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Genetic susceptibility to pregnancy-related venous thromboembolism: Roles of factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations
Genetic susceptibility to pregnancy-related venous thromboembolism: Roles of factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations Elvira Grandone, MD, Maurizio Margaglione, MD, Donatella Colaizzo, Biol Sc, Giovanna D’Andrea, Biol Sc, Giuseppe Cappucci, Biol Sc, Vincenzo Brancaccio, MD, and Giovanni Di Minno, MD Naples and Palermo, Italy OBJECTIVE: This study’s objective was to evaluate the association between venous thromboembolism during pregnancy and the postpartum period and the factor V Arg 506 Gln (factor V Leiden), the prothrombin G20210A, and methylenetetrahydrofolate reductase C677T polymorphisms. STUDY DESIGN: In this case-control study 42 case patients and 213 control subjects (parous age-matched women without history of thrombosis) were genotyped for all the polymorphisms. Moreover, antiphospholipid antibodies and protein C, protein S, and antithrombin III deficiencies were investigated in each case. RESULTS: Ten case patients (23.8%) and 4 control subjects (1.9%; odds ratio 16.3, 95% confidence interval 4.8-54.9) carried the factor V Leiden mutation; 13 case patients (31.0%) and 9 control subjects (4.2%; odds ratio 10.2, 95% confidence interval 4.0-25.9) were carriers of the prothrombin G20210A allele. Finally, 12 case patients (28.6%) and 34 control subjects (16.0%; odds ratio 2.1, 95% confidence interval 1.0-4.5) were homozygotes for methylenetetrahydrofolate reductase C677T. Overall, mutations were found in 25 case patients (59.5%) and 47 control patients (22.2%; odds ratio 5.2, 95% confidence interval 4.9-19.6). One patient carried the antithrombin III deficiency and 1 the protein S deficiency, whereas 2 women had a primary antiphospholipid syndrome. CONCLUSIONS: The significant risk estimates of having a pregnancy-related venous thromboembolism in the presence of the prothrombotic genetic risk factors analyzed suggest to screen for these mutations women with a personal history of thromboembolic events during pregnancy or the postpartum period. (Am J Obstet Gynecol 1998;179:1324-8.)
Key words: 5,10-Methylenetetrahydrofolate reductase C677T polymorphisms, factor V Leiden, pregnancy-related venous thromboembolism, prothrombin G20210A Estimates of the incidence of pregnancy-related venous thromboembolism vary between 1 in 1000 to 1 in 2000 deliveries.1 Pregnancy is a prothrombotic state in which increases in coagulation factors I, VII, VIII, and X, a decrease in protein S, and an inhibition of fibrinolysis are well documented. These phenomena are likely to contribute to the increased risk (5 times) of venous thrombosis during this period.2, 3 For women who have an inher-
From Unità di Aterosclerosi e Trombosi, Casa Sollievo della Sofferenza, S. Giovanni Rotondo, Divisione di Ematologia, Unità di Coagulazione, Ospedale A. Cardarelli, and Istituto di Medicina Interna e Geriatria, Università di Palermo. Supported by Telethon-Italy (grant E C 561). Received for publication December 31, 1997; revised March 16, 1998; accepted May 11, 1998. Reprint requests: Elvira Grandone, MD, Unità di Aterosclerosi e Trombosi, IRCCS Casa Sollievo della Sofferenza, Viale Cappuccini, S. Giovanni Rotondo (FG), 71013 Italy. Copyright © 1998 by Mosby, Inc. 0002-9378/98 $5.00 + 0 6/1/91595
1324
ited deficiency of a naturally occurring anticoagulant (antithrombin, protein C, or protein S), it has been reported that pregnancy and the postpartum period are associated with greatly increased risk of venous thromboembolism.4 However, the studies that produced these data lacked appropriate control groups and included many patients because they were seen with venous thrombosis either associated with or unrelated to pregnancy. The resistance to activated protein C is an autosomal dominant trait caused by a mutation in coagulation factor V gene that consists of a substitution of guanine with adenine at nucleotide position 1691 (Fig 1).5 This mutation results in a replacement of arginine residue 506 with a glutamine at 1 of the factor V cleavage sites for activated protein C.5 The role of this mutation, named factor V Leiden, in the occurrence of deep venous thrombosis is well known.6 Moreover, a role has been recently reported for this mutation in the occurrence of deep venous thrombosis in obstetric populations.7, 8 A common ge-
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netic variation (a guanine to adenine transition at position 20210) in the 3´-untranslated region of the prothrombin gene has been associated with an increased incidence of venous thrombosis.9 Individuals carrying this molecular variation have significantly higher prothrombin levels than do noncarriers. Regardless of sex and age, the relative risk of thrombosis associated with the G20210A allele has been estimated to be 2.7.9 On the other hand, there is recent evidence of the association among hereditary homocystinuria, factor V Leiden, and thrombosis.10 The prevalence of hyperhomocysteinemia in individuals with venous thrombosis has been found to be high, representing an independent risk factor for venous disease.11 A cause of moderate hyperhomocysteinemia is a relatively frequent mutation in the gene coding for 5,10methylenetetrahydrofolate reductase (MTHFR). The genetic defect is a cytosine to thymine missense mutation at nucleotide 677, which substitutes a valine for an alanine residue.12 The role of the TT genotype in arterial or venous thrombosis is still a matter of investigation.13-16 It has been suggested that this molecular variant may have a role only when associated with other risk factors for venous thrombosis, such as factor V Leiden16 or antiphosholipid antibodies.17 To evaluate the roles of the mutations described (prothrombin G20210A, factor V Leiden, and MTHFR TT genotype) as genetic risk factors predisposing toward pregnancy-related thromboembolism, we evaluated these risk factors in a population of white women from Southern Italy. Patients and methods Subjects. We performed a case-control study in 2 thrombosis centers at Hospital “Casa Sollievo della Sofferenza,” S. Giovanni Rotondo, and at A. Cardarelli Hospital, Naples. Between January 1995 and March 1998 we observed 42 women with a previous deep venous thrombosis during pregnancy or in the postpartum period. A group of 213 parous women without venous or arterial thromboses served as a control group. Both case patients and control subjects were white women living in Southern Italy. The 2 groups were comparable in social status and age. A complete clinical summary, with emphasis on personal and family history for thromboembolic disease, was obtained by a specially trained staff. Deep venous thrombosis was diagnosed by phlebography or ultrasonography; pulmonary embolism was diagnosed by angiography or ventilation-perfusion lung scan. Mean age of the case patients was 35.9 ± 12.6 years; that of the control subjects was 33.7 ± 7.1 years (P not significant). After approval was obtained from the local ethics committees, the study was carried out according to the Principles of the Declaration of Helsinki. Informed consent was obtained from all the subjects. Blood collection and coagulation tests. Blood samples
A
B Fig 1. Simplified scheme showing blood coagulation cascade and inhibitory effects (parallel lines) of antithrombin III and of protein C (PC) and protein S (PS) system on factor V (FV) wild type (Arg 506) (A) and on factor V Leiden (Gln 506) (B). Because the activated protein C cleaves factor V at the arginine 506 site, replacement at that site with glutamine leads to a resistance to protein C. FX, Factor X; FXa, activated factor X; FVa, activated factor V.
were collected in 3.8% trisodium citrate and centrifuged at 2000g for 15 minutes to obtain platelet-poor plasma, which was immediately frozen and stored in small aliquots at –70°C until tested. Antiphospholipid antibodies–lupus anticoagulant and immunoglobulin G anticardiolipin antibodies (enzyme-linked immunosorbent assay [ELISA]; Byk Gulden, Italy), antithrombin III, protein C, amydolytic and immunologic (Behring, Marburg, Germany) and total and free (ELISA; Diagnostica Stago, Asnières, France) protein S antigen were determined in all subjects, as reported elsewhere.17, 18 Clotting assays were performed on a KC4 Amelung coagulometer (Amelung, Austria). Interassay and intra-assay coefficients of all the variables never exceeded 8.0% and 5.0%, respectively. Deoxyribonucleic acid extraction and analysis. Leukocyte deoxyribonucleic acid was obtained from frozen blood by the use of standard techniques. A 220–base pair (bp) fragment of the factor V gene that included nucleotide 1691 was amplified by polymerase chain reaction as previously described,19 with some modifications.20 To identify the G20210A mutation of the prothrombin gene, a 345-bp fragment was obtained and digested with the HindIII endonuclease, according to
1326 Grandone et al
November 1998 Am J Obstet Gynecol
Table I. Prevalence of inherited prothrombotic defects and family history in entire group and according to time of event Factor V Leiden mutation
Venous thromboembolism during pregnancy (n = 9) Venous thrombolism after cesarean delivery (n = 9) Venous thromboembolism after spontaneous delivery (n = 24) All patients (n = 42) Family history (n = 8)
Prothrombin G20210A mutation
No.
%
No.
1
11.1
0
1
11.1
2*
1
4.2
3 0
7.1
%
TT MTHFR genotype
Combined defects
No.
%
No.
%
2
22.2
1
11.1
22.2
4
44.4
1
11.1
3
12.5
2
8.3
7†
29.2
5 2
11.9 25.0
8 1
19.1 12.5
9 2
21.4 25.0
*One woman also had a protein S deficiency. †One woman was affected by primary antiphospholipid syndrome.
Poort et al.9 A fragment of 198 bp containing the C677T polymorphism of MTHFR gene was generated as described elsewhere.12 Untypability for factor V Leiden occurred in 1 control subject. Statistical analysis. All the analyses were performed according to the Statistical Package for Social Science (SPSS 6.1 for Macintosh; SPSS Inc, Chicago, Ill). The significance of differences in means was evaluated by nonparametric tests, whereas the significance of any difference in proportions was tested by χ2 statistics. The significance of the difference of alleles and genotypes observed between the groups was tested with the χ2 analysis, after grouping homozygous and heterozygous carriers of the prothrombin G20210A allele and homozygous and heterozygous carriers of the MTHFR C677T allele. Odds ratios and 95% confidence intervals were calculated. Results As shown in Table I, all deep venous thromboses and pulmonary embolisms were observed in the postpartum period, except for 9 that occurred during pregnancy. Among these postpartum events, 9 (27.3%) occurred after a cesarean delivery. One patient carried antithrombin III deficiency and 1 carried protein S deficiency; in 2 other cases a primary antiphospholipid syndrome was present (Table I). No case patient had arterial or venous thrombosis outside the pregnancy or the postpartum period. Mean age at the time of the events was 27.7 ± 4.9 years (range 16-39 years). On the whole, 10 case patients (23.8%) and 4 control subjects (1.9%; odds ratio 16.3, 95% confidence interval 4.8-54.9) were heterozygous for the factor V Leiden (Table II). As far as the prothrombin G20210A allele was concerned, 9 control subjects (4.2%) were heterozygous. Thirteen case patients (31.0%; odds ratio 10.2, 95% confidence interval 4.0-25.9) carried this mutation (Table II). Of these 13, 2 were homozygous for the mutation and were analyzed with the heterozygous group. Twelve (28.6%) of the 42 case patients were homozygous for the
MTHFR C677T allele, whereas in the control group this number was 34 (16.0%; odds ratio 2.1, 95% confidence interval 1.0-4.5; Table II). The coexistence of >1 mutation was found in 9 case patients (21.4%; Table I). Of them, 6 women in whom an episode of deep venous thrombosis or pulmonary embolism occurred in the postpartum period (n = 5) or during pregnancy (n = 1) carried both factor V Leiden and prothrombin G20210A mutations. Two patients with thrombotic episodes occurring in postpartum and after a cesarean delivery, respectively, were carriers of the MTHFR C677T genotype and the prothrombin G20210A mutation. In 1 case patient, who had a deep venous thrombosis in the postpartum period, the MTHFR C677T genotype and the factor V Leiden mutation were found. Only 1 woman, who had an episode of deep venous thrombosis plus a cerebral venous thrombosis when she was 22 years old, showed all the gene variants. Among control subjects, only 1 carrier of both the MTHFR C677T genotype and the factor V Leiden mutation was observed (0.5%). Altogether, 25 case patients (59.5%) carried ≥1 mutation, compared with 47 of the healthy control subjects (22.2%; odds ratio 5.2, 95% confidence interval 4.9-19.6). A family history of deep venous thrombosis or pulmonary embolism was reported for 8 case patients (19.0%). Of these, 3 did not show any mutation, 2 were heterozygous for the prothrombin G20210A mutation (1 also had protein S deficiency), 1 was TT MTHFR homozygous, and 2 showed the coexistence of the factor V Leiden and prothrombin mutations. Mean ages at the time of thromboembolic episodes were 27.8 ± 4.3 years (range 19-34 years) for deep venous thrombosis or pulmonary embolism in the absence of any mutation (n = 17), including a woman with antithrombin III deficiency and 1 case patient with primary antiphospholipid syndrome; 25.7 ± 2.5 years (range 23-28 years) for the factor V Leiden carriers (n = 3); 26.8 ± 8.8 years (range 16-35 years) for prothrombin G20210A carri-
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Table II. Prevalence of factor V Leiden and prothrombin G20210A mutations and of TT MTHFR genotype in case patients and control subjects Case
Factor V Leiden mutation Prothrombin G20210A mutation TT MTHFR genotype
Control
No.
%
No.
%
Odds ratio
95% Confidence interval
10/42 13/42 12/42
23.8 31.0 28.6
4/212* 9/213 34/213
1.9 4.2 16.0
16.3 10.2 2.1
4.8-54.9 4.0-25.9 1.0-4.5
*Untypability was seen for 1 control subject.
ers (n = 5), including a woman with protein S deficiency; and 28.6 ± 5.1 years (range 23-39 years) for patients with the TT MTHFR genotype (n = 8). Finally, in the presence of combined defects (n = 9), mean age at the time of the event was 28.1 ± 4.1 years (range 22-35 years), including 1 patient with primary antiphospholipid syndrome. These differences were not statistically significant (analysis of variance test F = 0.245, P = .91). The exclusion of the 4 women carrying other congenital or acquired defects did not affect the analysis (data not shown). Comment It has previously been reported that pregnancy and the postpartum period are associated with greatly increased risk for venous thromboembolism among women with an inherited deficiency of a naturally occurring anticoagulant.4 The prevalences of pregnancy-related deep venous thrombosis and pulmonary embolism in a low-risk obstetric population were 0.71 and 0.15 per 1000 deliveries, respectively.21 Several studies have been published on the occurrence of thrombosis during pregnancy and in the postpartum period. Friederich et al22 found a higher prevalence of deficiency in anticoagulant factor in a group of women whose pregnancies were complicated by venous thrombosis. In this setting the risk for venous thromboembolism was increased 8 times in women with deficiencies (antithrombin, protein C, or protein S) compared with women without deficiencies. In a larger sample with a different ethnic background, DizonTownson et al7 found a prevalence of deep venous thrombosis of 28% (4/14) among factor V Leiden carriers. Finally, a study conducted on 15 patients with both venous and arterial events8 showed a prevalence of 46.6% of factor V Leiden carriers in a sample of patients from different ethnic backgrounds. On the other hand, a large study found an increased incidence of fetal losses among carriers of thrombophilic defects.23 Moreover, an association between the factor V Leiden carrier status and the occurrence of complicated pregnancies (repeated or recurrent otherwise unexplained fetal losses and preeclampsia) has been reported.20, 24, 25 A weaker association between preeclampsia and the TT MTHFR genotype was also found in our previous study.24 In this study we selected a white population from
Southern Italy with documented deep venous thrombosis or pulmonary embolism and compared the frequency of 3 prothrombotic genetic risk factors (factor V Leiden, prothrombin G20210A, and MTHFR C677T polymorphisms) with a selected age-matched control sample formed of parous women with the same ethnic background. In this study the allele and genotype frequencies observed in control subjects for each polymorphism are comparable with those reported for other white populations.5, 9, 13 The estimated risk of venous thromboembolism in our sample was significantly higher in case patients carrying the factor V Leiden mutation than in control subjects. A similar figure was registered for the novel mutation G20210A in the prothrombin gene. A weaker but still significant association was found between deep venous thrombosis or pulmonary embolism and the TT MTHFR genotype, suggesting that this mutation is involved in the thromboembolism associated with the pregnant state. The overall incidence of the 3 mutations considered in our study (59.5%) suggests that these factors play a major role in the occurrence of deep venous thrombosis and pulmonary embolism associated with pregnancy. A family history of venous thromboembolism indicates a risk for the occurrence of venous thrombosis. Such a history represents an important selection factor when screening thrombophilic patients. In our population 8 of 42 women (19.1%) had such a family history, and 5 of these (62.5%) were carriers of ≥1 inherited defects predisposing them toward venous thromboembolism. However, lack of a family history of venous thromboembolism did not exclude women with genetic determinants of thrombophilia. Seventeen of the 25 case patients carrying inherited defects (68.0%) did not report having relatives with venous thrombosis. This may be because of the occurrence of a fresh mutation (which is uncommon) or because affected relatives were still free of symptoms. On the basis of these findings, most of the subjects who reported a family history of venous thromboembolism showed an inherited hypercoagulable state, although the ability of family history to predict venous thromboembolism appears quite low. These data further support findings from a previous study on the association between pregnancy-related venous thromboembolism and the factor V Leiden carrier
1328 Grandone et al
status. In addition, for the first time a significant relationship is now reported for 2 other genetic risk factors for pregnancy-related thromboembolism, carriage of the prothrombin G20210A allele and the MTHFR C677T homozygosity. It should be stressed that factor V, prothrombin, and MTHFR genes represent susceptibility loci, and that the carrier status of 1 of these gene variants is a condition neither necessary nor sufficient for the expression of the disease. In view of the fact that deficiencies of protein C, protein S, and antithrombin III are rarer than are factor V Leiden, prothrombin G20210A, and TT MTHFR carrier statuses, however, it would be worthwhile to screen for these mutations in patients with a personal or family history of deep venous thrombosis or pulmonary embolism in pregnancy or in the postpartum period.
November 1998 Am J Obstet Gynecol
11.
12.
13.
14.
15.
16. REFERENCES
1. Toglia MR, Weg JG. Venous thromboembolism during pregnancy. N Engl J Med 1996;335:108-14. 2. Greer IA. Haemostasis and thrombosis in pregnancy. In: Haemostasis and thrombosis in pregnancy. Bloom AL, Forbes CD, Thomas DP, Tuddenham EG, editors. Edinburgh: Churchill Livingstone; 1994. p. 987-1015. 3. Hellgren M, Blombäck M. Studies on blood coagulation and fibrinolysis in pregnancy, during delivery and in the puerperium. I. Normal condition. Gynecol Obstet Invest 1981;12:141-54. 4. Conard J, Horellou MH, Van Dreden P, Lecompte T, Samama M. Thrombosis and pregnancy in congenital deficiencies in ATIII, protein C or protein S: study of 78 women. Thromb Haemost 1990;63:319-20. 5. Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994;369:64-7. 6. Svennson PJ, Dahlback B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994;330:517-22. 7. Dizon-Townson DS, Nelson LM, Jang H, Varner MW, Ward K. The incidence of the factor V Leiden mutation in an obstetric population and its relationship to deep vein thrombosis. Am J Obstet Gynecol 1997;176:883-6. 8. Hallak M, Senderowicz J, Cassel A, Shapira C, Aghai E, Auslender R, et al. Activated protein C resistance (factor V Leiden) associated with thrombosis in pregnancy. Am J Obstet Gynecol 1997;176:889-93. 9. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3´-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996;88:3698-703. 10. Mandel H, Brenner B, Berant M, Rosenberg N, Lanir N, Jakobs
17.
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21. 22.
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25.
C, et al. Coexistence of hereditary homocystinuria and factor V Leiden: effect on thrombosis. N Engl J Med 1996;334:763-8. den Heijer M, Koster T, Blom HJ, Bos GM, Briêt E, Reitsma PH, et al. Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N Engl J Med 1996;334:759-62. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111-3. De Franchis R, Mancini FP, D’Angelo A, Sebastio G, Fermo I, De Stefano V, et al. Elevated total plasma homocysteine and C → T mutation of the 5,10-methylenetetrahydrofolate reductase gene in thrombotic vascular disease. Am J Hum Genet 1996;59:262-4. Arruda VR, von Zuben PM, Chiaparini LC, Annichino-Bizzacchi JM, Costa FF. The mutation Ala677 → Val in the methylene tetrahydrofolate reductase gene: a risk factor for arterial disease and venous thrombosis. Thromb Haemost 1997;77:818-21. Ma J, Stampfer MJ, Hennekens CH, Frosst P, Selhub J, Horsford J, et al. Methylenetetrahydrofolate reductase polymorphism, plasma folate, homocysteine, and risk of myocardial infarction in US physicians. Circulation 1996;94:2410-6. Cattaneo M, Tsai MY, Bucciarelli P, Taioli E, Zighetti ML, Bignell M, et al. A common mutation in the Methylenetetrahydrofolate reductase gene (C677T) increases the risk for deep-vein thrombosis in patients with mutant factor V (factor V:Q506). Arterioscler Thromb Vasc Biol 1997;17:1662-6. Ames PR, Tommasino C, D’Andrea G, Iannaccone L, Brancaccio V, Margaglione M. Thrombophilic genotypes in subjects with idiopathic antiphospholipid antibodies-prevalence and significance. Thromb Haemost 1998;79,46-9. Ames PR, Pyke S, Iannaccone L, Brancaccio V. Antiphospholipid antibodies, haemostatic variables and thrombosis: a survey of 144 patients. Thromb Haemost 1995;73:768-73. Koeleman BP, Reitsma PH, Allart CF, Bertina RM. Activated protein C resistance as an additional risk factor for thrombosis in protein C–deficient families. Blood 1994;84:1031-5. Grandone E, Margaglione M, Colaizzo D, d’Addedda M, Cappuci G, Vecchione G, et al. Factor V Leiden is associated with repeated and recurrent fetal losses. Thromb Haemost 1997;77:822-4. Mc Coll MD, Ramsay JE, Tait RC. Risk factors for pregnancy associated venous thromboembolism. Thromb Haemost 1997;78:1183-8. Friederich PW, Sanson BJ, Simioni P, Zanardi S, Huisman MV, Kindt I, et al. Frequency of pregnancy-related venous thromboembolism in anticoagulant factor–deficient women: implications for prophylaxis. Ann Intern Med 1996;125:955-60. Preston FE, Rosendaal FR, Walker ID, Briët E, Berntop E, Conard J, et al. Increased fetal loss in women with heritable thrombophilia. Lancet 1996;348:913-6. Grandone E, Margaglione M, Colaizzo D, Cappucci G, Paladini D, Martinelli P, et al. Factor V Leiden, C → T MTHFR polymorphism and genetic susceptibility to preeclampsia. Thromb Haemost 1997;77:1052-4. Dizon-Townson DS, Nelson LM, Easton K, Ward K. The factor V Leiden mutation may predispose women to severe preeclampsia. Am J Obstet Gynecol 1996;175:902-5.
Key words: 5,10-Methylenetetrahydrofolate reductase C677T polymorphisms, factor V Leiden, pregnancy-related venous thromboembolism, prothrombin G20210A Estimates of the incidence of pregnancy-related venous thromboembolism vary between 1 in 1000 to 1 in 2000 deliveries.1 Pregnancy is a prothrombotic state in which increases in coagulation factors I, VII, VIII, and X, a decrease in protein S, and an inhibition of fibrinolysis are well documented. These phenomena are likely to contribute to the increased risk (5 times) of venous thrombosis during this period.2, 3 For women who have an inher-
From Unità di Aterosclerosi e Trombosi, Casa Sollievo della Sofferenza, S. Giovanni Rotondo, Divisione di Ematologia, Unità di Coagulazione, Ospedale A. Cardarelli, and Istituto di Medicina Interna e Geriatria, Università di Palermo. Supported by Telethon-Italy (grant E C 561). Received for publication December 31, 1997; revised March 16, 1998; accepted May 11, 1998. Reprint requests: Elvira Grandone, MD, Unità di Aterosclerosi e Trombosi, IRCCS Casa Sollievo della Sofferenza, Viale Cappuccini, S. Giovanni Rotondo (FG), 71013 Italy. Copyright © 1998 by Mosby, Inc. 0002-9378/98 $5.00 + 0 6/1/91595
1324
ited deficiency of a naturally occurring anticoagulant (antithrombin, protein C, or protein S), it has been reported that pregnancy and the postpartum period are associated with greatly increased risk of venous thromboembolism.4 However, the studies that produced these data lacked appropriate control groups and included many patients because they were seen with venous thrombosis either associated with or unrelated to pregnancy. The resistance to activated protein C is an autosomal dominant trait caused by a mutation in coagulation factor V gene that consists of a substitution of guanine with adenine at nucleotide position 1691 (Fig 1).5 This mutation results in a replacement of arginine residue 506 with a glutamine at 1 of the factor V cleavage sites for activated protein C.5 The role of this mutation, named factor V Leiden, in the occurrence of deep venous thrombosis is well known.6 Moreover, a role has been recently reported for this mutation in the occurrence of deep venous thrombosis in obstetric populations.7, 8 A common ge-
Grandone et al 1325
Volume 179, Number 5 Am J Obstet Gynecol
netic variation (a guanine to adenine transition at position 20210) in the 3´-untranslated region of the prothrombin gene has been associated with an increased incidence of venous thrombosis.9 Individuals carrying this molecular variation have significantly higher prothrombin levels than do noncarriers. Regardless of sex and age, the relative risk of thrombosis associated with the G20210A allele has been estimated to be 2.7.9 On the other hand, there is recent evidence of the association among hereditary homocystinuria, factor V Leiden, and thrombosis.10 The prevalence of hyperhomocysteinemia in individuals with venous thrombosis has been found to be high, representing an independent risk factor for venous disease.11 A cause of moderate hyperhomocysteinemia is a relatively frequent mutation in the gene coding for 5,10methylenetetrahydrofolate reductase (MTHFR). The genetic defect is a cytosine to thymine missense mutation at nucleotide 677, which substitutes a valine for an alanine residue.12 The role of the TT genotype in arterial or venous thrombosis is still a matter of investigation.13-16 It has been suggested that this molecular variant may have a role only when associated with other risk factors for venous thrombosis, such as factor V Leiden16 or antiphosholipid antibodies.17 To evaluate the roles of the mutations described (prothrombin G20210A, factor V Leiden, and MTHFR TT genotype) as genetic risk factors predisposing toward pregnancy-related thromboembolism, we evaluated these risk factors in a population of white women from Southern Italy. Patients and methods Subjects. We performed a case-control study in 2 thrombosis centers at Hospital “Casa Sollievo della Sofferenza,” S. Giovanni Rotondo, and at A. Cardarelli Hospital, Naples. Between January 1995 and March 1998 we observed 42 women with a previous deep venous thrombosis during pregnancy or in the postpartum period. A group of 213 parous women without venous or arterial thromboses served as a control group. Both case patients and control subjects were white women living in Southern Italy. The 2 groups were comparable in social status and age. A complete clinical summary, with emphasis on personal and family history for thromboembolic disease, was obtained by a specially trained staff. Deep venous thrombosis was diagnosed by phlebography or ultrasonography; pulmonary embolism was diagnosed by angiography or ventilation-perfusion lung scan. Mean age of the case patients was 35.9 ± 12.6 years; that of the control subjects was 33.7 ± 7.1 years (P not significant). After approval was obtained from the local ethics committees, the study was carried out according to the Principles of the Declaration of Helsinki. Informed consent was obtained from all the subjects. Blood collection and coagulation tests. Blood samples
A
B Fig 1. Simplified scheme showing blood coagulation cascade and inhibitory effects (parallel lines) of antithrombin III and of protein C (PC) and protein S (PS) system on factor V (FV) wild type (Arg 506) (A) and on factor V Leiden (Gln 506) (B). Because the activated protein C cleaves factor V at the arginine 506 site, replacement at that site with glutamine leads to a resistance to protein C. FX, Factor X; FXa, activated factor X; FVa, activated factor V.
were collected in 3.8% trisodium citrate and centrifuged at 2000g for 15 minutes to obtain platelet-poor plasma, which was immediately frozen and stored in small aliquots at –70°C until tested. Antiphospholipid antibodies–lupus anticoagulant and immunoglobulin G anticardiolipin antibodies (enzyme-linked immunosorbent assay [ELISA]; Byk Gulden, Italy), antithrombin III, protein C, amydolytic and immunologic (Behring, Marburg, Germany) and total and free (ELISA; Diagnostica Stago, Asnières, France) protein S antigen were determined in all subjects, as reported elsewhere.17, 18 Clotting assays were performed on a KC4 Amelung coagulometer (Amelung, Austria). Interassay and intra-assay coefficients of all the variables never exceeded 8.0% and 5.0%, respectively. Deoxyribonucleic acid extraction and analysis. Leukocyte deoxyribonucleic acid was obtained from frozen blood by the use of standard techniques. A 220–base pair (bp) fragment of the factor V gene that included nucleotide 1691 was amplified by polymerase chain reaction as previously described,19 with some modifications.20 To identify the G20210A mutation of the prothrombin gene, a 345-bp fragment was obtained and digested with the HindIII endonuclease, according to
1326 Grandone et al
November 1998 Am J Obstet Gynecol
Table I. Prevalence of inherited prothrombotic defects and family history in entire group and according to time of event Factor V Leiden mutation
Venous thromboembolism during pregnancy (n = 9) Venous thrombolism after cesarean delivery (n = 9) Venous thromboembolism after spontaneous delivery (n = 24) All patients (n = 42) Family history (n = 8)
Prothrombin G20210A mutation
No.
%
No.
1
11.1
0
1
11.1
2*
1
4.2
3 0
7.1
%
TT MTHFR genotype
Combined defects
No.
%
No.
%
2
22.2
1
11.1
22.2
4
44.4
1
11.1
3
12.5
2
8.3
7†
29.2
5 2
11.9 25.0
8 1
19.1 12.5
9 2
21.4 25.0
*One woman also had a protein S deficiency. †One woman was affected by primary antiphospholipid syndrome.
Poort et al.9 A fragment of 198 bp containing the C677T polymorphism of MTHFR gene was generated as described elsewhere.12 Untypability for factor V Leiden occurred in 1 control subject. Statistical analysis. All the analyses were performed according to the Statistical Package for Social Science (SPSS 6.1 for Macintosh; SPSS Inc, Chicago, Ill). The significance of differences in means was evaluated by nonparametric tests, whereas the significance of any difference in proportions was tested by χ2 statistics. The significance of the difference of alleles and genotypes observed between the groups was tested with the χ2 analysis, after grouping homozygous and heterozygous carriers of the prothrombin G20210A allele and homozygous and heterozygous carriers of the MTHFR C677T allele. Odds ratios and 95% confidence intervals were calculated. Results As shown in Table I, all deep venous thromboses and pulmonary embolisms were observed in the postpartum period, except for 9 that occurred during pregnancy. Among these postpartum events, 9 (27.3%) occurred after a cesarean delivery. One patient carried antithrombin III deficiency and 1 carried protein S deficiency; in 2 other cases a primary antiphospholipid syndrome was present (Table I). No case patient had arterial or venous thrombosis outside the pregnancy or the postpartum period. Mean age at the time of the events was 27.7 ± 4.9 years (range 16-39 years). On the whole, 10 case patients (23.8%) and 4 control subjects (1.9%; odds ratio 16.3, 95% confidence interval 4.8-54.9) were heterozygous for the factor V Leiden (Table II). As far as the prothrombin G20210A allele was concerned, 9 control subjects (4.2%) were heterozygous. Thirteen case patients (31.0%; odds ratio 10.2, 95% confidence interval 4.0-25.9) carried this mutation (Table II). Of these 13, 2 were homozygous for the mutation and were analyzed with the heterozygous group. Twelve (28.6%) of the 42 case patients were homozygous for the
MTHFR C677T allele, whereas in the control group this number was 34 (16.0%; odds ratio 2.1, 95% confidence interval 1.0-4.5; Table II). The coexistence of >1 mutation was found in 9 case patients (21.4%; Table I). Of them, 6 women in whom an episode of deep venous thrombosis or pulmonary embolism occurred in the postpartum period (n = 5) or during pregnancy (n = 1) carried both factor V Leiden and prothrombin G20210A mutations. Two patients with thrombotic episodes occurring in postpartum and after a cesarean delivery, respectively, were carriers of the MTHFR C677T genotype and the prothrombin G20210A mutation. In 1 case patient, who had a deep venous thrombosis in the postpartum period, the MTHFR C677T genotype and the factor V Leiden mutation were found. Only 1 woman, who had an episode of deep venous thrombosis plus a cerebral venous thrombosis when she was 22 years old, showed all the gene variants. Among control subjects, only 1 carrier of both the MTHFR C677T genotype and the factor V Leiden mutation was observed (0.5%). Altogether, 25 case patients (59.5%) carried ≥1 mutation, compared with 47 of the healthy control subjects (22.2%; odds ratio 5.2, 95% confidence interval 4.9-19.6). A family history of deep venous thrombosis or pulmonary embolism was reported for 8 case patients (19.0%). Of these, 3 did not show any mutation, 2 were heterozygous for the prothrombin G20210A mutation (1 also had protein S deficiency), 1 was TT MTHFR homozygous, and 2 showed the coexistence of the factor V Leiden and prothrombin mutations. Mean ages at the time of thromboembolic episodes were 27.8 ± 4.3 years (range 19-34 years) for deep venous thrombosis or pulmonary embolism in the absence of any mutation (n = 17), including a woman with antithrombin III deficiency and 1 case patient with primary antiphospholipid syndrome; 25.7 ± 2.5 years (range 23-28 years) for the factor V Leiden carriers (n = 3); 26.8 ± 8.8 years (range 16-35 years) for prothrombin G20210A carri-
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Table II. Prevalence of factor V Leiden and prothrombin G20210A mutations and of TT MTHFR genotype in case patients and control subjects Case
Factor V Leiden mutation Prothrombin G20210A mutation TT MTHFR genotype
Control
No.
%
No.
%
Odds ratio
95% Confidence interval
10/42 13/42 12/42
23.8 31.0 28.6
4/212* 9/213 34/213
1.9 4.2 16.0
16.3 10.2 2.1
4.8-54.9 4.0-25.9 1.0-4.5
*Untypability was seen for 1 control subject.
ers (n = 5), including a woman with protein S deficiency; and 28.6 ± 5.1 years (range 23-39 years) for patients with the TT MTHFR genotype (n = 8). Finally, in the presence of combined defects (n = 9), mean age at the time of the event was 28.1 ± 4.1 years (range 22-35 years), including 1 patient with primary antiphospholipid syndrome. These differences were not statistically significant (analysis of variance test F = 0.245, P = .91). The exclusion of the 4 women carrying other congenital or acquired defects did not affect the analysis (data not shown). Comment It has previously been reported that pregnancy and the postpartum period are associated with greatly increased risk for venous thromboembolism among women with an inherited deficiency of a naturally occurring anticoagulant.4 The prevalences of pregnancy-related deep venous thrombosis and pulmonary embolism in a low-risk obstetric population were 0.71 and 0.15 per 1000 deliveries, respectively.21 Several studies have been published on the occurrence of thrombosis during pregnancy and in the postpartum period. Friederich et al22 found a higher prevalence of deficiency in anticoagulant factor in a group of women whose pregnancies were complicated by venous thrombosis. In this setting the risk for venous thromboembolism was increased 8 times in women with deficiencies (antithrombin, protein C, or protein S) compared with women without deficiencies. In a larger sample with a different ethnic background, DizonTownson et al7 found a prevalence of deep venous thrombosis of 28% (4/14) among factor V Leiden carriers. Finally, a study conducted on 15 patients with both venous and arterial events8 showed a prevalence of 46.6% of factor V Leiden carriers in a sample of patients from different ethnic backgrounds. On the other hand, a large study found an increased incidence of fetal losses among carriers of thrombophilic defects.23 Moreover, an association between the factor V Leiden carrier status and the occurrence of complicated pregnancies (repeated or recurrent otherwise unexplained fetal losses and preeclampsia) has been reported.20, 24, 25 A weaker association between preeclampsia and the TT MTHFR genotype was also found in our previous study.24 In this study we selected a white population from
Southern Italy with documented deep venous thrombosis or pulmonary embolism and compared the frequency of 3 prothrombotic genetic risk factors (factor V Leiden, prothrombin G20210A, and MTHFR C677T polymorphisms) with a selected age-matched control sample formed of parous women with the same ethnic background. In this study the allele and genotype frequencies observed in control subjects for each polymorphism are comparable with those reported for other white populations.5, 9, 13 The estimated risk of venous thromboembolism in our sample was significantly higher in case patients carrying the factor V Leiden mutation than in control subjects. A similar figure was registered for the novel mutation G20210A in the prothrombin gene. A weaker but still significant association was found between deep venous thrombosis or pulmonary embolism and the TT MTHFR genotype, suggesting that this mutation is involved in the thromboembolism associated with the pregnant state. The overall incidence of the 3 mutations considered in our study (59.5%) suggests that these factors play a major role in the occurrence of deep venous thrombosis and pulmonary embolism associated with pregnancy. A family history of venous thromboembolism indicates a risk for the occurrence of venous thrombosis. Such a history represents an important selection factor when screening thrombophilic patients. In our population 8 of 42 women (19.1%) had such a family history, and 5 of these (62.5%) were carriers of ≥1 inherited defects predisposing them toward venous thromboembolism. However, lack of a family history of venous thromboembolism did not exclude women with genetic determinants of thrombophilia. Seventeen of the 25 case patients carrying inherited defects (68.0%) did not report having relatives with venous thrombosis. This may be because of the occurrence of a fresh mutation (which is uncommon) or because affected relatives were still free of symptoms. On the basis of these findings, most of the subjects who reported a family history of venous thromboembolism showed an inherited hypercoagulable state, although the ability of family history to predict venous thromboembolism appears quite low. These data further support findings from a previous study on the association between pregnancy-related venous thromboembolism and the factor V Leiden carrier
1328 Grandone et al
status. In addition, for the first time a significant relationship is now reported for 2 other genetic risk factors for pregnancy-related thromboembolism, carriage of the prothrombin G20210A allele and the MTHFR C677T homozygosity. It should be stressed that factor V, prothrombin, and MTHFR genes represent susceptibility loci, and that the carrier status of 1 of these gene variants is a condition neither necessary nor sufficient for the expression of the disease. In view of the fact that deficiencies of protein C, protein S, and antithrombin III are rarer than are factor V Leiden, prothrombin G20210A, and TT MTHFR carrier statuses, however, it would be worthwhile to screen for these mutations in patients with a personal or family history of deep venous thrombosis or pulmonary embolism in pregnancy or in the postpartum period.
November 1998 Am J Obstet Gynecol
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