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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 866-875
Molecular Genetic Analysis of a Compound Heterozygote for the
Glycoprotein (GP) IIb Gene Associated With Glanzmann's Thrombasthenia:
Disruption of the 674-687 Disulfide Bridge in GPIIb Prevents
Surface Exposure of GPIIb-IIIa Complexes
By
Consuelo González-Manchón,
Marta Fernández-Pinel,
Elena G. Arias-Salgado,
Milagros Ferrer,
M.-Victoria Alvarez,
Soledad García-Muñoz,
Matilde S. Ayuso, and
Roberto Parrilla
From the Department of Pathophysiology and Human Molecular Genetics,
Centro de Investigaciones Biológicas (CSIC), Madrid, Spain; the
Unidad de Biofísica, Instituto de Química
Física (CSIC), Madrid, Spain; and the Laboratory of Analytical
Hematology, University Hospital "La Paz," Madrid, Spain.
 |
ABSTRACT |
This work was aimed at elucidating the molecular genetic lesion(s)
responsible for the thrombasthenic phenotype of a patient whose low
platelet content of glycoprotein (GP) IIb-IIIa indicated that it was a
case of type II Glanzmann's thrombasthenia (GT). The parents did not
admit consanguinity and showed a reduced platelet content of
GPIIb-IIIa. Polymerase chain reaction (PCR)-single-stranded conformational polymorphism analysis of genomic DNA showed
no mutations in the patient's GPIIIa and two novel mutations in the GPIIb gene: one of them was a heterozygous splice junction mutation, a
C A transversion, at position +2 of the exon 5-intron 5 boundary [IVS5(+2)CA] inherited from the father. The
predicted effect of this mutation, insertion of intron 5 (76 bp) into
the GPIIb-mRNA, was confirmed by reverse transcription-PCR
analysis of platelet mRNA. The almost complete absence of this mutated
form of GPIIb-mRNA suggests that it is very unstable. Virtually all of
the proband's GPIIb-mRNA was accounted for by the allele inherited
from the mother showing a T2113C transition that
changes Cys674Arg674 disrupting the
674-687 intramolecular disulfide bridge. The proband showed a platelet
accumulation of proGPIIb and minute amounts of GPIIb and GPIIIa.
Moreover, transfection and immunoprecipitation analysis demonstrated
that [Arg674]GPIIb is capable of forming a heterodimer
complex with GPIIIa, but the rate of subunit maturation and the surface
exposure of GPIIb-IIIa are strongly reduced. Thus, the intramolecular
674-687 disulfide bridge in GPIIb is essential for the normal
processing of GPIIb-IIIa complexes. The additive effect of these two
GPIIb mutations provides the molecular basis for the thrombasthenic phenotype of the proband.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
GLANZMANN reported a case of a bleeding
disorder, starting immediately after birth, characterized by prolonged
bleeding time and abnormal clot retraction.1 Based on these
features, he named this disease "thrombasthenia." The platelets
from these patients would not spread or aggregate, indicating that they
were defective.2,3 The finding that the fibrinogen content
in platelets was low or absent2,4,5 led to the conclusion that the fibrinogen receptor was either defective or deficient in
thrombasthenic phenotypes.6,7 The fibrinogen receptor, expressed only in platelets, is a noncovalent, calcium-dependent, heterodimeric complex, formed by glycoprotein (GP) IIb and
GPIIIa, that belongs to the superfamily of cell surface adhesive
receptors termed integrins8 and so is also referred to as
integrin  IIb 3. This receptor
binds adhesive proteins other than fibrinogen, such as von Willebrand
factor, vitronectin, and fibronectin.9,10
Caen11 classified Glanzmann thrombasthenia (GT) into types
I or II based on their platelet fibrinogen content and clot-retracting capability: type I GT platelets lack fibrinogen and clot retraction; in
type II GT, platelets showed low to moderate clot retraction capability
and detectable amounts of fibrinogen. In agreement with these
observations, GPIIb-IIIa was found to be absent in the platelet
membrane of type I GT and present at only 10% to 20% of the control
values in type II GT. Another type of GT, termed variants, representing
less than 10% of the total number of cases, has also been identified
in which platelets possess normal or near normal (60% to 100%)
expression of dysfunctional receptors.7,12
From a molecular biological point of view, GT is a heterogeneous
disease.13 As the cDNA and the structural organization of
the GPIIb and GPIIIa genes became available,14-18 it was
possible to establish that the molecular basis for this heterogeneity
is the result of distinct genetic lesions in the GPIIb and GPIIIa genes. Since then, point mutations leading to single amino acid substitutions, insertions or deletions, nonsense mutations, or splicing
abnormalities have been reported in both genes.19 The majority of these lesions are associated with type I GT.
The present work reports the studies on a patient of type II GT
resulting from compound heterozygosity for the GPIIb gene, who
inherited a mutation in the splicing junction of the exon 5-intron 5 boundary of GPIIb [IVS5(+2)CA] from her father and a
T2113C transition that changes Cys674
to Arg from her mother. Functional studies of these two mutations demonstrated their association with the thrombasthenic phenotype of the patient.
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MATERIALS AND METHODS |
Analysis of GPIIb-IIIa and fibrinogen content.
Platelet content of GPIIb, GPIIIa, and fibrinogen was determined by
competitive enzyme immunoassays, using monoclonal anti-GPIIb (M3),
anti-GPIIIa (P37), and antifibrinogen (F2)
antibodies20,21 and pure GPIIb, GPIIIa, and
fibrinogen as standards.
For Western blot analysis of the GPIIb-IIIa content, sodium dodecyl
sulfate (SDS)-solubilized platelet proteins were first separated on 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions, electrotransferred onto
nitrocellulose membranes, and then incubated with anti-GPIIIa (P37) and
anti-GPIIb (M3) specific antibodies. The immunoreactive bands were
digitized with a high resolution scanner and analyzed on a Macintosh
(Cupertino, CA) computer using the public domain NIH Image
program (developed at the US National Institutes of Health and
available on the Internet at http://rsb.info.nih.gov/nih-image/).
Flow cytometry.
Platelets or stably transfected CHO cells were harvested using
phosphate-buffered saline (PBS) containing 5 mmol/L or 0.5 mmol/L EDTA, respectively, incubated with a monoclonal antibody (MoAb)
directed against either GPIIb (M3) or GPIIIa (P37) and then exposed to
fluorescein isothiocyanate-conjugated (FITC) F(ab')2 fragment of rabbit antimouse Ig (Dako A/S, Glostrup,
Denmark) and the surface fluorescence was analyzed in a
Coulter flow cytometer (model EPICS XL; Coulter, Hialeah, FL).
Reverse transcription-polymerase chain reaction
(RT-PCR) analysis of GPIIb- and GPIIIa-mRNA from platelet
and stably transfected CHO cells.
Platelet total RNA was prepared by the guanidinium thiocyanate
method,22 reverse-transcribed with the primer
GPIIb-(1275-1255) 5'-ACCCAGGAACACCAGCACTTG-3', and used as
template for the amplification of a 779-bp fragment encompassing exons
5 to 13 of GPIIb with the following oligonucleotides: sense,
GPIIb-(497-517) 5'-CAGAGAGCGGCCGCCGCGCCG-3', and antisense,
GPIIb-(1275-1255). To search for mutant forms of GPIIb-mRNA with
insertion of intron 5, a sense primer specific for sequences of intron
5, GPIIb-(intron 5, 1-19) 5'-GCGAGTAGGGAGCAAAAGC-3', and
the antisense primer GPIIb-(497-517) were used. Hybridization analysis
of the PCR products was performed as previously described23 using as probes GPIIb DNA fragments lacking the oligonucleotide sequences used for amplification.
Total RNA from the proband and her parents was also reverse-transcribed
with the primer GPIIb-(3154-3133),
5'-CAACCCTCCTGCTAGAATAGTG-3', and used as template for the
PCR amplification of a 1,190-bp fragment encompassing exons 20 to 30 of
GPIIb with the oligonucleotides: sense, GPIIb-(1965-1985)
5'-AGTTGGGGCAGATAATGTCCT-3'; and antisense, GPIIb-(3154-3133).
Total RNA from CHO cells stably transfected with GPIIIa and either
normal or mutant [IVS5(+2)CA]GPIIb was extracted as
described above and reverse-transcribed using the antisense
oligonucleotide GPIIb-(1513-1489)
5'-CACAGCTCTTCACAGCAGGATTCAG-3'. A 479-bp fragment of
normal GPIIb-cDNA was amplified using the oligonucleotides: sense,
GPIIb-(497-517) 5'-CAGAGAGCGGCCGCCGCGCCG-3', and antisense, GPIIb-(975-956) 5'-AGCCACTGAATGCCCAAAAT-3'. The predicted
size of the amplified fragment with insertion of intron 5 is 555 bp.
PCR-based quantitation of platelet GPIIb- and GPIIIa-mRNA with the
TaqMan system.
The instrumentation and the fluorogenic probes of the Perkin-Elmer
Cetus (Norwalk, CT) LS-50B TaqMan System were used for PCR-based
quantitation of GPIIb and GPIIIa mRNA in platelets from the proband,
her parents and sister, and normal individuals, as previously
described.24 Specific oligonucleotide probes,
R-CTCTGGCGCGTTCTTCCTCAAATTTAGC-Q, R-AGTGACCACGGAGCTGAAGCCCG-Q, and R-ATGCCCT-Q-CCCCCATGCCATCCTGCGT, were designed to anneal to targets located within PCR-amplified fragments of 132 bp (2157-2288) of GPIIIa, 194 bp (582-776) of GPIIb,
or 295 bp (2141-2435) of -actin cDNA, respectively. The location of
the reporter and quencher dyes are indicated by R and Q, respectively.
We first assessed the range of both the number of PCR cycles and the
amounts of template that would yield a linear response between DNA
target doses and fluorescence signal from amplified products. Different
dilutions of RNA samples were used for amplification of GPIIIa, GPIIb,
and -actin DNA fragments using the rTth polymerase XL from
Perkin-Elmer Cetus. Briefly, in a first-step mRNA was
reverse-transcribed with the antisense primers in the presence of 1.1 mmol/L Mn(OAc)2 during 30 minutes at 60°C. Then, the
PCR amplification was performed by chelating the Mn2+ and
adding 0.8 mmol/L Mg(OAc)2, the sense primer, and the
specific TaqMan probe. Twenty-five or 30 amplification cycles were
performed consisting of 15 seconds at 95°C and 15 seconds at
65°C. After PCR cycling, 40 µL portions were taken from each
sample and the fluorescence was measured using a 488 nm excitation
wavelength and 518 and 580 nm emission wavelengths for the reporter and
quencher dyes, respectively. Values were corrected for internal
quenching and expressed as GPIIIa/-actin and GPIIb/-actin
fluorescence ratios.
Single-stranded conformational polymorphism (SSCP) analysis,
cloning, and sequencing of PCR-amplified genomic DNA fragments.
PCR amplification of DNA fragments encompassing one or more exons of
GPIIb or GPIIIa was performed as previously described.25 Screening for mutations in GPIIb and GPIIIa was performed by
"cold" SSCP analysis.26,27 A 442-bp DNA fragment
encompassing exons 5 to 7 of GPIIb was amplified with the
oligonucleotides: sense, 5'-GGCTGACCCCTCCTCCTTGT-3', and
antisense, 5'-CTGGAAGTCTGGAATGGCGGT-3'. Portions of the PCR
product were analyzed by agarose gel electrophoresis, purified,
digested with Apa I to yield fragments of 321 and 121 bp, and
used for SSCP analysis. A 193-bp DNA fragment encompassing exon 21 of
GPIIb was amplified with the oligonucleotides: sense, 5'-TATATGATGCTCTGTAATTTC-3', and antisense,
5'-TCTGGTTATTCATGAGCCCCT-3'. DNA products showing altered
electrophoretic mobility patterns of single-stranded bands were cloned
into a T vector28 and their primary nucleotide sequence was
determined. DNA sequence analysis was performed as described by
Marck.29
Construction of mammalian expression vectors with normal or mutant
GPIIb cDNAs.
A GPIIb cDNA with intron 5 inserted, [intron 5]GPIIb, was prepared by
the splicing by overlap extension (SOE) PCR procedure.30 First, genomic DNA from the patient was used as template for PCR amplification of a fragment comprising exons 5 to 7 of GPIIb. This
product was digested to generate a 93-bp Alu I/Hpa II
fragment and a 54-bp Ban II fragment that overlap at the intron
5 region. Second, normal GPIIb-cDNA was used as template to generate
two PCR overlapping fragments, hereafter referred to as 5' and
3' segments: the 5' segment was amplified using the
oligonucleotide GPIIb-(497-517) 5'-CAGAGAGCGGCCGCCGCGCCG-3'
as sense primer and the Alu I/Hpa II genomic fragment
as antisense primer; the 3' segment was obtained using the
Ban II digestion product as sense primer and the
oligonucleotide GPIIb-(975-956)
5'-AGCCACTGAATGCCCAAAAT-3' as antisense primer. Third, the
5' and 3' PCR products were used as template in a new round
of PCR amplification with the oligonucleotides Not I and
Bsm I described above; the amplified DNA comprising intron 5 was digested with Not I and Bsm I, and the digestion product cloned in a vector containing the normal GPIIb cDNA previously digested with the same restriction enzymes. Normal or mutated [intron
5]-GPIIb-cDNA were subcloned into the HindIII site of pCEP4
(Invitrogen, San Diego, CA) to yield the plasmids pCEP4-GPIIb and
pCEP4-[intron 5]GPIIb, respectively.
GPIIb cDNA with a T to C substitution at position 2113 was prepared by
SOE PCR as described using two sets of oligonucleotide primers:
GPIIb-sense-(955-979) 5'-TATTTTGGGCATTCAGTGGCTGTCA-3', GPIIb-antisense-(2123-2103)
5'-TTCTGATTACGGATGAGTCTC-3'; and
GPIIb-sense-(2103-2123) 5'-GAGACTCATCCGTAATCAGAA-3',
GPIIb-antisense-(3154-3133) 5'-CAACCCTCCTGCTAGAATAGT-3'. Bases substituted to generate a mutation in overlapping fragments are
underlined. The final PCR product carrying the mutation was digested
with Bsm I and Nae I and ligated in a vector containing the wild-type GPIIb cDNA previously digested with the same enzymes. Wild-type and mutated cDNA were subcloned into the HindIII site of pcDNA3 (Invitrogene).
Cell culture and transfection.
CHO cells stably expressing GPIIIa (CHO-GPIIIa
cells)31 were grown in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal calf serum. Cells were transfected
with 5 µg of either plasmid pCEP4-GPIIb or pCEP4-[intron 5]GPIIb
using the calcium phosphate precipitation procedure.23 The
transfected cells were fed with medium containing 200 µmol/L
hygromycin every 3 to 4 days.
Transient transfection analysis of pcDNA3-GPIIIa and either
pcDNA3-GPIIb or pcDNA3-(2113C)GPIIb into CHO cells was
performed by the diethyl aminoethyl (DEAE)-dextran
method.32
Selective cloning of exons by the Exontrap vector system.
Exontrap (Mo Bi Tec, Göttingen, Germany) is a vector system
designed to selectively clone exon sequences from large genomic fragments.33 A 442-bp DNA fragment encompassing exons 5 to
7 of GPIIb was amplified with the primers for the intronic flanking regions (sense, 5'-GGCTGACCCCTCCTCCTTGT-3', and antisense,
5'-CTGGAAGTCTGGAATGGCGGT-3') using as template genomic DNA
from the proband or a control. The resulting PCR products were
blunt-end ligated into the Sma I site of the pET01, and
selected clones carrying DNA from either the normal or the mutant
alleles were transiently transfected into CHO cells. RT-PCR analysis of
RNA from the transfected cells was performed with primers for the exons
of the pET01 vector: sense, 5'-GAGGGAATCCGCTTCCTGCCCC-3',
and antisense, 5'-CCGTGACCTCCACCGGGCCCTC-3'. The PCR
products were cloned in a T vector and their DNA sequence was
determined in both directions.
Labeling and immunoprecipitation analysis of GPIIb-IIIa complexes
from platelets and CHO cells cotransfected with cDNAs encoding GPIIIa
and either normal or mutant forms of GPIIb.
To analyze surface expression of GPIIb-IIIa complexes, CHO
cells were transfected with normal GPIIIa and either normal or (C2113)-GPIIb cDNA and, 72 hours after transfection, were
washed twice with PBS, incubated in 2 mL of PBS containing 2.5 mmol/L
biotin-NHS (D-biotin-N-hydroxysuccinimidester; Boehringer Mannheim,
Mannheim, Germany) for 30 minutes followed by
immunoprecipitation of GPIIb-IIIa complexes with the specific
anti-GPIIIa (P37) or anti-GPIIb (M3) MoAb.21 The
immunoprecipitates were processed as previously described.34
Total (surface and intracellular) GPIIb-IIIa complexes were detected by
incubation of detergent lysates of platelets or transiently transfected
CHO cells with biotin-NHS, followed by immunoprecipitation with specific MoAbs.34
Materials.
Restriction enzymes and hygromycin were obtained from Boehringer
Mannheim, and DNA sequencing reagents were from Pharmacia Biotech
(Uppsala, Sweden). The pCEP4 and pcDNA3 expression vectors were from
Invitrogen, and the Exontrap vector system was purchased from Mo Bi
Tec. Most other reagents were purchased from Sigma Chemical Co (St
Louis, MO) or from Merck (Darmstadt, Germany). [35S]-dATP
(specific activity, 1,000 Ci/mmol) was from Amersham
Ibérica (Madrid, Spain).
| |
RESULTS |
Case report.
The patient is an 11-year-old girl clinically diagnosed with GT whose
history of bleeding episodes and unprovoked bruising started
immediately after birth. Her parents, who are clinically asymptomatic,
are not aware of any consanguinity or bleeding disorders in their
relatives. Clinical hematological studies showed a bleeding time  15
minutes, a platelet count of 286,000/µL, and a clotting time of 7 minutes with very low clot retraction capability. Platelets failed to
aggregate either spontaneously or in response to adenosine diphosphate
(ADP), epinephrine, or collagen, but showed a normal response to
ristocetin. Platelet adhesion to glass was 9%. The parents of the
proband showed decreased platelet aggregation in response to ADP or
epinephrine but normal responses to collagen or ristocetin.
Platelet GPIIb-IIIa content.
The surface level of platelet GPIIb-IIIa was analyzed by flow
cytometry, using anti-GPIIb or anti-GPIIIa MoAbs. The histograms in
Fig 1 show representative tracings of
fluorescence intensities of platelets from the proband and family
members. The proband showed GPIIb and GPIIIa mean fluorescence
intensities minus the background of about 10% of the control values.
Both parents showed a marked decrease in the mean fluorescence
intensity of both GPIIb and GPIIIa, whereas the platelets from the
sister of the proband showed values similar to those of the control
platelets. Enzyme immunoassay and immunoblotting analysis confirmed the
results obtained by flow cytometry (Table
1). The proband's platelet fibrinogen content is 25% of the control,
which is greater than the reported values for patients with GT showing
no surface expression of GPIIb-IIIa.35
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| Fig 1.
Flow cytometric analysis of GPIIb-IIIa content in
platelets from the proband, her father, mother, and sister. Washed
platelets were incubated with anti-GPIIIa or anti-GPIIb MoAbs as
described in Materials and Methods. Results are expressed as semilog
plots of cell number versus fluorescence intensity. The upper panel
shows the surface expression of GPIIb, using the MoAb M3, in platelets
from the patient, from her parents and sister, and from a normal
unrelated individual. The lower panel represents the surface expression
of GPIIIa using the MoAb P37. The negative control represents the
fluorescent signal of platelets treated only with the second antibody.
The plot corresponding to the sister's platelets overlaps with the
control and, therefore, only one tracing was represented. For the sake
of clarity, the original plots have been redrawn.
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Figure 2 depicts the immunoprecipitation
analysis of GPIIb-IIIa from platelet lysates using anti-GPIIIa (P37) or
anti-GPIIb (M3) MoAbs. Both antibodies yielded bands migrating like
GPIIIa and GPIIb, but a band migrating like proGPIIb was detected only with the anti-GPIIb MoAb. The density of GPIIb and GPIIIa bands is
lower and the ratio of proGPIIb to GPIIb intensity is higher in the
proband than in her relatives. This observation indicated that,
whatever the molecular lesion(s) could be, the formation of GPIIb-IIIa
complexes was not prevented. The parents showed similar pattern of
bands, although less intense, than the sister of the proband whose
surface GPIIb-IIIa content is normal (Fig 1).
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| Fig 2.
Immunoprecipitation analysis of platelet GPIIb and GPIIIa
from the proband, her mother, father, and sister. Proteins in total
platelet lysates were labeled with biotin-NHS, and GPIIb-IIIa complexes
were immunoprecipitated using MoAbs specific for GPIIIa (P37) or GPIIb
(M3) and processed as described in Materials and Methods.
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Identification of GPIIb mutations.
The coding sequences of GPIIb and GPIIIa were studied by SSCP analysis
and almost entirely sequenced. The GPIIb exon 5 from the proband and
her father showed distinct SSCP patterns
(Fig 3A). Sequencing of exon 5 and its
flanking regions showed the existence of a heterozygous CA
base substitution at position +2 of intron 5 (Fig 3C). This mutation,
[IVS5(+2) CA], changes the sequence of the donor splicing
site of intron 5, predicting the incorporation of the unspliced intron
(76 bp) into the mRNA, resulting in frameshift and premature
polypeptide chain termination. The translational product of this
messenger would be a truncated protein (~30 kD) one third of the
normal size. A second abnormal SSCP pattern was observed in exon 21 from the patient and her mother (Fig 3B), whose sequence identified a
heterozygous TC transition that changes
Cys674Arg (Fig 3D). The
T2113C transition creates a Fok I restriction
site. None of these mutations was found in genomic DNA from more than
100 unrelated individuals, suggesting that they might be involved in
the etiopathogenesis of the thrombasthenic phenotype. To verify the
presence of these mutations and to assess the carrier status of kindred
members, we performed allele-specific amplification (ASPCR) using
antisense oligonucleotides whose 3' end bases were complementary
to either the normal or the mutant sequence of GPIIb intron
536 and restriction analysis of exon 21 (results not
shown).
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| Fig 3.
Identification of the patient mutations in genomic DNA.
(A) A genomic DNA fragment of 442 bp comprising exons 5 to 7 of GPIIb
was PCR-amplified, digested with Taq I, denatured, and used to
search for mutations by SSCP analysis, using a 16% acrylamide-8.7%
glycerol gel at 14°C. (B) A 193-bp genomic DNA fragment comprising
exon 21 of GPIIb was subjected to SSCP analysis in a 14%
acrylamide-8.7% glycerol gel at 15°C. The arrows point to distinct
bands shown by the patient and her father or mother, respectively. (C)
and (D) show fragments of the sequencing ladders of the sense strands
(5' at the bottom). The arrows point to a heterozygous C to A
transversion at position +2 of exon 5-intron 5 boundary of GPIIb (C)
and a T to C transition in exon 21 of GPIIb (D).
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RT-PCR analysis of platelet mRNA-GPIIb.
The platelet contents of -actin-mRNA, GPIIb-mRNA, and GPIIIa-mRNA
were determined by the PCR-based TaqMan system24 under predetermined conditions of cycle number and amount of RNA in which the
TaqMan fluorescence emission was a function of the target DNA
concentration. The GPIIIa/-actin ratio was similar to the control in
all members of the family. In contrast, the GPIIb/-actin ratio
indicated that the platelet GPIIb-mRNA in the patient and her father
was reduced to 52% and 67% of the control value, respectively (results not shown).
To determine the allelic origin of the GPIIb transcripts in the
proband, we performed direct sequencing of a 1,190-bp PCR-amplified cDNA fragment encompassing exons 20 to 30, as described in Materials and Methods. Figure 4 (top panel) shows
that the proband contains only a cytidine at nucleotide position 2113, whereas her mother, who is heterozygous for the
T2113C transition, shows in the same position a
cytidine and a thymidine. According to this observation, virtually all
of the proband's GPIIb-mRNA is derived from the mother's allele
carrying the T2113C substitution.
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| Fig 4.
RT-PCR analysis of platelet GPIIb-mRNA. (Top panel) Total
RNA from the proband and her mother was reverse-transcribed and used as
template for the PCR amplification of a 1,190-bp fragment encompassing
exons 20 to 30 of GPIIb as described in Materials and Methods. Direct
DNA sequencing of the amplification products was performed in a model
ABIprism 377 DNA sequencer (Perkin-Elmer Cetus). (Bottom panel) (A)
779-bp DNA fragments encompassing exons 5 to 13 of GPIIb were amplified
using as template reverse-transcribed platelet RNA as described in
Materials and Methods. (B) Portions of the amplification products were
reamplified using a sense primer complementary to sequences of intron 5 of GPIIb. (C) and (D) depict the hybridization analysis of the PCR
products shown in (A) and (B), using a GPIIb probe lacking the
oligonucleotide sequences used for amplification. N, PCR products from
normal platelet RNA.
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To analyze the effect of the IVS5(+2)CA mutation on the mRNA
processing, platelet total RNA from the proband and her relatives was
reverse-transcribed with the oligonucleotide GPIIb (1305-1285), 5'-ACCCAGGAACACCAGCACTTG-3', and used as template for the
amplification of a 779-bp cDNA fragment encompassing exons 5 to 13. Figure 4A (bottom panel) shows a single DNA fragment of 779 bp from
normal DNA (N), the proband, and her parents. The lack of amplification products other than the 779-bp fragment confirmed the absence or
extremely low availability of transcripts from the IVS5(+2)CA mutated allele as a result of either insertion of intron 5 (76 bp) or
alternate splicing. This point was further investigated by reamplifying
portions of the first PCR product with a sense primer complementary to
a sequence of intron 5, GPIIb-(intron 5, 1-19)
5'-GCGAGTAGGGAGCAAAAGC-3', that could only be found in transcripts from the mutant allele. This reamplification (Fig 4B,
bottom panel) yielded a product of the same size as that of the primary
PCR product, most probably due to carryover of the primary sense
primer, and a new band of 727 bp seen only in the proband and her
father. When the first PCR was performed with the sense primer for
intron 5, there was no amplification. The identity of the amplification
products shown in Fig 4A and B (bottom panel) was established by
hybridization analysis using a GPIIb probe lacking the oligonucleotide
sequences used for the PCR amplification. The two hybridization signals
so obtained (Fig 4C and D, bottom panel) corresponded to the
amplification products shown in Fig 4A and B. Sequencing of the 727-bp
band showed that it was the result of insertion of intron 5 (76 bp)
into the GPIIb-mRNA. The minute representation of this transcript
indicates instability of the transcriptional product of the
[IVS5(+2)CA]-GPIIb allele.
Exontrap analysis.
Because of initial difficulties in obtaining platelets from this
kindred, we studied the influence of the [IVS5(+2)CA]GPIIb mutation on the processing of mRNA by the exontrap analysis system. Figure 5 depicts the results obtained in
the analysis of a fragment of genomic DNA encompassing exons 5 to 7 from either normal or mutant [IVS5(+2)CA]GPIIb alleles in
the expression vector pET01. RT-PCR analysis of RNA from CHO cells
transiently transfected with the normal genomic DNA yielded a GPIIb
fragment of 471 bp. In contrast, a 548-bp major product and a less
abundant one of 481 bp were detected in cells transfected with mutant
DNA. Sequencing of these products showed that the 471-bp fragment
amplified from the control comprised exons 5 to 7 of GPIIb flanked by
the pET01 exons. The major 548-bp product from cells transfected with
the mutated cDNA was shown to be the result of insertion of intron 5 (76 bp) into the normal sequence. In the smaller product (481 bp), we
found insertion of intron 5 and a partial deletion of exon 7 due to
activation of a cryptic splicing site and recovery of the normal
reading frame. The translation of this transcript would yield a mutated
polypeptide similar in size to the normal GPIIb. However, this form of
messenger was not detected by RT-PCR analysis in the proband's
platelet.
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| Fig 5.
Exontrap analysis of a fragment of genomic DNA
encompassing exons 5 to 7 of GPIIb. A fragment of genomic DNA
encompassing exons 5 to 7 of GPIIb from the proband and a control were
cloned in the expression vector pET01 and transfected into CHO cells as
described in Materials and Methods. RT-PCR analysis of RNA from the
transfected cells yielded a fragment of 471 bp in the control and a
product of 548 bp and a second smaller, less represented product of 481 bp in cells transfected with DNA from the
[IVS5(+2)CA]-GPIIb allele of the proband. Sequencing of
the amplification products demonstrated that the 471-bp fragment
comprised exons 5 to 7 of GPIIb and the vector exons, the 548-bp
fragment is the result of insertion of intron 5 into the normal
sequence, and the 481-bp product has intron 5 inserted and exon 7 partially deleted.
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Heterologous expression of normal or mutated forms of GPIIb.
The low representation of transcripts from the
[IVS5(+2)CA]GPIIb allele suggested a poor functionality of
its translational product. However, because the possibility existed
that the abnormal GPIIb form could accumulate, we performed a
heterologous overexpression of [intron 5]GPIIb to analyze its
interaction with GPIIIa. CHO cells inherently expressing human GPIIIa
(CHO-GPIIIa cells) were stably transfected with either the construct
pCEP4-GPIIb or pCEP4-[intron 5]GPIIb. Cells transfected with the void
plasmid showed surface expression of GPIIIa associated with endogenous
alpha subunits (Fig 6A). Cells transfected
with normal GPIIb showed surface exposure of GPIIb accompanied by a
significant increase in the fluorescence signal of GPIIIa (Fig 6B),
whereas no surface fluorescence changes in GPIIb-IIIa were observed in
cells transfected with the mutant [intron 5]GPIIb cDNA (Fig 6C).
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| Fig 6.
Flow cytometric analysis of CHO-GPIIIa cells stably
transfected with cDNAs encoding normal or mutant [intron 5]GPIIb. CHO
cells derived from hygromycin-resistant clones were analyzed for
surface expression of GPIIb-IIIa by flow cytometry. The cells were
incubated with either anti-GPIIb (M3) or anti-GPIIIa (P37) MoAbs and
then treated with FITC-conjugated rabbit F(ab') antimouse IgG.
(A) Surface expression of GPIIIa and GPIIb in cells inherently
expressing human recombinant GPIIIa (CHO-GPIIIa cells). (B) Surface
expression of GPIIb and GPIIIa in CHO-GPIIIa cells stably transfected
with cDNA encoding normal human GPIIb. (C) Surface expression of GPIIb
and GPIIIa in CHO-GPIIIa cells stably transfected with cDNA encoding
[intron 5]GPIIb. NC, negative control, fluorescence of cells
incubated only with the second antibody.
|
|
To analyze the function of the Cys674Arg
mutation, we performed transient cotransfections of CHO cells with
pcDNA3 plasmids containing GPIIIa and either normal or mutant
[C2113]GPIIb cDNA. The cells coexpressing normal GPIIb
and GPIIIa showed surface exposure of GPIIb-IIIa complexes
(Fig 7A), but surface expression was
strongly attenuated in cells coexpressing normal GPIIIa with the mutant
GPIIb (Fig 7A).
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[in a new window]
| Fig 7.
Immunoprecipitation analysis of GPIIb-IIIa complexes from
CHO cells transiently cotransfected with cDNAs encoding GPIIIa and
either normal or [Arg674]GPIIb. CHO cells were
transiently transfected with cDNAs encoding GPIIIa and either normal or
[Arg674]GPIIb. Surface (upper panel) or total (surface
and intracellular, lower panel) labeling of cells was performed with
biotin-NHS and the GPIIb-IIIa complexes were immunoprecipitated with
anti-GPIIIa (P37) or anti-GPIIb (M3) MoAbs and processed as described
in Materials and Methods.
|
|
To estimate the intracellular GPIIb-IIIa content, we incubated total
cell lysates with biotin before the immunoprecipitation procedure. The
pattern of bands from cells coexpressing normal subunits was similar to
that shown in surface labeled cells, except that proGPIIb was clearly
identified when the immunoprecipitation was performed with the
anti-GPIIb MoAb (Fig 7B). Immunoprecipitation of biotin-labeled lysates
from cells coexpressing normal GPIIIa and mutant
[Arg674]GPIIb with anti-GPIIb MoAb showed the presence of
a clear proGPIIb band and weaker bands migrating like GPIIb and GPIIIa.
When the anti-GPIIIa MoAb was used, GPIIIa and a faint band migrating
like GPIIb were observed (Fig 7B). These observations agree with the platelet immunoprecipitation analysis and seem to suggest that [Arg674]GPIIb is able to complex GPIIIa, but it fails to
maintain a normal rate of maturation and surface expression.
| |
DISCUSSION |
The low platelet GPIIb-IIIa complex (~10%) and fibrinogen (23%)
content indicate that the proband is a type II case of GT. The present
study demonstrates that she inherited a splicing site mutation
[IVS5(+2)CA] from her father and a point mutation, a T2113C transition that changes
Cys674Arg, from her mother and, therefore, is a
compound heterozygote for the GPIIb gene (Fig 3). Both mutations
cosegregate with reduced platelet content of GPIIb-IIIa and neither of
them was found in genomic DNA from 100 normal unrelated individuals,
ruling it out as being due to polymorphisms. As far as we know, this is
the sixth reported case of a GPIIb mutation associated with type II GT.19 However, functional studies have been performed only
in two of them.25,37,38 Theoretically, the
[IVS5(+2)CA]-GPIIb mutation should lead to insertion of
intron 5 into the mRNA resulting in a frame shift and appearance of a
premature termination codon. This prediction was verified by exontrap
analysis and, later on, confirmed by platelet GPIIb-mRNA sequencing.
The presence of a premature stop codon is accompanied by changes in the
amount of transcript. In our case, the extremely low amount of
messenger from the [IVS5(+2)CA]-GPIIb allele is consistent
with the idea that the amount of transcript is position related, so
that the closer to the 5' end of the coding sequence, the lower
the amount of transcript.39 The -thalassemia syndrome
was the first reported case of a human disease associated with splicing
junction mutations.40 Splice mutations in GPIIb or GPIIIa
associated with thrombasthenic phenotypes have been recently
reported.41-46 However, the [IVS5(+2)CA]-GPIIb mutation by itself cannot explain the thrombasthenic phenotype of the
patient, because her father, who is also heterozygous for this
mutation, shows a diminished platelet GPIIb-IIIa receptor but is
clinically asymptomatic.
The patient shows a decreased (~50%) platelet content of GPIIb-mRNA.
The possibility that part of this messenger was formed by normal
splicing of the [IVS5(+2)CA]-GPIIb allele, which could contribute to the platelet expression of GPIIb-IIIa receptor, is
improbable. This assertion is based on the exclusive finding of the
mutant [Arg674]GPIIb allele, inherited from her mother,
in the direct sequencing of the PCR-amplified platelet GPIIb-cDNA from
the patient.
The pathogenic significance of the [Arg674]GPIIb mutation
is suggested by (1) its association with reduced platelet content of GPIIb-IIIa receptor in the heterozygous states; (2) very low surface exposure of GPIIb-IIIa receptors and elevated ratio of proGPIIb to
GPIIb in the platelets from the proband who carries only the [Arg674]GPIIb-mRNA; and (3) accumulation of proGPIIb
and failure to show a normal level of GPIIb-IIIa surface exposure when
[Arg674]GPIIb was coexpressed with GPIIIa in CHO cells.
These observations indicate that subunit dimerization is not prevented,
but the maturation of GPIIb and/or intracellular transit
proceeds at an abnormally low rate. Cys674 forms an
intrachain disulfide bond with Cys687.47
Because no mutations other than [Arg674]GPIIb have been
found by RT-PCR analysis of GPIIb and GPIIIa-mRNAs in the proband and
her mother, it seems plausible to assume that disruption of the
intrachain 674-687 disulfide bond in GPIIb impedes the appropriate
subunit conformation to confer either heterodimer stability
and/or a normal rate of maturation and surface expression of
the GPIIb-IIIa receptor. The endoproteolytic cleavage of GPIIb requires
its association with GPIIIa.48 Thus, the increased intracellular proGPIIb to GPIIb ratio in either platelets or in cotransfected CHO cells may reflect either a limited availability of
heterodimers or a decreased affinity of the cleavage enzyme(s) for the
abnormal complexes. The failure to immunoprecipitate proGPIIb with an
anti-GPIIIa MoAb in cells coexpressing GPIIIa and
[Arg674]GPIIb (Fig 7B) seems to indicate an altered rate
of association of pro[Arg674]GPIIb with GPIIIa. The
functional importance of intrachain disulfide bonds in GPIIb-IIIa has
been previously noticed. Deletion of Cys107, which forms
the 107-130 intrachain disulfide bond in GPIIb,47 prevents
heterodimerization and surface expression of GPIIb-IIIa complexes.49 Moreover, disruption of the 374-386 disulfide
bond by substitution of Cys374 by Tyr in GPIIIa is
associated with type II thrombasthenia.50 However, removal
of Cys655 has no apparent effects on the surface expression
of GPIIb-IIIa,51 indicating that not all of the highly
conserved cysteine residues in this integrin are essential for the
function of the complex.
The parents of the proband, who are heterozygous for the
[IVS5(+2)CA]GPIIb and the [C2113]GPIIb
mutations, respectively, are both clinically asymptomatic and show a
marked reduction in the platelet content of GPIIb-IIIa. A reduced
expression of the platelet GPIIb-IIIa receptor has been observed in
heterozygous states for other GPIIb mutations, but the recessive
character of GT may be the reason for the lack of attention paid to the
biochemical features of these clinically asymptomatic states. Two
possibilities could be considered to explain the reduced platelet
content of GPIIb-IIIa in the heterozygous states: (1) a quantitative or
qualitative change in the mRNAs and (2) the translational products of
mutated messengers could have distinct functional properties, as has
been suggested before for other human diseases.52-56
PCR-based determination showed a reduction of platelet GPIIb-mRNA in
the proband and her father and a normal content in her mother. Because
the contribution of the mutated [IVS5(+2)CA]GPIIb allele to
the total mRNA is negligible, the reduced platelet expression of
GPIIb-IIIa receptor seems to correlate with a decreased availability of
messenger in the proband's father. At least 50% of the platelet
GPIIb-mRNA is accounted for by the mutated [T2113]GPIIb
allele in the proband's mother. Thus, the diminished platelet GPIIb-IIIa receptor in both heterozygous states seems to correlate with
a reduced availability of normal GPIIb messenger. Nevertheless, the
differences in the surface expression of GPIIb-IIIa in both cases,
taken together with the repression of the GPIIIa surface exposure
associated with endogenous subunits by the mutated
[Arg674]GPIIb (results not shown), suggest the operation
of mechanisms other than mRNAs availability in controlling the surface
exposure of the GPIIb-IIIa receptor.
To conclude, this work reports a case of a compound heterozygote for
the GPIIb gene associated with type II GT. The proband presents two
novel GPIIb mutations: a heterozygous CA base substitution at
position +2 of the exon 5-intron 5 boundary [IVS5(+2)CA], and a T2113C transition that changes
Cys674Arg674 disrupting the 674-687 intrachain disulfide bond. The following observations support the
etiopathogenic significance of these two mutations: (1) Neither of them
was found in a large number of normal unrelated individuals. (2) The
clinically asymptomatic heterozygous states for each of these mutations
show reduced platelet content of GPIIb-IIIa. (3) Cotransfection of
cDNAs encoding normal GPIIIa and [Arg674]GPIIb showed
accumulation of proGPIIb and markedly diminished surface exposure of
GPIIb-IIIa. Thus, the thrombasthenic phenotype of the patient is the
result of the additive effect of both mutations. The splicing junction
mutation acts primarily by limiting the availability of GPIIb-mRNA,
whereas the translational product of [C2113]GPIIb forms
heterodimers with GPIIIa unable to undergoing the pathway of
maturation, intracellular trafficking, and/or membrane assembly
at normal rates.
| |
ACKNOWLEDGMENT |
The authors thank Dr N. Kieffer for the gift of the CHO-GPIIIa cells.
| |
FOOTNOTES |
Submitted July 17, 1998; accepted September 21, 1998.
Supported in part by grants from Dirección General de
Investigación Científica y Técnica (PB94-1544),
Fondo de Investigaciones Sanitarias (96/2014), CAM: CO7191, and
European Community concerted action contract no. BMH1-CT93-1685.
M.F.-P., M.F., and E.G.A.-S. were recipients of predoctoral fellowships
from the Council of Research of the Autonomous Community of Madrid, the
Spanish Secretary of Education and Science, and the Fundación
Areces, respectively.
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 Roberto Parrilla, MD, Centro
de Investigaciones Biológicas (CSIC), Velázquez 144, 28006-Madrid, Spain; e-mail: rparrilla{at}fresno.csic.es.
| |
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Abstract
Introduction