College of American Pathologists
Printable Version

Mutations add up to thrombophilic risk

Making the tough decisions on FVL

January 2002
Cover Story

William Check, PhD

A substantial fraction of venous thromboembolic events are due to mutations in genes that directly affect coagulation. Finding mutations that increase the risk of venous thromboembolism, or VTE, has made it possible to identify a heritable predisposition to thrombosis in many VTE patients who have the hypercoagulable state called thrombophilia, says Douglas A. Triplett, MD, vice president and director of medical education at Ball Memorial Hospital, Muncie, Ind.

"The thrombophilia approach [to thrombosis] is very new," Dr. Triplett says. "We have talked about it for years, but have had very little to measure except proteins C and S and antithrombin. Now that we can do a lot more," he says, "thrombophilia is finally being appreciated by the medical community, though perhaps not yet by all."

Being able to detect thrombophilic mutations in the clinical laboratory can prevent recurrent events in some patients who have suffered a first VTE and possibly prevent first events in family members. Before this promise can be realized, wrinkles remain to be worked out. A CAP consensus conference in November 2001 addressed four basic questions: What tests should be done? When should testing be implemented? Who should be tested? And how should testing be done?

Who should be tested for genetic risk factors for thrombophilia and how testing should proceed is "a somewhat controversial issue," says Richard Press, MD, PhD, director of molecular pathology at the Oregon Health Sciences University, Portland. "In some cases conclusions changed from what people thought at the outset."

Although the conference touched on a long list of risk factors for thrombophilia, most are of minor clinical importance. For instance, says Kandice Kottke-Marchant, MD, PhD, section head of hemostasis and thrombosis in the Department of Clinical Pathology at the Cleveland Clinic Foundation, one might look for elevated fibrinogen or dysfibrinogen "if a patient with thrombosis is normal for all major risk factors for thrombophilia." And plasminogen has lost favor because, she says, "its clinical association with thrombosis is tenuous." Five heritable risk factors would be on most laboratories' core thrombophilia panel: genetic deficiencies in proteins C and S and antithrombin, along with factor V Leiden and prothrombin G20210A.

Some labs also test for the C677T polymorphism in the gene for methylenetetrahydrofolate reductase (MTHFR C677T), which affects VTE indirectly through homocysteine metabolism. "We do this test," says Dr. Press, "but mostly for research purposes. There is very little clinical demand. The evidence that MTHFR C677T is an independent risk factor for homocysteinemia is not great."

In addition to the four fundamental questions about testing for heritable thrombophilia—what, when, who, and how—Ronald McGlennen, MD, medical director of Esoterix Molecular Genetics and associate professor of laboratory medicine and pathology at the University of Minnesota, raises more complex issues. "Looking for heritable causes of thrombophilia represents a central challenge to the use of the clinical molecular laboratory, in contrast to what has been a very conventional algorithmic use of the coagulation laboratory to work up patients with a bleeding disorder," says Dr. McGlennen. In his view, this applies particularly to the two single-nucleotide mutations, factor V Leiden and prothrombin G20210A.

In addition, he says, "We have a challenge to educate our ordering physicians that gene-based tests give simple, straightforward answers that are immediately applicable to patient management. In contrast to the cystic fibrosis model, there is no consistent need for genetic counseling. The clinical scenario plays out directly."

Finally, he says, "We have a challenge to prove that we can do these tests for economically sound reasons, which in my strong opinion we can."

More than 100 years ago, Rudolf Virchow, MD, known as the father of pathology, formulated "Virchow's triad"—stasis, vascular injury, and hypercoagulability—as the basis of what we now know as thrombosis. "Virchow was a very astute observer and recorder," says Dr. Triplett. During the 20th century, with the biochemical elucidation of the complex homeostatic system that regulates clotting, the stage was set for a molecular genetic understanding of factors that predispose individuals
to VTE.

Discovery of antithrombin deficiency in 1965 marked the first identification of a heritable cause for predisposition to thrombosis. Finding genetic deficiencies of proteins C and S followed in the 1980s. However, all three of these heritable clotting defects are rare. "If we take 100 individuals with thrombosis who walk through the door, we would find no more than five percent who would have a variation of antithrombin, protein C, or protein S genes," Dr. Triplett says. "Nonetheless, for a long time these three heritable forms of thrombophilia were all we could measure."

Even now they remain part of the first-line screen for thrombophilia. Although rare, Dr. Marchant says, these three deficiencies explain an important, if minor, fraction of VTE: About four percent of thrombosis patients will have protein C deficiency, three percent will have antithrombin deficiency, and three percent protein S deficiency. "So together these deficiencies comprise five to 10 percent of thrombosis patients," she says.

More recently, clinical researchers discovered single-nucleotide mutations in two genes for coagulation factors that are more common and more potent in predisposing to VTE. In 1994, clinical scientists at the University of Leiden identified a single mutation in the gene for factor V. Named factor V Leiden (FVL), this mutation is considered the classic example of a genetic variant that predisposes to VTE. FVL is found in three to four percent of the U.S. Caucasian population. In Europe, its frequency varies with geography, ranging from 10 percent in areas of Scandinavia to two percent in the Mediterranean region. "Factor V Leiden is by far the most common hereditary cause predisposing to venous thrombosis," Dr. Triplett says.

In 1996, the Leiden group reported a second single-nucleotide mutation predisposing to venous thrombosis, prothrombin G20210A, which affects one to two percent of the U.S. Caucasian population.

(Both FVL and prothrombin G20210A are unique to Caucasian populations, not being found in Africa or Asia. The leading hypothesis for this observation, Dr. Triplett says, is that both mutations arose no more than 35,000 years ago, after early humans migrated out of Africa and split to populate the Far East and Europe.)

When present simultaneously, FVL and prothrombin G20210A impart a particularly high risk of VTE, Dr. McGlennen says. "What you find is that there is a considerable number of individuals who harbor both abnormalities," he says.There is synergy between FVL and protein C as well, Dr. Marchant adds, with a prevalence of thrombosis up to 80 percent in persons harboring both mutations.

Dr. Press, who coordinated the section on FVL testing in the CAP conference report, says the conference recommended testing for FVL and prothrombin G20210A simultaneously in the workup of thrombophilia. However, the report does not recommend routine testing for MTHFR C677T.

"Most data show that the mutant genotype works in a folate-dependent manner," Dr. Press says. "So if you are not folate-deplete, there is really no evidence that the homozygous MTHFR C677T genotype is a risk factor for thrombosis." Says Dr. Marchant: "Elevated homocysteine is a more established risk factor for VTE than the MTHFR C677T mutation." Many would include measuring plasma homocysteine level and, if elevated, go to gene-based study, according to Dr. Triplett.

Testing for FVL and prothrombin G20210A is recommended for persons with:

  • recurrent venous thrombotic events.
  • a venous thrombotic event at a young age (<50 years).
  • an unprovoked VTE at any age.
  • events at unusual anatomic sites (not leg, pelvis, or lung).
  • a first event in persons who have a family member <50 years with an     event.

    Testing for FVL and prothrombin G20210A should also be considered in women with a first VTE related to pregnancy, the puerperium, or oral contraceptive use; women with events related to hormone replacement therapy; and those with unexplained pregnancy loss during the second or third trimester. A good interview in a woman requesting oral contraceptives or HRT is important, Dr. Press notes. Testing is advised if justification is found in the personal or family history.

    The conference created a separate category for controversial applications of testing, chiefly persons over age 50 who have a first provoked VTE in the absence of cancer or an intravascular device.

    "A minority believe you should test for Leiden in virtually everybody with a venous thrombotic event," Dr. Press says. "That was not our consensus. Personally, I think we should be testing only people with thrombophilic predispositions." Accordingly, testing was not recommended as a general population screen or as a routine screening test during pregnancy or before prescribing oral contraceptives.

    Test methods include functional assays for activated protein C(APC) resistance (some FDA-approved), which are either chromogenic or based on clotting time, and DNA-based tests that directly detect the Leiden mutation. Appropriately validated DNA-based methods are extremely accurate and precise. "It was the conclusion of the group that first-generation APC resistance functional assays, in which plasma is not prediluted before addition of APC, have variable sensitivity and specificity that precludes their routine clinical use," Dr. Press says.

    Second-generation APC resistance assays, with dilution of patient plasma into FV-deficient plasma, look about equivalent to DNA-based tests, at least in some laboratories. "If you are going to test for Leiden, use a second-generation functional test or a DNA-based mutation detection method," Dr. Press sums up.

    An initial DNA method is preferred to screen patients with a strong lupus inhibitor and family members of patients known to have FVL. And confirmatory DNA-based testing should be done in patients with "borderline" APC resistance values, as defined in each laboratory. DNA confirmatory testing is also recommended in patients with positive APC tests to distinguish heterozygotes from homozygotes.

    "I don't think this varies much from what people are doing," Dr. Press says. "In my experience, most practitioners are either using a DNA test initially or a functional assay followed by DNA confirmation."

    Among DNA-based methods, each laboratory must decide which method best suits its situation. Many in-house assays for FVL have been developed. "As with any other in-house test," Dr. Press says, "the laboratory needs to validate the assay." Commercial DNA-based methods for Leiden detection are under development.

    In his laboratory, Dr. Press uses the Magna Pure instrument to prepare DNA, followed by real-time PCR analysis for Leiden and G20210A mutations on the Light-Cycler using allele-specific hybridization probes for amplification and detection.

    At the Cleveland Clinic, Ilka R. Warshawsky, MD, PhD, associate staff in the Department of Clinical Pathology, also tests for FVL and prothrombin G20210A by DNA extraction on the Magna Pure and real-time PCR on the Light-Cycler. "We use the Magna Pure to extract DNA from 200 ┬ÁL of blood," Dr. Warshawsky says. Blood and reagents are placed into cartridges on the Magna Pure and bar-coded patient information is input. Preparation takes about 40 minutes. After extraction, the instrument pipettes DNA samples and MasterMix (Roche's proprietary reagent mix containing primers and probes specific for the mutation, taq polymerase, nucleotide precursors, etc.) into capillaries in a carousel. Capillaries are centrifuged to move the reaction solution to the bottom of the capillaries, and the carousel is manually carried into the PCR room and put into the Light-Cycler, along with the disc containing bar-coded information.

    "We do runs of 28 samples on preset days each week," says Dr. Warshawsky. (Four slots are used for controls.) "We haven't gotten any complaints about turnaround time." Over the past eight months, 77 to 91 percent of FVL samples were normal, nine to 21 percent were heterozygous for FVL, and zero to two percent were homozygous. Testing for prothrombin G20210A was introduced in July. Of 500 samples tested since then, 12 were heterozygous for the mutation.

    Light-Cycler assay can detect new mutations in the region where probes hybridize if they have atypical melting curves, triggering sequencing. "We have found one new prothrombin mutation," Dr. Warshawsky says, "which has the same melting curve as another reported mutation. That would be
    a concern."

    Dr. Warshawsky now tests for MTHFR C677T, a much lower-volume assay, by PCR and restriction enzyme digestion. But she plans to set it up on Magna Pure and Light-Cycler.

    Dr. McGlennen also tests for FVL, prothrombin G20210A, and MTHFR C677T. He considers the contribution of MTHFR C677T controversial, calling it a "risk modifier"—a mutation that by itself does not increase thrombotic risk, but one that can augment risk in combination with other mutations. In addition, he probes for another mutation in the factor V gene, HR2, which also results in a factor V protein resistant to inactivation.

    Many CAPconference recommendations for testing flow from a patient's personal and family history, Dr. McGlennen points out. "Given that a patient has a series of blood clots under age 50," he says, "we really do need to have some family history or other evidence to conclude that testing is worthwhile." In this sense, he asserts, "Testing for thrombophilia has reinvigorated taking a good family history. Clinicians must go beyond simply asking, Has anybody in your family clotted?"

    While acknowledging that the standard of care is to do a thrombophilia panel only in patients whose personal or family history suggests thrombophilia, Dr. McGlennen says, "I have added a handful of additional scenarios in which identifying a thrombophilic tendency is helpful." For example, a person who has surgery is immobilized for a time and may develop a blood clot. When that patient resumes walking, the clot can migrate, generating a pulmonary embolism.

    "Our job is to find those individuals before they get into bed," Dr. McGlennen says, "so we can prevent clot formation by anticoagulation." As a result, he says, "I am looking at a transition from thrombophilia panels being ordered primarily by internists or hematologists, to ordering by surgeons who are deciding what to do with post-operative patients and gynecologists making decisions about oral contraceptives or HRT."

    Even so, he agrees that generalized screening cannot be justified.

    As for methodology, Dr. McGlennen aims to find the technology platforms that allow them to test inexpensively. "My goal is to make gene-based testing for thrombophilia as common as hematology-based coagulation assays," he says. He uses an in-house DNA microarray bearing probes for the four mutations for which he tests. "We have shown that we can do those four reactions for about the same cost as doing one test," he says. "And we could probably expand to one or two additional markers for minimal incremental cost." Although testing by microarray is unusual, Dr. McGlennen says, "All commercial microarray manufacturers are working assiduously to come up with their own thrombophilia panels."

    He focuses the commentary in his lab report on how gene-based defects create increased risk. "I try to leave the clinician with a sense of numerical proportionality of risk relative to the general population," he says. "And I may include one sentence stating the recommendation about therapy." Physicians know the risk of long-term anticoagulation. Before they consider initiating this therapy, they need to see a multiple of risk—however arbitrary—that justifies it.

    Dr. Triplett, too, stresses the role of history-taking in decisions about whether to test for thrombophilia. "For our baseline evaluation," he says, "we would, if possible, get a history in more than one generation and in both men and women." Women who carry the FVL gene are the principal group at increased risk of VTE. "Women are challenged as men are not," he says. Women in their reproductive years take oral contraceptives, pregnancy predisposes a woman with FVL to deep-vein thrombosis and other thromboembolic events, and many women take hormone replacement therapy later in life.

    Perhaps Dr. Triplett's heightened awareness of FVL's impact on women is due to the first FVL patient he saw—a female college freshman who had just started on oral contraceptives. She had a massive DVT and was admitted to the hospital with nonfatal pulmonary embolism. "That kind of scenario tells you that educating people about the relative risks of FVL, especially its increased risk to women, is very important," Dr. Triplett says. He believes that any clinician evaluating a woman for an OC prescription should ask, Have you or any members of your family, especially women, had
    a thrombosis?

    Of population screening, he says simply, "We really do not want to do that."

    As director of the Midwest Hemostasis and Thrombosis Laboratory, Dr. Triplett offers testing for FVL, prothrombin G20210A, and MTHFR C677T. "In most laboratories, these three tests are performed by molecular testing," he says. In his laboratory, gene-based testing is done using the Invader system from Third Wave Technologies (Madison, Wis.), which he finds easy to use.

    Dr. Marchant, who coordinated the section in the CAP conference report on testing for deficiencies of proteins C and S and antithrombin, says that these tests are reserved for persons who have had an episode of venous thrombosis under age 50 with no apparent inciting factors, such as malignancy, immobilization, surgery, or obesity—VTE that occurs "out of the blue." The local coroner recently called Dr. Marchant about a 22-year-old man who died suddenly of pulmonary embolism; on autopsy a large metastatic testicular tumor was found. She recommended against testing for thrombophilic mutations because of the documented tumor.

    Functional assays, rather than gene-based tests, are used for initial testing for proteins C and S and antithrombin deficiencies, because, unlike FVL and prothrombin G20210A, these deficiencies are due to multiple mutations. Protein C deficiency, for instance, can be due to any of more than 160 mutations. "Any one genetic test is unlikely to detect all those mutations," Dr. Marchant says.

    It is best to measure protein C remote from an acute thrombotic event, when the patient has been off oral anticoagulation for a month. However, many thrombosis patients come from a distance and can be tested only in the hospital. A practical recommendation is to measure protein C when you can. "If it is normal, that can exclude a deficiency," Dr. Marchant says. An abnormal result in the setting of an acute event or during oral anticoagulant therapy is uninterpretable.

    Recommended screening tests for protein C deficiency include chromogenic (amidolytic) assays that are widely available for automated coagulation instruments and have good sensitivity and reproducibility. Because these assays measure activity, they detect deficiencies due to gene deletions and point mutations.

    A clotting-based assay is also widely available. "I don't recommend this as strongly for general testing," Dr. Marchant says, "since it is subject to multiple interferences, including heparin, high levels of factor VIII or FVL, and lupus anticoagulant." The clotting assay does measure the ability of protein C to bind to protein S or factor V, which could be missed with a chromogenic assay unless the mutation is at the active site. "However," Dr. Marchant says, "most protein C mutations are not missed by chromogenic assays."

    Testing for protein S deficiency "remains problematic," she says. There is no direct functional assay, because protein S is not an enzyme, but a cofactor for protein C activity. A clotting assay is available, but it is subject to the same interferences as for protein C. Dr. Marchant uses the clotting assay and screens the sample for all possible interfering substances if the initial clottable protein S result is decreased—heparin, lupus anticoagulant, and elevated factor VIII or FVL. "That's a lot of testing just to make sure your result is valid," she says.

    Another complication with protein S is that it is present in plasma both in the free state and bound to a complement regulatory protein, C4bBP. Only free protein S is active. Persons with high C4bBP activity may have low plasma protein S activity. An antigenic assay that measures total and free protein S with polyclonal antiserum, polyethylene glycol precipitation, and centrifugation is "notoriously inaccurate," Dr. Marchant says. "In the conference, we recommended that probably the best screening assay is one of the new antigenic assays that use a monoclonal antibody to detect free protein S, which are not subject to as many interferences." Her laboratory is switching to a monoclonal assay.

    Because the activity of both protein C and protein S is vitamin K-dependent, protein C or S cannot be measured accurately in patients on anticoagulant therapy. Waiting one month after cessation of therapy is recommended. Also, antithrombin levels are decreased in patients on heparin therapy. Unfortunately, Dr. Marchant says, "As a reference laboratory, we get many samples with no information on whether a patient is on an anticoagulant. In these cases, results can be difficult to interpret." For this reason, she often does a pro-time, aPTT, or anti-Xa assay to determine whether a patient is on coumadin or heparin.

    Antithrombin assays, in contrast, are fairly straightforward. Chromogenic or amidolytic assays are most widely used and have fairly good sensitivity and precision, though patients on heparin may have decreased antithrombin activity.

    With one set of tests (proteins S and C and antithrombin) being done in the coagulation laboratory and the others (FVL, prothrombin G20210A, and MTHFR C677T) in the molecular laboratory, how are results coordinated? Typically, they are not.

    "Unless a clinician requests a clinical pathology consult, which they rarely do, individual test results are sent back with individual interpretations," Dr. Press says. "No one in the laboratory puts the whole picture together." At Oregon Health Sciences University, many patients getting thrombophilia workups are seen in the thrombophilia clinic by hematologists, who can usually integrate results themselves.

    However, Dr. Press says, about three-fourths of his testing is done on cases from community hospitals. He offers an interpretive report with molecular test results. "But I am completely in the dark about protein S and C and antithrombin results," he says, "as well as about the patient's clinical status. As in most of pathology, the appropriate clinical information usually doesn't accompany the specimen. So we are often interpreting these tests in the dark. I worry a bit about that and I think many of my colleagues do
    as well."

    Midwest Hemostasis and Thrombosis Laboratory does a full spectrum of testing for patients who bleed or have thromboembolic disorders. In other places, Dr. Triplett says, "Even among those with a substantial degree of sophistication in a coagulation laboratory, many offer only antithrombin and protein C and S. They may be sending genetic tests to a reference laboratory." They would need sufficient volume to justify incorporating gene-based tests into their own laboratory, and they would need a coagulation specialist who is comfortable with those mutations. If not, it might be wise to refer patients and families with thrombophilic mutations to another hospital.

    Therapeutic implications of thrombophilia assays are uncertain. "One of the things we said up front in the recommendations," Dr. Press says, "is that there is really no evidence that any of these test results are going to impact therapy." It is not clear that you would treat a patient who is positive for FVL or prothrombin G20210A longer or more intensively. However, some test results may affect prophylaxis in family members, especially female family members. "We believe that the knowledge of carrying the Leiden mutation may, and perhaps should, impact decisions such as choice of contraception and perhaps use of prophylactic anticoagulation during and/or after pregnancy," Dr. Press says.

    Dr. Marchant calls therapeutic decisions in patients with thrombophilic mutations, especially those with a combination of mutations, "an area of great clinical controversy."

    Amid all this uncertainty, Dr. McGlennen says, "We have a remarkable opportunity." But what can laboratorians do to get primary care physicians and surgeons to think about gene-based testing for thrombophilia? Coagulation laboratories, which primarily work up patients with a tendency to bleed, are always in the minds of clinicians doing a workup. "Along come gene-based tests to identify individuals who have a tendency to form abnormal clots, which appear to be orders of magnitude more common than bleeding problems," Dr. McGlennen says. "Now we have gene-based markers to look for more common diseases and a conventional laboratory algorithm that is well ensconced in routine practice but typically only for rare events. What I am suggesting is that we have an opportunity to look at a common thing commonly with DNA-based testing for FVL and prothrombin mutations," he says. "This is a challenge that I am working on."

    William Check is a medical writer in Wilmette, Ill.