© 1999 American Heart Association, Inc.
Original Contributions |
Phenotype and Genotype Expression in Pseudohomozygous Factor VLEIDEN
The Need for Phenotype Analysis
From the Department of Biochemistry, College of Medicine, University of Vermont, Burlington (M.K., K.G.M.); the Institute of Medical Semeiotics, University Hospital Padua Medical School, Padua, Italy (P.S., A.G.); and the Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy (F.B., B.L.).
Correspondence to Michael Kalafatis, Department of Chemistry, Science Building, 2351 Euclid Ave, Cleveland State University, Cleveland, OH 44115. E-mail m.kalafatis{at}popmail.csuohio.edu
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Abstract |
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Abstract—The presence of a DNA mutation is frequently used to define a disease or a risk state. Because DNA typing has become easy and convenient in contrast to protein characterization, it is generally assumed that a mutation if present (or not) at the DNA level will be also present (or not) in the corresponding protein. However, discrepancies between phenotype and genotype can occur. A point mutation in the coagulation factor V gene (G1691
A, resulting in an
Arg506Gln amino acid substitution in the factor V
molecule [factor VLEIDEN], leading to activated
protein C resistance) is the most common genetic risk factor for
familial thrombophilia. A pseudohomozygous factor VLEIDEN
phenotype would occur if a heterozygous individual for factor
VLEIDEN also did not express the "normal" (non-Leiden)
factor V allele. However, to date, no data have been available to
confirm the presence of only the factor VLEIDEN form in the
plasma of these individuals. Platelet mRNA from 2 presumed
pseudohomozygous patients and their family members was isolated, the
amplified partial cDNAs were sequenced or restricted, and the allelic
bands were quantified. Both patients were found to be heterozygous for
the G1691A substitution at both the DNA and mRNA levels.
The presence of either the normal or mutated form of factor V in the
patients' plasmas was investigated using a monoclonal antibody to
factor V that recognizes an epitope located between residues 307 and
506 of the factor Va heavy chain. No normal factor V could be detected
in the plasmas of the 2 propositi. The present data demonstrate
absence of a correlation between genotype at position 1691 (at
the DNA and mRNA levels) and the corresponding phenotype data
found in the plasmas of patients with pseudohomozygous factor
VLEIDEN. Overall, these data suggest the existence of
heterogeneous genetic "lesions," which interfere with
factor V expression, processing, secretion, and/or stability. Because
the presence of the factor VLEIDEN molecule in plasma is
directly related to pathology, identification and quantification of the
circulating forms of factor V in plasma may be required for the
diagnosis of individuals with activated protein C
resistance.
Key Words: factor VLEIDEN • thrombophilia • phenotype • genotype • pseudohomozygous
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Introduction |
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Factor V circulates in plasma as a single-chain procofactor (Mr 330 000) that requires proteolytic cleavage for expression of its function in the procoagulant enzyme complex that activates prothrombin to thrombin.1 Factor Va is composed of a heavy chain (Mr 105 000; residues 1 to 709; A1 and A2 domains) and a light chain (Mr 74 000; residues 1546 to 2196; A3, C1, and C2 domains) that are noncovalently associated in the presence of divalent metal ions1 2 (Figure 1
). Factor Va inactivation by activated protein
C (APC) is associated with 3 cleavages of the heavy chain at
Arg506, Arg306, and
Arg679.3 Cleavage at
Arg306 occurs only on the membrane-bound cofactor
(Figure 1) and is responsible for complete
inactivation.3 Individuals with a GA substitution at
nucleotide 1691 in the factor V gene (resulting in an
Arg506Gln mutation in the factor V molecule;
factor VLEIDEN) have a poor anticoagulant
response to APC (APC resistance), which is associated with a
significant increase in risk for deep venous thrombosis (7-fold for
heterozygotes and 80-fold for homozygotes).4 5 6 7 8 9 APC
resistance has been suggested to be the most common risk factor for
developing deep venous thrombosis, most likely because factor
VaLEIDEN is inactivated by APC at a
slower rate than is normal factor Va, thus leading to prolonged
thrombin generation (Figure 1, right).3 10 11
Pseudohomozygous APC resistance is a recently hypothesized condition
predisposing to thrombosis that is presumed to occur as a consequence
of the combination of 2 defects in factor V that are associated with a
quantitative reduction in factor V and heterozygous inheritance of the
factor VLEIDEN mutation.12 13 14 15 The
main laboratory findings are the presence of a low APC sensitivity
ratio (APC-SR) as well as factor V antigen and activity at the low
level of normal plasma values.16 Presently, no direct
evidence describing the molecular form of the factor V molecule in
these patients has been reported, although thrombotic episodes have
occurred at a relatively young age in 5 of 6 patients with presumed
pseudohomozygous APC resistance.12 13 14 15 The present
study was undertaken to compare the phenotypic and genotypic expression
of factor V in 2 pseudohomozygous factor VLEIDEN
patients.
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Methods |
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DNA Analysis
The presence or absence of the G
A substitution at
nucleotide 1691 in the factor V gene in normal individuals,
in homozygous carriers of factor VLEIDEN, in a
true heterozygous individual, and in the 2 pseudohomozygous patients
was performed as described with the use of peripheral
leukocytes.5 12 Primers and experimental conditions for
amplification of introns 9 and 16, as well as of exons 10, 13, and 16,
have been previously described.17 For detection of factor
V gene polymorphisms,17 polymerase chain reaction
(PCR) products were digested with MnlI,
HinfI, EcoRI, TaqI, and
RsaI under the conditions recommended by the suppliers (MBI
Fermentas, Boehringer, and Promega).
mRNA Analysis
Platelets from both pseudohomozygous APC-resistant
propositi (patients A and B) were separated from whole blood and the
RNA extracted as described.18 Total RNA, isolated with the
RNAfast method (Molecular Systems) from platelets, was used as a
template for cDNA synthesis. RNA was incubated at 42°C for 1 hour
with 200 U of reverse transcriptase (SuperScript II RT, GIBCO-BRL) and
1 µg of primer A from exon 11 (5'-CTGTTCGATGTCTGCTGC-3';
nucleotides 1722 to 1705) or primer B from exon 13
(5'-AAGAATAATTTGAACCAACAAT-3'; nucleotides 2425 to 2404).
Numbering of primers was in accordance with Jenny et al.2
The RT reaction mixture (1/10) was amplified (first round) as follows:
primer A with a forward primer from exon 10
(5'-ACAAC-ACCATGATCAGAGC-3'; nucleotides
1493 to 1511) and primer B with a forward primer from exon 12
(5'-TGACCCTC-TTCCCCATG-3';
nucleotides 2012 to 2028). A nested amplification was
performed with primers from exon 10
(5'-CCAGTGC-TTAACAAGACCA-3';
nucleotides 1584 to 1602) and exon 12
(5'-ACGGTCACAATGGATAATGT-3'; nucleotides 2044 to 2063). The
amplification conditions were as follows: denaturation at 93°C for
20 seconds; annealing at 57°C for 15 seconds (exons 10 and 11)
or at 52°C for 25 seconds (exons 12 and 13); extension at 70°C for
9 seconds (exons 10 and 11) or for 40 seconds (exons 12 and 13). The
amplified fragments (exons 10 and 11) were excised from
low-melting-point agarose gel and sequenced with Sequenase (US
Biochemical) using 
-35S–dATP as the
radiolabel. All sequences were determined at least twice, and the
allelic bands (1691 GA, resulting in the
Arg506Gln substitution) were evaluated by
densitometric scanning (GS-700 Imaging densitometer, Bio-Rad
Laboratories) as previous described.18 The allelic ratios
were also determined for polymorphic bands after cDNA amplification
and restriction analysis.
Analysis of Factor V in Whole Plasma
Citrated plasma (100 µL) from normal individuals or from
suspected pseudohomozygous patients was diluted 10-fold in 20
mmol/L HEPES, 0.15 mol/L NaCl, and 5 mmol/L
CaCl2, pH 7.4. Synthetic phospholipid vesicles
(20 µmol/L) to initiate the intrinsic pathway of the blood
coagulation cascade were added, and the plasma was incubated at 37°C
and allowed to clot.19 Clot formation was detected
visually. After clotting, the solution was centrifuged for 30
seconds at 10 000 rpm. Purified human plasma APC (5 nmol/L) was added
to the supernatant. At selected time intervals, aliquots of the mixture
were withdrawn and analyzed by SDS–polyacrylamide gel
electrophoresis followed by transfer to nitrocellulose. Immunoreactive
fragments were detected by using the monoclonal antibody HFVaHC No.
17 (described in Figure 1
).10 19 This antibody
recognizes an epitope located between residues 307 and 506 of the
factor Va heavy chain (Figure 1).19 The terminal
product after APC inactivation of membrane-bound normal factor Va
recognized by this antibody is an Mr
30 000 fragment (Figure 1, left), whereas the terminal
product derived from factor VaLEIDEN
inactivation by APC is an Mr 54 000
fragment. As a consequence, the presence of an
Mr 30 000 fragment in normal plasma after
addition of a membrane surface and APC is correlated with the presence
of normal factor Va, whereas the presence of an
Mr 60 000/54 000 doublet is correlated
with the presence of factor VaLEIDEN (Figure 1).
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Results and Discussion |
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We have studied 2 patients. Patient A is a 51-year-old man who experienced several episodes of superficial vein thrombosis (SVT) from the age of 40. His detailed clinical history has been described elsewhere.12 Patient B is a 36-year-old woman who experienced bilateral SVT to both legs during the second of 2 pregnancies at the age of 27 years. In the first pregnancy, at 23 years of age, no thrombotic events occurred; however, she subsequently developed recurrent SVT to both legs. No other thrombotic or hemorrhagic event has occurred in the proposita or in her family members. Routine coagulation tests including prothrombin time, activated partial thromboplastin time, antithrombin III, protein C, protein S, factor VIII:C, and fibrinogen determinations excluded any other abnormalities in these patients. Factor V antigen and activities were measured in the 2 propositi as described.12 Both patients had decreased but within-normal levels of both factor V antigen and activity (the Table
). The
tests for functional APC resistance and cDNA analysis for the
factor VLEIDEN mutation in both patients'
plasmas were performed as described with the use of
peripheral leukocytes,5 12 and the results are
shown in Figure 2 and the Table, along with the values
for normal individuals. Both probands display a low, normalized APC-SR
(see column n-APC-SR in the Table), suggesting homozygosity for
APC resistance. However, both propositi are heterozygous for the
1691 G/A substitution at the DNA level (factor
VLEIDEN mutation; Figure 2, lanes 5 and
6).
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Platelets from both propositi were separated from whole blood, and
the RNA extracted from these cells18 was studied by RT-PCR
of exons 10 and 11, producing the expected cDNA fragment (139 bp).
After sequencing, the allelic bands (1691 G/A, exon 10; Figure 3) were evaluated by densitometric analyses and their
ratios determined. The cDNA from the "normal" (non-Leiden) factor V
gene was compared with that carrying the
G1691A mutation (Figure 3, upper left
corner) and was found to be present in normal or slightly increased
amounts (ratio of 1.3 to 1.5) in patient A, whereas in patient B, the
amount of normal factor V gene was clearly reduced (ratio of 0.2 to
0.3). The cDNA ratios were also studied by restriction analysis
of polymorphic sites in exon 13 (cDNA of exons 12 and 13) in the
family members of patient B. In subjects I2 and II5 (carriers of factor
V deficiency, as indicated by the reduced levels of factor V antigen
found in their plasma; the Table), the reduced intensity of the
290-bp band from EcoRI digestion and of the 382-bp band from
TaqI digestion confirmed the reduced amount of mRNA produced
by the factor V–deficient gene (non-Leiden allele; Figure 3, upper right). In contrast, in subjects II6 (TaqI
pattern) and II7 (EcoRI pattern), who have normal levels of
factor V, as well as in a normal control individual (C), similar
intensities of the allelic bands indicate balanced expression of both
factor V genes (Figure 3 and the Table).
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The form of factor V expressed in the patients' blood was evaluated
after clotting and APC–phospholipid vesicle cleavage with monoclonal
antibody HFVaHC No. 17.10 This antibody recognizes an
epitope located between residues 307 and 506 of the factor Va heavy
chain (Figure 1).10 19 The terminal product
after APC inactivation of membrane-bound normal factor Va recognized by
this antibody is an Mr 30 000 fragment
(Figure 1, left), whereas the terminal product derived from
factor VaLEIDEN inactivation by APC is an
Mr 60 000/54 000
doublet.10 As a consequence, the presence in plasma
of an Mr 30 000 fragment after addition of
a membrane surface and APC is correlated with the presence of normal
factor Va, whereas the presence of an Mr
60 000/54 000 doublet is correlated with the presence of factor
VaLEIDEN (Figure 1). The cleavage
product analysis for the normal control (Figure 4A)
shows a product with an Mr of 30 000,
which corresponds to the fragment resulting from cleavage at
Arg306 and Arg5063
(residues 307 and 506; Figure 1). Similar analysis of an
individual genetically defined as homozygous for the factor
VLEIDEN mutation is shown in Figure 4B.
Fragments corresponding to cleavages at Arg306
and Arg679 are observed, resulting in a doublet
of Mr 60 000/54 000 (composed of residues
307 to 709 and 307 to 679). An individual heterozygous for the
Arg506Gln mutation (shown in panel C) displays
products of Mr 30 000 and
Mr 60 000/54 000. The data from the 2
potentially pseudohomozygous patients are presented in Figure 4D and 4E. Both patients have only the product derived from
factor VaLEIDEN cleavage at
Arg306/709/Arg306/679,10
because only the Mr 60 000/54 000 doublet
is present. No Mr 30 000 fragment was
detected in the plasmas of the propositi, demonstrating the absence of
circulating normal factor V. These data explain the thrombotic
tendencies in these 2 probands and prove the existence of homozygous
phenotypic expression of factor VLEIDEN in
individuals who are genetically heterozygous for the defect.
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Our data demonstrate unequivocally the existence of pseudohomozygous factor VLEIDEN patients with thrombotic disorders. The present data also show for the first time the presence of sufficient factor V mRNA in human platelets for RT-PCR amplification that allows for informative restriction analysis and family studies. Because proteolytic cleavage of factor Va by APC is required for inactivation of the cofactor and arrest of the procoagulant process, abnormalities in the mechanism of inactivation of factor Va by APC are associated with thrombotic episodes due to impaired inhibition of coagulation. The risk for thrombosis increases 7-fold in heterozygous and 80-fold in homozygous individuals for factor VLEIDEN.9 The low, normalized APC-SR and factor V genotype at nucleotide 1691 usually define the presence of factor VLEIDEN in the homozygous or heterozygous state in cohorts of patients with thrombotic episodes. However, correlation between the presence of the mutation at the gene level and actual thrombotic episodes varies between 20% and 60%. In some cases, these variations may reflect the use of different reagents involved in the execution of the APC resistance assay.19 20 21 22 23 24 It has been also shown that a number of polymorphisms within the factor V gene are associated with atherosclerotic disease in the elderly.25 Discrepancies between laboratories with respect to the correlation between thrombotic tendencies and APC resistance (ie, presence of the factor VLEIDEN mutation) for a given individual can be explained by either different results obtained from the APC resistance assay or from genetic analyses. Furthermore, although a recent study showed that the APC resistance assay was far from informative in the general population,24 it has also been established that a specific factor V gene haplotype (HR2) that was defined by 6 polymorphisms was able to contribute to the generation of thrombosis.26 Thus, it has become evident that both the APC resistance assay and PCR amplification and digestion with MnlI are not sufficient to establish the presence and proportion of factor VLEIDEN molecules in some patients. DNA restriction analysis detects only the gene mutation (and cannot account for the presence of the protein in plasma), and mRNA studies, as demonstrated in this article, are not informative in all patients and indicate the presence of heterogeneous lesions that interfere with factor V expression, processing, secretion, and/or stability. However, only the existence of an abnormal circulating factor VLEIDEN molecule can be correlated with an individual's symptoms (ie, resistance to APC and greater risk of venous thrombosis). Furthermore, because the level of factor V in normal plasma (as assessed by radioimmunoassay) varies from 57% to 200%,16 a value near 50%, though borderline, cannot be considered either indicative of a factor V deficiency per se or as prima face evidence of a "silent" gene. Thus, analyses of the quality and quantity of factor V molecules in an individual's plasma, which is ultimately responsible for the normal or disease state of the individual, is necessary for the evaluation of an individual's phenotype.
Unlike typical heterozygous individuals, most pseudo-homozygous
patients studied thus far (5 of 6) are symptomatic.
Dahlbäck and coworkers27 originally suggested that
APC resistance may be partially corrected by the addition of factor V.
They have further shown that factor V acts as a "cofactor" for the
APC/protein S–mediated inactivation of factor VIIIa.28 We
have recently reported a 2-fold increase in the inactivation rate of
factor VIII by the APC/protein S complex in the presence of normal
plasma factor V.29 We have further demonstrated that a
portion of the B region of the cofactor is most likely responsible for
the "cofactor" effect of factor V.29 In contrast,
factor VLEIDEN was found to have impaired
"cofactor" activity for the APC/protein S–mediated inactivation of
factor VIII.30 A heterozygous APC-resistant
individual has 
50% factor VLEIDEN and 50%
normal factor V, the latter being available to act as a cofactor for
the APC/protein S inactivation of factor VIII. A true homozygote for
factor VLEIDEN differs from a pseudohomozygous
patient in the level of circulating total factor V, the former having a
normal factor V level in plasma. Pseudohomozygous patients have 1
allele expressing factor VLEIDEN and, as
documented in the present report, a second allele with a gene
mutation resulting in the absence of circulating normal factor V.
Therefore, although they have 50% total factor V circulating in
their plasma, no normal factor V is available to act as a cofactor for
the APC/protein S–mediated inactivation of factor VIII. On the other
hand, while most of factor V is activated to factor Va during
clotting, only a small amount of factor Xa is generated.31
As a conclusion, the limiting step for prothrombinase assembly and
-thrombin generation is factor Xa and not factor Va formation. This
is the reason that individuals with circulating levels of factor V
between 50% and 100% have no major bleeding problems. Therefore, if
heterozygosity for factor VLEIDEN increases the
risk for thrombosis 7-fold whereas homozygosity for the same mutation
increases the risk 80-fold, it is reasonable to postulate that
pseudohomozygous patients have a >7-fold increase in thrombotic risk
(because these patients lack the protective effect of normal
circulating factor V) and a 
80-fold increased thrombotic risk as the
true homozygote. Thus, the possibility of an impaired "cofactor"
effect of factor VLEIDEN during APC-mediated
inactivation of factor VIII in these individuals' plasmas will result
in prolonged intrinsic tenase activity and may be an additional risk
leading to thrombosis.
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Acknowledgments |
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This work was supported by Merit Award HL 34575 (to K.G.M.), by Telethon-Italy grant E0.675 (to F.B.), and by a grant from the Regione del Veneto, Giunta Regionale-Ricerca Sanitaria Finalizzata No. 483/03/94, Venezia, Italia (to P.S.).
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Footnotes |
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This work was presented at the XVIth Congress of the International Society on Thrombosis and Haemostasis, Florence, Italy, June 6–12, 1997, and published in abstract form (Thromb Haemost. 1997;77:2440.).
Received April 2, 1998; accepted July 7, 1998.
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TopAbstractIntroductionMethodsResults and DiscussionReferences |
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