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Clinical Chemistry 48: 1406-1411, 2002;
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(Clinical Chemistry. 2002;48:1406-1411.)
© 2002 American Association for Clinical Chemistry, Inc.

Determination of the Factor V Leiden Single-Nucleotide Polymorphism in a Commercial Clinical Laboratory by Use of NanoChip Microelectronic Array Technology

Jess G. Evansa1 and Cindy Lee-Tataseo1

1 American Medical Laboratories, 4230 Burnham Ave., Las Vegas, NV 89119-5410.

aAuthor for correspondence. Fax 702-733-7589; e-mail Jess.Evans{at}aml.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Methods for analysis of the single-nucleotide polymorphism (SNP) known as factor V Leiden (FVL) are described. The technique provides rapid, highly accurate detection of the point mutation that encodes for replacement of arginine-506 with glutamine. After formal assay qualification, 758 clinical samples that had previously been analyzed by the InvaderTM Monoplex Assay were tested as research samples in a commercial clinical laboratory.

Methods: Primers specific for factor V (FV) were prepared, and PCR was performed. Samples were analyzed using the NanoChip® Molecular Biology Workstation with fluorescently labeled reporters for wild-type and SNP sequences.

Results: Of the 635 samples classified by the Third WaveTM assay as FV wild type, 10 were identified as heterozygous FVL by the NanoChip technique. Similarly, of the 114 putative heterozygous samples, 4 were wild type, and of the 9 reported homozygous samples, 6 were homozygous, 2 were heterozygous, and 1 was FV wild type by the NanoChip assay. All 17 results that were discordant with the Third Wave analysis were confirmed by DNA sequencing to be correctly classified by the NanoChip technology. The Nanochip system was 100% accurate in characterizing wild-type, heterozygous, and homozygous samples compared with accuracies of 99.2%, 90.2%, and 100% for the comparable Third Wave analysis.

Conclusions: The NanoChip microelectronic chip array technology is an accurate and convenient method for FVL screening of research samples in a clinical laboratory environment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hemostasis requires a delicate balance of coagulation and fibrinolysis. Perturbation of this balance can predispose to pathologic states, including thrombosis. An important component of this system is the relationship of factor V (FV)1 with activated protein C (APC). Wild-type FV serves as a substrate for APC and is inactivated after proteolytic cleavage at three different arginine residues, which is part of the normal coagulation regulatory mechanism. A common clinical manifestation, termed APC resistance, occurs when a single G-to-A point mutation replaces a critical arginine with glutamine at position 506 in the FV sequence, decreasing its susceptibility to APC cleavage. This single-nucleotide polymorphism (SNP), termed factor V Leiden (FVL) (1), has a prevalence of ~2–5% in US and European populations with wide variation among nationalities.

In wild-type FV, residues 493–506 provide factor Xa and protein S binding sites (2). Cleavage of Arg506 alone does not inactivate FV but decreases the rate at which Arg306 and, perhaps, Arg679 are cleaved (3). Cleavage of Arg306 appears to be responsible for most of the FV inactivation.

Published data (4)(5) have documented the prevalence and cardiovascular implications of the FVL mutation. The presence of the FVL mutation has been linked with increased risk for venous thrombotic events such as deep vein thrombosis and pulmonary embolism (4)(5), although the quantitative risk factors in patients with FVL vary dramatically. Rosendaal et al. (6) reported a 7-fold increase for deep vein thrombosis in heterozygotes and a 79-fold increase for homozygotes. Similarly, Hainaut et al. (7) reported 7-fold and 42-fold increases for the two groups. Although 129 of 624 patients with deep vein thrombosis were heterozygotes for FVL in a study reported by De Stefano et al. (8), the rate of recurrent thrombotic events in this group was not higher than for those patients who did not have the mutation. Molecular assessment of thrombosis risk will eventually require analysis of several point mutations and environmental factors (8)(9)(10)(11) that may interact in a complex manner. If this is the case, then analytical accuracy for each of the eventual SNP assays required is paramount in that small errors in each could contribute to a significant error in the final combined result.

Methods used to evaluate APC resistance related to FVL rely on thromboplastin clotting times in the presence and absence of APC. These tests are usually performed in the presence of FV-deficient plasma to increase specificity (12). The most commonly used and most accurate of the clotting time-based tests is the Russell viper venom test (13)(14). It is sensitive, provides a specificity of >90%, and can produce acceptable results on samples from patients on anticoagulant therapy and those containing lupus anticoagulants (13)(14). A comparison of 13 APC inhibition-based tests with DNA analysis using 80 frozen plasma samples showed that the functional tests are useful in initial screening (15). However, a subsequent study of 370 patients, using both APC resistance and molecular analysis, showed that the functional tests were useful but subject to high false-positive rates that required confirmation by molecular techniques (16).

Molecular analysis of SNPs such as FVL provides a convenient and cost-effective method for assessing the contribution of individual genetics to thrombosis. SNPs are the most common form of human sequence variation, occurring, on average, every 1000–2000 bases (17). They are very useful for human haplotype analysis and are well suited to automated, high-throughput genotyping. SNPs are much less likely to undergo additional mutation in comparison to other markers, such as microsatellites (17).

Molecular methods for detection of the FVL SNP have primarily depended on PCR to amplify genomic DNA followed by restriction enzyme digestion and mapping (PCR-restriction fragment length polymorphism analysis) and may include multiplexed techniques (18). Direct sequencing, either single pass or bidirectional, usually provides comparison for assessing the concordance of other methods. Several other methods for detection of FVL have been reported and include spectrophotometric evaluation of binding to allele-specific oligonucleotides in microtiter plates (19), an enzyme immunoassay for PCR product on a clinical analyzer (20), luciferase-linked DNA polymerase-mediated depolymerization (21), PCR-independent InvaderTM oligonucleotide-generated fluorescence (22), and a direct fluorogenic probe-based PCR assay (23). Although each method offers unique advantages, the most critical component required for any clinical application is high accuracy. The compounding of small discordant classifications in individual SNP evaluations will exponentially impact screening applications where additional or other factors, each with its own error rate, are present.

Alternatively, the method we describe is highly accurate and uses automated microelectronic array technology analysis. The NanoChip® Molecular Biology Workstation is fully automated and uses a proprietary semiconductor microchip to provide a flexible tool for the rapid identification and analysis of test samples containing charged molecules. It electronically deposits and addresses target oligomers and samples, hybridizes targets and/or reporter oligomers, and detects by fluorescence the hybridization on the NanoChip cartridge. The cartridge is composed of a 10 x 10 array of microelectrodes with a thin hydrogel permeation layer containing streptavidin, allowing binding of the biotinylated PCR amplicon. Stabilizers and either Cy3- or Cy5-labeled oligonucleotide reporters for each locus are hybridized, and the chip is then washed and imaged. The fluorescence signal ratios of the reporters allow discrimination between homozygous, heterozygous, and wild-type samples. The technology provides an open platform for flexibility in the assay design—either the amplicon down format to screen one or more patients for one or more SNPs or the capture down format to screen many patients for one or more SNPs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Except for differences in discrimination temperatures, the materials for blood DNA purification, and the PCR materials that are detailed below, all other procedures were performed as described in research application notes provided to us by Nanogen, Inc. (24)(25). AmpliTaq Gold® DNA Polymerase and AmpliTaq Gold Master Mix were obtained from Applied Biosystems. Millipore® MultiScreen®-PCR desalting plates and the associated vacuum manifold and pump were obtained from Millipore. The Gentra-PureGene reagent set was obtained from Gentra Systems, Inc. NanoChip cartridges were obtained from Nanogen.

sample preparation and analysis by the invader monoplex assay
Genomic DNA was extracted from whole blood by use of the Gentra-PureGene reagent set and submitted for clinical evaluation using the Invader Monoplex Assay (Third WaveTM Technologies, Inc.). The Invader Monoplex Assay was performed at American Medical Laboratories, using the standard protocol provided by Third Wave Technologies. Control samples for the wild type, heterozygotes, homozygotes, and no-target blanks (provided with the assay) were run with the test samples with expected results. Samples were stored at -20 °C. Results from initial testing classified the samples as 635 wild-type, 114 heterozygous, and 9 homozygous for FVL.

sample preparation and analysis by the NanoChip SYSTEM
Sample amplification.
Residual samples of DNA from blood that had been submitted for clinical evaluation using the Invader Monoplex Assay were available as research specimens for concordance studies using the NanoChip System described here. Samples were amplified by exponential PCR using AmpliTaq Gold Master Mix. The sequences for the forward (biotinylated) and reverse PCR primers flanking the SNP sequence are listed in Table 1 . During amplification, PCR reagents and template DNA were added in separate rooms to avoid contamination. In addition, a negative (no-template) control was included in the PCR amplification. The amplicons were desalted using a Millipore MultiScreen-PCR desalting plate to assure their subsequent optimal electronic manipulation and washed once with deionized H2O. Amplicons were resuspended in 200 µL of 50 mmol/L histidine, mixed by orbital shaker, and transferred to a 96-well plate.


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  		Table 1. Specific reagents used for FVL determination.

Loading the NanoChip cartridge.
A FVL Loader map file was created, and the desalted amplicons were transferred into a 96-well plate specific for the Loader. A ratio reference sample, for signal normalization, was prepared in 50 mmol/L histidine and added to empty wells in the 96-well plate. Two wells containing 0.3 mol/L NaOH and 0.1 mol/L NaOH solutions were also included on the plate. The Loader was programmed to electronically address each desalted amplicon to a specific pad on the cartridge, where the biotinylated amplicons were rapidly concentrated over a pad and bound to the permeation layer containing streptavidin. Samples were loaded in duplicate and separately analyzed.

Sample detection and analysis.
Samples genotyping was performed as an automated function on the Reader of the NanoChip System, using differences in hybridization energies of two fluorescently labeled, allele-specific reporter oligonucleotides (Table 1Up ). The difference in hybridization energy between the matched and mismatched reporters is enhanced by a stabilizer oligonucleotide (Table 1Up ) that provides base-stacking interaction to improve discrimination. The cartridge was heated to a predetermined discrimination temperature (30 °C, as opposed to the 31 °C in the Nanogen application note), washed, returned to temperature, and scanned using a two-laser system. Data were analyzed, and samples with a signal-to-noise ratio >5 were classified as wild-type, heterozygous, or homozygous for FVL (17 samples were discordant with the results from the Invader assay).

A diagrammatic representation of the oligonucleotide reporters/stabilizers used and their relationship to the PCR amplicon is shown in Fig. 1 .



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  		Figure 1. FV sequence and method-related oligonucleotides.

The FV sequence shows the relationship of PCR primers and stabilizer and reporter oligonucleotides. WT, wild type; Mut, mutant.

discordant sample analysis
Those samples determined to be discordant with the previous calls from the Invader Monoplex Assay were subjected to repeat testing on the NanoChip System and the Invader Assay (samples were tested in duplicate if sufficient DNA was available). Samples were reamplified for repeat testing on the NanoChip System and sent to Third Wave Technologies for testing using the Invader Biplex Assay. In addition, new amplicons were generated and subjected to bidirectional DNA sequencing (Sequetech Corp.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Of the 635 samples initially classified by the Third Wave assay as FV wild type, 10 were identified as FVL heterozygotes by the NanoChip technique. Similarly, of the 114 putative heterozygous samples, 4 were wild type, and of the 9 reported homozygous samples, 6 were identified as homozygous, 2 as heterozygous, and 1 as wild type by the NanoChip assay. All 17 results that were discordant with the Third Wave analysis were confirmed by DNA sequencing to be correctly classified by the NanoChip technology. The NanoChip System was 100% accurate in characterizing wild-type, heterozygous, and homozygous samples on initial testing, compared with accuracies of 99.2%, 90.2%, and 100% for the comparable Third Wave analysis. Table 2 and Fig. 2 detail the results of this study. Five additional samples were eliminated from the study because of very low DNA concentrations after the PCR. Subsequent testing using the Invader Biplex Assay produced nine concordant samples with sequencing and the NanoChip System, four equivocal or invalid samples, and four samples with insufficient DNA to perform the assay (Table 3 ).


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  		Table 2. Microelectronic array technology results for patient samples previously classified using the Invader Monoplex Assay.



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  		Figure 2. Histogram representation of discordant data (first set).

The second data set matched results from the first set. Columns represent fluorescence of reporters remaining after thermal denaturation (green, wild type; red, homozygous sequence).


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  		Table 3. Discordant results.1


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
FVL is one of the most important SNPs related to the failure to maintain hemostasis. Its detection in concert with other mutations is paramount in the study of hereditary contributions to thrombosis. As the most common cause of APC resistance, FVL has been the subject of numerous large clinical studies, and its relationship to pathologic states has been well documented. However, the role of FVL is complex in that it most likely is one of several factors that may work in concert to increase the risk of thrombotic events. Traditional activity-based assays that are useful in detecting APC resistance will continue to be of value in initial screening, but they lack the accuracy to support determination of FVL in a clinical diagnostic setting.

The NanoChip microelectronic array technology for analysis of the FVL SNP provides extremely high accuracy, readily discriminating between wild types, heterozygotes, and homozygotes, as confirmed by comparison with DNA sequencing. It is likely that molecular assessment of thrombosis risk will eventually require analysis of several point mutations that may interact in a complex manner. Even small errors encountered in the assay of an individual SNP that produce false-positive and -negative rates or misclassification of wild-type, heterozygous, or homozygous samples can have an immense impact when multiplied by the number of factors evaluated. In this study, the NanoChip system was 100% accurate on the first pass in classifying 630 wild-type, 122 heterozygous, and 7 mutant samples, compared with accuracies of 99.2%, 90.2%, and 100%, respectively, for the Third Wave method. The data document that the NanoChip microelectronic array method provides superior accuracy for the analysis of FVL samples. The data also suggest that, for certain samples, amplification of the DNA is necessary to obtain correct genotyping calls.

These research studies also demonstrate that the NanoChip system is compatible with conditions encountered in a typical commercial laboratory setting. The ability to select a minimal signal-to-noise ratio as a system suitability requirement allows the operator to quickly identify any sample analysis that might lead to a questionable call. The few failures encountered were unrelated to which analytical method was used and were the result of a failed PCR reaction. This dependence on the PCR indicates that the PCR conditions as optimized and reported in the application notes (24)(25) must be closely followed.

In conclusion, the ease with which samples are prepared and analyzed, the cost per sample, the ability to quickly assess suitability of the analyses by the signal-to-noise ratio, and the highly accurate results obtained demonstrate that this method is suited to use in a commercial clinical laboratory setting.


   Acknowledgments
 
Nanogen personnel provided initial assistance in defining specific method conditions, designing reagents, and technical review of this manuscript, for which we are grateful.


   Footnotes
 
1 Nonstandard abbreviations: FV, factor V; APC, activated protein C; SNP, single-nucleotide polymorphism; and FVL, factor V Leiden.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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