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Technical Briefs |
1 Istituto Clinico Humanitas, Rozzano, Italy;2 Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy;3 Dipartimento di Chimica e Biochimica Medica, Università di Milano, Segrate, Italy;
aaddress correspondence to this author at: Istituto Clinico Humanitas, via Manzoni, 56, 20089 Rozzano, Italy; fax 39-02-82244790, e-mail annalisa.verri{at}humanitas.it
Venous thrombotic events are quite common; they affect 
1 in every 1000 persons per year and have a lifetime clinical prevalence of 5%. The pathogenesis of venous thrombotic events is complex, involving the interaction of acquired risk factors with some genetic predisposition.
A wide array of methods and technologies have been used for screening of prothrombotic mutations (1)(2)(3)(4). However, it is known that when mutation detection methods other than direct sequencing are used to identify a particular sequence change, there is always some risk that other sequence alterations occurring at the recognition site could lead to allele misclassification.
This issue has been discussed, for example, for the silent A1692C polymorphism in the factor V gene, which is erroneously identified as factor V Leiden by restriction enzyme digest detection (5). Other genotyping methods could also be affected by adjacent sequence alterations, including allele-specific amplification (6), single-strand conformational polymorphism analysis (7), oligonucleotide ligation (8), heteroduplex analysis (9), and methods based on melting curve analysis, in which unexpected results should be clarified definitively by sequencing (10).
Although DNA sequencing is still considered the "gold standard" for characterizing specific nucleotide alterations and improved technology has made automated DNA sequencing available to the clinical molecular diagnostics laboratory, DNA sequencing remains too expensive and time-consuming for most applications.
Recent studies demonstrating the robustness and speed of pyrosequencing technology, as well as its possible use for multiplex genotyping (11), have led to its use in an increasing range of genetic research areas (12)(13)(14)(15).
The aim of our work was to establish a multiplex protocol for direct pyrosequencing analysis of a panel of coagulation factors mutations: the 3 single-nucleotide polymorphisms (SNPs) most commonly associated with thrombophilia—G1691A in factor V Leiden, G20210A in factor II, and C677T in methylenetetrahydrofolate reductase (MTHFR)—for a first-tier screening, and 3 additional polymorphisms—A1298C in MTHFR, Val34Leu in factor XIII, and 4G/5G in plasminogen activator inhibitor-1 (PAI-1)—which are believed not to have an independent effect on venous thrombosis but could be investigated in a second-tier screening because they may act synergistically with the previously mentioned factor mutations (16)(17) or, in the case of factor XIII Val34Leu, exert a protective effect (18).
We used pyrosequencing to genotype 100 individuals, previously analyzed by LightCycler (Roche) or by direct sequencing, for all 6 polymorphisms. Pure genomic DNA from EDTA-anticoagulated blood was isolated either by use of the semiautomated Magna Pure instrument with the Magna Pure LC DNA Isolation Kit (Roche) or manually, with the High Pure PCR Template Preparation Kit (Roche). These extraction procedures gave the same yield and PCR performance.
The 6 genomic segments containing the SNPs of interest were amplified in triplex PCR reactions with 3 pairs of primers (Eurogentec; see Table 1
).
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PCR conditions were optimized in preliminary experiments in which amplified products were analyzed by electrophoresis on agarose gel (4%) to optimize template concentration, magnesium concentration, and number of cycles to enhance PCR yield and specificity, which improve the success of the sequencing reaction.
Optimal PCR conditions for triplex amplification of factor V Leiden, factor II G20210A, and MTHFR C677T were as follows: 10 ng of pure genomic DNA, 10 pmol of each primer, 200 µM each deoxynucleotide triphosphate (dNTP), 3 mM MgCl2 and 0.5 U of HotGoldStar Taq polymerase (Eurogentec) in 20 µL (final volume) of the buffer supplied (Eurogentec). Thermal cycling started with 10 min at 95 °C followed by 35 cycles of 95 °C for 30 s, 59 °C for 30 s, and 72 °C for 60 s, with a final step at 72 °C for 5 min.
Triplex amplification of factor XIII Val34Leu, PAI-1 4G/5G, and MTHFR A1298C was performed with the following conditions: 10 ng of pure genomic DNA, 4.5 pmol of factor XIII primers, 25 pmol of MTHFR primers, 9 pmol of PAI-1 primers, 200 µM each dNTP, 1.5 mM MgCl2, and 0.5 U of HotGoldStar Taq polymerase in 30 µL (final volume) of the buffer supplied (Eurogentec). Thermal cycling started with 10 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 58 °C for 45 s, and 72 °C for 60 s, with a final step at 72 °C for 5 min.
At the end of PCR, 20–30 µL of each biotinylated PCR products was immobilized on 3 µL of streptavidin-coated Sepharose beads (Amersham Biosciences) to obtain single-stranded DNA suitable for sequencing. The immobilization, denaturation, washing, and primer annealing steps were performed with a vacuum preparation workstation according to the manufacturer’s instructions (Biotage AB).
The single-stranded biotinylated PCR products were subjected to a multiplex minisequencing reaction on a PSQ96MA instrument (Biotage AB) to interrogate 3 polymorphic loci simultaneously. The multibase reading capability of pyrosequencing facilitates optimal positioning of the sequencing primers (Table 1
). Sequencing was performed as described previously (19). The dispensation order to analyze the 3 sequences at the same time was selected by use of the SNP Entry module of the SNP Analysis Software (Biotage AB).
Parallel processing of 96 samples markedly reduced the handling time and pipetting steps needed. The maximum throughput was limited only by the number of PCR reactions because the multisequencing reaction lasted only 23 min for each 96-well microtiter plate. In addition, the results from the 96 completed sequencing reactions were analyzed by the pyrosequencing software in 2 min and were displayed as shown in Fig. 1
and in Fig. 1
of the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue7/. Sequences are determined by computer-automated comparison of predicted patterns with raw data.
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Generally, samples did not require time-consuming manual interpretation. Failure to make a genotype call at the first attempt was infrequent (5%) and was mostly attributable to insufficient signal-to-noise ratios caused by poor PCR amplification. The accuracy, robustness, and reproducibility of the assay were very high: 100% of the results obtained with pyrosequencing (Table 1 of the online Data Supplement) were confirmed by LightCycler or sequencing analysis.
In conclusion, this method is rapid and cost-effective when compared with traditional sequencing; it is also suitable for the present challenge of high-throughput SNP genotyping: including all reagents and PCR, the cost per sample was 3 
. This study shows, as has been shown previously, how a technology originally introduced into the field of basic biomedical research can be successfully adapted to the clinical laboratory.
Acknowledgments
We are grateful to Biosense for supplying the instrument and reagents and, in particular, to Dr. Elvira Meroni for excellent technical support. This work was partially supported by MURST-FIRB Grant RBAU01LSR4_001 (to F.F.).
References
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) and homozygous wild type for factor V G1691A (
) and factor II G20210A (
).