Download FV R506Q, FV H1299R, FV Y1702C, PT 20210G/A

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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Combinations of 4 mutations (FV R506Q, FV H1299R, FV Y1702C, PT
20210G/A) affecting the prothrombinase complex in a thrombophilic family
Elisabetta Castoldi, Paolo Simioni, Michael Kalafatis, Barbara Lunghi, Daniela Tormene, Domenico Girelli,
Antonio Girolami, and Francesco Bernardi
The study of the molecular bases of
thrombophilia in a large family with 4
symptomatic members is reported.
Three thrombophilic genetic components (FV R506Q, FV H1299R, and PT
20210G/A), all affecting the activity of
the prothrombinase complex, were detected alone and in combination in various family members. In addition, a newly
identified missense mutation (factor V
[FV] Y1702C), causing FV deficiency,
was also present in the family and appeared to enhance activated protein C
(APC) resistance in carriers of FV R506Q
or FV H1299R by abolishing the expres-
sion of the counterpart FV allele. The
relationships between complex genotypes, coagulation laboratory findings,
and clinical phenotypes were analyzed
in the family. All symptomatic family
members were carriers of combined defects and showed APC resistance and
elevated F1 ⴙ 2 values. Evidence for the
causative role of the FV Y1702C mutation, which affects a residue absolutely
conserved in all 3 A domains of FV,
factor VIII, and ceruloplasmin, relies on
(1) the absolute cosegregation between
the mutation and FV deficiency, both in
the family and in the general population;
(2) FV antigen and immunoblot studies
indicating the absence of Y1702C FV
molecules in plasma of carriers of the
mutation, despite normal levels of the
FV Y1702C messenger RNA; and (3)
molecular modeling data that support a
crucial role of the mutated residue in the
A domain structure. These findings help
to interpret the variable penetrance of
thrombosis in thrombophilic families
and to define the molecular bases of FV
deficiency. (Blood. 2000;96:1443-1448)
© 2000 by The American Society of Hematology
Introduction
The heterogeneity of clinical phenotypes and the variable manifestations of thrombosis observed in thrombophilic families have led
to the hypothesis that predisposition to venous thrombosis results
from the combination of several genetic defects.1-4 Functional
polymorphisms that are relatively frequent in the population, such
as factor V (FV) R506Q (Leiden mutation)5 and the prothrombin
(PT) 20210G/A mutation,6 have been found to play a major role as
risk factors.7-8 Both act by enhancing thrombin generation by the
prothrombinase complex,9 via different mechanisms. FV R506Q
impairs activated protein C (APC)-mediated FVa inactivation,10-12
a condition termed APC resistance,13 and the PT 20210G/A
mutation causes an increase in the concentration of circulating PT.6
Combinations of FV R506Q with the PT mutation and other
thrombophilic defects have been repeatedly documented in thrombophilic patients.14-17
The crucial role of FV, strategically placed at the crossroads of
the procoagulant and anticoagulant pathways,18-19 has prompted the
quest for new candidate determinants of venous thromboembolism
in the FV gene. A peculiar FV allele (FV H1299R, also known as
R2), marked by an A3G transition at position 4070,20 has been
described and found to be linked to a number of other FV gene
polymorphisms (the HR2 haplotype), which encode several amino
acid changes.21 Carriership of the FV H1299R allele is associated
with mild APC resistance21 and with a relative excess22 of the more
thrombogenic23 FV isoform24 (FV1) in plasma. Accordingly, it has
been reported that the FV H1299R mutation increases the risk of
venous thrombosis in carriers of the FV R506Q mutation25 and
represents per se a thrombotic risk factor.26
Moreover, quantitative FV deficiency,27 a potentially anticoagulant defect,28 has been shown to enhance APC resistance in
heterozygous carriers of the FV R506Q.29-32
We report here the molecular characterization of a thrombophilic family encompassing 3 thrombophilic mutations (FV R506Q,5
FV H1299R,20 and PT 20210G/A6) and quantitative FV deficiency.
The relationships between combinations of mutations affecting
components of the prothrombinase complex and coagulation and
clinical phenotypes were evaluated in the family.
From the Department of Biochemistry and Molecular Biology, Ferrara University,
Ferrara, Italy; Department of Medical and Surgical Sciences, University of Padua
Medical School, Padua, Italy; Department of Chemistry, Cleveland State University,
Cleveland OH; Department of Molecular Cardiology, Cleveland Clinic Foundation,
Cleveland OH; and Institute of Medical Pathology, Chair of Internal Medicine, Verona
University, Verona, Italy.
from the Department of Chemistry at Cleveland State University (M.K.).
Submitted October 4, 1999; accepted April 18, 2000.
Supported by Telethon, Italy (grant E.675) and MURST. Also supported in part
by a grant from Veneto Region, RSF No. 783/01/97 (P.S.) and by start-up funds
BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
Patients, materials, and methods
Patients
The thrombophilic family (Figure 1) came to clinical observation via
subject II4, a 51-year-old male who has experienced recurrent spontaneous
superficial vein thrombosis (SVT) and a deep vein thrombosis (DVT) in the
right leg. Three additional family members across 2 generations have
Reprints: Francesco Bernardi, Department of Biochemistry and Molecular
Biology, Via L. Borsari 46, 44100 Ferrara, Italy; e-mail: [email protected].
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.
© 2000 by The American Society of Hematology
1443
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1444
BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
CASTOLDI et al
Figure 1. Family pedigree and coagulation laboratory data. The propositus is
subject II4 (arrow). Open circle, FV Leiden (R506Q); gray circle, FV R2 (H1299R);
closed circle, FV defect (Y1702C); open triangle, PT 20210G/A. Symptomatic family
members are hatched. The numbers reported below each family member express the
FV antigen and activity levels (normal range, 70%-130%), the normalized APC ratio
(APC resistance is defined by an APC ratio less than 0.84), the PT antigen and
activity levels (normal range, 75%-125% and 80%-120%, respectively), and the
F1 ⫹ 2 values in nmol/L (normal range, 0.4-1.1 nmol/L), respectively.
Figure 2. Detection of the 5279A/G mutation (FV Y1702C). (A) Detection by direct
sequencing: The heterozygous sequencing pattern of the propositus is shown. The
“R” in the nucleotide sequence indicates that either an A or a G may be present at this
position. (B) Detection by restriction analysis: Mutagenized (bold) primer P1, which
creates an AccI restriction site (highlighted in gray) in the normal (A, underlined)
allele, is used in combination with reverse primer P2 to amplify a 120-bp DNA
fragment. The fragments obtained by AccI digestion of the polymerase chain reaction
products are visualized by agarose gel electrophoresis. C43, individual from the
general population; M, molecular weight marker.
experienced both SVT and DVT (I2, I3, II8) and, 2 of them (I2 and I3), also
pulmonary embolism.
FV mRNA studies
Normal controls from the general population
A sample of 252 subjects from northern Italy was recruited to perform a
population-based screening for the FV Y1702C mutation. Informed consent
to participate in the study was obtained from all subjects.
Coagulation laboratory investigations
The presence of PC, PS, and antithrombin III deficiencies in the family was
excluded by conventional coagulation tests. PT antigen levels were
determined by a specific enzyme linked immunosorbent assay (ELISA) as
previously described.33 PT activity was detected in a thromboplastin-based
assay using factor II–deficient plasma on a ACL 3000Plus analyser
(Instrumentation Laboratories, Milan, Italy). Normal ranges, obtained from
100 healthy subjects of both sexes 15 to 75 years old, were 75% to 125%
and 80% to 120% for PT antigen and activity, respectively. FV antigen
(FV:Ag) and activity (FV:c) levels, as well as APC resistance, were
measured as previously described.30 The normal ranges were 70% to 130%
for FV:Ag and FV:c and more than 0.84 for the normalized APC ratio.
F1 ⫹ 2 levels were determined in the family members using Enzygnost
F1 ⫹ 2 micro (Dade-Behring, Marburg, Germany) as previously described.34 The normal range was 0.4 to 1.1 nmol/L.
Total RNA was extracted from platelets, which are known to contain trace
amounts of FV messenger RNA (mRNA), by conventional methods
(RNAfast, Molecular Systems, San Diego, CA). FV mRNA was reverse
transcribed using primer P5 (5⬘-AAGAATAATTTGAACCAACAAT-3⬘, nt.
2425-2404), located in exon 13 (Figure 3), and Super Script II RT reverse
transcriptase (GIBCO-BRL, Life Technologies). A 382-bp FV complementary DNA (cDNA) fragment spanning exons 12 to 13 was synthesized in 2
rounds of polymerase chain reaction using forward primers P3 (5⬘TGACCCTCTTCCCCATG-3⬘, nt. 2012-2028) and P4 (5⬘-ACGGTCACAATGGATAATGT-3⬘, nt. 2044-2063), both in exon 12, and reverse
primer P5 as described31-32 (Figure 3). This region contains a neutral
TaqI restriction polymorphism (2391 G/A),36 the A allele of which marks
both the FV R506Q allele and the FV-deficient allele (Y1702C) in this
family. Heterozygosity for this marker in the propositus’ daughters who
carry the FV R506Q (III4) and Y1702C (III5) mutations, respectively,
offers the opportunity to evaluate the relative expression at the mRNA level
of both these alleles with respect to a normal FV allele. The cDNA
fragments obtained from family members III4 and III5 were digested
with TaqI and the products run on a 2.5% agarose gel and stained
DNA studies
Exon scanning of the FV gene was performed by direct sequencing as
described.31 Primers located in introns 14 (5⬘-AACCAGCCATTTTGACTTA-3⬘) and 15 (5⬘-GAAATAACCCCGACTCTTC-3⬘), respectively,
were used to amplify and sequence (Figure 2A) a 410–base pair (bp) DNA
fragment spanning the whole exon 15. A restriction protocol for the
detection of the 5279A/G mutation (Figure 2B) was obtained by means of a
mutagenized primer (5⬘-CTGTCGGGCTTGGGTCT-3⬘, P1, nucleotides
[nt.] 5262-5278) introducing an AccI restriction site in the normal allele.
Forward primer P1 was used in combination with reverse primer P2
(5⬘-GAAATAACCCCGACTCTTC-3⬘, intron 15) to amplify a 120-bp
DNA fragment, which was subsequently digested with AccI (New England
Biolabs, Beverly, MA) under the conditions recommended by the manufacturer. The FV H1299R and PT 20210G/A polymorphisms were detected as
reported.20,35
Figure 3. Expression at the mRNA level of the FV genes bearing the Leiden
mutation (R506Q) and the FV defect (Y1702C), respectively, relative to a normal
(Nor) FV allele. Primers P3, P4, and P5 are used to amplify a nested FV cDNA
fragment spanning exons 12 to 13 and containing a polymorphic TaqI restriction site.
The 382-bp uncleaved cDNA, corresponding to the A allele of the TaqI polymorphism,
marks the FV R506Q allele in subject III4 and the FV Y1702C allele in subject III5; the
346-bp TaqI restriction product, corresponding to the G allele of the TaqI polymorphism, marks the normal (Nor) FV allele in both subjects. The gene and cDNA
schemes are drawn to different scales. IVS 12, intron 12.
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BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
FOUR DIFFERENT MUTATIONS IN A THROMBOPHILIC FAMILY
1445
with ethidium bromide (Figure 3). The expression at the mRNA level of
different FV alleles was evaluated by densitometric analysis of the
obtained bands.
Protein studies
FV molecules present in plasma of the propositus and of family members
II2, II5, and III1 were characterized by Western blotting (Figure 4) as
described.32 Briefly, 100 ␮L of plasma was diluted 10-fold with HBS Ca⫹⫹
(20-mmol/L HEPES, 0.15-mol/L NaCl, 5-mmol/L CaCl2, pH 7.4) and
incubated at 37°C in the presence of synthetic phospholipid vesicles (PCPS,
20 ␮mol/L). After clotting, the solution was centrifuged at 10 000 rpm for
30 seconds, and purified human APC (5-20 nmol/L) was added to the
supernatant. Aliquots were drawn from the mixture at regular intervals and
run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to
monitor FV inactivation. The gel was transferred to nitrocellulose and
stained with monoclonal antibody ␣HFVaHC#17, which recognizes an
epitope located between residues 307 and 506 of the FVa heavy chain. The
FVa inactivation pattern obtained for the propositus was compared with
those of normal and FV R506Q homozygous controls.
Figure 5. Conservation and structural role of the mutated tyrosine (FV Y1702) in
the A domains. (Top) Alignment of a portion of the A domains of human FV, FVIII, and
CP. The conserved tyrosine and proline residues are highlighted. The tyrosine
residue affected by the FV 5279A/G mutation is boxed. (Bottom) Three-dimensional
model of a portion of the A3 domain of FV based on the coordinates of CP. The highly
conserved Y1702 residue forms 2 hydrogen bonds with Pro1618. The substitution of
the tyrosine residue by a cysteine, predicted by the FV 5279A/G mutation, causes the
loss of these interactions.
Molecular modeling
The FV Y1702C mutation in the A3 domain of FV was investigated using
the coordinates of the highly homologous A3 domain of ceruloplasmin
(CP)37 (PDB 1KCW), by means of the shareware software Swiss PdbViewer v3.5b3, developed by Nicholas Guex, Torsten Schwede, and
Alexander Diemand for the Glaxo Wellcome Experimental Research.
Results
A complete coagulation screening in the propositus revealed the
presence of marked APC resistance, elevated PT levels, and
quantitative FV deficiency (Figure 1). Accordingly, molecular
genetics investigations showed that he was a heterozygous carrier
of both FV R506Q and PT 20210G/A (Figure 1).
Identification and characterization of a new missense mutation
causing FV deficiency
Figure 4. Time courses of APC-mediated FVa inactivation. (A) Time course of FVa
inactivation in the FV R506Q/Y1702C doubly heterozygous propositus. The inactivation end-point patterns of normal (N) and R506Q (L) FVa are reported for comparison.
In the normal control, the FV heavy chain (HC) is cleaved by APC at Arg506 and
Arg306, resulting in a 30-kd immunoreactive fragment (HC307-506). In the FV R506Q
homozygote, cleavage of the FV heavy chain does not generate the HC307-506 fragment,
and a 60/54-kd doublet (HC307-679/709) appears instead. The propositus’ FV shows an
inactivation pattern indistinguishable from that of the R506Q FV homozygote, which
indicates that the Y1702C FV is not expressed in plasma. HCIIa is a shorted heavy
chain of FVa, due to the prolonged exposure of FVa to the thrombin generated
following clot formation and characterized by considerably lower cofactor activity. It
suggests a potentially alternative method for FVa inactivation in FVR506Q individuals.50,51 Lane 1, FVa in the presence of phospsholipid vesicles before adding APC;
lanes 2 to 4, FVa 5, 10, and 20 minutes after the addition of APC. Molecular weights (kd) are
reported on the left. (B) Time course of FVa inactivation in family members carrying the FV
Y1702C mutation as a single heterozygous defect. The normal FVa inactivation
pattern (presence of the 30-kd band and absence of the 60/54-kd doublet) indicates
that the Y1702C FV molecules do not contribute to the altered FVa inactivation pattern
(propositus, Figure 4A). Lane 1, FVa in the presence of phospholipid vesicles before adding
APC; lanes 2 to 5, FVa 3, 5, 10, and 20 minutes after the addition of APC.
To spot the mutation responsible for FV deficiency in the propositus, coding regions and splicing junctions of the FV gene were
scanned by direct sequencing. A novel A3G transition, which
predicts the substitution of Y1702 by a cysteine in the A3 domain
of FV, was identified at nucleotide 5279 in exon 15 in the
heterozygous state (Figure 2A). Alignment of the FV A domains
with the homologous counterparts38-39 of factor VIII (FVIII) and
CP showed the absolute conservation of the tyrosine residue
affected by the mutation and a high degree of homology among the
surrounding amino acid sequences (Figure 5).
Because the FV Y1702C mutation is not detectable by restriction enzyme digestion, a mutagenized primer introducing an AccI
restriction site in the normal (A) allele (Figure 2B) was designed to
allow rapid screening for the mutation (see “Materials and methods”). The FV Y1702C substitution was found (Figure 2B) in all
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1446
BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
CASTOLDI et al
family members whose FV levels (Figure 1) were compatible with
heterozygous FV deficiency, but it was absent in all others.
To substantiate the causative role of the FV Y1702C mutation,
252 healthy individuals from the general population were tested for
the presence of the Y1702C substitution and, in parallel, characterized for FV:c levels (mean 99%, SD 24%, range 30%-187%). One
of these controls (C43 in Figure 2B) was found to be a carrier, which
was confirmed by direct sequencing, and showed reduced FV
levels (FV:c 58%).
The expression of the FV allele bearing the Y1702C mutation
was investigated both at the mRNA and protein levels. Total RNA
extracted from platelets was reverse transcribed and a FV cDNA
fragment, spanning exons 12 to 13 and containing a TaqI restriction
polymorphism in linkage with both the FV R506Q and the
FV-deficient (Y1702C) alleles, was amplified (Figure 3). Heterozygosity for this marker in the propositus’ daughters who carry the FV
R506Q (III4) and the Y1702C (III5) mutations, respectively, made
it possible to compare the expression at the mRNA level of both
mutant genes with that of a normal FV allele. Densitometric
analysis of the TaqI-restricted FV cDNA fragments obtained from
subjects III4 and III5 indicated that the FV genes carrying either
mutation (Y1702C or R506Q) were normally expressed at the
mRNA level (Figure 3).
The expression of the FV Y1702C mutation at the protein level
was studied in the propositus, who carries the R506Q mutation on
the counterpart FV allele. This doubly heterozygous condition
offers the opportunity to evaluate the protein expression of the FV
Y1702C allele, because plasmatic FV bearing the R506Q substitution is clearly recognizable on an immunoblot by its characteristic
APC-mediated inactivation pattern.40 The time course of APCmediated FVa inactivation in vitro showed a pattern indistinguishable from that of a FV R506Q homozygote (Figure 4A), providing
direct evidence for the impaired secretion of FV bearing the
Y1702C substitution. However, to further investigate the possibility that FV molecules encoded by the FV Y1702C allele contribute
to the abnormal FVa inactivation pattern observed in the propositus, family members who carry the FV Y1702C mutation as a
single defect (II2, II5, and III1) were also studied as controls
(Figure 4B). These experiments indicate completely normal APCmediated FVa inactivation patterns for all 3 subjects (Figure 4B),
which confirms that the FV allele bearing the Y1702C mutation is
not expressed at the protein level in plasma and thus does not
participate in the abnormal FVa inactivation pattern.
findings,20 carriers of this mutation had FV levels (Figure 1) in the
lower end of the normal range (mean FV:Ag 87, mean FV:c 85).
The FV Y1702C substitution was found as a single defect in 3
family members, in combination with FV H1299R in the propositus’ mother, and in combination with PT 20210G/A in one of his
daughters (Figure 1). In accordance with the results of the Western
blot, it caused (Figure 1) a 50% reduction of plasma FV antigen and
activity levels in all carriers (mean FV:Ag 51, mean FV:c 51). The
lowest FV levels (FV:c 36%) were observed in the propositus’
mother who, in addition to the FV Y1702C mutation, carried the
FV H1299R variant.
The presence of the PT 20210G/A mutation was detected in the
propositus and in 2 of his daughters, and PT antigen and activity
levels were measured in all family members (Figure 1). All subjects
showed PT levels within the normal range, and only a weak
correlation was observed between carriership of the PT 20210G/A
mutation and high PT levels. F1 ⫹ 2 levels (Figure 1) were
also determined as an integrated measure of prothrombinase
activity,41 and the highest values were observed in carriers of
double defects and particularly in those who had experienced
venous thromboembolism.
Discussion
Thrombin generation by the prothrombinase complex represents a
key step of the coagulation cascade. The rate of PT conversion
depends on the concentration and activity of the various components of this macromolecular complex.9,42 A prominent regulatory
role is played by FV and FVa,19 which are subject to43-44 and themselves
participate in45-46 the negative control by the APC system.
Three thrombophilic mutations (FV R506Q, FV H1299R, and
PT 20210G/A) were found in different combinations in various
members of a large thrombophilic family. All of them affect the
activity of the prothrombinase complex (Figure 6), by increasing
either the substrate concentration (PT 21210G/A)6 or the survival
of FVa in plasma (FV R506Q and FV H1299R). Elevated PT levels,
Genotype, phenotype relationships in family members
The presence of FV gene mutations (R506Q, H1299R, and
Y1702C) and of the PT gene variant (PT 20210G/A) was investigated and coagulation data collected in the propositus’ family
(Figure 1).
FV R506Q was detected in the heterozygous condition in 7
family members, once in combination with the PT 20210G/A
mutation and 3 times with FV H1299R (Figure 1). The presence of
FV R506Q was also inferred in the deceased propositus’ father,
again in combination with PT 20210G/A (paternity was confirmed
by FV microsatellite analysis in the family). As expected, all
carriers of the FV R506Q mutation showed APC resistance (Figure
1) that was particularly marked in double heterozygotes for FV
Y1702C (the propositus, APC ratio 0.39) or for FV H1299R (mean
APC ratio 0.57), which is known to enhance APC resistance in FV
R506Q carriers.21
The FV H1299R substitution was present in 7 family members,
both alone and in combination. In accordance with previous
Figure 6. Mutations affecting the prothrombinase complex in the family under
study. (Top) Schematic representation of FV molecules characterized by the Leiden
mutation (FV R506Q), the R2 haplotype (FV H1299R), and the FV defect (FV
Y1702C), respectively. The amino acid substitutions linked to the FV H1299R
mutation are indicated by the smaller triangles. (Bottom) Expected combined effects
of the FV and PT mutations on the activity of the prothrombinase complex. The
arrows indicate the increased cofactor activity for the R506Q and H1299R FV
molecules and the increased PT levels for the 20210G/A mutation. The crossed arrow
indicates the lack in plasma of the FV molecules bearing the Y1702C substitution.
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BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
FOUR DIFFERENT MUTATIONS IN A THROMBOPHILIC FAMILY
predicted by the PT 21210G/A mutation, have been shown to
enhance thrombin generation significantly in a reconstructed
plasma system.42 Differently, both FV mutations predict a delay in
the APC-mediated inactivation of FVa, either (FV R506Q) by
suppressing one of the APC cleavage sites on FV5,10-12 or (FV
H1299R) by determining a relative increase22 of the poorly
phospholipid-binding FVa1 form in plasma.23-24 In addition, they
might also act via reduced cofactor activity in the APC-mediated
inactivation of FVIIIa.46-47
Quantitative FV deficiency, a potentially anticoagulant defect
that might in principle decrease the activity of the prothrombinase
complex and that actually prolongs the initiation phase of coagulation,42 was also present in the propositus and other family
members. The molecular investigations led to the identification of a
candidate defect (5279A/G, Y1702C) in the FV gene. Genetic,
functional, and structural evidence supports the association of the
FV Y1702C mutation with FV deficiency. First of all, the family
studies and general population screening indicate a complete
cosegregation between the mutation and low FV levels; in fact, the
only carrier of the mutation detected in the general population had
FV levels reduced by half. However, to exclude that the FV
Y1702C mutation is a mere neutral variant in absolute linkage
disequilibrium with the causative mutation, the role of the mutated
tyrosine residue, absolutely conserved in all 3 A domains of FV,
FVIII, and CP (Figure 5), was investigated by inspection of the
3-dimensional structure37 of CP. This analysis revealed that the
homologous tyrosine residue (Y860) in this protein is buried in the
domain core, belongs to a conserved ␤-sheet, and interacts via 2
hydrogen bonds with a proline residue (P791), which is also
absolutely conserved (Figure 5) in all A domains. Molecular
modeling of the FV A domains,48 based on the approximate 40%
homology with the A domains of CP, indicates that FV Y1702 has
the same features. Substitution of the tyrosine residue by a cysteine
would lead to the loss of these interactions (Figure 5), potentially
1447
disrupting the domain scaffold. Although a disulfide bridge formed
between this cysteine and one of the other free cysteines in factor
V49 would stabilize the abnormal structure, the FV antigen levels
and Western blot analyses indicate that the product of the FV
Y1702C allele is not detectable in plasma, although its expression
at the mRNA level is normal.
Although the Y1702C FV does not participate in the abnormal
FVa inactivation, FV deficiency caused by the Y1702C mutation
turned out to contribute to shape the thrombotic risk profile in the
family. In carriers of the FV R506Q mutation (propositus) and of
the FV H1299R mutation (propositus’ mother), quantitative FV
deficiency due to the FV Y1702C mutation, far from protecting
from thrombosis, proved to increase APC resistance and thus
thrombotic risk via a pseudohomozygous APC resistance mechanism.30-32 As suggested by the Western blot, increased APC
resistance is attributable to the exclusive presence of R506Q
(propositus) or H1299R (propositus’ mother) FV molecules in
plasma. In this respect, the propositus’ mother is the first case of
“FV H1299R pseudohomozygous APC resistance” ever described.
Plasma levels of F1 ⫹ 2 represent an integrated measure of the
activity of the prothrombinase complex. Although the family-based
evaluation of F1 ⫹ 2 levels is poorly reliable because of the assay
variability, F1 ⫹ 2 values were found to be higher in carriers of a
double defect and appeared to correlate with thrombotic risk in this
family. It was also observed that all symptomatic family members
showed more or less pronounced APC resistance (Figure 1).
The relationships between clinical phenotypes and combined
genotypes indicated that all family members who had developed
thrombosis were carriers of at least 2 defects (Figure 1), the
combination of APC resistance and high PT levels being associated
with recurrent thrombotic episodes. This valuable information is of
potential help for the prevention of thrombosis in the young
combined heterozygotes of the third generation of this family.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2000 96: 1443-1448
Combinations of 4 mutations (FV R506Q, FV H1299R, FV Y1702C, PT
20210G/A) affecting the prothrombinase complex in a thrombophilic
family
Elisabetta Castoldi, Paolo Simioni, Michael Kalafatis, Barbara Lunghi, Daniela Tormene, Domenico
Girelli, Antonio Girolami and Francesco Bernardi
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