Mutations add up
to thrombophilic risk
Making the tough decisions on FVL
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
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
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
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
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
"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
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
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
"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
"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
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 µL 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
"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
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
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
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,
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
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
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
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
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
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
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.