Testing for genetic predisposition to venous thrombosis

Testing for genetic predisposition to venous thrombosis By Marisa B. Marques, M.D. CONTINUING EDUCATION To earn CEUs, see test on page 20. LEARNING OBJECTIVES Upon completion of this article the reader will be able to: 1. Describe the inherited genetic defects associated with thrombosis. 2. Define the mechanisms and clinical manifestations for hypercoagulability. 3. Describe testing used to evaluate hypercoagulable states. 4. Define the laboratorian s role in providing guidance to physicians for cost-effective testing for patients with thrombosis. CE test published through an educational grant from 8 January 2002 MLO www.mlo-online.com

VENOUS THROMBOSIS When the German pathologist Rudolf Virchow postulated more than a century ago that venous thrombosis occurred as a consequence of a triad of factors: stasis of the blood, vessel injury, and/or hypercoagulability, he was unaware of genetic predisposing conditions associated with the latter. Amazingly, his postulate is still true, and several discoveries of the last decade have continued to confirm his observations. This discussion will focus on the genetic abnormalities that can be detected in the laboratory and predispose a patient to develop venous thrombosis. From the start, it is important to emphasize that, when put in perspective, many more patients have acquired rather than inherited risk factors for thrombosis. Furthermore, the former are often stronger than the genetic conditions. It is the combination of genetic and acquired, temporary or permanent, thrombotic risk factors that increases a person s chance to develop thrombotic clinical manifestations. 1 Understanding of synergistic effect of risk factors underlies the need for a complete evaluation, both clinical and in the laboratory. Overview of the discoveries Until 1993, only three genetic defects that predisposed to venous thromboembolism had been identified. These were deficiencies of antithrombin (1965) 2, protein C (1981) 3, or protein S (1984). 4,5 Combined, these phenotypes have been found in only 7.9 percent of unselected patients with venous thrombosis. 6 In 1993, a group of Swedish scientists published a newly discovered hypercoagulability condition that was named Activated Protein C Resistance or APCR. 7 A year later, the underlying defect leading to the phenotypic manifestation of APCR was described as a mutation in the coagulation factor V gene. This was named Factor V Leiden 8, which was found in as much as 10 percent of the population of some European countries, and in 5 percent of Caucasians in the United States. 9,10 Since 1995, high plasma level of factor VIII has also been known to predispose to thrombosis. 11,12 Although its genetic basis has not been confirmed, it is most probably an inherited condition. In 1996, a point mutation in the prothrombin gene that is associated with hypercoagulability was described by Poort, et al. 13 With the ability to test thrombosis patients for the presence of so many new risk factors, many more cases of spontaneous thrombosis can now be at least partially explained. However, since the prevalence of these abnormalities is much higher in Caucasians than in people of any other ethnic background, many events remain unexplained. 14-16 Amid so much information, there is great opportunity for laboratory personnel to elucidate which tests are appropriate for individual patients and when in their clinical presentation they should be performed. Mechanisms of hypercoagulability The mechanisms involved in thrombosis development induced by genetic risk factors discovered so far can be summarized in two groups: 6 1) Decreased control of thrombin generation: activated protein C resistance (due to Factor V Leiden or other causes), prothrombin mutation (20210), elevated factor VIII level, and deficiencies of proteins C and S; or 2) Decreased neutralization of thrombin: antithrombin deficiency. These conditions can also be classified based on their prevalence as seen in Table 1. Clinical manifestations suggestive of venous hypercoagulability There is no difference between the clinical manifestation of venous thrombosis in patients who have inherited versus acquired predisposing conditions. Deep vein thrombosis, especially of the lower extremities, is the most common manifestation of venous thrombophilia. However, the degree of suspicion for a genetic tendency for thrombosis increases when: 1) The thrombotic episode appears to be spontaneous ; 2) There is a history of recurrent episodes; 3) There is a family history of venous thrombosis; 4) The event occurs in a patient younger than 45-50 years of age; or 5) The thrombosis occurs in an unusual site. Am J Hum Genet 63:569-576, 1998 Table 1: Genetic causes of venous thrombosis Common Activated protein C resistance (APCR)/ Factor V Leiden Prothrombin 20210 mutation Methylenetetrahydrofolate reductase gene mutation Increased factor VIII activity Rare Deficiencies of antithrombin and proteins C and S Very Rare Dysfibrinogenemia Risk factor assessment versus diagnostic test An important distinction between the majority of the tests offered by a clinical laboratory and those for assessment of hypercoagulability is the purpose of the information. Results of thrombophilia tests aid in the determination of prognosis Continues on page 10 www.mlo-online.com MLO January 2002 9

COVER STORY Table 2 Acquired conditions affecting functional hypercoagulability test results This color-flow Doppler ultrasound shows a vein with a noncompressible filling defect (arrow) consistent with deep-vein thrombosis. for long-term planning rather than for modification of current patient management. Although much has been discussed in terms of clinical significance of the presence of genetic hypercoagulability defects, the jury is still out. The odds ratio for the development of thrombosis varies amongst the different conditions discovered so far. 16 Protein S deficiency www.medscape.com Antithrombin APCR Factor VIII Homocysteine Protein C Protein S Decreased in heparin use, recent thrombosis, liver disease, DIC 1, nephrotic syndrome First generation assays: High factor VIII level, LA 2, acute phase response Second generation assays: Strong LA 2, pregnancy Elevated in acute phase reaction; falsely decreased in the presence of LA 2 Elevated in recent thrombosis, B12, folate, and B6 deficiencies, renal failure, hypothyroidism, medications such as methotrexate, phenytoin, or theophyline, malignancy, pregnancy, menopause Decreased in warfarin therapy, recent thrombosis, surgery, DIC 1, liver disease, vitamin K deficiency, or L-asparaginase therapy; Decreased in warfarin therapy, pregnancy, oral contraceptives or hormone replacement therapy, acute phase reaction, liver dysfunction, proteinuria, vitamin K deficiency, active thrombosis, DIC 1, recent surgery, or L- asparaginase therapy 1 Disseminated intravascular coagulation 2 Lupus anticoagulant 10 January 2002 MLO www.mlo-online.com

VENOUS THROMBOSIS confers the highest relative risk, 10 17, followed by Factor V Leiden 18 and protein C deficiency 19 at 7, antithrombin 20, 5, and the prothombin mutation, 13 with a ratio of 2. For the most part, asymptomatic people are not evaluated unless there are circumstances that make the information critical for the prevention of complications. An example of that would be the relative of a Factor V Leiden carrier who is subjected to acquired prothrombotic situations such as pregnancy or prolonged immobilization. In these individuals, the knowledge of a genetic thrombophilic state will most likely affect management and followup. Another reason for testing would be to predict risk of thrombosis recurrence. Independent of the demonstration of a known hypercoagulability risk factor, however, all patients who have had venous thrombosis are prone to have a recurrent event. 6 Furthermore, patients who have antithrombin, protein C or protein S deficiencies, multiple inherited risk factors, or homozygosity for Factor V Leiden, were found to have increased risk of recurrence compared with patients without them. 21-24 Regarding factor VIII, Kyrle has demonstrated that patients with levels at or above the 90 th percentile are at increased risk of recurrence than those below the 90 th percentile. 25 Based on these findings, patients who harbor one or more of these risks are candidates for more prolonged anticoagulation following a thrombotic event. However, the decision to proceed is dependent on a number of other clinical factors that affect the risk-benefit ratio of long-term anticoagulation. The uniqueness of risk factor assessment rather than diagnostic testing affects the way a laboratory operates. Turnaround time is secondary in this arena and should be accommodated to drive costs and labor down. When offering any of the tests described here, it is perfectly reasonable to let physicians know that the assays are batched and performed only a few times a week depending on demand. In our institution, since we started this policy, we have had significant savings each year. How to test for hypercoagulability defects Even when physicians know which conditions to investigate in their thrombosis patients, the laboratory should still provide guidance. The main reasons for the laboratory involvement include the large number of factors that affect the different assays (Table 2) as well as the importance of cost-effective testing. An excellent set of algorithms prepared by Laposata and Van Cott was recently published in CAP Today. 26 The following paragraphs will describe the consensus regarding which tests to order, combined with our experience in saving resources in the performance of these assays. Continues on page 12 Circle 10 for more information. www.mlo-online.com MLO January 2002 11

COVER STORY Antithrombin and proteins C and S deficiencies The initial evaluations for these states are functional assays available from various manufacturers. Normal results rule out deficiencies of these natural anticoagulants and preclude the need for further testing. Abnormal results have to be investigated thoroughly since deficiencies can be secondary to current medical conditions or therapeutic agents such as warfarin and heparin (Table 2). If no explanation exists for the low functional level of these proteins, their plasma concentration should be measured in an immunologic assay. In type I deficiency, both antigen and activity are proportionally low, and in type II deficiency, there is normal antigen but decreased activity (qualitative defect). Protein S deficiency can also be of type III, in which free protein S antigen is low but total protein S antigen is normal. Laboratories performing these assays can also save considerable resources by running the samples in singles rather than in duplicate, providing that validation of this practice is completed and documented. Our policy includes repeating the determinations only in those samples that yield results below the reference range. Table 3: Appropriate tests for the investigation of venous thrombosis Patients on heparin, warfarin or recent thrombosis (< 7 days): Anticardiolipin antibodies APC Resistance (functional test for Factor V Leiden) Prothrombin 20210 mutation Patients NOT on anticoagulation > 10 days: Anticardiolipin antibodies Lupus anticoagulant APC Resistance (functional test for Factor V Leiden) Prothrombin 20210 mutation Factor VIII Protein C Protein S Antithrombin Homocysteine Activated protein C resistance and Factor V Leiden The screening test for Factor V Leiden is a modification of the functional assay described in 1993 by Dahlbäck et al. 7 The name describes the phenotype conferred by the mutation in factor V, activated protein C resistance (APCR), and it is available as first and second generation assays. In the first generation assay, the patient s plasma is mixed with a contact activator and Ca ++ with or without activated protein C (APC), and the time to clot is determined. In normal individuals, clotting should be at least twice as prolonged in the reaction containing APC compared with that without APC (APCR ratio > 2.0). In this assay system, however, high factor VIII activity, lupus anticoagulant, or factor deficiencies (such as induced by warfarin) can produce an abnormally low APCR ratio in addition to Factor V Leiden. The second generation assay was developed to be more specific for Factor V Leiden by diluting the patient s plasma 1:5 in Factor V-deficient plasma prior to the addition of the reagents. The modified assay also contains polybrene to inactivate heparin. Abnormal APCR ratios in the second generation assay are almost diagnostic of Factor V Leiden and a genetic test for the demonstration of the mutation should be performed to check for zygosity (homozygous versus heterozygous carriers). This determination can be done by polymerase chain reaction (PCR), or the Invader or the microarray technologies. In our experience, strong lupus anticoagulants and pregnancy may still lead to falsely low APCR ratios. A year ago, we adopted a policy of performing the APCR assay as a screening step in all samples with an order for Factor V Leiden. Only those samples that have an abnormally low ratio are referred for genetic testing. Based on the number of orders and the costs of the two tests, we calculate monthly savings of $2,467on average, compared with what it would cost to perform only genetic tests. This is a legitimate comparison because data gathered before we started this intervention showed that more than 90 percent of the orders were for Factor V Leiden instead of APCR and that only 5 percent of the tests were abnormal. Of interest, we have seen exceptional acceptance of this policy by our staff physicians, an example of cost-effectiveness that everyone understands and agrees to. Prothrombin 20210 Since there is no screening test for this mutation, whenever clinically indicated it should be investigated by molecular techniques. Of note, carriers have increased prothrombin activity, but this finding is not specific or sensitive enough to be used as a diagnostic tool. 13 Increased factor VIII activity The presence of this hypercoagulability risk factor is assessed by a functional factor VIII assay classically performed in the evaluation and/or management of hemophilia. Although this test is readily available in many clinical laboratories, it is not to be ordered indiscriminately and the result should be interpreted with caution. This is because factor VIII, as an acute phase reactant, increases in many physiologic and pathologic 12 January 2002 MLO www.mlo-online.com

VENOUS THROMBOSIS conditions (Table 3). For this reason, assessment of its baseline value may be difficult. If the result is high, it is advisable that it be measured again in different circumstances to check for persistence of the elevation. An alternative approach is to correlate the factor VIII level with that of C-reactive protein, fibrinogen, or erythrocyte sedimentation rate as surrogate markers of acute phase response. Despite much discussion, consensus has not been reached on how to interpret high factor VIII activity. Hyperhomocysteinemia and the C677T mutation in the Methylenetetrahydrofolate reductase (MTHFR) gene We recommend measuring a fasting homocysteine level as the first test in the investigation of abnormalities of this pathway. If the level is elevated, it could be the result of several other causes besides the genetic alteration (Table 2). Furthermore, since the treatment for hyperhomocysteinemia is the same independent of its cause, we do not recommend the genetic test. For the determination of plasma levels of homocysteine, the specimen has to be centrifuged within one hour of collection to avoid artificially elevated level following leakage from blood cells. Dysfibrinogenemia Qualitative abnormalities of fibrinogen can be a risk factor for thrombosis as well as bleeding. This condition can be inherited or acquired, the latter being commonly associated with endstage liver disease. Independent of the cause, dysfibrinogenemia is diagnosed by determination of the plasma concentration of fibrinogen and a functional test such as thrombin or reptilase time. When there is a discrepancy between antigen and activity of fibrinogen, dysfibrinogenemia is diagnosed. How can the laboratory aid physicians in hypercoagulability testing? In the computer system used by our physicians to place laboratory orders, we created links to lists of tests for thrombophilia investigation. Two lists describe the appropriate tests for a patient with a current thrombotic event and/or warfarin or heparin treatment, or for an asymptomatic patient who is also off anticoagulants (Table 3). In the former situation, only a few tests will yield interpretable and meaningful results. In someone with a remote personal or family history of thrombosis, the complete workup is indicated. Physicians can access the lists in the computer screen where the coagulation laboratory tests are displayed, in a separate section designated Hypercoagulability/Thrombosis. Alternatively, the lists can be found in the alphabetical list of tests, under the letters H for hypercoagulability or T for thrombosis. Physicians have to then choose the tests they want from the lists, which are only guides to proper laboratory utilization. Since the implementation of this system, we have seen improvement in the appropriateness of testing for thrombophilia in our hospital. In summary, genetic hypercoagulability risk testing is a dynamic area where discoveries are fast accumulating, offering the laboratory a great opportunity to enhance physician awareness and education. l Dr. Marques is a member of the Department of Pathology, University of Alabama at Birmingham, Birmingham, Al. References 1. Rosendaal FR: Venous thrombosis: a multicausal disease. Lancet 353:1167-1173, 1999. 2. Egeberg O: Inherited antithrombin deficiency causing thrombophilia. Thromb Diath Haemorrh 13:516-530, 1965. 3. Griffin JH, Evatt B, Zimmerman TS Kleiss AJ, Wideman C. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 68:1370-1373, 1981. 4. Comp PC, Esmon CT: Recurrent venous thromboembolism in patients with a partial deficiency of protein S. N Engl J Med 311:1525-1528, 1984. 5. 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