Genotyping of thrombotic risk factors by MALDI-TOF mass spectrometry

Objectives: Individual risk of thrombotic disease is highly associated with allelic sequence variations in the blood clotting factor V (F5) and factor II (F2) genes. The polymorphism (G 1691 A) in F5 causing the amino acid substitution R506Q and a genetic variation in the 3' untranslated region of F2 (G20210A) are highly prevalent in the Caucasian population. These findings raise the need for an efficient, cost-effective screening technique. Design and methods: Thrombotic risk alleles were determined by allele specific elongation and termination of extension primers. Primer extension products were analyzed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Results: Using MALDI-TOF-MS, the polymorphisms F5 (G1691A) and F2 (G20210A) were unequivocally and reliably detectable either alone or in combination (multiplex). Results were confirmed by conventional sequence analysis and, in each case, fully matched data obtained by gel-based genotyping. Conclusions: Due to its accuracy and sensitivity, this procedure can serve as an alternative method for clinical genotyping. It also possesses the potential for high throughput screening.