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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:511-518

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:511-518.)
© 1999 American Heart Association, Inc.

Original Contributions

Single and Combined Prothrombotic Factors in Patients With Idiopathic Venous Thromboembolism

Prevalence and Risk Assessment

Ophira Salomon; David M. Steinberg; Ariella Zivelin; Sanford Gitel; Rima Dardik; Nurit Rosenberg; Shlomo Berliner; Aida Inbal; Amira Many; Aharon Lubetsky; David Varon; Uriel Martinowitz; Uri Seligsohn

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.


*    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


*    Introduction
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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.


*    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
{chi}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. {chi}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.


*    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 1Down). 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 2Down). 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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  		Table 1. Frequency of Prothrombotic Polymorphisms in Patients With VTE and Controls


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  		Table 2. Frequency Distribution of Prothrombotic Polymorphisms in Patients and Controls and Estimated OR For VTE

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 2Up, 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 2Up.

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 3Down). 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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  		Table 3. Frequency of Prothrombotic Polymorphisms in 3 Subgroups of Patients With VTE

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 FigureDown and Table 4Down.



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  		Figure 1. Frequency of single and combined prothrombotic predispositions in 109 patients in whom a complete work-up of prothrombotic factors was performed. The 16 patients in the box labeled "Thrombophilia" represent 11 patients with a hereditary deficiency of protein C, protein S, or antithrombin III and 5 patients with an acquired lupus anticoagulant.


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  		Table 4. Distribution of Prothrombotic Parameters in 80 of 109 Patients in Whom a Complete Work-Up Was Done1

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 5Down). 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 6Down) 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 {chi}2 test. The hypothesis that the groups had identical frequencies was rejected (P=0.017; see Table 7Down). 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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  		Table 5. Frequency of Prothrombotic Polymorphisms Among Subgroups of Women With VTE


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  		Table 6. ORs With (95% CIs) For Each Polymorphism in Subgroups of Women With VTE


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  		Table 7. Frequency of None, Single, or Combined Prothrombotic Polymorphisms Among Males and Subgroups of Female Patients

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 8Down. 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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  		Table 8. Frequency of Prothrombotic Markers in Patients With and Without a Family History of VTE


*    Discussion
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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 1Up and 2Up). 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 2Up). 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 2Up). 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 6Up). 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 6Up), 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 7Up). 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 {approx}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 FigureUp and Table 4Up 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.


*    Acknowledgments
 
We are indebted to Anna Ostrovsky and Bruria Ravid for their skilled laboratory assistance.


*    Footnotes
 
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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