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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Clinical Chemistry, University
Medical Center, Utrecht; Department of Hematology, University Medical
Center, Utrecht; Van Creveldkliniek, Utrecht Department of Crystal and
Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht
University, Utrecht, The Netherlands; Department of Clinical Chemistry,
Ziekenhuis Eemland, Amersfoort, The Netherlands.
Coagulation factor V (FV) plays an important role in maintaining
the hemostatic balance in both the formation of thrombin in the
procoagulant pathway as well as in the protein C anticoagulant pathway. FV deficiency is a rare bleeding disorder with variable phenotypic expression. Little is known about the molecular basis underlying this disease. This study identified 5 novel mutations associated with FV deficiency in 3 patients with severe FV deficiency but different clinical expression and 2 unaffected carriers. Four mutations led to a premature termination codon either by a nonsense mutation (single-letter amino acid codes): A1102T, K310Term. (FV Amersfoort) and C2491T, Q773Term. (FV Casablanca) or a
frameshift: an 8-base pair deletion between nucleotides 1130 and
1139 (FV Seoul1) and a 1-base pair deletion between
nucleotides 4291 and 4294 (FV Utrecht). One mutation was a novel
missense mutation: T1927C, C585R (FV Nijkerk), resulting in the absence
of mutant protein despite normal transcription to RNA. Most likely, an
arginine at this position disrupts the hydrophobic interior of the FV
A2 domain. The sixth detected mutation was a previously reported missense mutation: A5279G, Y1702C (FV Seoul2). In all
cases, the presence of the mutation was associated with type I FV
deficiency. Identifying the molecular basis of mutations underlying
this rare coagulation disorder will help to obtain more insight into
the mechanisms involved in the variable clinical phenotype of patients with FV deficiency.
(Blood. 2001;98:358-367) Human coagulation factor V (FV) is a single-chain
glycoprotein that plays an important role in maintaining the hemostatic balance. It circulates in blood as an inactive procoagulant with a
Mr of 330 kd and a structure consisting of 3 homologous
A-type domains and 2 homologous C-type domains connected by a heavily glycosylated B domain in the order A1-A2-B-A3-C1-C2. Proteolytic cleavage by thrombin at R709, R1018, and R1545 (single-letter amino
acid codes) results in removal of the B domain and converts the
procofactor into the fully active cofactor FVa, which consists of a
Mr 105-kd heavy chain (A1-A2) and a Mr 74- or
71-kd light chain (A3-C1-C2), associated via a single Ca++
ion.1-3 The difference in molecular weight of the light
chain reflects the presence of 2 isoforms of FVa (FVa1 and
FVa2) due to alternative glycosylation of the C2 domain,
which leads to different affinities for biologic membranes and
subsequent overall procoagulant activity.4,5 In its active
form, FVa forms an essential part of the prothrombinase complex that
catalyzes the conversion of prothrombin to thrombin by factor Xa in the
presence of calcium and a phospholipid membrane.1-3
Activated protein C (APC) inactivates FVa through cleavage of the
active cofactor at R306, R506, and R679 and requires FV as a cofactor
in the APC-mediated inactivation of factor VIIIa
(FVIIIa).6,7 Thus, FV plays an important role in the
procoagulant pathway as well as in the protein C anticoagulant
pathway. The structure of FV is similar to FVIII (both
cofactors share approximately 40% homology in their heavy and light
chains) and ceruloplasmin, the copper-binding protein in
plasma.8,9 Recently, the crystal structure of the C2
domain of FV has been established10 and molecular models for the A and C domains of FV have been proposed.11,12
The gene for coagulation FV has been mapped to chromosome
1q2313 and spans more than 80 kilobases (kb). It consists
of 25 exons and the messenger RNA (mRNA) encodes a leader peptide of 28 amino acids and a mature protein of 2196 amino acids. Roughly, the
heavy chain is encoded by exons 1 to 12 and the light chain by
exons 14 to 25. The entire B domain is encoded by exon 13, which
contains 2 tandem repeats of 17 amino acids and 31 tandem repeats of 9 amino acids that are absent in the B domain of FVIII.14,15
Deficiency of FV, or parahemophilia, was first described in 1947 by
Owren.16 It is a rare autosomal recessive bleeding
disorder with an estimated frequency of one in one million. The
phenotypic expression of FV deficiency is variable; heterozygotes are
usually asymptomatic, whereas homozygous patients show mild, moderate, or severe bleeding symptoms. Identifying the molecular basis underlying this disease will help to obtain more insight into the mechanisms involved in this variable clinical expression. The recently published complete nucleotide sequence of the FV gene (GenBank
accession number Z99572) has facilitated the molecular characterization underlying FV deficiency and reports have identified mutations in the
FV gene that result in FV deficiency.17-30 To
date, no compound heterozygous patients have been described. In the
present study we used DNA sequence analysis to detect 5 novel mutations
and one previously reported mutation in the FV gene of 3 patients, one of whom is the first characterized compound heterozygote, with severe FV deficiency and 2 asymptomatic carriers. All mutations resulted in type I FV deficiency.
Patients
Patient 2 is a 5-year-old boy of Turkish ancestry. He was diagnosed at
the age of 1 year, because of soft tissue bleed in his mouth that did
not stop after conservative treatment. His laboratory tests
demonstrated an FV activity less than 1%. His consanguineous parents
both had lowered FV activity and antigen levels, suggesting a FV
deficiency carrier status (Table 1). No clinical bleeding problems in
the parents were apparent. The following years he frequently had bleeds
of his mouth, hematomas, and epistaxis. Severe bleeding episodes were
treated with fresh frozen plasma. Until now he has not had muscle or
joint bleeds.
Patient 3 is a 15-year-old girl from Morocco. The diagnosis of severe
FV deficiency, FV activity less than 1% (Table 1), was made following
family screening. She does not have a bleeding tendency, but so far she
has not had any operative procedures or injuries. Her parents are
first-degree cousins. Her 23-year-old brother also has a severe FV
deficiency. Other than prolonged bleeding after injuries, he has had no
bleeding problems. He has been described before.31
Patients 4 and 5 are both asymptomatic Dutch individuals who were
discovered during routine laboratory testing. They presented with
normal to slightly prolonged activated partial thromboplastin time
(APTT), respectively, 31 and 34 seconds (reference values, 24-32 seconds), and slightly prolonged prothrombin time (PT), respectively,
13.6 and 13.8 seconds (reference values, 11.0-13.0 seconds). Liver
failure and all other possible causes of aberrant APTT and PT results
were excluded. FV activity and antigen levels were decreased (Table 1).
Informed consent was obtained from all patients and family members.
Control group
Blood collection for coagulation tests Venous blood was collected into plastic tubes in 1/10 volume of 3.8% sodium citrate. Platelet-poor plasma was obtained by centrifugation at 1200g for 20 minutes at room temperature and aliquots were immediately stored at 80°C until testing.
FV coagulant activity and FV antigen Activity of FV was measured on an STA coagulation analyzer (Roche, Mannheim, Germany) by performing a PT in a plasma sample diluted with FV-deficient plasma. FV antigen was determined by Professor Rogier M. Bertina by measuring FV light chain by an enzyme-linked immunosorbent assay (ELISA) as described.20Isolation of DNA and RNA DNA was isolated from peripheral white blood cells using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's instructions. Lymphocytes were isolated from whole blood using Ficoll-Pague (Pharmacia, Uppsala, Sweden) and total RNA was extracted as described.32Amplification of the FV gene Nucleotides are numbered according to Jenny and colleagues.14 Nucleotides in the putative promoter region are numbered relative to the initiation codon. The primers used for polymerase chain reaction (PCR) amplification and DNA sequence analysis of the promoter and the individual exons are listed in Table 2. Intronic primers were chosen at appropriate distance from the splice sites. The large exon 13 was divided into 5 fragments (a-e). The PCR reactions were carried out with 50 to 100 ng DNA in 100 µL volumes containing 10 mM Tris-HCL, pH 8.3, 50 mM KCL, 1.5 mM MgCl2, 0.01% (wt/vol) gelatin, 0.2 mM of each dNTP, 0.3 µM of each primer, and 2.5 U AmpliTaq DNA polymerase. All reagents were obtained from Perkin Elmer (Roche Molecular Systems, Branchburg, NJ). The samples were subjected to 30 or 35 cycles of amplification with denaturation at 94°C for 30 seconds (5 minutes at 95°C prior to the first cycle), annealing for 30 seconds at 50 to 64°C (Table 2) and extension at 72°C for 30 to 60 seconds followed by an elongated extension time of 10 minutes after the last cycle.
DNA sequence analysis Automated DNA sequence analysis was performed with the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems, Warrington, England), according to the manufacturer's instructions. Sequencing reactions were all carried out in forward and reverse direction, and samples were analyzed on an Applied Biosystems ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). The PCR products were purified prior to DNA sequence analysis with the QIAquick PCR purification kit (Qiagen, Valencia, CA) or excised from the agarose gel and purified using the Prep-A-Gene DNA Purification Kit (Biorad Laboratories, Hercules, CA) according to instructions.Reverse transcription-PCR Reverse transcription-PCR (RT-PCR) to detect the T1927C mutation was performed using the GeneAmp RNA PCR Kit from Perkin Elmer (Roche Molecular Systems, Branchburg, NJ) according to the instructions of the manufacturer. Briefly, 0.3 to 1.0 µg total RNA was reverse transcribed using random hexamers as primers. After addition of 30 pmol of primers HCFV-C11F 5'-ATGAGGTGAAACGTGATGACC-3', exon 11 nts 1802 to 1822 and HCFV-C12R 5'-ACCGTCACAGATTCTCCACG-3', exon 12 nts 2048 to 2029, the samples were subjected to 35 cycles of amplification with denaturation at 94°C for 30 seconds (5 minutes at 95°C prior to the first cycle), annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds, followed by an elongated extension time of 10 minutes after the last cycle. Total liver RNA was used as a positive control and controls without RNA as well as controls in which the reverse transcription step was omitted were included.Restriction enzyme analysis All mutations were confirmed by restriction enzyme analysis on a newly amplified PCR product (Table 3). The 8-bp deletion in exon 7 was confirmed by MboII digestion. The PCR product as amplified with primers HCFV-7F and HCFV-7R is 241 bp and contains one restriction site for MboII. Digestion of the wild-type allele results in fragments of 134 and 107 bp. The deletion abolishes this site, rendering a single fragment of 233 bp.
To confirm the T1927C mutation, exon 12 was amplified with primers HCFV-12F and HCFV-12R and subjected to BslI digestion. The 286-bp PCR product from the wild-type allele contains one restriction site for BslI, yielding fragments of 214 and 72 bp. The mutation creates a second restriction site resulting in additional fragments of 117 and 97 bp. The 247-bp PCR product containing exon 12, as obtained by RT-PCR with primers HCFV-C11F and HCFV-C12R, yields fragments of 228 and 19 bp for the wild-type allele and 131, 97, and 19 bp for the T1927C allele, following digestion with BslI. The C2491T nonsense mutation in exon 13 was confirmed by HphI digestion of the 5' end of exon 13 as amplified with primers HCFV-13aF and HCFV-13aR. The 605-bp PCR product normally contains one recognition site for HphI, producing fragments of 458 and 147 bp, which is abolished by this base change and thus results in an uncut PCR product from the mutant allele. To detect the A5279G mutation, the PCR product (600 bp) amplified with primers HCFV-15F2 and HCFV-15R, containing exon 15, was subjected to digestion with Cac8I. The mutation adds an extra restriction site to the one normally present in this fragment; 338-, 55-, and 207-bp fragments are produced in contrast to the wild-type allele, 338 and 262 bp. In general, 20 µL of the appropriate PCR product was incubated with 10 to 20 U of the respective enzymes. All enzymes were purchased from New England Biolabs (Beverly, MA). Molecular modeling A 3-dimensional diagram of the cysteine at residue 585 in FV was generated from an FV homology model12 using computer programs Molscript33 and Raster3D.34
FV activity and antigen Factor V activity and antigen results of the propositae and relevant family members are summarized in Table 1. In all subjects, excluding patient 3 from whom plasma was unavailable, the reduction of FV activity correlated with a similar reduction of FV antigen, indicating a type I FV deficiency.Molecular analysis During the course of the DNA sequence analysis of the complete FV gene of the patients, relevant relatives, and a control subject, a number of novel base changes as compared to the published sequence were identified. One was located in the promoter, 15 in the coding region, and 19 in the noncoding region. The intronic base changes were excluded from being the (primary) genetic defect underlying the FV deficiency, based on the fact that they were not located in or near consensus sequence motifs considered critical for RNA processing, neither did they create any such sequence.35,36 They were therefore considered to be polymorphisms. Likewise, the promoter base change at nt426 was
considered to be polymorphic given its high frequency in the control
group. We report the allelic frequencies of all applicable exonic base
changes, except for the 2 frameshift mutations and nonsense mutants, as
determined in the control group. The results obtained from the
molecular analysis are summarized in Table 3.
Patient 1 By DNA sequence analysis, this patient was found to be heterozygous for a novel deletion in exon 7, where 8 bp ((TG)AAGAGG(TG)) were deleted between nts 1130 and 1139 (Figure 1), resulting in a frameshift and a premature stop codon at residue 351. This mutation was confirmed by MboII digestion. Because the patient had undetectable levels of FV activity and antigen (Table 1), we postulated her to be a probable compound heterozygote. Further DNA sequence analysis revealed, apart from 6 previously published polymorphisms (G1628A,37,38 (A2663G, A2684G, A2863G),14,15,39,40 C4279T41 and A5380G,42-44) one other base change in the heterozygous state: exon 15: A5279G, Y1702C. Because she was an orphan without any known relatives, it was not possible to determine which changes occurred in trans of the 8-bp deletion by linkage analysis. However, the A5279G mutation has recently been associated with FV deficiency25 and is therefore most likely the causative mutation on the other FV allele of this patient. This missense mutation was confirmed by Cac8I digestion. We named this compound heterozygous variant FV Seoul1,2.
Patient 2 DNA sequence analysis revealed a novel homozygous deletion in exon 13 where one cytosine was deleted from a series of 4 cytosines present between nts 4291 and 4294 (Figure 1). This resulted in a frameshift leading to a premature translation stop at codon 1381. Both parents, who were first cousins, were heterozygous for the same deletion thereby excluding the possibility of hemizygosity in the propositus due to a large deletion. We designated this variant FV Utrecht.Patient 3 The FV gene of this patient showed a novel homozygous nonsense mutation in exon 13 where a C T substitution at nt 2491 predicted a stop codon at residue 773 instead of the wild-type
glutamine (Figure 1). This mutation was confirmed by HphI
digestion and we named this variant FV Casablanca.
Patient 4 This patient was found to have a single heterozygous AT base
change at nt 1102 in exon 7 (Figure 1), thereby changing the codon for
lysine at residue 310 into a termination codon. All available family
members were tested for this mutation and the nonsense mutation
cosegregated with reduced FV activity and antigen levels (Table 1).
This mutant was designated FV Amersfoort.
Apart from this nonsense mutation and a previously described heterozygous polymorphism (G409C),45 we identified a novel homozygous base substitution in exon 24: T6533C, M2120T. Because this substitution concerns a conserved residue in the C2 domain of FV, its occurrence was investigated in the control group (Table 3). T6533C appeared to be a rare polymorphism of which all of the children in this family are obligate carriers, whereas the spouse of the patient is a wild-type homozygote (data not shown). The 2 children in this family who lack the A1102T mutation had normal FV activity levels (Table 1), so any (additional) effect of this amino acid substitution to the more pronounced reduction in FV activity of the propositus compared to his 2 sons (34% versus 62% and 60%) was not likely to be due to the fact that he was homozygous for this rare polymorphism. Patient 5 We identified 6 nucleotide substitutions linked to lowered activity and antigen levels in this family. Five were previously reported polymorphisms: (A2663G, A2684G, A2863G),14,15,39,40 C4300T,41 and A5380G,42-44 whereas one was a novel TC substitution at
nt 1927 in exon 12, coding for a CR change at residue 585. We
investigated the occurrence of T1927C in a normal population but found
no control subjects carrying this allele (Table 3). We postulated the
T1927C base change to cause FV deficiency (see "Discussion") in
this family (Table 1 and Figure 2) and
named this variant FV Nijkerk. The mutant allele was transcribed to RNA, because the mutant RNA could be detected, by RT-PCR, in both the
patient and his mother (Figure 2).
Little is known about the molecular basis underlying FV deficiency due to its low frequency in the population combined with the complexity of the gene itself. In this study we investigated 3 patients with severe FV deficiency and 2 asymptomatic carriers from 5 different families. We identified 5 novel mutations and a recently published mutation associated with FV deficiency. Patients 1, 2, and 3 all had severe FV deficiency and undetectable levels of FV. DNA sequence analysis of the FV gene revealed 2 novel frameshift mutations and a novel nonsense mutation associated with an FV null allele. Patients 2 and 3 were both homozygous for a mutation in exon 13, respectively, a 1-bp deletion between nts 4291 and 4294 and a C2491T nonsense mutation. Patient 1 was compound heterozygous for an 8-bp deletion in exon 7 and a missense mutation in exon 15: A5279G, Y1702C (FV Seoul2). The missense mutation was recently described in the heterozygous state as causative for FV deficiency.25 Our study provides further evidence for the postulation that the Y1702C change leads to FV deficiency, because neither FV activity nor FV antigen could be detected in our patient. The 2 small deletions and the nonsense mutation in the FV
genes of patients 1, 2, and 3 would, if translated, result in
truncated FV molecules. The 8-bp deletion between nts 1130 and 1139 in
exon 7, In general, nonsense mRNAs resulting from frameshift and nonsense
mutations are highly unstable, because they are subjected to
nonsense-mediated mRNA decay (NMD),46-48 preventing the
potentially deleterious effects of truncated proteins. Recent studies
of FV RNA metabolism demonstrated the absence of RNA of the in
trans non-FV Leiden allele in patients who are "pseudo
homozygous" for FV Leiden due to either a nonsense
mutation21 or a frameshift mutation19 in exon
13. Consequently, the frameshift and nonsense mutations in patients 1, 2, and 3 most likely are associated with an FV null allele. The
patients presented with different phenotypes: patients 1 and 2 had
moderate bleeding symptoms, whereas patient 3 was asymptomatic.
Furthermore, patient 1 suffered from spontaneous muscle bleedings,
whereas these were absent in patient 2. This variability in phenotypic
expression has previously been observed in other FV-deficient patients.
Moreover, as mentioned in earlier reports,49,50 there is a
striking discrepancy between mice and man. FV null mice die either in
utero or of fatal perinatal hemorrhage,49 whereas human
patients with undetectable FV levels do survive. Table
4 summarizes the clinical phenotype of
all patients with severe FV deficiency described to date who have been
characterized at the molecular level. There is a marked difference in
age at diagnosis and clinical presentation. As shown by patients I, VI,
XI, and XII in Table 4, similar residual amounts of FV are still
associated with a different phenotypic expression. Also, individuals
with the same mutation, between families (patients I and VI) and even
within families (patients II and VII), show a different clinical
bleeding pattern. Therefore, the clinical expression of FV deficiency
most likely does not depend solely on the type of mutation but also on
other, yet unknown, modulating factors.
Previously it has been proposed50,51 that in patients with
severe FV deficiency a very low level of expression of FV may occur.
Because trace amounts of FV are capable of generating a significant amount of thrombin,52 this would then result
in a level of FV synthesis sufficient to mitigate the clinical
phenotype. For instance, "ribosomal frameshifting" has previously
been recognized as a mechanism able to partially rescue protein
synthesis from genes harboring frameshift mutations,53-55
and it has also been proposed to play a role in the phenotypic
expression of FV deficiency caused by a frameshift mutation in exon
13.50 Mutations in exon 13 occur frequently in the
FV gene (Table 5) and because
the B domain is relatively unimportant for FV procoagulant function, "ribosomal frameshifting" may thereby reflect one of the modulating factors involved in the phenotypic differences observed in these patients. As shown in Table 4, however, mutations in exon 13 still can
lead to a severe phenotypic expression of the disease.
In mammals, the basic mechanism by which nonsense transcripts are
recognized and subjected to NMD is still poorly
understood.48 Therefore, it cannot be excluded that any
residual amount of stable nonsense FV mRNA could actually produce small
amounts of (truncated) protein. These molecules could escape detection
by the methods used, but still be of interest for their remaining
function or potentially harmful effects in the procoagulant and
anticoagulant pathways. In humans, the role of NMD as a modifier of the
phenotypic consequences of nonsense mutations is becoming increasingly
evident, for example, in Patients 4 and 5 were both asymptomatic and carriers of a novel FV null allele. However, because the presence and function of these FV variants could be studied only in individuals heterozygous for the mutation, we cannot exclude the presence of small amounts of mutant protein that would go by undetected because of the wild-type background. In patient 4, a novel nonsense mutation was identified: A1102T, K310Term (FV Amersfoort) that would code for a truncated protein lacking the A2 domain of the heavy chain, the complete B domain and the entire light chain (Figure 1). This mutation was found to cosegregate within the affected family with decreased FV activity and antigen levels. In patient 5, a novel base change was detected: T1927C, C585R (FV Nijkerk) that most likely represents the mutation associated with the decrease in FV activity and antigen levels. This missense mutation cosegregated with FV deficiency in the affected family and was not detected among 100 normal alleles. Although mutant RNA was detected, indicating transcription from the FV Nijkerk allele, FV antigen and activity levels were consistent with heterozygous type I FV deficiency. Apparently, substitution of C585 by arginine results in rapid degradation of FV, either before or immediately after secretion into the circulation. C585 is conserved among human, bovine, and murine FV, but not in FVIII and ceruloplasmin. Analysis of disulfide bridges and free cysteines in bovine FVa have not yielded conclusive data on the state of C589 (C585 in human FV).59 However, homology modeling of FV A domains based on the crystal structure of ceruloplasmin indicates that C585 is not involved in disulfide-bond formation.12 In the FV homology model C585 is completely buried inside the hydrophobic interior of the A2 domain. Substitution of a small and hydrophobic cysteine by a large and hydrophilic arginine would disrupt the tight packing interactions typical of protein interiors. As a consequence, the protein might be unstable and prone to intracellular proteolysis. If stable FV Nijkerk would be synthesized, functionality is doubtful because a potential thrombin cleavage site is created by the introduction of an arginine at residue 585, adjacent to S586. Cleavage by thrombin at this site would result in excision of a fragment from amino acids 585 to the next thrombin cleavage site at R709. This would disrupt the C-terminal end of the A2 domain, resulting in a loss of function or unstable protein. Noteworthy with respect to the sister of the propositus in the family carrying the FV Nijkerk variant is the fact that her FV levels were consistently lower than in the other carriers of the T1927C mutation (Table 1). This may be explained by the fact that she inherited a FV HR2 allele from her father (data not shown). This variant FV, characterized by the H1299R substitution in the FV B domain, has been associated with reduced FV levels.60-62 Phenotypically, she is therefore "pseudo homozygous" for FV HR2, a rare FV genotype that has been reported previously once by Castoldi and coworkers.25 They reported a family in which one member carried a FV missense mutation (Y1702C), associated with absence of the mutant protein in plasma, on the allele in trans of the FV HR2 allele. This family member also had lower FV levels as compared to carriers of only the missense mutation in the family.25 This study brings the total number of mutations associated with FV deficiency described to date to 17. An overview is presented in Table 5 where the mutations are listed according to their proposed mode of action. The majority of these mutations have been found in unique families; only the C1690T,22,23 C3571T,28 A5279G (this study and reference 25) and G6395A29 have been found in more than one (unrelated) family. FV Seoul1,2 in this study is the first compound heterozygous patient described to date who is fully characterized at the molecular level and in whom 2 rare FV deficiency-associated mutations are combined that completely abolish FV synthesis. Identifying the molecular basis underlying this rare coagulation disorder will help to obtain more insight into the mechanisms involved in the variable clinical phenotype of FV-deficient patients.
The authors wish to sincerely thank Richard Dirven and Rogier Bertina (Department of Hematology, Hemostasis and Thrombosis Research Center, University Medical Center, Leiden, The Netherlands) for providing the FV light chain data and Bruno Villoutreix (Department of Bioinformatics-Blood Coagulation, University of Paris, France) for kindly providing the PDB file containing the model structure of the 3 FV A domains.
Submitted January 23, 2001; accepted March 23, 2001.
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: Wouter W. van Solinge, Department of Clinical Chemistry, University Medical Center, Postbus 85500, 3508 GA, Utrecht, The Netherlands; e-mail: wsolinge{at}lab.azu.nl.
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© 2001 by The American Society of Hematology.
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  M. S. Mahadevan and P. V. Benson Factor V Null Mutation Affecting the Roche LightCycler Factor V Leiden Assay Clin. Chem., August 1, 2005; 51(8): 1533 - 1535. [Full Text] [PDF]
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 P. M. Mannucci, S. Duga, and F. Peyvandi Recessively inherited coagulation disorders Blood, September 1, 2004; 104(5): 1243 - 1252. [Abstract] [Full Text] [PDF]
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M. Steen, E. A. Norstrom, A.-L. Tholander, P. H. B. Bolton-Maggs, A. Mumford, J. H. McVey, E. G. D. Tuddenham, and B. Dahlback Functional characterization of factor V-Ile359Thr: a novel mutation associated with thrombosis Blood, May 1, 2004; 103(9): 3381 - 3387. [Abstract] [Full Text] [PDF]
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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]
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M. Steen, M. Miteva, B. O. Villoutreix, T. Yamazaki, and B. Dahlback Factor V New Brunswick: Ala221Val associated with FV deficiency reproduced in vitro and functionally characterized Blood, August 15, 2003; 102(4): 1316 - 1322. [Abstract] [Full Text] [PDF]
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S. Duga, M. C. Montefusco, R. Asselta, M. Malcovati, F. Peyvandi, E. Santagostino, P. M. Mannucci, and M. L. Tenchini Arg2074Cys missense mutation in the C2 domain of factor V causing moderately severe factor V deficiency: molecular characterization by expression of the recombinant protein Blood, January 1, 2003; 101(1): 173 - 177. [Abstract] [Full Text] [PDF]
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R. van Wijk, G. Rijksen, E. G. Huizinga, H. K. Nieuwenhuis, and W. W. van Solinge HK Utrecht: missense mutation in the active site of human hexokinase associated with hexokinase deficiency and severe nonspherocytic hemolytic anemia Blood, January 1, 2003; 101(1): 345 - 347. [Abstract] [Full Text] [PDF]
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I. Schrijver, M. A. Koerper, C. D. Jones, and J. L. Zehnder Homozygous factor V splice site mutation associated with severe factor V deficiency Blood, April 15, 2002; 99(8): 3063 - 3065. [Abstract] [Full Text] [PDF]
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G. A.F. Nicolaes and B. Dahlback Factor V and Thrombotic Disease: Description of a Janus-Faced Protein Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 530 - 538. [Abstract] [Full Text] [PDF]
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E. Ajzner, I. Balogh, T. Szabo, A. Marosi, G. Haramura, and L. Muszbek Severe coagulation factor V deficiency caused by 2 novel frameshift mutations: 2952delT in exon 13 and 5493insG in exon 16 of factor 5 gene Blood, January 15, 2002; 99(2): 702 - 705. [Abstract] [Full Text] [PDF]
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G. A.F. Nicolaes and B. Dahlback Factor V and Thrombotic Disease: Description of a Janus-Faced Protein Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 530 - 538. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
-thalassemia.