© 1999 American Heart Association, Inc.
Original Contributions |
Single and Combined Prothrombotic Factors in Patients With Idiopathic Venous Thromboembolism
Prevalence and Risk Assessment
From the Institute of Thrombosis and Hemostasis, Department of Hematology, Sheba Medical Center, Tel-Hashomer, and Sackler Faculty of Medicine (O.S., A.Z., R.D., N.R., S.G., A.I., A.M., U.M., A.L., D.V., U.S.), Tel-Hashomer; the Department of Statistics and Operations Research, Raymond and Beverly Sackler Faculty of Exact Sciences (D.M.S), Tel-Aviv University, Tel-Aviv; and the Anticoagulant Clinic, Maccabi Health Care Services, (S.B., U.S.), Tel-Aviv, Israel.
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Abstract |
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Abstract—The inherited thrombophilias—deficiencies of protein C, protein S, and antithrombin III—and the prothrombotic polymorphisms factor V G1691A and factor II G20210A predispose patients toward venous thromboembolism (VTE). The aim of this study was to determine the prevalence of single and combined prothrombotic factors in patients with idiopathic VTE and to estimate the associated risks. The study group consisted of 162 patients referred for work-up of thrombophilia after documented VTE. The controls were 336 consecutively admitted patients. In all subjects factor V G1691A, factor II G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T were analyzed by specific polymerase chain reactions and restriction enzymes. Activities of antithrombin III and protein C, free protein S antigen, and lupus anticoagulant were determined in a subset of 109 patients who were not receiving oral anticoagulants. The prevalences of heterozygotes and homozygotes for factor V G1691A and factor II G20210A among patients and controls were 40.1% versus 3.9% and 18.5% versus 5.4%, respectively (P=0.0001). The prevalence of homozygotes for MTHFR C677T in patients was 22.8% and in controls, 14.3% (P=0.025). Heterozygous and homozygous factor V G1691A, factor II G20210A, and homozygous MTHFR C677T were found to be independent risk factors for VTE, with odds ratios of 16.3, 3.6, and 2.1, respectively. Two or more polymorphisms were detected in 27 of 162 patients (16.7%) and in 3 of 336 controls (0.9%). Logistic regression analysis disclosed odds ratios of 58.6 (confidence interval [CI], 22.1 to 155.2) for joint occurrence of factor V and factor II polymorphisms, of 35.0 (CI, 14.5 to 84.7) for factor V and MTHFR polymorphisms, and of 7.7 (CI, 3.0 to 19.6) for factor II and MTHFR polymorphisms. Among 109 patients in whom a complete thrombophilic work-up was performed, 74% had at least 1 underlying defect. These data indicate that in most patients referred for evaluation of thrombophilia due to idiopathic VTE, 1 or more underlying genetic predispositions were discernible. The presence of >1 of the prothrombotic polymorphisms was associated with a substantial risk of VTE.
Key Words: thrombophilia • factor V G1691A (Leiden) • factor II G20210A • methylenetetrahydrofolate reductase • venous thromboembolism
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Introduction |
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Inherited thrombophilias are genetically determined tendencies toward venous thromboembolism (VTE).1 The 3 major thrombophilias that have been identified are deficiencies of antithrombin III, protein C, and protein S. Recently, 2 prothrombotic polymorphisms were found to be associated with VTE. The first to be discovered was factor V G1691A, which renders factor V resistant to activated protein C and confers an enhanced risk of VTE,2 3 with odds ratios (ORs) of 3 to 8 in heterozygotes4 5 6 and of 30 to 140 in homozygotes.7 8 The second prothrombotic polymorphism, factor II G20210A, is associated with an increased level of factor II, and it exerts in heterozygous carriers an increased risk of VTE, with ORs of 2.8 to 3.8.9 10 Both factor V and factor II prothrombotic polymorphisms are relatively frequent in white populations, with allele frequencies of .01 to .0811 12 and of .01 to .03, respectively,9 13 and a founder effect has been demonstrated for each polymorphism.14 15 Another apparent prothrombotic polymorphism is 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T. Homozygotes for this polymorphism manifest a 50% reduction in the specific activity of MTHFR, which gives rise to reduced conversion of homocysteine to methionine through the transmethylation pathway.16 The resultant mild hyperhomocysteinemia observed in homozygotes for MTHFR C677T is accentuated when such patients have reduced plasma folic acid levels.17 Evidence linking homozygous MTHFR C677T per se with an increased risk of VTE was recently provided by several groups of investigators18 19 20 but not by others.21 22 23
The simultaneous occurrence of hereditary thrombophilias and prothrombotic polymorphisms was shown to substantially increase the risk of VTE.24 25 26 27 28 29 30 31 This enhancement was demonstrated in subjects bearing factor V G1691A and deficiencies of protein C,24 25 protein S,26 27 or antithrombin III28 and in subjects affected by these thrombophilias or factor V G1691A in conjunction with factor II G20210A.29 Similarly, an increased risk of thrombosis was recently observed by us in patients with factor V G1691A and homozygous homocystinuria30 and by Ridker et al31 in male patients in the Physicians Health Study who had hyperhomocysteinemia due to unspecified causes and factor V G1691A. Finally, the common occurrence of heterozygous factor V G1691A and homozygous MTHFR C677T was shown to confer a greater risk of VTE than factor V G1691A alone.21
This information prompted us to determine the prevalence of the classic hereditary thrombophilias, prothrombotic polymorphisms, lupus anticoagulant, and their concomitant occurrence in a retrospective cohort of patients referred for evaluation of thrombophilia after idiopathic VTE. The risk of thrombosis caused by a single and >1 prothrombotic predisposition was estimated.
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Methods |
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Study Group
The study group consisted of 162 unrelated patients (67 men and 95 women; 159 Jews and 3 Arabs) ranging in age from 19 to 83 years with a median age of 41.5. The patients were referred by their primary care physicians to the Institute of Thrombosis and Hemostasis for evaluation of thrombophilia between March 1993 and September 1997 after experiencing at least 1 episode of VTE. Patients belonging to the study group were questioned regarding the circumstances under which they manifested the first event of VTE, and evidence for VTE was documented from their medical records. Seventy-one patients had experienced >1 event of VTE. The diagnosis of deep-vein thrombosis (DVT) was established by compression ultrasonography, Doppler ultrasonography, or contrast venography; pulmonary embolism was diagnosed by angiography or ventilation-perfusion lung scans. One hundred forty-four patients had DVT of a lower limb involving popliteal, femoral, or ileofemoral veins; 6 patients had DVT in an upper limb involving axillary or brachial veins; 4 subjects had DVT in an upper and lower limb; and 8 patients had pulmonary emboli without documentation of DVT at the time of the event. History of VTE in siblings, parents, and grandparents was also recorded. Excluded from the study group were patients with malignant disorders, myeloproliferative disorders, systemic lupus erythematosus, systemic infections, trauma, or prolonged immobilization. Also excluded were patients whose liver function tests were abnormal and patients undergoing in vitro fertilization. Blood samples were drawn from patients into EDTA for DNA extraction and into buffered citrate for coagulation assays.
Control Group
Three hundred thirty-six subjects (153 male and 183 female)
whose age ranged between 17 and 97 years (median, 71) constituted the
control group. The subjects were patients consecutively admitted to the
hospital who had no history of VTE. DNA was extracted from blood
samples sent for routine blood counts. The human subject ethics
committee of the Medical Center approved this procedure.
Detection of Classic Thrombophilias
Antithrombin III was measured by a standard
chromogenic assay based on inhibition of activated
factor X (Chromogenix). Protein C activity was determined by a
chromogenic assay (Diagnostica Stago) and free
protein S antigen by a commercially available kit
(Diagnostica Stago). Normal ranges (±2SD of the mean) for
antithrombin III, protein C, and free protein S were 80% to 125%,
70% to 130%, and 60% to 135% of normal, respectively. Lupus
anticoagulant was assessed by a method described
elsewhere.32
Detection of Prothrombotic Polymorphisms
DNA extraction was performed by a standard
method.33 Factor V G1691A was detected by polymerase chain
reaction (PCR) and MnII digestion,25 MTHFR
C677T by PCR and HinfI digestion,16 and
factor II G20210A by a slight modification of the originally described
method.9 The modification involved an amplification of a
253-bp DNA segment instead of a 345-bp segment and digestion by both
HindIII and Mspl restriction enzymes rather than
by HindIII only. This provided a positive control, because
both G20210 and A20210 alleles were simultaneously
digested and yielded typical fragments.
Statistical Analyses
2 tests were used to compare the
frequency of prothrombotic polymorphisms between patients and
controls; among subsets of female patients; and among patients with
none, single, or combined prothrombotic polymorphisms. Logistic
regression analysis was used to estimate the OR (with 95%
confidence intervals [CIs]) effects for each possible combination of
prothrombotic polymorphisms. 2 tests were
also used to assess whether patients bearing prothrombotic
polymorphisms or hereditary thrombophilic conditions were more
likely to have a closely related family member who had experienced VTE.
Fisher's exact test was used for calculating the significance of
differences when relatively small numbers of observations were
available.
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Results |
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Prevalence of Prothrombotic Polymorphisms and Risk of VTE
The proportions of subjects who bore factor V G1691A or factor II G20210A or who were homozygous for MTHFR C677T were significantly higher among patients with VTE than among controls (Table 1
).
For each prothrombotic polymorphism manifested as a single defect,
we computed the direct OR relative to the group not bearing that
particular polymorphism and estimated the OR from a logistic
regression model (Table 2). The similarity of the model-based
ORs to the direct ORs indicated that a multiplicative model was
appropriate and provided no evidence of interaction among the
prothrombotic polymorphisms. These data indicated that the 3
prothrombotic polymorphisms acted as independent risk factors for
VTE.
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Among the 65 patients bearing factor V G1691A, 14 were homozygotes for
this prothrombotic polymorphism. Of these 14 homozygotes, 6 had
additional defects as follows: 1 was a homozygote for factor II
G20210A, 2 were heterozygotes for factor II G20210A, and 3 were
homozygotes for MTHFR C677T. Among the 30 patients bearing factor II
G20210A, 3 were homozygotes. No subject in the control group was
homozygous for factor V G1691A or for factor II G20210A. In view of the
greater risk of VTE in homozygotes versus heterozygotes for factor V
G1691A7 8 and with no comparable data available for
homozygous versus heterozygous factor II G20210A, we repeated the
analyses after excluding the 14 homozygous patients for factor
V G1691A. In the analyses shown in Table 2
, the ORs from
the logistic regression analysis were very close to the direct
ORs. For example, for heterozygotes of factor V G1691A, the direct OR
was 12.6 and the OR derived from the logistic model was 12.8; for
heterozygosity for both factor V G1691A and factor II G20210A, the
respective ORs were 39.0 versus 46.0 and for factor V G1691A
heterozygotes and MTHFR C677T homozygotes, 30.3 versus 27.3. As
expected, after exclusion of factor V G1691A homozygotes, all of the
ORs mentioned above were somewhat lower than those shown in Table 2.
Forty-seven of the 162 VTE patients were receiving oral anticoagulants
at the time of the study, and thus, protein C and protein S
deficiencies could not be evaluated with certainty. For 6 additional
patients, values of antithrombin III and of lupus anticoagulant
were unavailable. We were concerned that the distribution of
prothrombotic polymorphisms in the study group might be quite
different among subjects with and without these additional risk
factors. However, the frequency distribution of the prothrombotic
polymorphisms remained stable after division of the entire study
group into subsets of patients (Table 3). Moreover, logistic
regression analysis performed on each of the subsets in
comparison with the control group remained almost the same, except for
a larger probability value due to the reduction in sample size (data
not shown).
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Prevalence of All Examined Prothrombotic Predispositions
In 109 patients, the whole set of prothrombotic markers was
examined, ie, the 3 polymorphisms, proteins C and S, antithrombin
III, and lupus anticoagulant. Eighty of the 109 patients (73.6%)
were found to have at least 1 defect. Fifty patients had a single
defect, of whom 16 had a deficiency of protein C, protein S, or
antithrombin III, and 34 bore 1 of the prothrombotic polymorphisms.
Thirty patients had combined defects: 17 with 2 or more
prothrombotic polymorphisms and 13 with at least 1 prothrombotic
polymorphism combined with deficiencies of protein C, protein S,
antithrombin III, or lupus anticoagulant. The breakdown of these
various subcategories of patients is illustrated in the Figure
and Table 4.
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Frequency of Prothrombotic Polymorphisms in Subsets of
Women
At the time of their first VTE, 38 of 95 women (40%) were
taking oral contraceptives, 15 (16%) were pregnant, and 18 (19%) were
in the post partum state. None of the patients received hormone
replacement therapy at the time of VTE. Reliable information was
unavailable for 1 woman. The frequencies of the prothrombotic
polymorphisms were higher in women who belonged to any 1 of these
subsets than in the other 23 female patients (median age, 47), none of
whom was receiving hormone replacement therapy (Table 5).
However, only factor V G1691A was significantly more frequent,
especially in women on contraceptives or during puerperium. The risk of
thrombosis exerted by each prothrombotic polymorphism was estimated
in all subcategories of women. The ORs (Table 6) showed that the
highest values were for factor V G1691A in women during the post partum
period or while on contraceptives and that the lowest values were in
women with MTHFR C677T homozygosity who were pregnant or on
contraceptives. The frequencies of single, combined, or no
prothrombotic polymorphism in the 4 subsets of women were compared
by a 2 test. The hypothesis that the groups
had identical frequencies was rejected (P=0.017; see Table 7). Similar results were obtained when men were included in the
latter comparison. These data indicated that the presence of 1, >1, or
none of the prothrombotic polymorphisms was group dependent.
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Frequency of Familial VTE
Information was obtained on VTE among family members for 159 of
162 patients. Fifty patients (31.4%) had at least 1 close relative
with a history of VTE. The proportions of the prothrombotic
polymorphisms and thrombophilic markers among patients with and
without a familial history of VTE are presented in Table 8. More patients with 1 of the 3 prothrombotic polymorphisms
had relatives with VTE than did patients not bearing these
polymorphisms, but the differences were not statistically
significant. Among the thrombophilic markers, only antithrombin III
deficiency was associated with a significantly higher number of
patients who had relatives with VTE (Fisher's exact test).
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Discussion |
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The data presented here indicate that patients with idiopathic VTE and who are suspected of having a thrombophilia by their primary care physicians carry a heavy burden of prothrombotic polymorphisms irrespective of the presence of 1 of the classic thrombophilias or lupus anticoagulant. In 102 of 162 patients (63.0%), we found 1 or more of the following prothrombotic genotypes: homozygous or heterozygous factor V G1691A, homozygous or heterozygous factor II G20210A, and homozygous MTHFR C677T. Among 336 controls, only 76 (22.6%) bore 1 or more of the 3 prothrombotic polymorphisms, and none of them was homozygous for either factor V G1691A or factor II G20210A. An inadvertently low prevalence of the 3 prothrombotic polymorphisms in the control group was ruled out by finding similar frequency distributions of the polymorphisms in 3 other control groups examined by us in other studies currently performed in Israel, viz, 96 healthy subjects belonging to the laboratory personnel of the Medical Center (median age, 46 years; range, 25 to 72), 140 healthy male subjects of the regular army (median age, 40; range, 26 to 48), and 191 Ashkenazi-Jewish newborns screened for Gaucher's disease (data not shown).
Each 1 of the 3 polymorphisms examined was found in the present
study to be an independent risk factor, with factor V G1691A
manifesting the greatest risk and homozygous MTHFR C677T the lowest
(Tables 1 and 2). The OR of 16 for factor V G1691A in
this study was higher than the OR determined in previous
studies.4 5 6 These inconsistencies could have resulted
from the relatively large percentage (21.5%) of homozygous patients in
our study, because these individuals have been found to have an
enhanced risk for VTE.8 However, the OR for heterozygous
individuals (after exclusion of homozygotes) was still high (12.6)
compared with other studies. Another possible explanation is that the
different ORs reflect the variable modes by which patients were
enrolled. Several studies were population based,31 34
whereas others,21 including our study, recruited patients
referred for work-up of thrombophilia. However, we do not think that
selective referral alone can explain the higher OR observed in this
study. For example, if we restricted our attention to only those
patients at their first VTE episode, then the OR for our study would be
11.6, higher than those reported in other studies.4 5 6
Moreover, the frequency of factor V G1691A was not significantly
related to other possible criteria for referral, such as age, family
history, pregnancy, or the use of oral contraceptives.
With respect to factor II G20210A, our results are in accord with previous reports.9 10 29 35 36 37 38 For homozygous MTHFR C677T, our conclusions are similar to those in several reports18 19 20 but dissimilar to others.21 22 23 Conceivably, the latter inconsistency reflects variable plasma folic acid levels, which have been shown to exert a significant effect on plasma homocysteine level in homozygotes for MTHFR C677T17 ; hyperhomocysteinemia, in turn, was identified as an independent risk factor for VTE.39 No significant relations were found between the frequency of MTHFR C677T and any of the patient referral criteria mentioned above, so our OR for MTHFR C677T does not appear to have been affected by selective patient referral.
Two or more prothrombotic polymorphisms were observed in 27 of 162
patients (16.7%) and in only 3 of 336 controls (0.9%). Because the
number of patients affected by combined defects was rather small, we
estimated the attributed risks of VTE by a logistic regression model
(Table 2). The highest risk was exerted by the common occurrence
of factor V G1691A and factor II G20210A (OR, 58.6), and a lower risk
was observed for patients with a common occurrence of factor V G1691A
and homozygous MTHFR C677T (OR, 35.0). Only 3 patients in the
present study bore all 3 prothrombotic polymorphisms, and thus,
no definitive conclusions can be drawn from the high estimated OR of
125 (Table 2). Recent reports have also described an enhanced
risk of VTE in heterozygotes for factor V G1691A when associated with
heterozygous factor II G20210A29 40 or homozygous MTHFR
C677T.21
Seventy-five percent of women enrolled in the study were either
pregnant, at the post partum stage, or using contraceptives. We found
an increased risk of VTE in subjects bearing factor V G1691A who used
contraceptives or who were in the post partum period (Table 6).
This finding supports and extends the results of previous
studies.41 42 The risk associated with factor II G20210A
was also higher among these women (Table 6), but the difference
was less pronounced than for factor V G1691A. Among female patients
bearing at least 1 of the prothrombotic polymorphisms, 11 of 27
contraceptive users and 7 of 13 post partum women had double defects.
The proportions of subjects with combined defects were lower in men and
other subcategories of female subjects (Table 7). Thus, our data
suggest that subjects with factor V G1691A or with >1 prothrombotic
polymorphism are particularly predisposed to VTE while using
contraceptives or during the post partum period. However, larger
cohorts of patients will have to be studied to substantiate these
associations.
A history of familial VTE has been considered an important issue in deciding whether or not a patient with VTE should undergo a detailed search for a hereditary thrombophilia. However, the data presented by Heijboer et al43 and those presented in this study cast doubt on the validity of such an approach. In probands with a familial history of VTE, the frequency of thrombophilias (except for antithrombin III deficiency) and of prothrombotic polymorphisms was higher but not significantly different from the frequency observed in probands who had a negative familial history of VTE. Thus, a work-up for hereditary thrombophilia should not be denied to patients with idiopathic VTE who do not have a family history.
Until 1993, searches for underlying causes in cohorts of patients
presenting with idiopathic VTE were frustrating. Our own
study44 and those of others recently reviewed by
Koeleman et al45 identified deficiencies of
protein C, protein S, and antithrombin III in 4.5% to 19.9% of
patients with VTE. Testing for lupus anticoagulant further
increased the yield of finding an underlying cause by
4%.44 After the seminal discoveries of
activated protein C resistance2 due to factor V
G1691A3 as a risk factor for VTE and the identification of
factor II G20210A9 and homozygous MTHFR C677T as
additional independent risk factors, the proportion of identifiable
causes could be substantially increased. The Figure and Table 4 illustrate that we discerned a prothrombotic predisposition in
74% of patients with idiopathic VTE who were referred for work-up of a
possible inherited or acquired thrombophilia. The proportion of
identifiable causes for VTE could perhaps be further increased by
studying in similar cohorts of patients additional prothrombotic
factors not examined in the present study, like elevated
homocysteine levels due to causes other than MTHFR C677T
homozygosity,31 39 46 47 increased plasma factor II not
related to factor II G20210A,9 increased plasma level of
factor VIII,48 dysfibrinogenemia,49 and
mutations in the thrombomodulin gene.50 It should,
however, be borne in mind that the proportion of identifiable
prothrombotic factors may vary significantly in different populations.
For example, in Africans, Orientals, and native Americans bearing
neither factor V G1691A11 51 52 53 54 nor factor II
G20210A,13 55 56 57 the yield of such searches is expected
to be low. In contrast, in populations like southern
Swedes58 or Palestinian Arabs,12 the
anticipated yield would be high due to a high prevalence of factor V
G1691A.
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Acknowledgments |
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We are indebted to Anna Ostrovsky and Bruria Ravid for their skilled laboratory assistance.
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Footnotes |
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Reprint requests to Dr Uri Seligsohn, Institute of Thrombosis and Hemostasis, Department of Hematology, Sheba Medical Center, Tel-Hashomer, Israel 52621.
Received May 21, 1998; accepted July 30, 1998.
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