SUMMARY OF ISSUES AND RECOMMENDATIONS
Issue 1: Which methodology should be used: Factor V Leiden DNA testing or functional
activated protein C (APC) resistance testing?
Recommendation 1
When appropriate clinical care requires testing for the factor V Leiden allele, either
direct DNA-based genotyping or a factor V Leiden-specific functional assay is recommended.
Patients who test positive by a functional assay should then be further studied with
the DNA test for confirmation and to distinguish heterozygotes from homozygotes. Patients
on heparin therapy or with known lupus anticoagulant should proceed directly to molecular
testing if the modified functional assay is not used. When relatives of individuals
known to have factor V Leiden are tested, the DNA method is recommended.
Issue 2: Who should be tested?
Recommendation 2
Opinions and practices regarding factor V Leiden testing vary. Some physicians advocate
testing of all patients with venous thrombosis except when active malignancy is present.
Others exclude testing in patients over age 60 in the absence of a family history
of thrombosis or a previous thrombotic event.
There is growing consensus that testing should be performed in at least the following
circumstances (these are the same general recommendations for testing for any thrombophilia):
Age <50, any venous thrombosis.
Venous thrombosis in unusual sites (such as hepatic, mesenteric, and cerebral veins).
Recurrent venous thrombosis.
Venous thrombosis and a strong family history of thrombotic disease.
Venous thrombosis in pregnant women or women taking oral contraceptives.
Relatives of individuals with venous thrombosis under age 50.
Myocardial infarction in female smokers under age 50.
Testing may also be considered in the following situations:
Venous thrombosis, age >50, except when active malignancy is present.
Relatives of individuals known to have factor V Leiden. Knowledge that they have factor
V Leiden may influence management of pregnancy and may be a factor in decision-making
regarding oral contraceptive use.
Women with recurrent pregnancy loss or unexplained severe preeclampsia, placental
abruption, intrauterine fetal growth retardation, or stillbirth. Knowledge of factor
V Leiden carrier status may influence management of future pregnancies.
Random screening of the general population for factor V Leiden is not recommended.
Routine testing is not recommended for patients with a personal or family history
of arterial thrombotic disorders (e.g., acute coronary syndromes or stroke) except
for the special situation of myocardial infarction in young female smokers. Testing
may be worthwhile for young patients (<50 years of age) who develop acute arterial
thrombosis in the absence of other risk factors for atherosclerotic arterial occlusive
disease.
Neither prenatal testing nor routine newborn screening is recommended.
Issue 3: Should testing be offered to individuals with environmental risk factors?
Recommendation 3
Factor V Leiden testing is recommended in women with venous thromboembolism during
pregnancy or oral contraceptive use. In contrast to general screening before administration
of oral contraceptives, targeted testing of women with a personal or family history
of venous thrombosis is advisable. Routine screening for factor V Leiden in asymptomatic
womencontemplating or using oral contraceptives is not recommended, except for those
with a personal history of thromboembolism or other medical risk factors. Those women
with a family history of thromboembolism, APC resistance, or documented factor V Leiden
mutation should be counseled about their risks and options and considered for testing
depending on the overall clinical situation. Women with a history of recurrent late-trimester
fetal loss should also be considered for testing. Whether or not the woman smokes
would not alter these recommendations. Screening of asymptomatic individuals with
other recognized environmental risk factors such as surgery, trauma, paralysis, and
malignancy is not necessary or recommended, since all such individuals should receive
appropriate medical prophylaxis for thrombosis regardless of carrier status.
Issue 4: Should patients found to be positive for factor V Leiden or APC resistance
be tested for any of the other heritable thrombophilic risk factors?
Recommendation 4
Patients testing positive for factor V Leiden or APC resistance should be considered
for molecular genetic testing for the most common other thrombophilias with overlapping
phenotype for which testing is easy and readily available. At present, only the prothrombin
20210A variant fits these criteria. It is present in 1–2% of the general population,
its involvement in venous thromboembolism is well-established, and the DNA test is
as simple as that for factor V Leiden (with which it can even be multiplexed). Protein
S, protein C, and antithrombin III deficiencies are too genetically heterogeneous
for routine molecular genetic testing, but testing by functional coagulation assays
may be considered, especially if there is a strong family history of venous thrombosis.
Hyperhomocysteinemia should be considered and tested (in most cases by measuring plasma
homocysteine levels) as another potential risk factor in those found to be positive
for factor V Leiden. Patients with classic homocystinuria are at extremely elevated
risk of thromboembolism and should probably be tested for other available thrombophilic
risk factors.
Issue 5: Should testing for other heritable thrombophilic factors be performed simultaneously
with factor V Leiden testing?
Recommendation 5
Physicians ordering factor V Leiden on a venous thrombosis patient for any of the
indications recommended here should also consider the utility of functional, biochemical,
and molecular screening for other heritable thrombophilic factors, especially prothrombin
20210A and plasma homocysteine levels.
Issue 6: Are there any other factor V mutations in addition to factor V Leiden which
should be tested?
Recommendation 6
The factor V Leiden (R506Q) mutation is currently the only molecular analysis of the
factor V gene indicated in the routine workup of thrombotic risk.
Issue 7: What are the recommended methodologies and quality assurance standards for
performing these tests?
Recommendation 7
The factor V Leiden mutation test should be performed using any of the accepted technical
approaches as long as they have been properly validated by the laboratory, while adhering
to current ACMG/CAP quality assurance guidelines for molecular genetic testing.
Issue 8: What are the appropriate pre- and postanalytic procedures to be followed
in factor V Leiden testing?
Recommendation 8
Formal informed consent should not be required for factor V Leiden testing, but individuals
being tested should be made aware that this is a genetic test, that test results have
implications about risk in other family members, and that there may be attendant issues
of confidentiality and possible insurance discrimination. The laboratory’s report
should state explicitly the relative risk implications for factor V Leiden heterozygotes
and homozygotes, the risk that other relatives may have the mutation, and the recommendation,
if indicated, for testing for other inherited hypercoagulabilities.
It is important for individuals testing positive for factor V Leiden to understand
the risk implications and genetic implications of their result. Patients should be
counseled about these implications by their physician or genetic counselor.
BACKGROUND
Normal hemostasis requires a delicate balance between the natural procoagulant and
anticoagulant systems. Both of these systems are subject to disruption by either inherited
or acquired (including both intrinsic and environmental) defects. Inherited defects
associated with clinical bleeding disorders (e.g., hemophilia A and B and von Willebrand
disease) have been known and studied for centuries. In contrast, inherited defects
causing thrombosis have been recognized more recently. These heritable thrombophilias
(hypercoagulabilities) include factor V Leiden (R506Q mutation), the prothrombin 20210A
mutation, antithrombin III deficiency, and deficiencies of protein C and protein S.
The incidence of venous thrombosis is about 1 per 1,000 person-years1 and leads to
50,000 deaths annually in this country.2 It is a multifactorial disorder, involving
one or a combination of genetic risk factors and acquired or environmental conditions
such as pregnancy, oral contraceptive use, estrogen therapy, malignancy, stroke with
extremity paresis, trauma, surgery, or immobility.3 The risk increases with the number
of genetic and/or environmental conditions present, though venous thromboembolism
can occur in the absence of known risk factors. Known genetic causes are present in
approximately 25% of unselected venous thrombosis cases and up to 63% of familial
cases.4
Factor V R506Q (Leiden), causing activated protein C (APC) resistance, was discovered
in 1994 and is the most common genetic risk factor for venous thrombosis. It is present
in 5% of Caucasian Americans, 20% of idiopathic first venous thrombosis cases, and
60% of venous thrombosis cases in pregnant women. Knowledge of the presence of factor
V Leiden in patients and relatives can influence management and prevention of venous
thrombosis in some cases. Factor V Leiden has also been associated with increased
risk of recurrent pregnancy loss and placental infarction.
The procoagulant system
The normal function of the procoagulant system is to target a hemostatic plug containing
platelets and fibrin into a breach within the inside lining of “injured” blood vessels.
The procoagulant system has been characterized as a “cascade” of amplifying enzymatic
reactions leading to the final serine protease, thrombin. The procoagulant system
is triggered by exposure of the coagulation activator, tissue factor, to circulating
blood. Tissue factor normally is sequestered from the circulation within the wall
of blood vessels and only exposed to blood after vessel wall injury. Exposed (or expressed)
tissue factor binds circulating factor VIIa, and in the presence of anionic phospholipid
and divalent cations (e.g., calcium), forms a factor X-ase complex. This factor X-ase
complex either directly cleaves (“activates”) factor X to Xa, or activates factor
IX to IXa, by limited proteolysis. Factor IXa binds factor VIIIa to form a second
factor X-ase complex, which activates factor X to Xa. Factor Xa binds factor Va to
form the prothrombinase complex, which activates prothrombin to thrombin. Thrombin
produces a hemostatic plug by cleaving fibrinogen to form fibrin monomers, by activation
of platelets, and by activating factor XIII to XIIIa which crosslinks strands of fibrin
monomers to form a stable hemostatic plug. In a feedback amplification loop, thrombin
also increases its own production by activating factors V, VIII, and XI. Together
factors Va and VIIIa can potentially increase the rate of thrombin generation by one
million-fold and provide major control points for the regulation of thrombin generation.
The anticoagulant system
The function of the natural anticoagulant system is to confine a normal hemostatic
plug to the site of vessel wall injury and to prevent the beneficial thrombus from
propagating to form a pathologic thrombus, which occludes the lumen of the vessel
or embolizes to occlude distant vessels. The recognized anticoagulant components of
this system include antithrombin III, protein C, and protein S. The anticoagulant
system is activated in parallel with the procoagulant system. Protein C is a circulating
vitamin K-dependent zymogen, which is activated to APC, the active enzyme, by the
thrombin-thrombomodulin complex. APC functions as a natural anticoagulant by inactivating
(via proteolysis) procoagulant factors Va and VIIIa in the presence of protein S.
Antithrombin III is a serine protease inhibitor (SERPIN) and acts as a pseudosubstrate
to irreversibly inhibit thrombin by covalently binding the thrombin enzymatic active
site. The rate of thrombin inhibition by antithrombin III is increased markedly by
glycosaminoglycans (e.g., heparin). Familial reductions in plasma antithrombin III,
protein C, or protein S activity due to either reduced plasma protein levels (i.e.,
altered protein expression), or normal levels of a dysfunctional protein (i.e., altered
protein structure), are strongly associated with deep vein thrombosis and pulmonary
embolism (venous thromboembolism), and validate the important role of these proteins
in the natural anticoagulant system. Altogether, however, the prevalence of these
previously recognized familial thrombophilias among venous thromboembolism patients
ranges from 5 to 20%, depending on cohort selection.5
Activated protein C resistance and the factor V Leiden mutation
The report of three unrelated probands and their families with idiopathic recurrent
venous thromboembolism whose plasma was resistant to the anticoagulant effect of exogenously
added APC6 has provided exciting new insights into the etiology of venous thromboembolism.
Early epidemiologic data suggested that an abnormally low anticoagulant response to
APC, termed “activated protein C resistance (APC-R),” was familial, with an autosomal
dominant or semidominant inheritance pattern.7 Factor Va isolated from APC-R patient
plasma was resistant to inactivation by APC.8 Subsequent work identified a single
point mutation (G to A) at nucleotide 1691 of the factor V gene, which results in
substitution of a glutamine for arginine at residue 506 (R506Q), one of three APC
cleavage sites (R306, R506, R679).9–13 This mutation is known as factor V Leiden.
Initial APC cleavage at the R506 position is required for optimal exposure and subsequent
rapid inactivation of factor V by APC cleavage at positions R306 and R679.14,15 Depending
on the APC resistance functional assay used and the cut-off values for defining an
abnormal result, the factor V Leiden mutation may account for 85–95% of patients with
APC resistance. Recent studies show that APC-R with normal factor V R506 genotype
is a risk factor for venous thrombosis.16,17
APC resistance is the most common recognized abnormality of coagulation among patients
with venous thromboembolism. The factor V Leiden mutation is carried in heterozygous
form by about 5% of the Caucasian population; it is rarer in Hispanic-Americans, rarer
still in African-Americans, and virtually absent in Africans and Asians.18,19 It is
believed to produce a relative risk of venous thrombosis of about 7-fold in the heterozygous
state and about 80-fold in the homozygous state. It is the most common hereditary
thrombophilia and is found in roughly 11–20% of individuals of all ages presenting
with their first episode of venous thrombosis.20,21 When venous thrombosis patients
are selected to be under 50 years old and/or to have recurrent thrombosis, up to 40%
have the factor V Leiden genotype. Because only a single mutation is involved, testing
by any number of simple and inexpensive molecular genetic methods is possible.
CHARGE TO THE WORKING GROUP
Given the high allele frequency of the factor V Leiden mutation and some of the other
inherited thrombophilia defects, questions regarding criteria for testing and the
possibility of population screening have been raised. The issues are quite complex
and were referred to a specially appointed working group of the ACMG for deliberation.
It should be noted at the outset that the group’s proposed recommendations as presented
here were based on the best scientific evidence available at the time. However, the
heritable thrombophilia field is changing rapidly as more clinical studies to further
define the genetic risks are completed and additional interacting factors are identified,
making the attempt to develop recommendations something of a moving target. As such,
these recommendations should be understood as being subject to change as new knowledge
accrues.
ISSUES AND RECOMMENDATIONS
1.
Which methodology should be used: Factor V Leiden DNA testing or functional APC resistance
testing?
APC resistance due to factor V Leiden can be diagnosed by functional analysis of the
intrinsic or extrinsic coagulation pathway or by direct molecular genetic testing
for the R506Q mutation in the factor V gene. The coagulation assay for APC resistance
is based on a functional analysis of the anticoagulant effect on patient plasma of
exogenously added APC. As an anticoagulant, APC normally decreases the rate of thrombin
generation in plasma. Available APC resistance assays test for the APC anticoagulant
effect via either prolongation of clotting time (e.g., activated partial thromboplastin
time [aPTT], prothrombin time [PT], etc.), or by direct measurement of thrombin generation
using a chromogenic substrate. The traditional functional test, based on the partial
thromboplastin time (aPTT), casts a wider net, since not all cases of clinical APC
resistance are due to the factor V Leiden mutation. However, it lacks specificity
for factor V Leiden and is subject to perturbation by acute phase reactants, pregnancy,
oral contraceptives, the acquired lupus anticoagulant syndrome (antiphospholipid antibody),
and yet undefined factors.22 In addition, it cannot be used in patients receiving
heparin or warfarin sodium anticoagulant therapy, and it is much less efficient at
distinguishing factor V Leiden heterozygotes from homozygotes due to extensive overlap
in the assay values. Making this distinction is clinically important since homozygotes
have about a 10-fold higher risk of thrombotic events than heterozygotes. A recent
modification of the functional assay, involving dilution of patient plasma into factor
V-deficient plasma, provides quite reliable differentiation of heterozygotes and homozygotes
and little or no interference by the other clinical factors, but narrows the specificity
to that of the mutation assay, so that cases of APC resistance due to other causes
will be missed.8,23,24 Despite this drawback, the modified assay has been adopted
widely; therefore, any consideration of the relative merits of molecular versus coagulation
testing must take this into account.
Other points of comparison between the tests are cost and convenience of specimen
handling. Currently, the cost for the DNA test is higher than that for the functional
test (though this is likely to change with the advent of new automated DNA technologies).
The DNA test requires blood at room temperature, while the APC resistance test requires
citrated frozen plasma, which must be prepared using centrifugation.
Recommendation 1
When appropriate clinical care requires testing for the factor V Leiden allele, either
direct DNA-based genotyping or a factor V Leiden-specific functional assay is recommended.
Patients who test positive by a functional assay should then be further studied with
the DNA test for confirmation and to distinguish heterozygotes from homozygotes. Patients
on heparin therapy or with known lupus anticoagulant should proceed directly to molecular
testing if the modified functional assay is not used. When relatives of individuals
known to have factor V Leiden are tested, the DNA method is recommended.
2.
Who should be tested?
Although factor V Leiden is detected in an appreciable percentage of patients, opinions
differ as to the usefulness of identifying the mutation and the clinical criteria
for testing. Testing would clearly be helpful if it identified individuals with increased
recurrence risk who could then be considered for long-term antithrombotic therapy.
In general, for patients with a first, objectively documented venous thromboembolism,
the risk of recurrence is highest during the first 6–12 months after the event, with
a cumulative recurrence rate of about 30% by 8–10 years.25,26 Patients with persistent
risk factors for venous thromboembolism (e.g., cancer, stroke with extremity paresis,
obesity) and patients with idiopathic venous thromboembolism are at highest risk for
recurrence.27,28 It is not yet clear whether factor V Leiden heterozygosity increases
risk of recurrent venous thrombosis. A few studies21,29 found increases in recurrence
risk of 4- to 5-fold and 2-fold, respectively, but other studies found no increase.30,31
Currently, identification of factor V Leiden heterozygosity does not change the therapeutic
approach to venous thrombosis or subsequent prophylaxis in most patients. For patients
with recurrent venous thromboembolism, some clinicians recommend lifelong anticoagulation
therapy, regardless of whether a genetic risk factor is present,32 while other clinicians
would test patients to assist in decision-making about indefinite anticoagulant therapy
and genetic counseling of patients and their families.
Despite the reservations listed above, there are several arguments in favor of testing
for factor V Leiden. In some circumstances, knowledge of the factor V Leiden status
strongly influences patient management. Testing will identify factor V Leiden homozygosity
in 1.5% of patients under age 70 with a first episode of venous thromboembolism in
the absence of malignancy.33 Lifetime antithrombotic prophylaxis should be considered
for homozygotes after a thrombotic event.34,35 This approach is also the case for
venous thrombosis patients heterozygous for both factor V Leiden and the prothrombin
20210A mutation, which is not an uncommon finding, in whom recurrence risk has been
shown to be high.36 However, the decision must take into account the coexistence of
bleeding tendencies and other contraindications. The risk of major bleeding with chronic
warfarin therapy may reach 8% per year,37 and there are no studies which provide good
estimates of the absolute risk of venous thromboembolism among homozygous carriers.
Thus, we do not know what the true risk-benefit ratio is of life-long warfarin anticoagulation
for these patients.
A benefit of identifying the factor V Leiden mutation in patients with venous thrombosis
is that asymptomatic family members can opt to determine whether they are at increased
risk for venous thrombosis due to this risk factor. The lifetime risk for venous thrombosis
in factor V Leiden heterozygotes is approximately 10%38 and for homozygotes is >80%.33
Knowledge of factor V Leiden status in asymptomatic relatives can be useful in guiding
antithrombotic prophylaxis during periods of risk, particularly postpartum,39 and
might allow for heightened awareness of presenting signs of deep vein thrombosis.
Female relatives may also wish to know their status before deciding to use oral contraceptives.
Factor V Leiden increases the risk for recurrent fetal loss, possibly due to placental
thrombosis.40,41 Testing in women with recurrent pregnancy loss may be important,
since antithrombotic therapy may be effective in allowing these women to carry a pregnancy
to term.42 Factor V Leiden has also been associated with increased risk of severe
preeclampsia, placental abruption, unexplained intrauterine fetal growth retardation,
and stillbirth.41,43,44 On the other hand, given that factor V Leiden-associated thrombophilia
is an adult-onset disorder of low penetrance, fetal testing is not indicated. For
similar reasons, routine newborn screening for factor V Leiden is not recommended.
Increasing age is a strong independent risk factor for venous thrombosis, and for
this reason, many physicians do not attempt to identify genetic risk factors in elderly
patients with venous thrombosis. However, at least two studies have shown that among
factor V Leiden carriers, the first lifetime episode of VTE usually occurs after age
50 years, suggesting that testing for this mutation should not be limited to young
patients.38,45 In another study, 26% of men over age 60 with a first episode of idiopathic
venous thromboembolism had factor V Leiden.21
The weight of currently available evidence suggests that arterial thrombosis, myocardial
infarction, and stroke are not associated with factor V Leiden.21 An exception is
myocardial infarction in young (<45 years old) female smokers, in whom the combination
of the two factors increases the relative risk 32-fold.46 Factor V Leiden has also
been implicated in younger adults (<50) who develop arterial thrombosis in the absence
of other risk factors for atherosclerotic disease.47,48
Recommendation 2
Opinions and practices regarding factor V Leiden testing vary. Some physicians advocate
testing of all patients with venous thrombosis except when active malignancy is present.
Others exclude testing in patients over age 60 in the absence of a family history
of thrombosis or a previous thrombotic event.
There is growing consensus that testing should be performed in at least the following
circumstances (these are the same general recommendations for testing for any thrombophilia):
▪ Age <50, any venous thrombosis.
▪ Venous thrombosis in unusual sites (such as hepatic, mesenteric, and cerebral veins).
▪ Recurrent venous thrombosis.
▪ Venous thrombosis and a strong family history of thrombotic disease.
▪ Venous thrombosis in pregnant women or women taking oral contraceptives.
▪ Relatives of individuals with venous thrombosis under age 50.
▪ Myocardial infarction in female smokers under age 50.
Testing may also be considered in the following situations:
▪ Venous thrombosis, age >50, except when active malignancy is present.
▪ Relatives of individuals known to have factor V Leiden. Knowledge that they have
factor V Leiden may influence management of pregnancy, and may be a factor in decision-making
regarding oral contraceptive use.
▪ Women with recurrent pregnancy loss or unexplained severe preeclampsia, placental
abruption, intrauterine fetal growth retardation, or stillbirth. Knowledge of factor
V Leiden carrier status may influence management of future pregnancies.
Random screening of the general population for factor V Leiden is not recommended.
Routine testing is not recommended for patients with a personal or family history
of arterial thrombotic disorders (e.g., acute coronary syndromes or stroke) except
for the special situation of myocardial infarction in young female smokers. Testing
may be worthwhile for young patients (<50 years of age) who develop acute arterial
thrombosis in the absence of other risk factors for atherosclerotic arterial occlusive
disease. Neither prenatal testing nor routine newborn screening is recommended.
3.
Should testing be offered to individuals with environmental risk factors?
Some individuals are at increased risk of venous thromboembolism due to environmental
exposures, and some of these risks are synergistic with factor V Leiden if both are
present, with combined relative risk values many times higher than those for either
condition alone. Examples include oral contraceptive use, pregnancy, and estrogen
therapy. Patients facing commonly recognized environmental risks such as surgery,
trauma, paralysis, and malignancy should be receiving appropriate venous thromboembolism
prophylaxis regardless of genetic status. However, involvement of an environmental
trigger for venous thrombosis does not preclude the possible presence of factor V
Leiden or other genetic risk factor.
The environmental factor most extensively discussed in this context is oral contraceptive
use in women, which produces a 30-fold increase in thrombotic risk when the factor
V Leiden mutation is also present. Some have, therefore, proposed that women contemplating
oral contraceptive therapy be screened for factor V Leiden and that counseling be
provided and an alternative method of birth control be offered to those who test positive.
On the other hand, convincing arguments can be made that widespread screening on such
a large population would not be cost-effective based on number of lives actually saved
and the increased risk of pregnancy and other complications in those women obligated
to turn to other methods of contraception. Despite the popular concept, it remains
controversial whether or not smoking while on oral contraceptives increases the relative
risk; recent evidence suggests a synergistic effect on risk of myocardial infarction
and cerebral thromboembolic stroke, but not on venous thromboembolism, which is the
primary phenotype of factor V Leiden.49,50
Converting these hypothetical risks into probabilistic numbers is illustrative of
the complexities involved in this sort of decision-making. The increased risk of thrombosis
caused by oral contraceptive use alone is about 4-fold; in the setting of factor V
Leiden heterozygosity, this risk increases to 30-fold.51,52 While these relative risks
may seem high, the absolute risk of thrombotic events in this patient population (primarily
young women) is quite low.53 The baseline incidence of venous thromboembolism in women
under age 44 is about 5 events per 100,000 woman-years. Given a mortality rate from
venous thromboembolism in this age group of 1%, and the increase in relative risk
from 4-fold to 30-fold caused by factor V Leiden heterozygosity, it is estimated that
the combined risk would produce 15 deaths per million woman-years (compared to 4 deaths
per million woman-years caused by oral contraceptives alone).51,54 Based on the population
frequency of the factor V Leiden allele, some have estimated that it would require
screening as many as 2 million women to prevent one death.54 Furthermore, it would
result in withholding oral contraceptives from 90,000 carriers identified in the screening
process. These women would be obligated to turn to alternative, typically less effective,
forms of contraception, with a resulting increase in pregnancy rate and its attendant
complications (including, ironically, intrapartum and postpartum venous thromboembolism,
as well as preeclampsia, placental abruption, and fetal growth retardation, which
are also associated with factor V Leiden43). In addition, they would be exposed to
all the potential psychosocial and insurance discrimination risks inherent in any
genetic screening program.
Recommendation 3
Factor V Leiden testing is recommended in women with venous thromboembolism during
pregnancy or oral contraceptive use. Routine screening for factor V Leiden in asymptomatic
women contemplating or using oral contraceptives is not recommended, except for those
with a personal or family history of thromboembolism or other medical risk factors.
Those women with a family history of thromboembolism, APC resistance, or documented
factor V Leiden mutation should be counseled about their risks and options and considered
for testing, depending on the overall clinical situation. Women with a history of
recurrent late-trimester fetal loss should also be considered for testing. Whether
or not the woman smokes would not alter these recommendations. Screening of asymptomatic
individuals with other recognized environmental risk factors such as surgery, trauma,
paralysis, and malignancy is not necessary or recommended, since all such individuals
should receive appropriate medical prophylaxis for thrombosis regardless of carrier
status.
4.
Should patients found to be positive for factor V Leiden or APC resistance be tested
for any of the other heritable thrombophilic risk factors?
A growing constellation of heritable thrombophilic factors, some more accurately described
as variants or polymorphisms than mutations, are becoming recognized. These include
protein S deficiency, protein C deficiency, antithrombin III deficiency, the prothrombin
20210A variant, hyperhomocysteinemia, and classical homocystinuria. The allele frequencies
of some of these conditions are high enough that combined states of two or even three
risk factors have been reported, with synergistic effects on relative risk.55–60 Thus,
if a patient tests positive for factor V Leiden, it does not exclude the possibility
that other genetic risk factors may be at work also. Some of these other defects are
as easy to test for as factor V Leiden and can even be multiplexed in a single assay.61–63
After factor V Leiden, the most common of the heritable thrombophilias are the prothrombin
20210A variant and hyperhomocysteinemia. The prothrombin variant is a single nucleotide
change in the 3&cjs1227;-untranslated region of the gene that results in elevated
circulating prothrombin levels through an unknown mechanism. It is present in 1–2%
of the general Caucasian population and produces a phenotype similar to that of factor
V Leiden. It is found in 6–8% of unselected patients with a first episode of venous
thromboembolism.64 In addition, it has been associated with myocardial infarction
in young women, cerebral vein thrombosis in oral contraceptive users, preeclampsia
and other complications in pregnancy, and miscellaneous infarctions at other sites.43,64–67
Among patients with a first episode of venous thromboembolism, 10% of those identified
as factor V Leiden heterozygotes will also have the prothrombin 20210A variant.
A flurry of recent work has addressed the possible relationship of elevated plasma
homocysteine levels with risk of both venous thromboembolic and cardiovascular disease.
It has been known for a long time that these are common complications of homocystinuria,
an inborn error of metabolism in which homocysteine levels are dramatically increased.
Moreover, there is evidence that the risk of such events is heightened in homocystinuric
patients who are also factor V Leiden carriers.55 More recently a more proportional
gradation of risk has been associated with moderate physiologic elevations of plasma
homocysteine in otherwise healthy adults, with relative risk beginning to increase
as fasting plasma homocysteine concentration exceeds 10 μmol/liter.
A product of methionine metabolism, homocysteine is maintained within a narrow range
of concentrations through a complex series of reactions involving several enzymes
and cofactors (the latter including vitamin B6, vitamin B12, and folic acid). Levels
may rise as a result of subclinical deficiency of any of the enzymes involved, dietary
deficiency of one of more of the cofactors, or a variety of other acquired medical
conditions and lifestyle factors.68 Of the dietary factors, most recent attention
has focused on folate intake, which is essential for metabolism of homocysteine via
the remethylation pathway, catalyzed by N,5N10-methylenetetrahydrofolate reductase
(MTHFR). Folate supplementation can lower homocysteine levels by enhancing this pathway,
even in states of mild relative deficiency, such as that due to a common thermolabile
variant (677C→T) of the MTHFR enzyme found in heterozygous form in 30–40% of the general
population and homozygous form in 10–15%.69 Because folic acid deficiency is also
associated with risk of neural tube defects, dietary supplementation on a population-wide
basis through fortification of grain products in the U.S. is now in progress; this
action may yield secondary benefits on cardiovascular disease incidence as well. Still,
the precise relation between hyperhomocysteinemia and cardiovascular disease or venous
thromboembolism remains controversial.70–73 Homozygosity for the 677C→T variant increases
the risk for hyperhomocysteinemia, which in turn increases the risk of arterial thrombosis;
but the variant by itself is not associated with arterial thrombosis in the absence
of hyperhomocysteinemia, and is not associated with venous thrombosis in any case.
As a simple point mutation (or point polymorphism), the 677C→T variant is easy to
screen for using molecular methods; however, homozygosity for this mutation accounts
for only about a third of cases of hyperhomocysteinemia. Therefore, many authorities
feel plasma homocysteine measurement is more informative than molecular testing. Hyperhomocysteinemia
interacts synergistically with coexisting factor V Leiden to increase the relative
risk of venous thrombosis to 20-fold greater than in individuals without either risk
factor.54
Recommendation 4
Patients testing positive for factor V Leiden or APC resistance should be considered
for molecular genetic testing for the most common other thrombophilias with overlapping
phenotype for which testing is easy and readily available. At present, only the prothrombin
20210A variant fits these criteria. It is present in 1–2% of the general population,
its involvement in venous thromboembolism is well-established, and the DNA test is
as simple as that for factor V Leiden (with which it can even be multiplexed). Protein
S, protein C, and antithrombin III deficiencies are too genetically heterogeneous
for routine molecular genetic testing, but testing by functional coagulation assays
may be considered, especially if there is a strong family history of venous thrombosis.
Hyperhomocysteinemia should be considered and tested (in most cases by measuring plasma
homocysteine levels) as another potential risk factor in those found to be positive
for factor V Leiden. Patients with classical homocystinuria are at extremely elevated
risk of thromboembolism and should probably be tested for other available thrombophilic
risk factors.
5.
Should testing for other heritable thrombophilic factors be performed simultaneously
with factor V Leiden testing?
Venous thrombosis is multifactorial, and the presence of more than one genetic risk
factor is not uncommon. It could be argued that anyone presenting for factor V Leiden
or APC resistance testing because of a thrombotic event already carries a risk factor
for recurrent thrombosis even if found to be negative for factor V Leiden. Recurrent
thrombosis carries a significant morbidity and mortality and is readily prevented
by oral anticoagulant therapy, though not without significant risk of bleeding events.
Therefore, it is important to identify patients at risk but to target anticoagulation
therapy to those at highest risk. Such risk stratification is possible through panel
testing of several common hereditary thrombophilic factors as well as acquired conditions
such as lupus anticoagulant and/or anticardiolipin antibody. Standard functional coagulation
assays performed on such patients are useful to detect defects in antithrombin III,
protein C, and protein S; consideration should thus be given to supplementing factor
V Leiden DNA testing with testing for prothrombin 20210A, biochemical measurement
of plasma homocysteine, and functional coagulation assays for antithrombin III, protein
C, and protein S.
Recommendation 5
Physicians ordering factor V Leiden on a venous thrombosis patient for any of the
indications recommended here should also consider the utility of functional, biochemical,
and molecular screening for other heritable thrombophilic factors, especially prothrombin
20210A and plasma homocysteine levels.
6.
Are there any other factor V mutations in addition to factor V Leiden which should
be tested?
Factor V Leiden appears to account for 90–95% of cases of APC resistance. Two rare
mutations in the factor V gene have been described and are of dubious clinical significance.
Factor V-Cambridge (R306T) is not strongly associated with venous thrombosis in controlled
epidemiologic studies.74 Factor V-Hong Kong (R306C) has been found in 1–2% of Chinese
patients but does not appear to be associated with APC resistance.75
The R2 allele (H1299R, or A4070G) of the factor V gene, associated with a haplotype
known as HR2, is present in 10% of the general population, and early studies indicate
that it increases the risk of venous thrombosis in individuals heterozygous for factor
V Leiden an additional 3-fold beyond their already 7-fold increased risk.76 Testing
for R2 in factor V Leiden heterozygotes could potentially become useful if further
larger studies support these early findings. R2 further reduces the sensitivity for
APC in factor V Leiden heterozygotes.77,78 R2 alone, without coinheritance of factor
V Leiden, neither reduces sensitivity for APC nor increases venous thrombosis risk.77,79
The R2 allele is not present in the same haplotype as factor V Leiden, so it is not
possible for factor V Leiden homozygotes to have the R2 allele.
Recommendation 6
The factor V Leiden (R506Q) mutation is currently the only molecular analysis of the
factor V gene indicated in the routine workup of thrombotic risk.
7.
What are the recommended methodologies and quality assurance standards for performing
these tests?
When performed properly using standard techniques, the factor V Leiden mutation test
has extremely low false-negative and false-positive rates, whether done by restriction
endonuclease digestion of PCR amplicons (AMP-FLPs), allele-specific PCR, allele-specific
oligonucleotide probe hybridization, or other validated manual or automated methods.
The traditional functional APC resistance test has very high sensitivity but suboptimal
specificity for factor V Leiden.
Recommendation 7
The factor V Leiden mutation test should be performed using any of the accepted technical
approaches as long as they have been properly validated by the laboratory, while adhering
to current ACMG/CAP quality assurance guidelines for molecular genetic testing.
8.
What are the appropriate pre- and postanalytic procedures to be followed in factor
V Leiden testing?
Factor V Leiden testing is well established in mainstream medicine and is used by
physicians from numerous specialties including hematology, internal medicine, primary
care, and obstetrics. It is important that the genetic implications of factor V Leiden
DNA test results be explained adequately by the health care professional conveying
the test results to the patient.
Recommendation 8
Specific informed consent should not be required for factor V Leiden testing, but
prior to testing, patients should be made aware that this is a genetic test, that
test results have implications about risk in other family members, and that there
may be attendant issues of confidentiality and possible insurance discrimination.
The laboratory’s report should state explicitly the relative risk implications for
factor V Leiden heterozygotes and homozygotes, the risk that other relatives may have
the mutation, and the recommendation, if indicated, for testing for other inherited
hypercoagulabilities.
It is important for individuals testing positive for factor V Leiden to understand
the risk implications and genetic implications of their result. Patients should be
counseled about these implications by their physician or genetic counselor.