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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
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.
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 expression 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 + 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) 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 A 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.
Patients
Normal controls from the general population
Coagulation laboratory investigations
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
FV mRNA studies 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 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 µL 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 µmol/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.
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.
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 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 A G 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 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 APC-mediated 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 APC-mediated 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. 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 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.
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, 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 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.
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 from the Department of Chemistry at Cleveland State University (M.K.).
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.
Reprints: Francesco Bernardi, Department of Biochemistry and Molecular Biology, Via L. Borsari 46, 44100 Ferrara, Italy; e-mail: ber{at}unife.it.
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  D. Scanavini, D. Girelli, B. Lunghi, N. Martinelli, C. Legnani, M. Pinotti, G. Palareti, and F. Bernardi Modulation of Factor V Levels in Plasma by Polymorphisms in the C2 Domain Arterioscler. Thromb. Vasc. Biol., January 1, 2004; 24(1): 200 - 206. [Abstract] [Full Text] [PDF]
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 M. C. Montefusco, S. Duga, R. Asselta, M. Malcovati, F. Peyvandi, E. Santagostino, P. M. Mannucci, and M. L. Tenchini Clinical and molecular characterization of 6 patients affected by severe deficiency of coagulation factor V: broadening of the mutational spectrum of factor V gene and in vitro analysis of the newly identified missense mutations Blood, November 1, 2003; 102(9): 3210 - 3216. [Abstract] [Full Text] [PDF] |
Top
Abstract
Introduction
-sheet, and interacts via 2 hydrogen bonds with a proline residue (P791), which is also absolutely
conserved (Figure 
A polymorphism associated to factor V Leiden: a report of two thrombophilic families.
Thromb Res.
1998;89:249-252