Two-Step Genetic Screening of Thrombophilia by Pyrosequencing

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 …