Clinical Chemistry 45: 41-46, 1999;
(Clinical Chemistry. 1999;45:41-46.)
© 1999 American Association for Clinical Chemistry, Inc.
Automated Detection of the Factor V Leiden Mutation Using the LCx Microparticle Enzyme Immunoassay
Mathilde Hunault1,
Camile Marsh-Scott2,
Cynthia Jou3,
Ron Marshall3,
Christi Scheffel3,
Louis D. Fiore4,
Kenneth A. Bauer1 and
Michael J. Mitchell2,a
1
Hematology-Oncology Section, Brockton-West Roxbury Veterans Affairs Medical Center, Brockton, MA 02410.
2
Division of Laboratory Medicine, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, MA 02215.
3
Abbott Laboratories, Molecular Diagnostics, Abbott Park,
IL 60064.
4
Hematology-Oncology Section, Boston Veterans Affairs
Medical Center, Boston, MA 02130.
a Address correspondence to this author at: Department of Hospital Laboratories, University of Massachusetts Medical Center, 50 Lake Avenue North, Worcester, MA 01655. Fax (508) 856-1537; e-mail Michael.Mitchell{at}banyan.ummed.edu.
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Abstract
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The factor V Leiden mutation, a G
A transition at position 1691 in
exon 10 of the gene that codes for factor V, produces an
Arg506Gln substitution and is the most common genetic risk
factor for venous thrombosis. We have developed a rapid, sensitive, and
specific method to detect the factor V Leiden mutation in genomic DNA
from whole blood by PCR amplification and microparticle enzyme
immunoassay detection using the Abbott LCx instrument. We compared this
automated method with the standard procedure using restriction
endonuclease digestion of PCR products followed by gel electrophoresis
in blinded experiments. In 130 patients (from Veterans Affairs medical
centers) with deep venous thromboses, including 24 heterozygotes with
the factor V Leiden mutation, there was complete agreement between the
two methods. The assay was also able to distinguish heterozygotes from
homozygotes. This method, which carries a low potential for
cross-contamination of samples, should be a useful routine test for the
factor V Leiden mutation in clinical laboratories with sufficient
demand for molecular diagnostic assays using the LCx instrument.
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Introduction
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The factor V Leiden mutation, a GA transition at position 1691
in exon 10 of the gene that codes for factor V, produces an
Arg506Gln substitution (1). The
Arg506Gln substitution eliminates one of the three sites in
the factor Va molecule that are cleaved by activated protein C
(2) and is responsible for almost all hereditary cases of
activated protein C resistance. Studies have shown that heterozygosity
for the factor V Leiden mutation confers a 3.5- to 8-fold increased
risk for an initial episode of deep venous thrombosis or pulmonary
embolism as compared with genetically unaffected patients
(3)(4). Homozygosity confers an 80-fold
increased risk for venous thrombosis (5). The prevalence of
this mutation in general Caucasian populations varies between 1% and
8.5%. The mutation is apparently not present in African, Chinese,
Japanese, or Native American populations (6). Among patients
with an initial episode of deep venous thrombosis or pulmonary
embolism, the prevalence of factor V Leiden is between 12% and 50%,
whereas <10% of patients have hereditary deficiencies of antithrombin
III, protein C, or protein S. The factor V Leiden mutation is,
therefore, the most common genetic risk factor found in thrombosis
patients.
Although many methods are available to detect the factor V Leiden
mutation, an automated assay procedure suitable for use in the routine
clinical laboratory would be desirable. We developed a rapid,
sensitive, and specific method for the factor V Leiden mutation, using
PCR amplification and microparticle enzyme immunoassay
(MEIA)1
detection, using the commercially available LCx instrument
(Abbott).
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Materials and Methods
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patients
One hundred and thirty male veterans from 21 Veterans Affairs
medical centers with at least one episode of deep venous thrombosis or
pulmonary embolism occurring between 1990 and 1995 were included in
this study. Their mean age was 63 years. Thromboembolism was
objectively confirmed by at least one of the following tests: positive
impedance plethysmography, venous duplex ultrasound, venogram, and
positive pulmonary angiogram, or a high-probability lung scan with
appropriate clinical history. Patients were ineligible if their factor
V Leiden genotype was known; if the thrombosis was precipitated by
surgery, immobilization, or malignancy; or if they were known to have a
lupus anticoagulant/antiphospholipid antibody syndrome or a deficiency
of antithrombin III, protein C, or protein S.
informed consent
Informed consent was obtained from all patients. The study was
approved by the Human Studies Committee of Brockton-West Roxbury
Department of Veterans Affairs Medical Center and Beth Israel Deaconess
Medical Center (Boston, MA).
dna isolation
Blood was collected by atraumatic venipuncture into tubes
containing 0.129 mol/L buffered trisodium citrate. The ratio of blood
to anticoagulant was 9:1 (by volume). Cellular elements were stored at
-80 °C after centrifugation at 4 °C for 15 min at
2000g. Genomic DNA from the 130 patients with venous
thromboses; two individuals known to be homozygous or heterozygous for
the factor V Leiden mutation, respectively; and a control subject
without the mutation was purified from leukocyte nuclei obtained from
whole blood by either phenol-chloroform extraction (103 samples)
(7), or the QIAamp Blood kit from Qiagen (27 samples)
according to the manufacturers' instructions. DNA concentrations were
measured spectrophotometrically at 260 nm.
detection of the factor v leiden mutation
Detection of the factor V Leiden mutation by PCR, restriction
digestion, and polyacrylamide gel electrophoresis.
The
oligonucleotides used for PCR (Table 1
) were synthesized on an Applied Biosystems 381A DNA
Synthesizer. To detect the factor V Leiden mutation, we modified the
restriction endonuclease method described by Gandrille et al.
(8), using the restriction enzyme HindIII. A
125-base pair (bp) fragment of exon 10 of the gene that codes for
factor V was amplified with an oligonucleotide introducing a cleavage
site for HindIII in the allele bearing the mutation, which
leads to the appearance of fragments of 33 and 92 bp. An 8-µL aliquot
of the PCR reaction mixture was digested with 0.3 U of
HindIII (New England Biolabs) at 37 °C for 16 h. The
products were then subjected to electrophoresis under nondenaturing
conditions in an 8% polyacrylamide gel containing 90 mmol/L
Tris-borate, pH 8.3, and 2 mmol/L EDTA. After completion of
electrophoresis, gels were stained in 0.5 mg/L ethidium bromide for 15
min and photographed under ultraviolet transillumination.
Detection of the factor V Leiden mutation by PCR and LCx MEIA.
Two adamantane-labeled oligonucleotide primers adapted from Ridker et
al. (4) (Table 1
) were used to amplify a 223-bp fragment,
which included the last 136 bp of exon 10 and the first 87 bp of intron
10. PCR amplifications were performed using a DNA Thermal Cycler
(Perkin-Elmer Cetus) (9). The reaction mixtures contained 50
ng of genomic DNA, 5 U of Taq DNA polymerase (Perkin-Elmer Cetus), 0.25
µmol/L of each adamantane-labeled oligonucleotide primer (Abbott),
150 µmol/L of each dNTP, 1 mmol/L MgCl2, 10 mmol/L
Tris-HCl, pH 8.3 (at 25 °C), 50 mmol/L KCl, and 0.01 g/L of
autoclaved gelatin. In addition, each reaction mixture contained 5
nmol/L of either the wild-type
(5'-GACAGGCGAGGAA-Biotin-TTTTTT-Biotin-3') or the mutant
(5'-GACAGGCAAGGAA-Biotin-TTTTTT-Biotin-3') biotinylated
probe, which had annealing temperatures 15 °C lower than the
adamantane-labeled primers. A polyT linker was introduced between the
two molecules of biotin to facilitate streptavidin capture in the MEIA.
Although both PCR primers were labeled with adamantane, it would have
been sufficient to label only the primer used to amplify the DNA strand
complementary to the biotin-labeled probe. Each sample was subjected to
a first cycle of 4 min denaturation at 97 °C, followed by 35 cycles
of denaturation (1 min at 94 °C) and annealing (1 min at 64 °C).
Single-stranded PCR product was obtained by a 5-min denaturation step
at 97 °C in the last cycle. The biotinylated probes in the samples
were preferentially annealed to single-stranded PCR product by rapid
cooling to 15 °C and incubation for 5 min. When convenient, samples
could be left overnight at 12 °C. PCR reactions were run in
duplicate for each probe type in a volume of 200 µL.
After PCR, the unopened tubes were transferred to the automated LCx
instrument for detection of the amplified product or amplicon by MEIA
(10)(11). Biotin-labeled probes that hybridized
to the wild-type or mutant single-stranded PCR product were captured on
microparticles coated with streptavidin. After extensive washing, the
captured probe-amplicon complex was detected by an
anti-adamantane-alkaline phosphatase conjugate that, in the presence of
the substrate methylumbelliferyl phosphate, generates a fluorescent
product at a rate proportional to the amount of captured PCR product.
The rate of increase of fluorescence is quantified by the LCx and
expressed as counts per second (cps)/s. The LCx can accommodate up to
24 patient specimens in a single analytical run. If the time for sample
preparation is included, results can be obtained in ~5 h, including
90 min for DNA extraction, 120 min for PCR amplification, and 60 min
for MEIA detection. The different steps of the MEIA method are
summarized in Fig. 1
.
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Figure 1. Schematic representation of the LCx MEIA after PCR
amplification.
PCR products spanning 223 bp of the factor V gene containing nucleotide
1691 were obtained using adamantane-labeled primers. After completion
of the PCR reaction, double-stranded products are denatured, and the
single adamantane-labeled strands hybridize with biotinylated probes
specific either for the wild-type or the mutant sequence. The
single-stranded wild-type PCR product that had annealed to the
biotinylated wild-type probe is then captured on streptavidin-coated
microparticles. The captured probe-amplicon complex is detected by an
anti-adamantane-alkaline phosphatase conjugate that, in the presence of
an alkaline phosphatase substrate (methylumbelliferyl phosphate;
MUP), generates a fluorescent product at a rate
(count/sec/sec) proportional to the amount of captured
amplicon. The single-stranded PCR product carrying the mutation does
not hybridize to the wild-type biotinylated probe (Normal
probe); it therefore is not linked to the microparticles coated
with streptavidin and is washed away during the extensive washing step,
generating fluorescence no greater than the background.
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Results
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optimization of reaction conditions
We first performed experiments to define PCR conditions that would
readily distinguish heterozygosity and homozygosity for the sequence
encoding factor V Leiden from the wild-type sequence. In patients with
and without the factor V Leiden mutation, optimal conditions were
defined by high and low rates of fluorescence with the mutant probe,
respectively, and conversely low and high fluorescence rates with the
wild-type probe, respectively. The experimental conditions evaluated
included number of PCR thermal cycles (30–40), annealing temperature
(55, 60, 62, and 64 °C), annealing time, Mg2+
concentration (0.5–4 mmol/L), and dNTP concentration (63–250
µmol/L). The best results were obtained using 35 cycles with 1-min
annealing at 64 °C, 150 µmol/L of each dNTP, and 1 mmol/L
Mg2+ (data not shown), and these conditions were used to
analyze all patient samples.
We also determined the lowest amount of genomic DNA that could be used
in the PCR reaction. No detectable amplification was observed when 1 ng
was used, but PCR product was detectable with as little as 5 ng; the
fluorescent signal reached a plateau at 50 ng (Fig. 2
). Similar results were obtained with the mutant probe (data not
shown); therefore, all additional experiments were performed with 50 ng
of genomic DNA.
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Figure 2. MEIA fluorescence signal relative to the amount of DNA in
the PCR reaction.
DNA from a subject without the mutation was used with the wild-type
probe. The detection of the fluorescent product in the MEIA assay is
expressed in cps/s. The error bars represent the SD of five
independent reactions performed in duplicate.
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meia results in patients with known factor v leiden status
Using the wild-type and mutant biotinylated probes and the
conditions described previously, we were able to accurately
differentiate patients who were heterozygous or homozygous for the
factor V Leiden mutation from a control who did not have the mutation
(Fig. 3
). The wild-type probe specifically hybridized with the
adamantane-labeled PCR product from the wild-type gene sequence and
gave a high fluorescent signal for a single patient without the
mutation (840 ± 175 cps/s, n = 10 determinations or two
independent PCR reactions performed in 5 separate assays) and for a
single heterozygote (550 ± 163 cps/s) with the factor V Leiden
mutation. However, the wild-type probe did not hybridize to the mutant
sequence, as can be seen in a homozygous patient, in whom the
fluorescent signal was equivalent to background obtained without PCR
product (21 ± 3 vs 18 ± 3 cps/s). Conversely, the
biotinylated mutant probe produced a high signal with amplified DNA
from homozygous (847 ± 260 cps/s) and heterozygous patients
(635 ± 235 cps/s) without hybridizing to the PCR product from the
patient without the mutation (24 ± 12 cps/s). We observed an SD
of ~30% in the fluorescent signals obtained for each patient;
however, the genotype of an individual patient was always readily
apparent when both the wild-type and the mutant probes were used. As a
system control, a sample from a known Factor V Leiden heterozygote was
included in each analytical run. The MEIA signals for identical samples
within a single assay were virtually identical (data not shown).
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Figure 3. Detection of homozygosity and heterozygosity for the
factor V Leiden mutation with wild-type (open column) and
mutant biotinylated probe (striped column) by the LCx MEIA
method.
The results obtained in an individual without the mutation
(Normal) and the background signals in the absence of DNA
are also shown. The error bars represent the SD of 10
determinations or two independent PCR reactions performed in five
separate assays.
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comparison of factor v leiden mutation detection by meia and
restriction enzyme digestion
We compared the MEIA LCx assay with a standard method in blinded
experiments, using restriction enzyme digestion and polyacrylamide gel
electrophoresis. Of the 130 samples from a cohort of patients with deep
venous thromboses, 24 were heterozygous for the factor V Leiden
mutation (18.5%), and 106 did not have the mutation, as determined by
restriction enzyme digestion and polyacrylamide gel electrophoresis.
With the LCx MEIA, we successfully identified the same 24 patients
heterozygous for the factor V Leiden mutation and the 106 patients
without the mutation. The background signals (negative control),
resulting from the detection of wild-type or mutant biotinylated probe
without any added genomic DNA were 18.1 ± 3.4 cps/s and 18.5
± 3.6 cps/s, respectively (mean ± SD, n = 130). In patients
without the mutation, the signals were high with the wild-type probe
(694 ± 256 cps/s, n = 106), but similar to background with
the mutant probe (21.2 ± 3.5 cps/s; Fig. 4
). Heterozygous patients were easily identified by intermediate
signals with both probes (480 ± 170 cps/s with the wild-type
probe and 598 ± 187 cps/s with the mutant probe, n = 24).
None of the patients in this cohort was homozygous for the factor V
Leiden mutation. The results of MEIA were therefore in perfect
agreement with the standard method, leading to a sensitivity and a
specificity of 100%.
To extract genomic DNA from leukocytes in blood, we used
phenol-chloroform extraction or the Qiagen kit. The magnitude of the
MEIA signal in patients without the mutation, using the wild-type
probe, were somewhat higher after phenol-chloroform extraction (after
adjusting the amount of added genomic DNA to 50 ng for the PCR
amplification; 727 ± 254 cps/s, n = 85) than with the Qiagen
kit (without measuring the amount of added DNA; 561 ± 222 cps/s,
n = 21). This difference was also observed in heterozygous samples
[612 ± 176 cps/s and 519 ± 162 cps/s (n = 18) when
phenol-chloroform was used, compared with 557 ± 232 cps/s and
362 ± 144 cps/s (n = 6) when the Qiagen kit with the mutant
and wild-type probes was used]. In Fig. 4
, one sample extracted with
the Qiagen kit was an outlier with lower, but clearly positive signals,
when either the wild-type or the mutant probe was used (99 and 150
cps/s, respectively), which suggested heterozygosity for the factor V
Leiden mutation. Increasing the amount of DNA for this sample increased
the signals to 152 and 455 cps/s with the two probes, suggesting that
the yield of DNA in this specimen was low. All other DNA specimens
prepared with the Qiagen kit gave signals >200 cps/s with both probes.
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Discussion
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Several methods are available for the detection of the factor V
Leiden mutation. Screening can be accomplished by performing a clotting
assay on patient plasma diluted in factor V-deficient plasma; the
presence of the mutation is suspected if there is inadequate
prolongation of the activated partial thromboplastin time after the
addition of exogenous activated protein C, compared with the baseline
activated partial thromboplastin time (12)(13).
Samples testing positive are then confirmed genetically. The initial
report of the factor V Leiden mutation used PCR amplification of the
region of the factor V gene containing the base substitution followed
by restriction digestion with MnlI and gel electrophoresis,
or dot-blot hybridization of the PCR product with biotinylated
wild-type or mutant oligonucleotide probes (1). Other
PCR-based assays have used primers that introduce a NlaIII
(14) or Hind III (8) restriction site
when the mutation is present or a TaqI site when the
wild-type sequence is present (15). PCR amplification using
sequence-specific primers (allele-specific PCR) avoids the need for a
lengthy restriction enzyme digestion followed by a potentially long
electrophoresis step to separate products of similar and low molecular
size (16)(17); the specificity of such methods
may, however, be lost if the discriminating 3' end is degraded in any
way. Mutation detection methods using single-strand conformation
polymorphism (18)(19) or heteroduplex technology
analysis (20) have also been developed. The above methods,
however, are not suitable for the routine clinical laboratory because
they are relatively time-consuming, require technical skills such as
electrophoresis, and do not lend themselves to the analysis of one or
only a few samples at the same time.
A few attempts have been made to produce automated assays.
Allele-specific PCR combined with analysis by capillary electrophoresis
led to the development of a nonradioactive assay that is reproducible
and semiautomated (21). An ELISA-based oligonucleotide
ligation assay (22) and colorimetric minisequencing assay
(23) could both be automated, but require working with
microtiter plates with the attendant risk of sample
cross-contamination.
The Abbott LCx system for the detection of amplicons after PCR or
ligase chain reaction amplification is in routine use in clinical
laboratories for the detection of pathogenic microorganisms like
Neisseria gonorrhoeae (24), Chlamydia
trachomatis (25), or GB virus C (26). This
system uses MEIA for signal detection after PCR and has not been used
previously to identify point mutations in human genomic DNA.
We have developed an assay that uses the LCx system to detect the
factor V Leiden mutation. The entire assay, including DNA extraction,
can be completed within 5 h. The procedure involves purification
of genomic DNA and PCR amplification, followed by transfer of the
closed PCR tubes to the automated LCx instrument for MEIA. The
agreement of this method with restriction enzyme digestion for the
factor V Leiden mutation is 100%. Determination of a patient's
genotype does not require professional interpretation of the data.
Homozygous patients are defined by high signals with the mutant probe
and signals equivalent to background with the wild-type probe, patients
without the mutation are identified by high signals with the wild-type
probe but signals similar to background with the mutant probe, and
heterozygous patients are identified by intermediate counts with both
probes. Because the detection method is extremely sensitive and
requires as little as 5 ng of genomic DNA per reaction, it can be
performed with DNA extracted from <200 µL of blood by either
phenol-chloroform or a commercial kit for DNA extraction. A major
advantage of this method is the low potential for cross-contamination
of samples because the reactions take place in self-contained reaction
vials.
We set up four tubes for each DNA sample, two with the biotinylated
probe specific for the wild-type sequence and two with the biotinylated
probe specific for the mutant sequence. It would, however, be possible
to initially screen patient DNA with only the mutant probe. A second
PCR reaction containing the wild-type probe could be run only for
patients who are positive with the mutant probe to differentiate
heterozygotes and homozygotes with factor V Leiden.
In summary, we have demonstrated the feasibility of using PCR in
conjunction with the LCx MEIA detection method for detecting factor V
Leiden, a frequently encountered point mutation in venous thrombosis
patients. The Abbott LCx instrument could be used to detect another
frequent prothrombotic risk factor, namely the GA 20210 mutation in
the 3' untranslated region of the prothrombin gene (27). It
is our belief that such assay procedures could be undertaken in routine
clinical laboratories provided the demand for molecular diagnostic
assays using MEIA detection was sufficient to make it cost-effective.
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Acknowledgments
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These studies were supported in part by the Medical Research
Service of the Department of Veterans Affairs and Abbott Laboratories,
Diagnostics Division. M. Hunault is supported by a grant from the
Fondation de France-Fédération Nationale des Centres de
Lutte contre le Cancer.
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Footnotes
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1 Nonstandard abbreviations: MEIA, microparticle enzyme immunoassay; bp, base pair; and cps, counts per second. 
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J. G. Evans and C. Lee-Tataseo
Determination of the Factor V Leiden Single-Nucleotide Polymorphism in a Commercial Clinical Laboratory by Use of NanoChip Microelectronic Array Technology
Clin. Chem.,
September 1, 2002;
48(9):
1406 - 1411.
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Abstract
Introduction
) or with the Qiagen kit (
). Triangles are
heterozygous patients with the mutation whose DNA has been extracted by
phenol-chloroform (
) or with the Qiagen kit (
). The
(+) represents a homozygous patient who has been included
for illustrative purposes but is not from the cohort of 130 patients
with venous thromboses.