Determination of the factor V Leiden (G1691A) and prothrombin (G20210A) mutations by using a dHPLC system. W.L. van Heerde¹, H. Kenis¹, P. Lux¹, K. Hamulyak³, D. Wi³, J.M.L.S. Lavergne², C.P.M. Reutelingsperger¹. 1) Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht, The Netherlands; 2) INSERM U143, Paris, France; 3) Haematology Department, University Hospital Maastricht, Maastricht The Netherlands.
   During the last five years two rather common mutations were discovered with a high prevalence of venous thrombo-embolitic events. These mutations, the factor V Leiden (G1691A) and the G20210A prothrombin mutation are nowadays routinely screened in all coagulation laboratories. Several approaches have been used to determine these mutations, such as restriction enzyme analysis, Taqman and FRET technologies. Here we present an alternative methodology to determine both mutations in one single assay,by using the dHPLC system. In this system mutations can be determined on basis of the melting behaviour of heteroduplexes which elute from the column by a combination of column temperature and acetonitrile gradient. 155 subjects were included both symptomatic patients with proven venous thrombo-embolism and asymptomatic first degree relatives. Genomic DNA was amplified in a multiplex reaction and assayed on the system. To determine homozygosity all samples were also mixed with a negative control in a second run. A 100 % sensitivity was obtained for both mutations when compared to routinely screened assays. Moreover, we observed additional polymorphisms; 4 for factor V exon 10 and 2 for prothrombin 3 untranslated region. In conclusion, this alternative methodology is a reliable technique to determine both mutations in a routinely setting. This technique is fast and cost effective and can be performed on a per patient basis. Furthermore, it allows high throughput screening. Due to the presence of possible other polymorphisms we suggest to perform sequencing on positive samples. Finally, this multiplex screening can be adapted to other genes involved in thrombo-embolism.