A Rapid Multiplexed Genotyping of Hereditary Thrombophilia by SELDI-TOF Mass Spectrometer

It is known that approximately 50% of patients presenting with venous thromboembolism are associated with polymorphic SNPs in the genes for coagulation factor V (factor V Leiden), prothrombin (prothrombin G20210A), and MTHFR (C677T). Genotyping these thrombophiliac SNPs helps clinicians to properly manage patients with thrombotic disorders. In this study, we reported a novel method to rapidly genotype all three thrombophiliac SNPs in a multiplexed fashion. First, patient DNA samples were subjected to PCR reactions to amplify and extend the DNA products with masses corresponding to genotypes for each of the three targeted thrombophiliac SNPs. PCR products were then applied to Q10 anionic Protein Chips, undergoing on-chip sample enrichment and clean-up. Finally, genetic variants were genotyped by Surface-enhanced laser desorption and ionization time-of-flight (SELDI-TOF) mass spectrometry based on their masses. This method offers a rapid turnaround time of less than five hours. The analytical accuracy of each SNP genotyping result has been confirmed by DNA sequencing. Additionally, the genotype results produced by this method were validated by comparing them to the results obtained by the approved method in the clinical reference laboratory. In summary, we have developed a novel approach of multiplex genotyping for known thrombophilic SNPs by SELDI-TOF mass spectrometer. This method is fast, accurate, reproducible, and is ready for immediate application in the clinical laboratory.