lane 4, mother, III.6. (B)
Diagrammatic representation of the fragment the Mlu I sites are
indicated.
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Characterization of the splicing events associated with this
mutation.
FVII is synthesized in the liver, a relatively inaccessible tissue.
However, total RNA isolated from lymphocytes has been shown to contain
mRNA transcripts for genes not normally expressed in these cells. The
analysis of these so-called "ectopic transcripts" has been used
in the analysis of mutations in a number of inherited disorders. To
determine the splicing pattern associated with this mutation we
isolated total polyadenylated RNA from lymphocytes of both parents
(III.5, III.6) and analyzed the ectopic transcripts by RT-PCR. cDNA
synthesis was primed using an oligonucleotide corresponding to the
antisense sequence of exon 6 and two rounds of PCR were primed using
nested primers from exons 2 and 5. The expected product after correct
splicing would be 350 bp. However, if either the intron was not removed
or exon skipping occurred, then fragments of 2064 and 237 bp,
respectively, would be predicted. After RT-PCR there were two bands
visible on agarose gel electrophoresis of about 220 and 344 bp, both of
which hybridized to FVII cDNA on Southern blot analysis. These
fragments were cloned and sequenced. The sequence of the larger
fragment corresponded to wtFVII sequence spanning exons 2, 3, 4, and 5, whereas the sequence of the smaller fragment corresponded to exons 2 and 3 directly spliced to exon 5 (Fig 5).
Therefore, the GT to AT transition at the 5 donor splice site of
intron 4 causes skipping of exon 4 in processed mRNA.
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| Fig 5.
Nucleotide sequence of the exon 3-exon 5 splicing variant
observed in RT-PCR analysis of ectopic transcripts in lymphocytes from
the parents. (A) Normal allele, (B) mutant allele.
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Expression of recombinant wt and EGF 1 FVII.
The skipping of exon 4 would produce an mRNA with an in-frame deletion
of 113 nucleotides which encode 38 amino acid residues (G47-H84)
comprising the entire first EGF domain of FVII. To determine whether
the EGF 1 FVII mRNA would direct the expression of a mutant protein,
we generated expression constructs containing either the wt or EGF 1 FVII cDNA sequences, transiently transfected COS-7 cells, and compared
the levels of secreted FVII:Ag in conditioned media by both ELISA and
Western blot analysis. The relative efficiency of transfection was
measured by cotransfecting with a -galactosidase expression vector
and measuring -galactosidase activity in the transfected cell
lysates. The values of FVII:Ag determined by the ELISA were corrected
for transfection efficiency. FVII was secreted by COS-7 cells
transfected with the wt cDNA (35 mU/mL); however, no detectable FVII
was secreted by cells transfected with the EGF 1 FVII cDNA even
after fourfold concentration (ie, <0.26 mU/mL). Western blot analysis
showed that the detection antibody from the ELISA kit recognized
predominantly the heavy chain of fully purified recombinant FVIIa. FVII
was detectable in the conditioned media from cells transfected with the
wt cDNA but not in the conditioned media from cells transfected with
the EGF 1 FVII cDNA (Fig 6).
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| Fig 6.
Western blot analysis of proteins secreted by COS-7 cells
into the growth media using a polyclonal antibody against FVII. From
concentrated conditioned media 2.5 µg of protein was electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to
nitrocellulose. The FVII was detected using a polyclonal rabbit anti-human FVII antibody (Stago Asserachrom; Shield Diagnostics) and
chemiluminescence detection reagents. Lane 1, vector alone; lanes 2 and
3, EGF 1 FVII; lanes 4 and 5, wt FVII; lanes 6 to 8, 5, 2, and 1 ng
purified recombinant wtFVII, respectively; lanes 9 to 12, 5, 10, 20, and 50 ng recombinant FVIIa, respectively. The relative sizes of
molecular weight markers are shown in kD.
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Mutation-specific prenatal diagnosis.
While this work was in progress, the mother became pregnant again and
requested prenatal diagnosis. Chorionic villus material obtained from
the fetus in the 10th week of pregnancy was shown by SSCP and
MluI restriction analysis of PCR-amplified exons 3 and 4 to be
homozygous for the wt FVII sequence (Fig
7). A baby boy was born after an uneventful
pregnancy and was clinically normal with FVII activity in the cord
blood sample of 62% (Table 1).
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| Fig 7.
(A) PCR-SSCP analysis. (B) Restriction endonuclease
analysis of exons 3 and 4 including intron/exon junctions. Lane 1, chorionic villus sample (IV.3); lane 2, normal control; lane 3, affected child (IV.2); lane 4, father (III.5); lane 5, mother (III.6). An arrow indicates extra band with altered migration, when compared with normal control bands.
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DISCUSSION |
In this paper, we describe the further characterization of a novel
mutation in a patient with homozygous lethal FVII deficiency which we
reported in a preliminary form.13 This mutation has recently been reported in an Italian family with severe but nonlethal FVII deficiency due to double heterozygosity for the mutation described
above and FVIIT359M.14 The mutation was identified by SSCP
analysis of PCR-amplified DNA and sequencing cloned PCR products. The
propositus was found to be homozygous for a G to A substitution in the
invariant GT dinucleotide in the 5 splice junction of intron 4. The
homozygosity was confirmed by restriction endonuclease digestion.
Splicing defects are not an uncommon cause of human genetic disease;
between 8% and 15% of known single-base pair substitutions disrupt
the normal splicing of an mRNA transcript.15 The vast
majority of known mutations that affect splicing are single-base pair
substitutions within 5 and 3 splice sites. Mutations within a 5 or
3 splice site usually reduce the amount of mature mRNA generated
and/or activate alternative "cryptic" splice sites in the
vicinity. The use of cryptic splice sites results in the production of
mRNAs which either lack a portion of the coding sequence or instead
contain additional intronic sequences. An alternative consequence of a
splice site mutation could be for an exon to be no longer recognized as
such, and as a result, to be excluded from the mature mRNA, a process
called exon skipping. The analysis of the actual consequence of this mutation for FVII mRNA processing could not be carried out in cells
which would normally express FVII because it is synthesized in the
liver, which is inaccessible without invasive biopsy. The existence of
extremely low background levels of correctly spliced mRNA transcripts
of tissue-specific genes has been shown in supposedly "nonexpressing" cell types such as lymphocytes. These so-called ectopic transcripts can be analyzed by PCR after RT.16-19
Analysis of the splicing pattern of ectopic transcripts in lymphocytes isolated from the parents of the propositus revealed that this mutation
is associated with skipping of exon 4. The skipping of exon 4 would
produce an mRNA with an in-frame deletion of 113 nucleotides which
encode 38 amino acid residues (G47-H84) comprising the entire first EGF
domain of FVII.
One might predict that this mutant protein would fold correctly and be
secreted from the cell. However, it seemed probable from the FVII
antigen-activity measurements made on plasma samples obtained from the
parents (Table 1) that they have little if any circulating EGF
1-deleted FVII. Transfection of COS-7 cells with expression constructs
containing either wt or EGF 1 FVII cDNAs confirmed that the mutant
protein is not expressed in detectable amounts, less than 0.26 mU/mL or
at least 135-fold less than wt, consistent with the observed phenotype.
Multiple lines of evidence suggest that the EGF domains of FVII are
important in the TF/FVII interaction. Chimeric FIX/FVII molecules only
bind to TF when FVII-EGF1-EGF2 modules are present.20 A
monoclonal antibody whose epitope has been mapped to residues 51-88 of
FVII (EGF 1) has been shown to inhibit FVII activation and its binding
to TF.21 Analysis of the molecular defects responsible for
FVII deficiency has identified two independent mutations at Arg 79 in
the first EGF domain, FVII-R79Q and FVII-R79W.9 Surface plasmon resonance studies of the interaction between recombinant FVII-R79Q and TF have shown a decrease in its affinity for
TF.22 The three-dimensional structure of the TF/FVIIa
complex determined by x-ray crystallography shows EGF1 interacting with
TF residues identified by mutational analysis as being responsible for
FVIIa affinity.23 Finally, alanine scanning mutagenesis of
FVII provides evidence that EGF1 tethers the enzyme to
TF.24 Plausibly, an FVII molecule lacking EGF1 would be
nonfunctional, or have severely reduced specific activity. However, the
data presented in this work show that such a molecule is not expressed
or expressed at extremely low levels.
The formation of the bimolecular complex of TF and FVII is thought to
be the primary event in the initiation of blood coagulation in vivo.
The mutation described in this paper results in severely reduced
expression of a probably nonfunctional FVII molecule. Consequently,
according to current coagulation theory, blood coagulation cannot be
initiated a situation incompatible with life. The fact that the
homozygous affected children in this family survived to term suggests
that the first serious hemostatic challenge occurs at parturition.
Furthermore, the fact that both children had no detectable
developmental abnormalities supports the hypothesis that FVII is not
required for normal fetal development, thus the embryonic lethal
phenotype associated with disruption of TF expression is independent of
fetal FVII expression. This conclusion is supported by the recent
description of the targeted disruption of the mouse FVII gene.
FVII/ mice develop normally to term but die at/or
shortly after birth due to abdominal and intracranial
hemorrhaging.25 It can be hypothesized that normal in utero
development in mice and humans lacking FVII is due to maternal rescue
by transplacental transfer of small but sufficient amounts of FVII.
However, there is no evidence for maternal-fetal transfer of FVII in
the FVII/ mouse embryos.25
In conclusion, we have identified a mutation in the donor splice site
of intron 4 of the gene encoding blood coagulation factor VII, which
results in skipping of exon 4 which encodes the EGF 1 domain. This
mutant protein is not expressed in vitro at detectable levels.
Identification of the genetic basis of FVII deficiency in this family
allowed mutation-specific prenatal diagnosis to be performed
successfully in a subsequent pregnancy.
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FOOTNOTES |
Submitted July 18, 1997;
accepted March 30, 1998.
Address reprint requests to John H. McVey, PhD, Haemostasis Research
Group, MRC Clinical Sciences Centre, ICSM, Du Cane Rd, London W12 0NN,
UK; e-mail: jmcvey{at}hgmp.mrc.ac.uk.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract