I CI =confidence interval

Transcription

1 Prothrombin G20210A Polymorphism and Thrombophilia ADRIAN NGUYEN, MD Recently, a single mutation in the 3'-untranslated region of the prothrombin gene was reported, resulting in a G-to-A substitution. This finding added to the growing list of genetic disorders thought to be responsible for familial thrombophilia. Although most studies generally agree about the increased risk of venous thrombosis in individuals carrying this mutation, its role in the first event of venous thromboembolism and in recurrent events is unclear. Even less clear is the role this mutation has in the formation of arterial thrombosis (including coronary artery disease and cerebral ischemia) due to contradicting results of studies. This mutation has important clinical implications since it is a common cause of genetic thrombophilia, second only to the factor V Leiden mutation. However, the mutation by itself may not be enough to trigger disease because thromboembolic disease is now generally accepted as a multifactorial disorder. Careful evaluation of this mutation will augment the clinician's ability to stratify systematically an individual's risk of developing spontaneous thrombosis. Mayo Clin Proc. 2000;75: I CI =confidence interval E ach year, venous thrombosis occurs in about 1 in 1000 persons in the United States, resulting in more than 50,000 deaths. I This disorder commonly manifests as deep venous thrombosis of the leg and varies sharply with age, from 1 per 100,000 children annually to almost 1 per 100 elderly persons annually. ' However, the incidence of venous thromboembolic events is difficult to estimate because even large studies have some degree of statistical uncertainty. In an article written almost 150 years ago, Virchow' postulated 3 main causes of thrombosis: stasis of the blood, changes in the vessel wall, and changes in the composition of the blood. This applies to thrombosis in both the arterial and the venous systems, but with regard to disorders causing familial thrombophilia, it is Virchow's third postulate that plays the primary role. (Acquired risk factors for thrombosis are well described and are not discussed in this article.) Patients with inherited thrombophilia typically present with deep venous thrombosis of the lower limbs and pulmonary embolus, similar to those who have an acquired disorder. However, patients with inherited disorders tend to have manifestation of the disease at an earlier age, have familial clustering of thrombosis, and exhibit a tendency for recurrent events. Reports have even described thrombosis occurring in atypical sites, such as the mesenteric vein or the cerebral vein, which has been attributed to a genetic predisposition for thrombosis. Since the 1960s, several genetic disorders that contribute to venous thrombosis have been characterized (Table 1). In 1965, Egeberg" described the first disorder, antithrombin deficiency. Inherited deficiency of protein C was described by Griffin et ap in Protein S deficiency associated with thrombotic disease was described by 2 groups in All 3 of these disorders are inherited in an autosomal dominant manner. In contrast to these 3 disorders, there are reports of several other genetic defects that are either extremely rare or not well established. Dysfibrinogenemia is caused by the generation of an abnormal fibrin resistant to fibrinolysis.' However, it is a highly uncommon cause of inherited thrombosis. A study in identified 1 patient with thrombomodulin deficiency and thrombosis, although an Italian study" of patients with thrombosis indicated that thrombomodulin deficiency due to this mutation is an uncommon cause of thrombosis. Anecdotal studies have reported thrombosis associated with heparin cofactor II deficiency,'" but a larger study concluded that cofactor II deficiency by itself may not be an inherited risk 'factor for thrombosis." During the past few years, however, we have seen an explosion of discoveries of inherited thrombophilia. Since the early 1990s, several groups have appreciated a link between hyperhomocysteinemia and thrombosis.p:'? In 1991, Kang et al'" reported a genetic disorder involving a cytosine-to-thymine substitution at nucleotide 677 of the methylenetetrahydrofolate reductase gene, resulting in a thermolabile variant. In 1994, Bertina et al" described factor V Leiden as the cause of resistance against activated protein C. This is due to a G-to-A transition in nucleo- From the Department of Internal Medicine. University of Texas. Southwestern Medical School. Dallas. Tex. Individual reprints of this article are not available. Address correspondence to Adrian Nguyen. MD. Department of Internal Medicine. University of Texas. Southwestern Medical School Harry Hines Blvd. Dallas. TX Mayo Clin Proc. 2000;75: Mayo Foundati on/or Medical Education and Resear ch

2 596 Prothrombin G20210APolymorphism and Thrombophilia Mayo Clin Proc, June 2000, Vol 75 Table 1. Inherited Thrombophilic Disorders Established Extremely rare Newly described Antithrombin deficiency Protein C deficiency Protein S deficiency Dysfibrinogenemia Thrombomodulin deficiency Heparin cofactorii deficiency Hyperhomocysteinernia Factor V Leiden Prothrombin G20210A tide 1691 of exon 10 of the factor V gene, which results in the substitution of arginine 506 by glutamine in 1 of 3 cleavage sites for activated protein C.1S Recently, Poort et ap6 described the prothrombin G20210A gene variant that is thought to be associated with an increased risk of thrombosis. Some disorders ofinherited thrombophilia do not have a substantial clinical effect in the general population (Table 2). Despite their high relative risk for thrombosis, deficiencies of protein C, protein S, and antithrombin are rare, which makes the true risk difficult to assess. Moreover, these genetic defects are caused by a large number of mutations; thus, DNA testing is infeasible. Although a high serum homocysteine level is common, most patients with this condition do not have inherited gene defects but rather have acquired causes due to deficiencies in folate, vitamin B 6, or vitamin B 12 The 2 most common inherited causes of hyperhomocysteinemia are homozygous defects in the cystathionine ~ - s y n tgene h a and s e the methylenetetrahydrofolate reductase gene, but their relative risk for thrombosis is minimal. The 2 most commoncauses of familial thrombophiliaare factor V Leiden and factor II G20210A. Factor V Leiden occurs in 5% of the general population16 and in about 20% of patients with venous thrombosis.'? Factor II G20210A occurs in about halfas many people as the factor V variant, and 6% of patients with a family history of venous thrombosis have the prothrombin mutation." About 32% of the causes of deep venous thrombosis can be attributed to known hereditary thrombophilia (Table 2). The rest of the cases are due to acquired risk factors or undiscovered genetic disorders. More causes of genetic thrombophilia will likely be discovered because familial clustering of venous thromboembolic events has been documented, even in the absence of any known genetic cause. PROTHROMBIN G20210A POLYMORPHISM Prothrombin, or factor II, is the precursorof thrombin. This protein is a vitamin K-dependent zymogen that is produced by the liver and has a central role in the conversion of fibrinogen into fibrin. Prothrombin also has other functions in the body, such as limiting the hemostatic process and initiating platelet aggregation. By sequencing the coding and flanking regions of the prothrombin gene systematically, Poort et al" found a single substitution of adenine for guanine at position in 18% of selected patients (5 of 28) with a personal and family history of venous thrombosis but in only I% in a group of 100 healthy controls. In a subsequent populationbased, case-control study, these investigators showed that 6.2% of 471 unselected consecutive patients with a first, objectively confirmed, episode of deep venous thrombosis carried the prothrombin G20210A allele compared with 2.3% of 474 control subjects matched for age and sex. This resulted in an odds ratio of 2.8 (95% confidence interval [CIl, ). These investigators also assessed prothrombin activity levels and found that a higher level of prothrombin activity itself was associated with venous thrombosis. Moreover, individuals with 1 copy of the G-to-A transition had a mean prothrombin level of 1.32 U/mL, whereas individuals with the GG genotype had a significantly lower mean prothrombin level of 1.05 UI ml. 16 Thus, the factor II polymorphism is thought to be associated with elevated levels of plasma prothrombin activity. This association between the plasma level of prothrombin and the G-to-A variant was confirmed in at least 2 other studies.f-" Recently, however, reports have challenged the theory that there is little, if any, correlation between plasma prothrombin levels and those harboring the gene mutation." Table 2. Prevalence ofinherited Risk Factors in Caucasians* Risk factor Factor V Leiden'? Prothrombin G2021OAi6 Protein C deficiency" Protein S deficiency" Antithrombin deficiency" Homocystinuria" *DVT = deep venous thrombosis. Prevalence in general population (%) :335,000 Prevalence in patients with DVT (%) <0.1 Relative risk

3 Mayo Clin Proc, June 2000, Vol 75 Prothrombin G20210APolymorphism and Thrombophilia 597 Table 3. Prevalence ofprothrombin G20210A in People of African Descent Region Washington, United States" Bahia, Brazil" Italy" Atlanta, Ga, United States" Benin, West Africa" Israel" Midwest, Southwest, and Pacific Northwest areas, United States" Total *All had sickle cell disease. Ethnicity African Americans Africans Somalians African Americans Africans Ethiopian Jews African Americans Prevalence 0/9 2/144 0/40 0/52 0/120* 0/147 1/185 3/697 (0.43%) GEOGRAPHIC DISTRIBUTION A recent review by Rosendaal et af5 combined data from II centers in 9 countries. Three of these centers included non-caucasians, whereas the rest were solely or mainly composed of Caucasians. The investigators examined a total of 5527 people and found that III were heterozygous carriers of the G20210A mutation. No homozygous individuals were found. Among these centers, the prevalence estimates varied from 0.7 to 4.0, and the overall prevalence was 2.3% when a weighted average was calculated. Among nonwhites, the factor II G20210Aheterozygous state is almost absent. When the data were pooled from different studies of black persons, including Africans, African Americans, and Israeli Ethiopian Jews, the prevalence was very low (Table 3). Of note, the 3 Brazilians with G20210A were from an area with many ethnic groups and hence interracial marriages." An analysis of people of Asian descent shows a similar pattern of low prevalence (Table 4). Like factor V Leiden, the G20210A gene variant is important only in white persons. Factor V Leiden is more common in northern Europe, but the prothrombin gene mutation is more common in southern Europe, particularly around the Mediterraneanarea (except for France). 30 Mediterranean countries such as Spain (6.5%),31 Italy (4.6%),32 Turkey (6.2%),33 and Greece (4.0%)22 have a relatively high prevalence, as does northern Africa (5.5%).34 In contrast, Iceland, a country settled by immigrants mostly from Ireland, Norway, and Scotland, has a very low prevalence (0.9% of healthy controls)." Recently, a study by Zivelin et ap4 found a large difference in the allele frequencies of 4 dimorphisms among several groups of Caucasians, indicating strong linkage disequilibrium and suggesting a founder effect. This supports the theory of a single founder (as opposed to recurrent mutational events) for factor II G20210A that probably occurred after the divergence of Africans from non-africans and of Caucasoid from Mongoloid subpopulations. VENOUS THROMBOSIS In the past several years, many groups from around the world have corroborated the initial observation made by Poort et al" between venous thrombosis and the variant allele (Table 5). In general, the patient groups consistently had a higher prevalence of the G20210A mutant gene than did the control groups. However, the patient groups are diverse with respect to their clinical presentation, number of events, and selection criteria. Moreover, each of the studies had different designs and goals. Table 4. Prevalence of Prothrombin G20210A in Healthy People of Asian Descent Region Taiwan" Washington, United States" Amazon region, Brazil" Italy" Milwaukee, Wis, United States, and Bangalore, India" Seoul, South Korea" Southwest region, United States" Japan" Total Ethnicity Taiwanese Native Americans Native Americans Asian Indians Asian Indians Koreans Native Americans Japanese Prevalence 0/149 0/20 0/83 0/41 1/89 0/94 0/87 0/210 1/773 (0.13%)

4 598 Prothrombin G20210A Polymorphism and Thrombophilia Mayo Clin Proc, June 2000, Vol 75 Table 5. Prevalence of Prothrombin G20210A in Venous Thrombotic Disease*t Clinical No. of Controls Patients Crude Reference presentation events Patient selection criteriat (%) (%) OR (CI) Brown et ap6 DVT,PE ~ l Consecutive blood samples from patients ( ) with a history of VTE (age, 9-89 y) Corral et ap7 DVT ~ l Consecutive patients referred to clinic (NR) (age, y) Cumming et ap8 DVT,PE ~ l Patients referred to clinic between 4/95-1/97, ( ) excluded if lupus anticoagulant or underlying malignant disease present (age, y) Ressner et ap9 DVT,PE ~ l Patients referred to clinic (enrollment period ( ) and age range not given) Hillarp et al 40 DVT ~ l Consecutive outpatients recruited from ( ) emergency department between 3/94-12/95 (age, y) Leroyer et apo DVT,PE ~ 1 Consecutive patients admitted between ( ) 7/94-8/97; those who developed VTE while in hospital were excluded (age, y) Margaglione DVT,PE Patients referred to clinic between 5/ ( ) et ap2 12/97, excluded if they had recurrent event (age, 3-81 y) Poort et ap6 DVT Unselected consecutive outpatients ( ) (age, y) Souto et api DVT,PE ~ 1 All patients referred to or who visited clinic ( ) between 10/96-11/97 (age, y) *CI = confidence interval; DVT = deep venous thrombosis; NR = not reported; OR = odds ratio; PE = pulmonary embolism; VTE = venous thromboembolism. tincludes only studies with predominantly Caucasian populations. tali patients had an objective diagnosis of DVT (by phlebography, compression ultrasonography, or contrast venography) or PE (by either ventilation-perfusion scan or pulmonary angiogram). The role of the G202lOA mutation in patients with only 1 thrombotic episode is unclear. A study by Souto et api showed that the G202lOA variant is likely a risk factor for venous thromboembolism, with an adjusted odds ratio slightly greater than 3, which in general concurs with most previous reports. However, when comparing patients after their first event with those who have recurrent events, differences were noted. In patients with only 1 episode of venous thrombosis, the prevalence of carriers was 7 of 62 (11.3%), with an adjusted odds ratio of 2.0, whereas in the group of patients with recurrent events, the prevalence of carriers was 13 of 54 (24.1%), with an adjusted odds ratio of Thus, this study failed to demonstrate that the G20210A variant is a risk factor among patients with only 1 thrombotic episode. Interpretation of this odds ratio as a relative risk should be done cautiously because it can be an overestimation due to bias. Based on these data, one could surmise that the role of G202lOA is stronger in recurrent thrombotic events. However, this issue is controversial because a recent report by Lindmarker et al" did not support this idea. These investigators followed up 534 patients for 48 months after their first episode of venous thromboembolism and found that the risk of recurrent events for carriers was not statistically different from noncarriers, with an odds ratio of 0.9 (95% CI, ). The caveat in interpreting these results is that the average risk for a second episode of venous thromboembolism in all patients who have had 1 event is approximately lo-fold higher than that in patients who carry the prothrombin gene G20210A polymorphism." Thus, other conditions have a more important role in the risk of recurrent events. These conditions likely represent interactions between genetic factors (known and unknown) and acquired conditions. One study that examined 116 unrelated patients with venous thromboembolism found no difference in age at first thrombotic event between G20210A carriers (n=20, mean age, 39 years; range, years) and noncarriers (n=96, mean age, 42 years; range, years)." Of the 96 noncarriers, 71 had normal findings on biological studies, whereas the remaining 25 had factor V Leiden, antiphospholipid antibody, moderate hyperhomocysteinemia, or protein S deficiency.

5 Mayo Clin Proc, J une 2000, Vol 75 Prothrombin G20210A Polymorphism and Thrombophilia 599 Table 6. Prevalence of Prothrombin G20210A in Arterial Disease* Age Controls Patients OR Reference Sex (y) (%) (%) (CI) Arruda et al 42 M,F /295 (0.7) 4nO (5.7) 8.4 ( ) Corral et ap7 M,F /101 (2.0) 4/101 (4.0) 2.0 ( ) Croft et a1 4J M,F <75 14/498 (2.8) 11/539 (2.0) 0.72 ( ) Doggen et al" M <70 8/646 ( 1.2) 10/560 (1.8) 1.5 ( ) Eikelboom et al" M,F /679 (3.2) 16/644 (2.5) 0.8 ( ) Ferraresi et ail2 7/176 (4.0) 3/90 (3.3) 0.77 ( ) Franco et al 46 M,F /400 (1.0) 7/173 (4.0) 4.2 ( ) Ridker et al" M /1774 (3.9) 12/404 (3.0) 0.8 ( ) Rosendaal et al" F /381 ( 1.6) 4/79 (5.1) 4.0 ( ) Watzke et al 49 M,F 2/102 (2.0) 5/98 (5.1) 2.7 ( ) *CI =confidence interval; OR =odds ratio. Ellipses indicate information was not provided. CORONARY ARTERY DISEASE Thrombin has regulatory function s that are important in the pathogenesis of arteriosclerosis, including platelet aggregation and endothelium activation. Thus, the recent demonstration that a single point mutation in the prothrombin gene is associated with increased prothrombin levels has generated considerable clinical interest in the role of the prothrombin G202 10A mutation and arterial thrombosis (Table 6). Some studies have shown that the mutation is a risk factor for coronary artery disease only in the presence of another known risk factor, where as other studies have concluded that no association exists. One of the first studies to investigate the role of the prothrombin G20210A gene variant in arterial thrombus examined women between the ages of 18 and 44 years with heart disease in a popu lation-based, case-control study." Rosendaal et al" compared this gene defect in a group of 79 women with early myocardial infarction vs 381 controls and found that the prevalence was 4 of 79 (5.1%) and 6 of 381 (1.6%), respectively. The odds ratio was 4.0 (95% CI, 1.1 to 15.1). These results did not change appreciably when the analyses were restricted to Caucasian women, premenopausal women, or women not taking oral contraceptives. Of note, among women who did not smoke or have a metabolic risk factor (obesity, hypertension, hyperlipidemia, or diabetes mellitus), the risk was not increased even in the presence of the gene variant. However, when the gene variant was present in addition to these acquired risk factors, the risk increased from 9-fold to 43-fold for smokers and from 5-fold to 34-fold for women who had other metabol ic risk factors." Myocardial infarcts are very rare in young women; thus, several risk factors must be present for infarcts to occur. Moreover, younger peop le tend to have clean coronary arteries, and therefore prothrombotic influences may be more evident in this group. Thus, these patients represent an excellent population for study of the effect of a new risk factor and the interaction between risk factors." Similarly, a study by Doggen et al" showed that, in the presence of traditional cardiac risk factors, the addition of the prothrombin mutation causes a synergistic effect on the risk of myocardial infarction. This study consisted of 560 men with a first myocardial infarction before age 70 years compared with 646 men who were matched by age. Patients who smoke and have metabolic risk factors in comb i nation with a coagulation defect have a higher risk of myocardial infarction compared with noncarriers with the same risk factors. For example, the odds ratio for smoking by itself was 3.3 (95% CI, ), but smoking combined with either a G20210A or factor V Leiden increased the odds ratio to 6.1 (95% CI, ). A similar synergistic effect was seen with metabolic risk factors." Opinions about the role of the G20210A gene defect in arterial thrombosis vary because the reports are conflicting. A study by Corral et ap7exam ined 101consecutive patients referred for myocardial infarction or unstable angina. These patients were compared with a group of age- and sex-matched control s, and the prevalence of the G20210A allele did not differin these 2 groups (P=.41). Furthermore, studies by Ferraresi et af2and Ridkeret al" showed that the frequency of carriers of the G2021OAallele in patients with coronary artery disease was similar to that observed among matched healthy contro l subjects. Of note, most studies on this topic have samples consisting of few participants. Therefore, the calculations were inevitably based on low numbers of mutations, with wide CIs for risk estimates. PROTHROMBIN G20210A AND CEREBRAL ISCHEMIA To date, there has not been a wealth of information on the role of G20210A in cerebral ischemia (Table 7). Three studies failed to demonstrate an association.p-"-" One of

6 600 Prothrombin G20210A Polymorphism and Thrombophilia Mayo Clin Proc, June 2000, Vol 75 Table7. Prevalence of Prothrombin G20210A in Cerebral Ischemia* Reference Country De Stefano et apo Martinelli et ap1 Ferraresi et af2 Corralet ap7 Italy Italy Italy Spain Controls Patients (%) (%) Crude OR (CI) 5/198 (2.5) 5/155 (3.2) 3/70 (4.3) 2/104 (1.9) 9/72 (l2.5)t 6/155 (3.9) 2/105 (1.9) 1/104 (1.0) 3.8 ( )t 1.2 ( ) 0.77 ( ) 0.50 (NR) *CI =confidence interval; NR =not reported; OR =oddsratio. ttwo patients werehomozygous. tadjusted OR for isolated heterozygous state. these was a retrospective case-control study comparing 155 consecutive patients who had a stroke or transient ischemic attack with 155 healthy subjects matched by age and sex." This study showed that the prevalence in the patient group, 6 of 155 (3.9%), was similar to that in the control group, 5 of 155 (3.2%). These results did not differ when the patients were adjusted for age. These data are in contrast with a study that investigated 72 young patients (age range, 2-50 years) without traditional metabolic risk factors such as diabetes, hypertension, and hyperlipidemia who had ischemic strokes." These individuals were compared with 198 controls. In the early stroke group, 7 heterozygotes (9.7%) and 2 homozygotes (2.8%) had the G20210A gene variant, whereas 2.5% of heterozygotes were found in the control group. This study showed that the risk associated with the heterozygous mutation was 3.8-fold, heterozygous plus homozygous was 5.5-fold, and homozygous only was 208-fold. so The seemingly opposing conclusions of these studies emphasize that further prospective studies, including large groups of unselected patients, are needed to clarify the true role of the prothrombin G20210A allele in cerebrovascular disease. HOMOZYGOUS PROTHROMBIN G20210A Only a few homozygous individuals have been described (Table 8) thus far. Assuming that the prevalence of heterozygotes in the general population is 2.3%,16 this leads to a mutant allele frequency of According to the Hardy-Weinberg equilibrium, the expected number of homozygous individuals in the normal population is 1.3 per 10,000. Not surprisingly, data pooled from 5527 healthy individuals recently detected no homozygous individuals." Poort et al" described a female patient from a 24-member family pedigree with thrombotic symptoms who had the homozygous genotype. However, this patient was also heterozygous for the factor V Leiden mutation, and the events surrounding her thrombotic state (surgery, oral contraception, etc) were not reported. In other studies that have reported homozygous carriers of the prothrombin mutation, the clinical manifestation varies substantially. An investigation by De Stefano et apo found 2 homozygous individuals among 72 young patients with ischemic stroke (2.8%). One was a 24-year-old man with an ischemic stroke in the thalamic region, and the other was a 26-year-old man with ischemic stroke involving the basilar artery. This strongly suggests a relationship between the factor II mutation and the thrombotic symptoms. In this study, the estimated risk associated with the homozygous genotype was exceedingly high at 208-fold, which is statistically significant but with a wide 95% CI ( ). In contrast, a report of 5 individuals with prothrombin G20210A in a 3-generation family showed that this homozygous state did not confer a high risk of thrombosis. 55 The mother of the proband was homozygous and despite 5 pregnancies, a state known to trigger thrombosis, was free of venous thromboembolism at the age of 74 years. Moreover, 3 of the 4 homozygous siblings had no thrombotic event despite several triggering conditions including pregnancy and surgery. The proband's father (not tested because he had died) had several episodes of deep venous thrombosis despite a high probability of being only heterozygous for the factor II mutation. Perhaps he had another risk factor, such as factor V Leiden or a yet undiscovered form of inherited thrombophilia. COSEGREGATION AND INTERACTION WITH OTHER RISK FACTORS Poort et al" studied 28 probands selected for hereditary thrombophilia and found that 5 (18%) carried the G20210A gene defect. Of these 5, 2 (40%) also carried the factor V Leiden mutation, suggesting an increased cosegregation frequency of heterozygosity for these 2 genetic variants in families with hereditary thrombophilia. This generated the impetus for other investigators to confirm the reports (Table 9). Recently, Makris et af3 compared the prevalence of the G20210A allele in 101 unrelated subjects with a history of venous thromboembolism to establish whether the additional inheritance of the G20210A allele is associated with an increased risk of venous thromboembolism. These re-

7 Mayo Clin Proc, June 2000, Vol 75 Prothrombin G20210A Polymorphism and Thrombophilia 601 Table 8. Persons With Homozygous Prothrombin G20210A Reported to Date Reference Poort et al" Scott et al" Ridker et al" Howard et ap3 De Stefano et apo Souto et ap4 Margaglione et ap2 Morange et a155 Clinical presentation Woman with thrombosis and heterozygous for factor V Leiden mutation 18-year-old woman with thrombosis during pregnancy Asymptomatic man in control group, age not given Patient with massive thrombosis Two men, mid-20s, with ischemic strokes Two asymptomatic relatives, ages 51 and 19 years Five patients with first episode of venous thromboembolism 44-year-old man with spontaneous deep venous thrombosis, 4 asymptomatic family members despite several acquired risk factors searchers found that 5% of patients carried both factor V Leiden and the factor II variant, which is much higher than the expected population frequency of 0.1%. However, a French study sought an association of the 2 risk factors in 288 individuals belonging to 26 families.57 The factor V mutation was present in 151 subjects, and 66 had thrombosis; however, none of the probands or family members had the factor II gene mutation. The researchers also analyzed 400 apparently healthy controls and found the G20210A mutation in 2.8%, a frequency that is comparable to the Dutch population studied by Poort et a1. 16 However, some investigators have criticized this study because of the low number of kindred (N=26),58 a low number of symptomatic patients," and bias in selection of patients. Except for the French study, the weight of evidence suggests that the factor II G2021O,A allele is frequently coinherited in carriers of the factor V Leiden mutation with thrombophilia. This concurrence raises the issue of whether both mutant alleles could have the same distribution in the Caucasian population. However, aforementioned population studies showed that the factor II polymorphism is more predominant around the Mediterranean area, whereas the factor V mutation occurs more frequently in northern Europe. Taken together, the data suggest that about 0.6% to 5.0% of symptomatic carriers of the factor V Leiden mutation also carry the prothrombin gene. The published data should be interpreted carefully since family studies will result in skewed data, and the expected frequency of carriers of both DNA alterations depends on whether the population was selected or unselected for venous thromboembolism. Why these 2 defects are coinherited more frequently than expected is unknown. Additionally, it is unclear whether the increased risk of recurrence of thrombosis among heterozygous carriers of factor V Leiden and the G20210A prothrombin mutation is due to an interaction between the 2 mutations or simply due to the presence of the mutant prothrombin gene." Recent studies that failed to find an association between the prothrombin mutation and an increased risk of recurrent events do not support the latter possibility.">' No study has shown that the G20210A gene variant is coinherited with any other known inherited thrombophilic disorder. However, this is difficult to interpret because many of the studies analyzed cosegregation of G20210A and factor V Leiden and did not examine all known inherited thrombophilic defects in the control group. Many investigators now believe that the G20210A genetic defect alone is usually not sufficient for the development of venous thromboembolism. Margaglione et ap2 ana- Table 9. Coinheritance of Factor V Leiden and Factor II G20210A* No. of Factor V Factor II Both Reference patients (%) (%) (%) Makris et af (50.0) 8 (7.9) 5 (5.0) Margaglione et ap (13.2) 26 (9.3) 14 (5.0) De Stefano et al" (20.7) NR 17 (2.7) Leroyer et apo (11.2) 12 (3.3) 5 (1.4) Brown et ap (15.9) 25 (5.0) 3 (0.6) Alhenc-Gelas et ap (52.4) 0(0.0) 0(0.0) *NR = not reported.

8 602 Mayo elin Proc, June 2000, Vol 75 Prothrombin G20210A Polymorphism and Thrombophilia Iyzed this issue by examining 281 patients referred to 2 thrombosis centers in San Giovanni Rotondo and Naples, Italy. This group was divided into patients with the G20210A mutant who had no additional risk factors and into those with the G20210A mutant who had additional risk factors. The additional risk factors were defined as protein C deficiency (4), protein S deficiency (2), antiphospholipid antibodies (32), or circumstantial risk factors (surgery, immobilization, pregnancy, postpartum period, trauma, oral contraceptive use, varicose veins, and cancer)." The comparison showed that the risk of venous thrombosis associated with prothrombin G20210A approached significance only in the subset of patients with additional risk factors (odds ratio, 3.58 and 1.86 in groups with and without additional risk factors, respectively)." Like other genetic thrombophilic disorders, the 0202 loa variant often needs the presence of additional acquired or inherited risk factors to manifest disease. However, asse ssing the relative contribution of each risk factor is difficult because thrombophilia is a multifactorial disorder. SCREENING AND MANAGEMENT Clinicians may be tempted to screen for the prothrombin G20210A mutation since the mutation is monogenic and has a high prevalence in the general Caucasian population. However, more data are needed on rates of primary thrombosis and recurrence rates of thromboembolism in affected individuals to determine the predictive value of the prothrombin G2021 OA mutation.l" Until this information is available, the low risk of thrombosis associated with this mutation does not necessarily justify its detection as part of the routine investigation of patients with an episode of venous thromboembolism." Because evidence has linked a synergistic effect between the presence of prothrombin A and acquired risk factors, some investigators have suggested that individuals who smoke or use oral contraceptives should be screened. Although the interactions between the prothrombin mutation and these acquired risk factors increase the risk of thrombotic events by manyfold, the absolute incidence is still low. Screening of individuals with these preexisting conditions for the presence of prothrombin G20210A is not recommended because it is costly and will not likely change the long-term management of venous thromboembolism. Screening of asymptomatic females with no family history of venous thromboembolism who are considering oral contraceptives is not recommended because this approach may deny thousands of women access to a safe and effective method of contraception.s Similarly, women who are taking estrogen for hormone replacement therapy have a 2fold to 3-fold risk of venous thromboembolism, but the absolute risk remains 10w.60 Since the protective effect of estrogen in preventing coronary artery disease is greater. than the risk of estrogen contributing to venous thrombosis, replacement therapy should not be denied to women who have the mutation. Thus, women in this group who have no history of thrombotic events should not be screened. However, there are certain subgroups that may benefit from testing. For example, a woman of childbearing age with a personal history of venous thromboembolism who is considering oral contraceptives can be advised of the increased risk of venous thrombosis. Screening individuals who have a clear family history of thrombophilia may be useful for identifying those who may benefit from antithrombotic prophylaxis during exposure to transient risk factors. Some investigators have suggested that individuals with a double heterozygous mutation for factor V Leiden and prothrombin OA have a higher risk of recurrent thrombosis and thu s need to receive lifelong anticoagulation. " However, the cost-effectiveness of this approach has yet to be proved, and this is likely impractical considering the risk of bleeding associated with anticoagulation. The management of acute thrombosis in the setting of the G2021 OA mutation should be similar to that for venous thromboembolism due to other causes. Moreover, primary prophylaxis in individuals with the prothrombin A mutation does not differ from that of other conditions of inherited thrombophilia. Lifelong prophylaxis should not be implemented because of the cost and risk of lifelong anticoagulation. The exception would be during conditions of prothrombotic states, such as surgery and childbirth. The duration of oral anticoagulation after an event was discussed in a recent prospective multicenter trial that followed up 492 patients with venous thromboembolism for 24±16 months after discontinuation of oral anticoagulation."' At 24 months, the probability of recurrence was 8% in patients with the mutation and 12.2% in patients without the mutation." Since the risk of early recurrent venous thromboembolism is not higher in patients with the A mutation than in those without the mutation, routine long-term secondary thromboprophylaxis with oral anticoagulants in heterozygous carriers of the mutant prothrombin allele is not justified. Lifelong anticoagulation in this group should be individualized based the patient's age, coexisting prothrombotic trait s, and site of the first thrombotic event. SUMMARY Based on the available data, several conclusions can be made about the prothrombin OAallele. Like factor V Leiden, the mutation is found mostly in Caucasians, with a prevalence between 2% and 3%. In other ethnic groups and races, the mutation frequency is much lower. It can be

9 Mayo Clin Proc, June 2000, Vo] 75 coinherited with factor V Leiden even though it does not share the same geographic distribution among white persons. Although it is the second most common cause of inherited thrombophilia, it is only a mild risk factor for venous thrombosis. With regard to coronary artery disease, it is at best expressed only in the presence of traditional risk factors. Unfortunately, its contribution to cerebral ischemia remains controversial, and more studies are needed. Most studies of this prothrombin polymorphism have been based on data collected from Caucasian populations, and the number of individuals tested has been limited; hence, the large CIs in the data. Although several issues about the management of patients with the prothrombin G202l OA mutation await further studies, the current recommendation does not differ substantially from that of other conditions of inherited thrombophilia because thrombophilia is a multifactorial disease that usually manifests due to the interaction of genetic disorders with acquired risk factors. As the understanding of the hemostatic and fibrinolytic system grows, discoveries of novel disorders at the genetic level will enable clinicians to assess rationally an individual's risk of thrombosis. REFERENCES II Van Cott EM, Laposata M. Laboratory evaluation of hypercoagulable states. Hematol Oncol Clin North Am. 1998;12: Rosendaal FR. Thrombosis in the young: epidemiology and risk factors: a focus on venous thrombosis. Thromb Haemost. 1997;78: 1-6. Virchow RLK. Phlogose und Thrombose im Gefassytem. In: Gesammelte Abhandlungen zur wissenschaftliche Medicin. 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