The mutation in the prothrombin gene was discovered

Transcription

1 Clinical and Laboratory Management of the Prothrombin G20210A Mutation Ronald C. McGlennen, MD; Nigel S. Key, MB, FRCP Objective. To make recommendations regarding the appropriate evaluation for the prothrombin G20210A mutation, as reflected by published evidence and the consensus opinion of recognized experts in the field. Data Sources. Review of the medical literature, primarily since Data Extraction and Synthesis. After an initial assessment of the literature, key points defining the condition, and review of the clinical study design, a draft manuscript was prepared and circulated to every participant in the College of American Pathologists Conference on Diagnostic Issues in Thrombophilia before the meeting. Each of the key points and associated recommendations were then presented for discussion at the conference. Recommendations were accepted if a consensus of 70% of experts attending the conference was reached. The results of the discussion were used to revise the manuscript into its final form. Conclusions. Consensus was reached on several recommendations concerning the criteria for testing for the prothrombin G20210A mutation and for the method of testing. First, a major point of consensus was that the prothrombin G20210A mutation is a significant risk factor for venous thromboembolism (VTE) and that testing should be considered in the initial evaluation of suspected inherited thrombophilia. Second, although several analytic methods are commonly used for genetic testing for the prothrombin mutation, all are generally robust and reliable. The recommendations for testing for the prothrombin mutation parallel those for the factor V Leiden mutation and include patients with a history of recurrent VTE, a first episode of VTE before the age of 50 years, a history of an unprovoked VTE at any age, thromboses in unusual anatomic sites, or an affected first-degree relative with VTE. A history of VTE related to pregnancy or estrogen use and unexplained pregnancy loss during the second or third trimesters were also considered to be indications for testing. Other scenarios remain controversial or not recommended, including general population screening. (Arch Pathol Lab Med. 2002;126: ) The mutation in the prothrombin gene was discovered in 1996 by Poort et al 1 after the prothrombin gene had been identified as a candidate gene for venous thrombosis in families with a history of venous thromboembolism (VTE). The mutation is due to a G to A transition at base pair in the 3 untranslated region of the prothrombin gene. Haplotype analysis suggests that the mutation arose from a single founder to years ago. 2 In Europe, the prevalence of the G20210A mutation is 2.0% (95% confidence interval [CI], 1.4% 2.6%) overall, with a range of 0.7% to 4.0%. The highest prevalence appears to be in southern European regions (approximately 3%), with the lowest prevalence in the northern parts of the continent (approximately 1.7%). In the United States, the overall prevalence estimates are between 1% and 2%, although this is highly dependent on race. The mutation is uncommon in African Americans Accepted for publication June 11, From the Department of Laboratory Medicine and Pathology (Dr McGlennen) and Department of Internal Medicine, Division of Hematology, Oncology, and Transplantation (Dr Key), University of Minnesota Medical School, Minneapolis. Presented at the College of American Pathologists Consensus Conference XXXVI: Diagnostic Issues in Thrombophilia, Atlanta, Ga, November 9 11, Reprints: Ronald C. McGlennen, MD, MMC Box 609, 420 Delaware St SE, Minneapolis, MN ( mcgle001@tc.umn.edu). (approximately 0.2%) and is also rarely seen in Asians and native Americans. 3 The survival advantage conferred by this polymorphism can only be hypothesized but presumably relates to a decreased risk of death from hemorrhage during childbirth or after traumatic injury. PATHOPHYSIOLOGY In the first description of the mutation, heterozygous carriers were found to have significantly higher mean plasma prothrombin levels (1.32 U/mL; range, U/mL) compared with individuals who were homozygous for the GG (wild-type) genotype (1.05 U/mL; range, U/mL). Mutant A/A homozygotes demonstrate factor II activity levels of about 1.70 U/mL. Because the odds ratio (OR) for VTE increased with increasing plasma prothrombin levels, it was surmised that hyperprothrombinemia was the pathophysiologic mechanism underlying the thrombotic propensity. This hypothesis is supported by a recent study 4 of families with the G20210A mutation, in which a close linkage between a quantitative trait locus determining plasma prothrombin levels and the G20210A mutation was demonstrated. Furthermore, other studies 2 have failed to uncover alternative polymorphisms in the promoter and coding region of the prothrombin gene that might be in linkage equilibrium with the G20210A mutation. A recent report 5 demonstrates that the G A mutation causes a gain of function due to increased recognition of the 3 end cleav- Arch Pathol Lab Med Vol 126, November 2002 Prothrombin G20210A Mutation McGlennen & Key 1319

2 Table 1. Thrombotic Risks Associated With the Prothrombin G20210A Mutation and Related Genetic and (Patho)physiologic Factors* Risk Relative Risk References VTE risk for prothrombin mutation alone VTE risk in combination with factor V Leiden VTE risk with use of OCPs Cerebral vein thrombosis with use of OCPs VTE risk with pregnancy MI risk for young women ( 50 y) MI risk for young women smokers MI risk in postmenopausal hypertensive women MI risk in postmenopausal hypertensive women undergoing HRT 2- to 3-fold 20-fold 16-fold 149-fold 15-fold 4-fold 43-fold 4-fold 11-fold * VTE indicates venous thromboembolism; OCPs, oral contraceptives; MI, myocardial infarction; and HRT, hormone replacement therapy. 1, age signal and increased 3 end processing. The net result is accumulation of messenger RNA and increased protein synthesis for prothrombin. The hypothesis that elevated prothrombin levels are crucial in the etiology of VTE is supported by studies by Butenas and colleagues. 6 Using an in vitro model of tissue factor initiated blood coagulation, it was shown that elevation of prothrombin levels to 150% of normal (while maintaining 100% levels of all other procoagulant and anticoagulant factors) resulted in significantly increased thrombin generation. In contrast, similar supplementation with factors V, VIII, IX, or X to a level of 150% had no such effect on thrombin generation. Similarly, Kyrle and colleagues 7 found increased thrombin generation after initiation of coagulation in the plasma of heterozygous and homozygous G20210A patients. Others have shown that high prothrombin levels may inhibit activated protein C mediated inactivation of factor Va, which would also result in enhanced thrombin generation after initiation of coagulation. 8 CLINICAL CONDITIONS VTE Risk With Prothrombin G20210A Mutation Numerous case-control studies have more or less consistently demonstrated an association between the prothrombin G20210A mutation and VTE (Table 1). Poort et al 1 reported that the 20210A allele increases the risk of VTE by almost 3-fold (OR, 2.8; 95% CI, ). Since that time, several other studies 9 have estimated a relative risk of VTE between 2 and 12, with the majority in the 2 to 3 range. As yet, there are only limited data from prospective studies confirming this association. In one of the few available, the US Physicians Health Study, a nonsignificant association of the A allele with VTE was found (OR, 1.7; 95% CI, ). 10 Despite this, most evidence to date suggests that the prothrombin mutation is a genuine, albeit relatively weak, risk factor for VTE. In keeping with this, homozygosity for the 20210A allele, although also associated with VTE, is clearly not as significant a risk factor for thrombosis as is homozygosity for proteins C or S or probably even the factor V Leiden mutation. 11 However, more data are needed before definitive conclusions can be drawn. VTE Risk When Prothrombin 20210A Cosegregates With Other Forms of Thrombophilia Significant attention has been paid to the concept that although the prothrombin 20210A allele on its own may be a relatively weak risk factor for VTE, it may interact with other known risk factors to significantly enhance the risk of VTE. Foremost among the interactions that have been examined is that with the factor V Leiden mutation. Several case-control studies have demonstrated that the prothrombin 20210A allele increases the risk of thrombosis when coinherited with the factor V 506Q allele. Importantly, coinheritance of these 2 mutations may also increase the risk of recurrent VTE after a first event, providing a compelling reason to consider simultaneous testing for both mutations in symptomatic individuals (see Specific Recommendations section). Although the risk of thrombosis in subjects with the prothrombin 20210A allele is reportedly increased by coinheritance of moderate hyperhomocyst(e)inemia and/or homozygosity for the C677T variant of the methylene tetrahydrofolate reductase (MTHFR) gene in some studies, 15,16 other studies 17,18 have been unable to confirm this association. The association between hyperhomocyst(e)inemia and VTE, which has been questioned by recent prospective studies (see article by Key and McGlennen in this issue, pp ), suggests that cosegregation of hyperhomocyst(e)inemia with prothrombin G20210A is unlikely to enhance the thrombotic propensity. Finally, whether coinheritance of prothrombin G20210A with heterozygous deficiency of antithrombin, protein C, or protein S leads to an enhanced risk of thrombosis, as seems to be the case for factor V Leiden, is more difficult to study because of the rarity of these disorders. Although an enhancing effect of cosegregation has been reported, 19 other studies, 20 for example, in a large, well-characterized protein C deficient kindred of French Canadian descent, have failed to confirm an enhanced risk of thrombosis in individuals with both mutations. Reproductive Issues Associated With the Prothrombin G20210A Mutation As with the factor V Leiden mutation, the risk of VTE in women during pregnancy or oral contraceptive (OCP) therapy appears to be increased in the presence of the prothrombin G20210A mutation. 21 Martinelli and colleagues 22 reported that the relative risk of deep vein thrombosis was increased 16.3-fold (95% CI, ) in women with prothrombin G20210A who used OCPs compared with noncarriers and nonusers. This relative risk was indicative of a multiplicative effect of the risks associated with OCPs and the prothrombin mutation. Interestingly, the same group reported a 149-fold increased risk of cerebral vein thrombosis (95% CI, ) in women with the prothrombin mutation taking OCPs. 23 The risk of 1320 Arch Pathol Lab Med Vol 126, November 2002 Prothrombin G20210A Mutation McGlennen & Key

3 Table 2. Measurement of plasma factor II activity Testing Methods in the Evaluation of the Prothrombin G20210A Mutation* Method Comment References Genetic detection of the prothrombin G20210A mutation PCR with restriction endonuclease digestion Simultaneous amplification of normal and mutant alleles by PCR Automated fluorescence detection Invader assay Multiplexed PCR-based assay DNA microarray * PCR indicates polymerase chain reaction. Standardized; potentially insensitive to diagnosis due to physiologic variation of plasma levels of factor II Most common method, requires no automated instrumentation; robust and reliable Potential for cost saving in labor and turnaround time but harbors technical artifacts that make the results less reliable Simple and robust; useful for large batch analysis; expensive equipment and validation process Simple to perform; occasional artifact with the technology; interpretation requires expertise Useful for screening panels or needs for higher throughput; technically difficult, particularly for routine use In-house based assay potentially as good as commercial types; requires specialized knowledge and equipment; demonstrated utility for multiplex genetic analysis at lower cost , , VTE appears to be highest during the first 6 to 12 months of OCP therapy in these individuals. 24 Pregnancy and the puerperium are both associated with an increased risk of VTE, with incidences that are approximately 8 and 25 times, respectively, the risk in nonpregnant women of the same age. 25 The relative risk of pregnancy and puerperal VTE in women with the G20210A prothrombin mutation was 15.2 (95% CI, ), and with both the 20210A allele and the 506Q factor V allele, the estimated OR increased to Recently, there has been considerable interest in the role that congenital and acquired thrombophilias may play in stillbirth and second-trimester pregnancy loss, abruptio placentae, intrauterine growth restriction, and preeclampsia. 27 These events are presumed to be sequelae of compromised placental perfusion. Nearly all published casecontrol studies demonstrate an association of one or more of these outcomes with factor V Leiden and/or the prothrombin 20210A allele. Finally, postmenopausal hormone replacement therapy (HRT) increases the risk of VTE by 2- to 4-fold It seems likely that this risk is increased further in women with the prothrombin G20210A mutation. This risk may offset some of the beneficial effect expected from HRT in women with atherosclerotic coronary artery disease undergoing this therapy. 35 Prothrombin 20210A as a Possible Risk Factor for Arterial Thrombotic Disease Following rupture of an atherosclerotic plaque, acute thrombosis ensues. Although the thrombus in this site is classically held to be platelet rich, the efficacy of heparins and thrombin inhibitors in acute coronary syndromes proves that it is clearly also dependent on activation of the plasma coagulation system. Therefore, it is certainly conceivable that, like other forms of thrombophilia, the prothrombin G20210A mutation might play a role in arterial thrombosis. 36 Although the prothrombin mutation has generally not been associated with myocardial infarction (MI) or stroke in older populations, several studies 11,37 39 suggest that it might be important in premature atherosclerotic disease, especially when MI is the outcome. However, not all studies 40,41 agree with this conclusion. A general theme of those studies 37,42 with a positive association is a significant interaction of the mutation with other risk factors for atherosclerotic disease, such as smoking and hypertension. When this situation arises in postmenopausal women, HRT may also contribute to an increased risk of MI. 42,43 TEST METHODS In consideration of the strong association of the nucleotide substitution G20210A with significant elevation of plasma prothrombin levels, any discussion regarding methods to detect this abnormality is highly focused on genetic testing (Table 2). A brief review of those methods follows. MOLECULAR GENETIC METHODS Robust methods for the genetic detection of the prothrombin alleles are well documented. Most literature citations describe the use of the polymerase chain reaction (PCR) to first amplify a segment of the 3 untranslated region of the factor II gene surrounding the polymorphism, followed by the detection of the nucleotide sequence(s) by methods of gel electrophoresis, radioisotopic probing, or following restriction endonuclease digestion (Table 2). Each of these methods provides an accurate and reliable test result. The choice of one or another, however, is typically based on the experience of the testing laboratory and the availability of the readout equipment. The prothrombin mutation positioned at nucleotide substitutes the nucleotide adenosine for guanosine. This single nucleotide change permits the introduction of an oligonucleotide primer containing the recognition site Arch Pathol Lab Med Vol 126, November 2002 Prothrombin G20210A Mutation McGlennen & Key 1321

4 for the restriction endonuclease HindIII. The strategy to incorporate this restriction site into the downstream PCR primer was first used by Poort and colleagues. 1 The assay permits the simultaneous amplification of the normal and mutant alleles that are discriminated by digestion with HindIII. The resulting DNA products are separated by gel electrophoresis, where the mutant A allele is identified as a shorter DNA fragment. 1 This method is commonly used in many molecular genetic laboratories across the United States and has been shown to be highly reliable in comparative interlaboratory proficiency surveys. Attempts to simplify the PCR-based strategy to detect the G20210A sequence focused on the development of methods that permit the simultaneous amplification of both alleles using competitive and noncompetitive downstream primers. These methods, referred to by a number of names, including allele-specific PCR, amplification resistant mutation system, simultaneous allele-specific amplification, and others, eliminate the step of using a restriction digestion of the PCR product before gel electrophoresis Using various strategies of single nucleotide mismatches within the downstream primer, the primer may anneal to either the normal or mutant allele. 48 Eliminating the restriction digestion steps after PCR improves the assay throughput and hence the cost of the test. However, it has been suggested that such 1-step methods are less reliable for day-to-day clinical application. One problem is the fact that long-term storage of primer stocks lead to a tendency of the sense and antisense oligonucleotides to multimerize, and their subsequent use in the PCR reaction leads to differential amplification of the mutant or wild-type alleles. This has been reported to result in erroneous interpretation of results. The advent of automated analysis systems has led to adaptation of the various PCR-based approaches to these instruments. Automated fluorescence detection, whether by slab gel or capillary electrophoresis, is achieved by fluorochrome labeling of the 5 end of one of the oligonucleotide primers, which in turn is size separated on the instrument. 49,50 The implied automation refers to the detection of the fluorochrome-labeled product by using a laser to scan the separation media and to record the position of the fluorescent peaks. Computerized algorithms that assess whether one or both alleles are present permit automated interpretation of the genotype. 51 Automated genotyping is one advantage of some of the newer gene chemical techniques, including the Invader assay for prothrombin and other markers of thrombophilic risk. The Invader technology is based on the generation of a fluorescent signal in the reaction solution following the cleavage of a synthetic oligonucleotide probe assembled with a so-called Invader probe and the DNA template that contains either the normal or mutation nucleotide. The thermostable enzyme Cleavase recognizes the formation of a 3-stranded complex at the position of the mutation and cleaves a flap of DNA noncomplemetary to the complex. The reaction takes place in a microtiter well, and the signal is detected in a fluorescent plate reader. 52 The resulting numerical data are compared to a set of positive and negative controls and the genotypes determined from the mathematical ratios of the sample and control signals. Genotyping is essentially automatic. Although this method is reliable, the points of vulnerability include reliance on higher amounts of template DNA, occasional problems with high background fluorescence from the no template control, and batch-to-batch variation of the fluorescent-labeled oligo probe reagent. The recognition that the prothrombin mutation may often coexist with other thrombotic risk factors such as factor V Leiden has inspired several investigators to developed multiplexed assays. The assembly of multiple primers in solution for the simultaneous amplification of the prothrombin, factor V Leiden, and MTHFR sequences has been reported for both manual and automated detection techniques In some reports, multiplexed PCR is combined with restriction endonuclease digestion or allelespecific amplification. 45,54,56,57 In other cases, the newer techniques of either TaqMan or the Lightcycler are used in a real-time fluorescent detection format. 51,58,59 In each of these reports, the comparison of the new techniques with the so-called gold standard of PCR followed by restriction endonuclease digestion resulted in perfect or near-perfect concordance. Hence, it is difficult to ascribe that one method is better than any other aside from the availability of such technologies in individual laboratories. Importantly, there is no study that compares the cost-effectiveness of these techniques. The cost of gene testing for prothrombin G20210A or any other markers of thrombophilia must be considered. Recent improvements in DNA microarray technology have provided the opportunity to perform assays combining multiple genetic markers at a cost per test well below that of the more conventional coagulation assays. Several commercial platforms are available that combine factor V Leiden, prothrombin G20210A, and the MTHFR C677T mutation as well as others. The basic differences between the various DNA chip platforms relate to the method by which the probes are affixed to the surface of the chip. The application of newer gene amplification strategies, such as strand displacement and ligase detection reaction, and the improvement in the in-house manufacture of DNA microarray chips are proving to be a viable alternative to the purchase of expensive commercial chip technologies for some larger clinical laboratories. 60 So-called low-density arrays that combine up to 10 to 20 independent gene markers have the advantage of combining the variety of genetic mutations associated with a clinical syndrome into a single test. One version of such a low-density array is based on the use of combined PCR and ligase detection reaction using primer modified on one end with a heterologous DNA sequence anchor called a zip code. 61 The DNA chip is configured with a series of geographically distributed probes composed of the complements to the ligase-derived zip codes. The advantage of using zip code probes for the hybridization steps is that each probe is designed to optimally anneal at the identical wash and temperature conditions. The result is a simple-to-read and straightforward genotype result for several related markers produced for less cost. TEST APPLICATIONS In Primary Prevention Although about half the episodes of VTE in carriers of the prothrombin G20210A mutation will occur in association with surgery, trauma, prolonged immobilization, or pregnancy or estrogen therapy, routine population screening before these events is not recommended. However, in an individual with a family history positive for VTE in at least one other family member, screening may be reason Arch Pathol Lab Med Vol 126, November 2002 Prothrombin G20210A Mutation McGlennen & Key

5 able if it is likely to alter the management of that event. For example, finding the mutation may lead to administration of short-term VTE prophylaxis in a situation where it would otherwise not be routinely used. Regarding this scenario, standard practice in the United States is to administer VTE prophylaxis after total hip or knee arthroplasty; therefore, in the absence of any data indicating that the duration of prophylaxis needs to be longer in individuals with thrombophilic risk factors, there is no apparent value to screening. However, in certain other surgical settings, for example, hysterectomy, VTE prophylaxis is not routinely used, and discovery of a thrombophilic mutation may tip the balance in favor of administration of prophylaxis. However, the impact of primary prophylaxis in asymptomatic family members has not yet been formally studied. Whether the small absolute increase in risk of VTE in young women who are carriers of the prothrombin mutation should result in withholding of OCP therapy (with the attendant increase in unwanted pregnancies) is a more contentious issue that, unfortunately, may be more influenced by fears of medicolegal action in the United States rather than a reasoned approach based on the medical evidence. 21 The annual incidence of thrombosis in first- and seconddegree relatives of probands with VTE has been estimated to be 0.13% (95% CI, ) for the prothrombin mutation, 0.19% (95% CI, ) for the factor V Leiden mutation, and 0.42% (95% CI, ) for those with both mutations. This compares to an annual incidence of 0.066% (95% CI, ) for noncarriers. 62 It is important to appreciate that this 2-fold risk of VTE in carriers of the prothrombin mutation, or even the 6-fold risk in a doubly heterozygous individual, does not merit the hemorrhagic risks associated with lifelong prophylaxis with oral anticoagulants in the absence of a prior history of VTE. It is recommended that screening of asymptomatic relatives be performed only in individuals from young adulthood and beyond. Prepubertal children have an extremely low risk of developing VTE, even in higher-risk situations. In Secondary Prevention In VTE, the finding of one or more thrombophilic defects, with the possible exception of antiphospholipid antibodies, does not imply that a higher intensity of initial heparin or warfarin therapy is needed in the treatment of that thrombotic episode. Therefore, this is generally not a valid reason to initiate screening. On the other hand, discovery of thrombophilic risk factor(s) may influence the duration of therapy. Much of the published evidence indicates that the prothrombin mutation as a solitary finding does not predict a higher risk of recurrent VTE and would not therefore indicate a need for more prolonged anticoagulation after a first event On the other hand, coinheritance of the prothrombin G20210A and factor V Leiden mutations appears to predispose to a higher rate of recurrence in a symptomatic individual and would likely indicate the need for prolonged, if not lifelong, therapy. 66,67 Therefore, once the decision to test has been reached, it is probably unwise to separate testing for the prothrombin G20210A and factor V Leiden mutations, since it is the combination of the 2 together that most clearly affects clinical decision making. Although the chance of finding the mutation in thrombophilic patients (those with a positive family history, early onset of VTE) is the most significant (approximately 18%), approximately 6% of unselected patients with a first episode of DVT can be expected to be positive for the mutation. 1 Arguably, therefore, it is probably not appropriate to set rigorous patient selection criteria (age, presence of family history, site of clot) in deciding which individuals to screen. Testing for the prothrombin G20210A mutation in young individuals with MI may be appropriate, especially if other risk factors, such as smoking, hypertension, or HRT, are applicable. However, other than providing genetic counseling advice to asymptomatic relatives, it is not clear whether discovery of the mutation would affect the patient s future therapy or prognosis with respect to a second event. Rather, treatment should focus on detection and management of primary risk factors for atherosclerosis, such as dyslipidemias and hypertension, and behavioral factors, such as smoking. Apart from the desire to provide an explanation for adverse events during pregnancy that may be associated with thrombophilic disorders, it is unclear whether testing contributes to the clinical management of these patients. There are a number of obstetricians who choose to treat women with thrombophilia and a history of adverse events during pregnancy with prophylactic anticoagulation during subsequent pregnancies based on preliminary data indicating favorable outcomes. 68 However, this strategy has not yet been subjected to rigorous prospective randomized clinical trials. CRITERIA FOR DIAGNOSIS The laboratory criteria for the diagnosis of the prothrombin G20210A mutation are based on the result of DNA-based testing. An individual is either normal for both alleles (both G nucleotides), heterozygous (one abnormal copy of the A allele), or homozygous mutant (both alleles hold the A nucleotide). Since the assay is DNA based, the actual timing of testing is relatively unimportant and may be performed at any time in relation to the thrombotic event. CONCLUSIONS There is currently no evidence that the short-term therapeutic management of VTE events (length or strength of anticoagulation) should be different in patients with prothrombin G20210A. We hypothesized that the primary advantages of prothrombin G20210A testing would be the identification of high-risk patients who could benefit from either long-term anticoagulant therapy or aggressive prophylaxis in temporary periods of high thrombotic risk. Other direct clinical benefits of prothrombin G20210A testing would include the opportunity to detect female probands and at-risk family members for whom future decisions as to antithrombotic prophylaxis, OCP use, HRT, or management of pregnancy complications could depend on prothrombin G20210A carrier status. Definitive, direct DNA-based methods to detect the prothrombin G20210A mutation are available in clinical diagnostic laboratories by any of several different in-house developed (non Food and Drug Administration approved) methods. These direct mutation assays, when appropriately validated in a licensed clinical laboratory, are Arch Pathol Lab Med Vol 126, November 2002 Prothrombin G20210A Mutation McGlennen & Key 1323

6 extremely accurate and precise for the detection of prothrombin G20210A. SPECIFIC RECOMMENDATIONS Prothrombin G20210A testing is recommended in patient populations with a mutation prevalence above that of the normal population, such as those with VTE events and a clinical suspicion of thrombophilia based on any of the following criteria. 1. Prothrombin G20210A testing is recommended in patients with the following: a history of recurrent VTE, Level 2 9 a first VTE at younger than 50 years, Level 1 1,9 a first unprovoked VTE at any age, Level 1 1,9 a first VTE at an unusual anatomic site, such as the cerebral, mesenteric, portal, or hepatic veins, Level 2 23 a first VTE at any age in a patient with a first-degree family member with a VTE before the age of 50 years, Level 1 1,9 a first VTE related to pregnancy, the puerperium, or OCP use, each Level ,26 a first VTE related to HRT, Level 3 women with unexplained pregnancy loss during second or third trimester. Level Testing for prothrombin G20210A is controversial in the following: young women smokers (age 50 years) with an MI, Level 2 37,38 older patients (age 50 years) with a first provoked VTE event in the absence of cancer or an intravascular device, Level 3 a first VTE related to serum estrogen receptor modifier or tamoifen, Level 3 selected cases of women with unexplained severe preeclampsia, placental abruption, or intrauterine growth restriction. Level 3 3. After appropriate counseling, testing for prothrombin G20210A also may be indicated in asymptomatic adult family members of probands with known prothrombin G20210A mutations, especially those with a strong family history of thrombosis at a young age, Level 2 62 asymptomatic female family members who are pregnant or are considering OCPs or pregnancy. Level Prothrombin G20210A testing is NOT recommended in the following: as a general population screen, Level 1 10 as a routine initial test during pregnancy, Level 2 21 as a routine initial test before or during OCP use (Level 2), 21 HRT, or serum estrogen receptor modifier therapy (Level 3), as a prenatal test, newborn initial test, or routine test in asymptomatic prepubescent children, Level 2 as a routine initial test in patients with arterial thrombotic events. Level 1 10 For an explanation of the levels cited in this article, refer to Olson. 69 References 1. 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: Zivelin A, Rosenberg N, Faier S, et al. A single genetic origin for the common prothrombotic G20210A polymorphism in the prothrombin gene. Blood. 1998;92: Dilley A, Austin H, Hooper WC, et al. Prevalence of the prothrombin G-to-A variant in blacks: infants, patients with venous thrombosis, patients with myocardial infarction, and control subjects. J Lab Clin Med. 1998;132: Soria JM, Almasy L, Souto JC, et al. Linkage analysis demonstrates that the prothrombin G20210A mutation jointly influences plasma prothrombin levels and risk of thrombosis. Blood. 2000;95: Gehring NH, Frede U, Neu-Yilik G, et al. Increased efficiency of mrna 3 end formation: a new genetic mechanism contributing to hereditary thrombophilia. Nat Genet. 2001;28: Butenas S, van t Veer C, Mann KG. Normal thrombin generation. Blood. 1999;94: Kyrle PA, Mannhalter C, Beguin S, et al. Clinical studies and thrombin generation in patients homozygous or heterozygous for the G20210A mutation in the prothrombin gene. Arterioscler Thromb Vasc Biol. 1998;18: Smirnov MD, Safa O, Esmon NL, Esmon CT. Inhibition of activated protein C anticoagulant activity by prothrombin. Blood. 1999;94: Girolami A, Simioni P, Scarano L, Carraro G. Prothrombin and the prothrombin G to A polymorphism: their relationship with hypercoagulability and thrombosis. Blood Rev. 1999;13: Ridker PM, Hennekens CH, Miletich JP. G20210A mutation in prothrombin gene and risk of myocardial infarction, stroke, and venous thrombosis in a large cohort of US men. Circulation. 1999;99: Girolami A, Simioni P, Girolami B, Scarano L. G to A prothrombin polymorphism and venous thrombosis: simple association or causal relationship? Clin Appl Thromb Hemost. 2000;6: Zoller B, Svensson PJ, Dahlback B, Hillarp A. The A20210 allele of the prothrombin gene is frequently associated with the factor V Arg 506 to Gln mutation but not with protein S deficiency in thrombophilic families. Blood. 1998; 91: Salomon O, Steinberg DM, Zivelin A, et al. Single and combined prothrombotic factors in patients with idiopathic venous thromboembolism: prevalence and risk assessment. Arterioscler Thromb Vasc Biol. 1999;19: Emmerich J, Rosendaal FR, Cattaneo M, et al. Combined effect of Factor V Leiden and prothrombin 20210A on the risk of venous thromboembolism. Thromb Haemost. 2001;86: De Stefano V, Zappacosta B, Persichilli S, et al. Prevalence of mild hyperhomocysteinaemia and association with thrombophilic genotypes (factor V Leiden and prothrombin G20210A) in Italian patients with venous thromboembolic disease. Br J Haematol. 1999;106: Tosetto A, Rodeghiero F, Martinelli I, et al. Additional genetic risk factors for venous thromboembolism in carriers of the factor V Leiden mutation. Br J Haematol. 1998;103: Alhenc-Gelas M, Le Cam-Duchez V, Emmerich J, et al. The A20210 allele of the prothrombin gene is not frequently associated with the factor V Arg 506 to Gln mutation in thrombophilic families [letter]. Blood. 1997;90: Brown K, Luddinton R, Baglin T. Effect of the MTHFRC677T variant on risk of venous thromboembolism: interaction with factor V Leiden and prothrombin (F2G20210A) mutations. Br J Haematol. 1998;103: Makris M, Preston FE, Beauchamp NJ, et al. Co-inheritance of the 20210A allele of the prothrombin gene increases the risk of thrombosis in subjects with familial thrombophilia. Thromb Haemost. 1997;78: Bovill EG, Hasstedt SJ, Callas PW, et al. The G20210A prothrombin polymorphism is not associated with increased thromboembolic risk in a large protein C deficient kindred. Thromb Haemost. 2000;83: Vandenbroucke JP, Rosing J, Bloemenkamp KW, et al. Oral contraceptives and the risk of venous thrombosis. N Engl J Med. 2001;344: Martinelli I, Taioli E, Bucciarelli P, Akhavan S, Mannucci PM. Interaction between the G20210A mutation of the prothrombin gene and oral contraceptive use in deep vein thrombosis. Arterioscler Thromb Vasc Biol. 1999;19: Martinelli I, Sacchi E, Landi G, Taioli E, Duca F, Mannucci PM. 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