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Clinical Chemistry 46: 1051-1056, 2000;
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(Clinical Chemistry. 2000;46:1051-1056.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Genotyping of Factor V G1691A (Leiden) without the Use of PCR by Invasive Cleavage of Oligonucleotide Probes

Martin J. Hessnera,1, Mary Ann Budish1 and Kenneth D. Friedman1

1 The Diagnostic Laboratories of The Blood Center of Southeastern Wisconsin, Milwaukee, WI 53201-2178.
a Address correspondence to this author at: The Blood Center, 638 North 18th St., PO Box 2178, Milwaukee, WI 53201-2178. Fax 414-937-6202; e-mail MJHessner{at}bcsew.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The factor V G1691A Leiden (FVL) mutation is the most common known hereditary risk factor for venous thrombosis.

Methods: Third Wave Technologies, Inc. (Madison, WI) has developed a new microtiter plate-based assay that does not require PCR, restriction digestion, or gel electrophoresis. This technology system, termed the InvaderTM assay, utilizes a 5' "invading" oligonucleotide and a partially overlapping 3' "signal" oligonucleotide, which together form a specific structure when bound to a complementary genomic DNA template. A thermostable flap endonuclease cleaves this structure, releasing the 5' flap from the signal oligonucleotide. Increased temperature and an excess of the signal probe enable multiple probes to be cleaved for each target sequence present without temperature cycling. The cleaved probes then direct cleavage of a secondary probe, which is 5' end-labeled with fluorescein but is quenched by an internal dye. Upon cleavage, the fluorescein-labeled product is detected using a standard fluorescence plate reader. Genotypes are determined by net wild-type/mutant signal ratio.

Results: Complete concordance was observed, after resolution of four discordances, when 1369 individuals (1264 wild type, 102 heterozygous, 3 homozygous) were FVL genotyped by both the Invader assay and by allele-specific PCR.

Conclusion: We conclude that FVL genotyping using invasive cleavage of oligonucleotide probes is a rapid and reliable alternative to genotyping by more traditional PCR-based methods.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Protein C is a serine protease, which when activated acts as an anticoagulant by degrading the procoagulant factors Va and VIIIa. Dahlback et al. (1) reported an inherited defect, termed activated protein C (APC)1 resistance, which was defined by a poor response of plasma to the addition of APC. This defect has been shown to be present in 20–60% of patients with venous thrombosis and 2–5% of the general population (2). Bertina et al. (3) demonstrated that the phenotype of APC resistance was attributable to the presence of a G->A transition at nt 1691 of the factor V gene and led to the substitution of glutamine for arginine at amino acid residue 506. This mutation, termed factor V Leiden (FVL), leads to the elimination of one of the three APC cleavage sites on factor Va (4). The factor V gene is localized on chromosome 1q21-25, possesses 25 exons, and spans ~80 kb of DNA (5)(6). Heterozygous carriers of the factor V 1691A allele have been determined to have an ~3- to 8-fold increased risk for venous thrombosis, whereas individuals homozygous for factor V 1691A have an estimated 91-fold increased risk for venous thrombosis (7)(8)(9).

The InvaderTM assay, a new mutation detection technology developed by Third Wave Technologies, Inc., has been applied to FVL G1691A genotyping (10)(11). A schematic representation of the Invader assay is shown in Fig. 1 . This microtiter plate-formatted assay uses fluorescence resonance energy transfer detection and does not require PCR, restriction digestion, or gel electrophoresis (11). The Invader assay is based on a novel linear signal amplification technology in which two oligonucleotides, a wild-type (1691G) or mutant (1691A) signal probe plus an upstream Invader probe, hybridize in tandem to a specific region of genomic DNA. Invader technology relies on the specificity of Cleavase® enzymes, a class of naturally occurring and engineered enzymes that recognize and cleave structures that form when the 3' end of an upstream oligonucleotide overlaps the hybridization site of the 5' end of a downstream oligonucleotide probe by at least one base pair (10)(12). This activity enables detection of single nucleotide mismatches immediately upstream of the cleavage site on the downstream DNA strand because mispairing leads to the formation of a noninvasive structure that the enzyme does not recognize as a substrate. Invader assays are conducted isothermally at an increased temperature to promote probe turnover, and an excess of the signal probe enables multiple probes to be cleaved for each target sequence present without thermal cycling, producing a linear increase in signal over time. Each cleavage product then serves as an Invader oligonucleotide in a secondary reaction, where it directs the cleavage of a combined labeled fluorescence resonance energy transfer probe/template construct. This secondary probe oligonucleotide is 5' end-labeled with the donor fluorophore (fluorescein), which is quenched by an internal acceptor dye (Cy3). Upon cleavage, the donor and acceptor dyes are no longer in close proximity, the quenching is abolished, and the fluorescein-labeled product is detected using a standard fluorescence plate reader. These sequential cleavage reactions produce 106–107 labeled cleavage products per target sequence per hour. Assays are read with a fluorescence plate reader, and genotypes are assigned after determining the net wild-type/mutant signal ratio for each sample.



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  		Figure 1. Schematic of Invader assay.

Single nucleotide discrimination requires a match between the primary probe (1691A or 1691G) and the target sequence at the base to be discriminated, as well as invasion of at least one base by the Invader probe. (A), formation of overlapping invasive complex between 1691A probe and 1691A template, releasing the 5' flap, which in turn serves as an invader probe in the secondary reaction, producing fluorescence. (B), the hybridization of 1691G probe to 1691A template does not generate the invasive structure recognized by the Cleavase VIII enzyme; therefore, cleavage of the primary probe does not occur. Mut, mutant; WT, wild type; F, fluorescein; Q, quencher.

To evaluate the suitability of the Invader assay for detection of the FVL mutation, we studied 1369 individuals who had been genotyped previously by allele-specific PCR (ASPCR). The results of our study demonstrate that FVL genotyping using the Invader assay is a rapid and reliable alternative to genotyping by more traditional PCR-based methods.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Collection of peripheral blood samples and DNA isolation
Peripheral blood was collected in Na2EDTA from unrelated individuals. A total of 1079 samples were drawn from apparently healthy Caucasian individuals living in the metropolitan Milwaukee area. The thrombosis patient group included 290 unrelated Caucasian individuals whose blood had been referred to the Hemostasis Reference Laboratory of The Blood Center of Southeastern Wisconsin after a first or recurrent objectively confirmed deep vein thrombosis or pulmonary embolism. All samples were numbered, unlinked, and tested anonymously. Genomic DNA was isolated from 200–250 µL of buffy coat using the QIAamp Blood Kit (Qiagen). DNA was quantified by measuring the absorbance at 260 nm using a Hitachi U2000 spectrophotometer (Hitachi) or a SPECTRAmax Plus (Molecular Devices). All DNA concentrations were adjusted to 25 ng/µL with distilled water.

Genotyping by ASPCR
Initial determination of FVL status was accomplished by subjecting samples to ASPCR analysis as described previously (13)(14)(15). Each amplified PCR product (20 µL) was analyzed by electrophoresis through 2% agarose gels stained with ethidium bromide.

Genotyping by the invader assay
All reagents for Invader genotyping were provided by Third Wave Technologies (Madison, WI), and assays were conducted in accordance with the manufacturer’s instructions. Each sample was assayed once in separate wild-type (1691G) and mutant (1691A) reactions. In each 96-well plate, there is capacity for 48 typings, including 1 wild-type, 1 heterozygous, 1 mutant, 1 no-target blank (yeast tRNA), and up to 44 samples. Target/Primary Invader Reaction Mix (5 µL; 128 g/L PEG 8000, 40 mmol/L MOPS, and 0.1 µmol/L Invader Oligo) was dispensed into each well of 96-well microplates (MJ Research); 10 µL of genomic DNA sample (250 ng) or the appropriate control was then added to the appropriate well with mixing. Mixtures were overlaid with 20 µL of clear Chill-Out 14 liquid wax (MJ Research). Samples were incubated at 95 °C for 5 min in a Perkin-Elmer GeneAmp® PCR System 9600 Thermal Cycler (PE-ABI). The temperature was lowered to 63 °C, and 5 µL of Cleavase VIII enzyme/Mg2+/Probe Reaction Mix was added to each well. This gave a reaction volume of 20 µL with final concentrations of 0.5 µmol/L wild-type or mutant probe, 0.5 µmol/L fluorescence resonance energy transfer probe, 7.5 mmol/L MgCl2, and 200 ng of Cleavase VIII enzyme. The assay plate was incubated at 63 °C for 4 h in a Perkin-Elmer GeneAmp PCR System 9600 Thermal Cycler or a water bath (Precision Scientific).

Direct measurement of fluorescence in the 96-well microtiter assay plate was accomplished using a Cytofluor Series 4000 Fluorescence MultiWell Plate Reader (PerSeptive Biosystems). The instrument settings were as follows: excitation, 485/20 nm (wavelength/bandwidth); emission, 530/25 nm (wavelength/bandwidth); gain, ~40 (such that "no-target" blanks read ~200 counts); reads per well, 30; SetTemp, 25 °C. Alternatively, fluorescence was read indirectly by stopping reactions with the addition of 100 µL of 10 mmol/L EDTA and transferring 100 µL of each reaction to a Costar solid black 96-well microtiter plate (Corning). Fluorescence was read in a CytoFluor Series 4000 Fluorescence MultiWell Plate Reader as described above with the gain set at ~60 (such that no target blanks read 200 counts).

Data analysis
The net signal counts for the wild-type (1691G) and mutant (1691A) Invader reactions of each sample were determined by subtracting the no-target blank or background counts from the total signal count. Determination of the sample genotype was based on the ratio of the net counts for the wild-type reaction to the net counts for the mutant reaction, as follows:

In cases where the net counts were equal to or less than zero, the net counts were set equal to one to rank all ratios as positive numbers and eliminate division by zero. The following ratio values, recommended by Third Wave Technologies, were used to interpret assay results:

Acceptable signal strength was defined as a net wild-type or net mutant signal count >30. Any sample that did not meet this minimum signal strength or yielded a ratio value within the equivocal range was repeated.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A total of 1369 samples were genotyped for the factor V (G1691A) Leiden mutation by both the Invader assay and ASPCR. A group of 290 unrelated Caucasian thrombosis patients whose blood had been referred to the Hemostasis Reference Laboratory of The Blood Center of Southeastern Wisconsin after a first or recurrent objectively confirmed deep vein thrombosis or pulmonary embolism was included in this evaluation. This patient group had previously been genotyped by ASPCR and was nonrandomly selected so that multiple individuals of each genotype could be evaluated with the Invader assay. Complete concordance was observed among the 245 wild-type (1691GG), 42 heterozygous (1691GA), and 3 homozygous (1691AA) individuals. Additionally, 1079 random Caucasian individuals were genotyped with both methods; 99.6% concordance was observed among 1015 wild-type (1691GG) and 100% concordance was observed among 64 heterozygous (1691GA) individuals. Four factor V 1691GG ASPCR-typed samples among the random Caucasian samples typed as 1691GA by the Invader assay; these four samples were retyped by both ASPCR and the Invader assay. Upon retesting, all samples typed 1691GG by both methods.

With the Invader assay, the factor V 1691GG, 1691GA, and 1691AA genotypes were clearly delineated with no overlap on the basis of wild-type:mutant reaction signal ratios (Table 1 ). The average net adjusted signal ratio for wild-type, heterozygous, and homozygous samples was 411, 1.25, and 0.002, respectively. A narrow equivocal ratio range (>=3 to <5) is used to avoid any ambiguity between heterozygous and wild-type genotypes, and samples generating ratios within this range must be retyped. In this study, seven samples (0.5%) were classified as equivocal; upon retesting, they generated ratios >5 and therefore were correctly typed as wild-type (1691GG). Additionally, 16 (1.2%) samples generated invalid results because of unacceptable signal strength, defined as net wild-type and mutant counts <=30. These 16 samples were repeated from the same DNA preparations, and all were successfully typed as wild-type. Overall, >98% (1346 of 1369) of the samples were evaluable and yielded the correct genotype after a single analysis.


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  		Table 1. Summary of FVL genotyping with the Invader assay.

Among the 1079 randomly genotyped Caucasian individuals, we observed a 3.0% factor V 1691A allele frequency (64 1691A alleles in a total of 2158 alleles), which is consistent with the reported 2–8% allele frequency among apparently healthy European and American populations of European descent (16)(17).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ASPCR is a powerful technique for the discrimination of alleles arising from single or multiple base substitutions. Over the past 10 years, this technique has been used by our laboratory for the typing of the numerous hematologic antigen systems, HLA, as well as genetic prothrombotic risk factors, including factor V G1691A Leiden, prothrombin G20210A, and methylene tetrahydrofolate reductase C677T (18)(19)(20)(21)(22)(23). Recently, an Invader assay has been developed by Third Wave Technologies to detect FVL and is the first commercially available diagnostic kit for this mutation. In this study, we evaluated this new assay by typing 1369 samples that previously had been typed by ASPCR.

Complete concordance was observed between ASPCR and Invader factor V genotyping among the 245 wild-type (1691GG), 42 heterozygous (1691GA), and 3 homozygous (1691AA) thrombosis patients. An additional 1079 random Caucasians were genotyped with both methods. Included in the random Caucasian group were 64 heterozygous (1691GA) individuals, for whom we observed complete concordance between the two methods. Among 1015 wild-type (1691GG) random Caucasians, a 99.6% (1011 of 1015) concordance rate between ASPCR and the Invader assay was observed in initial testing. Four samples in this set that had typed 1691GG by ASPCR typed 1691GA by the Invader assay. Retyping of these four samples by both methods was concordant, with all genotypes being unequivocally 1691GG. These four false-positive results, each observed in a different assay run, may have been the result of technical error during assay set-up. False-negative results were not observed with the Invader assay; however, the assay does not possess an internal positive control in its current format, so it is possible that a heterozygous sample could be mistyped as 1691GG or 1691AA if template or reagents are not properly dispensed into both reaction wells.

The net 1691G/1691A signal ratio is used by the Invader assay to establish genotypes. This approach reduces variations attributable to DNA quantity and quality, as well as variability between assays. We found that the Invader assay clearly differentiated factor V 1691GG, 1691GA, and 1691AA genotypes with no overlap in net signal ratios when the cutoff values provided by Third Wave Technologies were used. In this study, seven samples (0.5%) were classified as equivocal (ratio range, >=3 to <5), which upon retesting generated ratios >5 and were correctly typed as wild-type. The equivocal ratio range is useful to avoid any ambiguity between heterozygous and wild-type genotypes, although in general, we observed a large difference between factor V 1691GG and 1691GA genotype signal ratios (Table 1Up ). Likewise, heterozygous and 1691AA genotypes were also clearly differentiated; however, the manufacturer is now recommending that a second equivocal zone between heterozygous and homozygous genotypes be used. Among samples with the same genotype, some variation in signal strength was observed, presumably because of differences in template quality. A reaction signal of <30 was observed in 16 samples (1.2%) and required repeat analysis using the same DNA isolation; all 16 samples ultimately generated robust signals and typed concordantly with ASPCR. We also observed that 250 ng of genomic DNA per reaction was sufficient to generate robust reaction signals, which is consistent with the DNA requirements (>70 ng/reaction) of the Invader assay reported recently by Ryan et al. (11). The use of 250 ng of genomic DNA per reaction was selected purely for convenience because we routinely adjust all DNA to a concentration of 25 ng/µL and the Invader assay has capacity for the addition of 10 µL of sample.

Invader technology offers several advantages over traditional PCR-based technologies for the detection of point mutations. Invader technology offers a distinct specificity advantage over assays based solely on hybridization in that the Invader assay requires specific hybridization of two oligonucleotides to form the substrate structure recognized by the Cleavase VIII enzyme. Furthermore, many clinical laboratories may not possess the expertise to develop in-house assays or the proper facilities to conduct PCR-based genotyping. In the Invader assay, no target copies are generated; therefore, separate pre- and postreaction areas are not required, nor are special procedures needed to minimize product carryover. Additionally, the Invader assay does not require gel electrophoresis, requires <90 min of actual "hands-on" time, and results are available within 1 working day. Finally, the microtiter plate format makes possible the simultaneous analysis of multiple analytes for each patient sample.

Evidence continues to accumulate indicating the polygenic basis for inherited thrombotic tendency, e.g., the prothrombin G20210A mutation and possibly the methylene tetrahydrofolate reductase (MTHFR) C677T mutation (24)(25)(26). The ability to easily test patients genetically for multiple prothrombotic risk factors is becoming increasingly important. Numerous studies have revealed that certain prothrombotic risk factors, especially factor V 1691A and prothrombin 20210A, appear to work cooperatively with one another and that patients possessing multiple risk factors are at higher risk of venous thrombosis (27)(28)(29)(30). If Invader assays for the detection of multiple genetic mutations are designed to be performed under identical reaction conditions, then simultaneous testing of patients at multiple genetic loci will be possible. Invader oligonucleotides designed to use the same technology platform, reaction conditions, and instrumentation for detection of the prothrombin G20210A and MTHFR C677T are currently under development and are being evaluated by our laboratory.

In conclusion, the Invader platform is suitable for direct detection of the factor V G1691A Leiden mutation from genomic DNA and provides laboratories with an alternative to genotyping methods that require PCR.


   Footnotes
 
1 Nonstandard abbreviations: APC, activated protein C; FVL, factor V Leiden; and ASPCR, allele-specific PCR.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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