© 2000 American Heart Association, Inc.
Thrombosis |
Prospective Evaluation of the Risk Conferred by Factor V Leiden and Thermolabile Methylenetetrahydrofolate Reductase Polymorphisms in Pregnancy
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 dfitzgerald{at}rcsi.ie
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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.
Key Words: factor V Leiden polymorphism • thermolabile methylenetetrahydrofolate reductase polymorphism • pregnancy • venous thrombosis • recurrent fetal loss • genetic risk factors
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Introduction |
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Functional polymorphisms in genes controlling hemostasis contribute to an increased risk of thrombotic events. 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) methylenetetrahydrofolate reductase (MTHFR) polymorphisms have been implicated as risk factors for thrombosis.1 2 3 The FV Leiden variant arises as a result of a point mutation at nucleotide position 1691, resulting in an arginine to a glutamine substitution at position 5064 that reduces its sensitivity to inactivation by activated protein C. FV Leiden has been associated with familial thrombophilia5 and 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 to 100-fold.8
MTHFR is critical in the metabolism of homocysteine, as it effects the NADPH-linked reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. A C-to-T missense mutation at nucleotide 677 results in an enzyme that is thermolabile and exhibits reduced activity compared with the wild type. TT MTHFR homozygotes are predisposed to increased plasma homocysteine levels, particularly in individuals with low folate.9 10 Hyperhomocysteinemia has been implicated in premature vascular disease11 and more recently, in venous thrombosis12 and unexplained early pregnancy loss.13
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%,14 far 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.15 Because 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,3 consistent 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 examined the frequency in patients with a history of thromboembolic disease in pregnancy and in patients with recurrent fetal loss, which in some cases reflects a prothrombotic tendency.16
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Methods |
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Study Groups
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 primigravid subjects attending 2 specific booking clinics over a 4-month period were asked to participate. Patients with a history of hypertension 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 underwent 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.
DNA Analysis
Total genomic DNA was extracted from whole human blood by a
salting-out procedure.17 Care 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 amplification 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
MgCl2 at a concentration of 1.5 mmol/L in a
final volume of 50 µL. 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 µL containing 200
µmol/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).19 The 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 automated sequencer.20 The 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.
Statistical Analysis
The Pearson 
2 test probability was
computed by using STATEXACT 3 software for exact
nonparametric inference.21
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Results |
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Of the primigravid subjects asked to participate in the study, 94% agreed. The mean age (±SEM) at the time of presentation was 25±0.2 years, and the mean gestational age was 14.2±0.26 weeks. The allele frequencies of the 2 polymorphisms 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.
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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.
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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
2 analysis 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.
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Discussion |
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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%.14 Indeed, 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 throughout Europe, with the Irish gene pool reflecting the predominance of ethnic subgroups with a low FV Leiden frequency.
Both FV Leiden and T MTHFR have been implicated in several pregnancy-related conditions, including thrombosis,22 23 24 fetal loss,25 26 and preeclampsia,27 28 based on the frequency of the mutations in selected disease groups. However, 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 overestimated. As an example, a synergistic effect has been shown between the 20210 G/A prothrombin polymorphism, another risk factor for thrombosis, and FV Leiden.29 Similarly, interactions have been demonstrated between FV Leiden and T MTHFR.1 3 For example, the coexistence of both FV Leiden and T MTHFR has been associated with retinal arterial occlusion.30
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 other case-control and cohort studies that have implicated FV Leiden in second-trimester but not in first-trimester miscarriages.31 A 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 between 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. However, in some cases, there is an underlying prothrombotic disorder, such as the presence of lupus anticoagulant,33 and recently, mutations in the thrombin-binding domain of thrombomodulin result in recurrent fetal loss in mice.34 The frequency of FV Leiden in patients with a history of thrombosis was higher than expected, consistent with previous reports. However, the frequency of FV Leiden was only marginally higher in patients with recurrent fetal loss. Moreover, 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 findings were in agreement with recent findings that FV Leiden may contribute to the hypercoagulability of some subjects with antiphospholipid antibodies.35 In the population reported, we screened all cases for other prothrombotic factors, and in all cases these factors were absent. However, differences 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. Prospective 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.
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Acknowledgments |
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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.
Received March 24, 1999; accepted June 28, 1999.
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