Prospective Evaluation of the Risk Conferred by Factor V
Leiden and Thermolabile Methylenetetrahydrofolate
Reductase Polymorphisms in Pregnancy
Ronan P. Murphy, Catherine Donoghue, Ruth J. Nallen, Melwyn D’Mello, Carmen Regan,
Alexander S. Whitehead, Desmond J. Fitzgerald
Abstract—Factor V (FV) Leiden and thermolabile methylenetetrahydrofolate reductase (MTHFR) are 2 common
polymorphisms that have been implicated in vascular thrombosis. We determined whether these mutations predicted an
adverse outcome in pregnancy. Second, we looked for an interaction between these 2 mutations in patients with recurrent
fetal loss or thrombosis in pregnancy. Primigravid subjects at their booking visit to the National Maternity Hospital
(Holles Street, Dublin, Ireland) were screened for the polymorphisms. Thermolabile MTHFR and FV Leiden genotypes
were detected by either restriction fragment length polymorphism or heteroduplex capillary chromatography. The carrier
frequency of FV Leiden in the screened primigravid population was 2.7% (allele frequency 1.36%), all being
heterozygous for the mutation. This value was lower than expected from previous studies in European populations.
Forty-nine percent of the screened population (289 of 584) were heterozygous for thermolabile MTHFR, and 10.6%
were homozygous (62 of 584). The frequency of the 2 polymorphisms was no higher in those who subsequently
developed preeclampsia (n?12) or intrauterine growth retardation (n?9), and none of the screened population
developed thrombosis. However, the frequency of FV Leiden was higher in patients who subsequently miscarried after
the first trimester of pregnancy (allele frequency of 5.5%, P?0.0356). Among those positive for FV Leiden, 3 of 27
miscarried, compared with 24 of 572 of FV Leiden–negative patients (11% versus 4.2%). No interaction was found
between the 2 mutations in the control or patient populations. In patients with a prior history of venous thrombosis, the
carrier rate of FV Leiden was increased (4 of 33, allele frequency of 7.6%, P?0.0115). In contrast, the carrier frequency
for thermolabile MTHFR was no higher, and there was no interaction between the 2 mutations. Neither mutation
occurred at a significantly higher frequency in patients with a prior history of recurrent fetal loss. In conclusion, FV
Leiden is a risk factor for thrombosis in pregnancy and possibly for second-trimester miscarriage independent of
thermolabile MTHFR. However, prospective analysis suggests that the risk conferred by FV Leiden is low in a
primigravid population. The thermolabile MTHFR genotype was not implicated in any adverse outcome. (Arterioscler
Thromb Vasc Biol. 2000;20:266-270.)
Key Words: factor V Leiden polymorphism?thermolabile methylenetetrahydrofolate reductase polymorphism
?pregnancy?venous thrombosis?recurrent fetal loss?genetic risk factors
However, the penetrance of any single mutation is highly
variable, even in families with a history of thrombosis. One
explanation for the variable penetrance is that combinations
of polymorphisms in several genes may act synergistically to
increase the risk of thrombosis.
Factor V (FV) Leiden and the thermolabile (T) methylene-
tetrahydrofolate reductase (MTHFR) polymorphisms have
been implicated as risk factors for thrombosis.1–3The FV
Leiden variant arises as a result of a point mutation at
nucleotide position 1691, resulting in an arginine to a glu-
unctional polymorphisms in genes controlling hemostasis
contribute to an increased risk of thrombotic events.
tamine substitution at position 5064that reduces its sensitivity
to inactivation by activated protein C. FV Leiden has been
associated with familial thrombophilia5and indeed, is the
commonest inherited risk factor for venous thrombosis.
Compared with those without the mutation, heterozygous
carriers have a 7-fold increased risk of venous thrombosis,6,7
and homozygous individuals have a risk that is increased up
MTHFR is critical in the metabolism of homocysteine, as
it effects the NADPH-linked reduction of 5,10-methylenetet-
rahydrofolate to 5-methyltetrahydrofolate. A C-to-T missense
mutation at nucleotide 677 results in an enzyme that is
Received March 24, 1999; revision accepted June 28, 1999.
From the Department of Clinical Pharmacology (R.P.M., R.J.N., D.J.F.), Royal College of Surgeons in Ireland; the National Maternity Hospital
(M.D’M., C.R.); and the Department of Genetics and Biotechnology Institute (C.D., A.S.W.), Trinity College, Dublin, Ireland; and the Department of
Pharmacology and Center for Experimental Therapeutics (A.S.W.), University of Pennsylvania Medical School, Philadelphia, Pa.
Correspondence to Professor Desmond Fitzgerald, Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, 123 St. Stephen’s
Green, Dublin 2, Ireland. E-mail email@example.com
© 2000 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
thermolabile and exhibits reduced activity compared with the
wild type. TT MTHFR homozygotes are predisposed to
increased plasma homocysteine levels, particularly in indi-
viduals with low folate.9,10Hyperhomocysteinemia has been
implicated in premature vascular disease11and more recently,
in venous thrombosis12and unexplained early pregnancy
Although it has been suggested that FV Leiden increases
the risk of thrombotic events in pregnancy, the carrier
frequency of this mutation is 4% to 15%,14far in excess of the
risk of thrombosis (1 to 2 per 1000). Even in a family with the
mutation and a history of thrombosis, the penetrance is highly
variable, suggesting that other factors are involved.15Because
both of these mutations are common, we asked whether the
presence of T MTHFR increased the risk of FV Leiden. An
interaction has been shown between homocysteine and FV
Leiden in men who develop thromboembolic disease,3con-
sistent with this hypothesis. In this study, we prospectively
examined the frequency of FV Leiden and T MTHFR
polymorphisms in an unselected primigravid population and
assessed their effect on pregnancy outcome. We also exam-
ined the frequency in patients with a history of thromboem-
bolic disease in pregnancy and in patients with recurrent fetal
loss, which in some cases reflects a prothrombotic
The research protocol was approved by the Ethics Committee at the
National Maternity Hospital, Holles Street, Dublin, Ireland, and all
participants gave written, informed consent. All consecutive primi-
gravid subjects attending 2 specific booking clinics over a 4-month
period were asked to participate. Patients with a history of hyper-
tension or thrombosis were excluded. A venous blood sample was
drawn into EDTA for genetic analysis on their first clinic visit. All
of the patients were followed up until at least 6 weeks postpartum.
Preeclampsia was defined by the classification of Davey and
McGillivray on the basis of the following criteria: a systolic blood
pressure ?140 mm Hg and/or a diastolic blood pressure ?90 mm Hg
on at least 2 occasions 6 hours apart, or a single diastolic reading of
110 mm Hg or higher; associated proteinuria defined as ?1 by
Dipstick; and resolution of the hypertension by 6 weeks postpartum.
Intrauterine growth retardation was defined as a birth weight below
the 10th percentile for gestational age. No other thrombotic risk
factors were looked for in the primigravid group.
In addition, blood was obtained from patients with a prior history
of venous thrombosis and/or pulmonary embolism during pregnancy,
the diagnosis of which was based on peripheral Doppler studies
and/or a ventilation/perfusion lung scan. All of the patients under-
went a thrombophilia screen as part of their routine management,
which included lupus anticoagulant (diluted Russell viper venom
assay); anticardiolipin antibody; and antigenic or functional levels of
protein C, protein S, and antithrombin III. We also studied a group
of patients with recurrent fetal loss, defined as at least 2 previous and
unexplained events at any point during pregnancy. These patients
also underwent the thrombophilia screen, and all parameters tested
for were normal.
Total genomic DNA was extracted from whole human blood by a
salting-out procedure.17Care was taken to avoid contamination, and
a sterile-water blank was taken through each batch of isolations and
used as a polymerase chain reaction (PCR) control. PCR amplifica-
tion of human genomic DNA for the region containing the FV
Leiden mutation was performed as follows. Genomic DNA (500 ng)
and 25 pmol of sense and antisense primers (sense primer FV1-TGC
CCA GTG CTT AAC AGA CCA; antisense primer FV2A-TCT CTT
GAA GGA AAT GCC CCA TTA, to prime for fragment 1 [F1]; or
FV2B-AAG GAC AAA AGT ACC TGT ATT CCA, to prime for
F2) were used in a reaction containing 2 U of Taq polymerase
enzyme (Promega) and MgCl2at a concentration of 1.5 mmol/L in a
final volume of 50 ?L. The PCR program was as follows: initial
denaturation of 95°C for 5 minutes, followed by 95°C for 1 minute,
60°C for 1 minute, and 72°C for 1 minute in a 35-cycle reaction.
Dimethyl sulfoxide was added to a concentration of 3.5% in the PCR
for F2 to optimize amplification. PCR amplification for the region
containing the MTHFR mutation was as follows: 20 pmol of both
sense primer (5?-TGA AGG AGA AGG TGT CTG CGG GA-3?;
exonic sequence) and antisense primer (5?-AGG ACG GTG CGG
TGA GAG AGT G-3?; intronic sequence) were used in a reaction
volume of 50 ?L containing 200 ?mol/L dNTPs, 1.5 mmol/L MgCl2,
50 mmol/L Tris-HCl (pH 9), 50 mmol/L KCl, 2 U of Taq polymerase
(Promega), 200 ng of genomic DNA, and 1% Triton X-100 (for
MTHFR PCR only). The PCR cycling conditions were 94°C for 1
minute, 63°C for 1 minute, and 72°C for 1 minute for 40 cycles. PCR
products were electrophoresed on 2% agarose gel, stained with
ethidium bromide, and visualized under UV light.
The restriction fragment length polymorphism analysis for the FV
Leiden mutation was carried out as described previously by using
both restriction enzymes MnlI and NlaIII (New England Biolabs).4,18
A similar protocol was used to analyze the MTHFR T mutant by
using the restriction enzyme HinfI (New England Biolabs).19The FV
Leiden mutation is associated with the loss of an MnlI site in F1. This
results in a band shift from 118 to 155 bp on visualization of the
electrophoresed product. Conversely, the mutation combined with
the sequence of primer 2B used to amplify product F2 results in the
acquisition of a novel NlaIII site, resulting in a shift of 91 to 67 bp.
Amplification with the MTHFR-specific primers yields a 198-bp
product. On restriction analysis with HinfI enzyme and in the
presence of the mutant T allele, the PCR product is cleaved, giving
rise to a 175- and a 23-bp fragment. In the absence of the mutation,
no cleavage is observed.
FV analysis was also carried out using heteroduplex analysis and
capillary electrophoresis on a Perkin-Elmer ABI PRISM 310 auto-
mated sequencer.20The primers were as follows17: sense 5?-CAT
GAG AGA CAT CGC CTC TG-3? and antisense 5?-GAC CTA ACA
TGT TCT AGC CAG AAG-3? (labeled with a HEX fluor). The
heteroduplex generator probe was a kind gift from Dr Derrick
Bowen, Hemostasis Research Laboratory, The Arthur Bloom Center,
Cardiff, Wales. A nondenaturing matrix (native 3% polymer) and a
37-cm capillary were used for electrophoresis. Analysis was carried
out using GENESCAN software. All reagents and software were
supplied by Perkin-Elmer.
The Pearson ?2test probability was computed by using STATEXACT 3
software for exact nonparametric inference.21
Of the primigravid subjects asked to participate in the study,
94% agreed. The mean age (?SEM) at the time of presenta-
tion was 25?0.2 years, and the mean gestational age was
14.2?0.26 weeks. The allele frequencies of the 2 poly-
morphisms in the 3 study groups are shown in Table 1. The
allelic frequency of FV Leiden was lower than expected, at
1.36% (a carrier rate of 2.7%), all of the carriers being
heterozygotes. The allelic frequency for T MTHFR was
similar to that previously reported in Western populations.
Allele Frequencies of FV Leiden and T MTHFR in the
Primigravida (n?593)RFL (n?41)RT (n?33)
RFL indicates recurrent fetal loss; RT, recurrent thrombosis.
Murphy et al Risks in Pregnancy Due to FV Leiden and MTHFR
The relationship between mutations and adverse outcomes
in this prospective analysis of 593 primigravidas is shown in
Tables 2 and 3. There was no correlation between MTHFR
thermolabile homozygosity and the various outcomes
(?2?4.347, P?0.6351). However, the presence of FV Leiden
was shown to be significantly more frequent in those who
subsequently miscarried (?2?7.104, P?0.0356). Among
those positive for FV Leiden, 3 of 27 miscarried compared
with 24 of 572 of FV Leiden–negative patients (11% versus
4.2%). We used the same statistical model to determine
whether there was any association between TT MTHFR and
the FV Leiden allele. No association was found in any of the
groups, demonstrating that TT MTHFR did not add to the risk
of FV Leiden.
We also retrospectively studied patients with recurrent
fetal loss (n?41, 32?0.74 years) or a history of thrombosis in
pregnancy (n?33, 29?0.97 years). The genotype distribution
was compared with the prospectively studied patients. The
Pearson ?2analysis of the genotypes in these groups is shown
in Tables 4 and 5. There was a statistically significant
association between FV Leiden and recurrent thrombosis
(Table 4, ?2?12.04, P?0.0115) but not recurrent fetal loss.
There was no significant difference in the distribution of the
T MTHFR allele between normal primigravid subjects and
the groups characterized by thrombosis in pregnancy and
recurrent fetal loss. Moreover, there was no interaction
between the 2 polymorphisms in these groups.
The allele frequency of FV Leiden in our control population
of primigravid women was relatively low at 1.4% compared
with the frequency throughout Europe, which has an average
frequency of 4.4%.14Indeed, compared with another insular
population, Icelanders (2.6%), the frequency is still low.
Although it is possible that the primigravid subjects were in
some way selected (patients who were not pregnant were
excluded by the design of the study), the frequency was
similar to that of a nonpregnant hospital population that we
have studied (data not shown). The low frequency in the Irish
population probably reflects early migration patterns through-
out Europe, with the Irish gene pool reflecting the predomi-
nance of ethnic subgroups with a low FV Leiden frequency.
Both FV Leiden and T MTHFR have been implicated in
several pregnancy-related conditions, including thrombo-
sis,22–24fetal loss,25,26and preeclampsia,27,28based on the
frequency of the mutations in selected disease groups. How-
ever, estimates of gene frequency in diseased populations
may be confounded by other factors, either environmental or
genetic, so that the impact of a mutation may be overesti-
mated. As an example, a synergistic effect has been shown
between the 20210 G/A prothrombin polymorphism, another
risk factor for thrombosis, and FV Leiden.29Similarly,
interactions have been demonstrated between FV Leiden and
T MTHFR.1,3For example, the coexistence of both FV
Leiden and T MTHFR has been associated with retinal
We examined the impact of FV Leiden and T MTHFR on
pregnancy outcome in an unselected primigravid population,
as this provides a real estimate of risk. Pregnancy outcome
was largely unaffected by either mutation, although there was
a modest increase in the rate of second-trimester miscarriages
in patients with FV Leiden. This result is consistent with
for the Control Group of Primigravid Subjects (n?588)
Distribution of FV Leiden Genotypes and Outcomes
FVNormalPET MiscarriageFGRThrombosis Total
? ? ?
? ? ? ? ? ?
? ? ?
? ? ?? ? ? ? ? ?? ? ?
? ? ?
? ? ?
? ? ?
? ? ?
PET indicates preeclamptic toxemia; FGR, fetal growth retardation; and
?/?, ?/?, heterozygous and homozygous for FV Leiden, respectively. Five
cases were excluded owing to incomplete data.
the Control Group of Primigravidas (n?584)
Distribution of MTHFR Genotypes and Outcomes for
MTHFRNormalPET Miscarriage FGRThrombosisTotal
? ? ?
? ? ?? ? ?
? ? ?
? ? ?
? ? ?
? ? ?
PET indicates preeclamptic toxemia; FGR, fetal growth retardation; and
?/?, ?/?, heterozygous and homozygous for T MTHFR, respectively. Nine
cases were excluded owing to incomplete data.
Thrombotic Group and the Recurrent Fetal Loss Group
Compared With the Normal Outcomes of the Primigravid
Genotype Frequencies of FV Leiden Among Both the
2 13 (2.4%)
? ? ? ? ? ?
? ? ?
? ? ?
?/?, ?/? indicate heterozygous and homozygous for FV Leiden, respectively.
Thrombosis Group and the Recurrent Fetal Loss Group
Compared With the Normal Outcomes of the Primigravid
Control Group (n?540)
Genotype Frequencies of T MTHFR Among Both the
? ? ?
? ? ?
?/?, ?/? indicate heterozygous and homozygous for T MTHFR, respectively.
268Arterioscler Thromb Vasc Biol.
other case-control and cohort studies that have implicated FV
Leiden in second-trimester but not in first-trimester miscar-
riages.31A possible explanation for this fact is that recurrent,
first-trimester miscarriage reflects a failure in implantation,
and second-trimester miscarriage reflects a thrombotic event
in the placenta. There were no thrombotic events in the
screened population, although this was not unexpected, as
the frequency of thrombosis is 1 to 2 in 1000 pregnancies.32
The MTHFR TT genotype did not influence the rate of late
miscarriage, and importantly, no interaction was found be-
tween these 2 mutations in determining the risk of events.
However, homocysteine levels were not measured in our
study groups, and because folate supplementation during
pregnancy is common, the underlying effect of the MTHFR
TT genotype may have been masked. Our findings suggest
that widespread screening for these mutations in unselected
populations is unlikely to be clinically useful.
We also examined the allelic frequency of these mutations
in 2 groups of patients, 1 with a history of thromboembolic
disease in pregnancy and a second with recurrent fetal loss.
The cause of recurrent fetal loss is largely unknown. How-
ever, in some cases, there is an underlying prothrombotic
disorder, such as the presence of lupus anticoagulant,33and
recently, mutations in the thrombin-binding domain of throm-
bomodulin result in recurrent fetal loss in mice.34The
frequency of FV Leiden in patients with a history of throm-
bosis was higher than expected, consistent with previous
reports. However, the frequency of FV Leiden was only
marginally higher in patients with recurrent fetal loss. More-
over, there was no association between T MTHFR and these
2 disorders, and, as in the control population, no linkage was
found between the 2 mutations in these patient groups.
Therefore, the presence of T MTHFR did not increase the risk
attributable to FV Leiden.
It is difficult to explain why there was an association
between FV Leiden and late miscarriage, yet we could not
show a significant association with recurrent fetal loss. The
numbers of subjects were relatively small, and this may be a
factor. It is also possible that FV Leiden alone is insufficient
to cause fetal loss. During the course of the study, we
identified 2 sisters who were heterozygous for FV Leiden and
who had experienced recurrent fetal loss, with 4 losses in 1
case and 6 in the second. Both also were positive for lupus
anticoagulant and antiphospholipid antibodies. These find-
ings were in agreement with recent findings that FV Leiden
may contribute to the hypercoagulability of some subjects
with antiphospholipid antibodies.35In the population re-
ported, we screened all cases for other prothrombotic factors,
and in all cases these factors were absent. However, differ-
ences in genetic background may explain variations in risk of
FV Leiden between studies.36
In conclusion, FV Leiden occurred more frequently in
patients with prior thromboembolic disease but not in patients
with recurrent fetal loss. No association between these events
and T MTHFR was found, and there was no interaction
between the mutations in these patient populations. Prospec-
tive identification of FV Leiden and T MTHFR did not
predict adverse outcomes, other than a weak association
between miscarriage and FV Leiden. Thus, widespread
screening of the pregnant population for either of these
mutations is unlikely to be fruitful in identifying patients at
risk of adverse pregnancy outcomes.
This study was supported by grants from the Health Research Board
(to R.P.M., A.S.W., and D.J.F.), the Higher Education Authority (to
D.J.F.), and the Royal College of Surgeons in Ireland Research Fund
(to R.P.M.). We would like to thank all of the individuals who
participated and assisted in the study. We would also like to thank
Anthony Kinsella, MSc, for his invaluable help with the statistical
analysis of the results.
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