New real-time PCR-based method for the joint genotyping of JAK2 (V617F) with inherited thrombophilic F5 and F2 mutations J. Martínez-Serra a,c, ⁎ ,1 , E. Maffiotte b,1 , A. Gutierrez a,c , M.A. Durán a , J.C. Amat a , J. Besalduch a a Servicio de Hematología, Hospital Universitario de Son Dureta, Palma de Mallorca, C/Andrea Doria, n°. 55, 07014, Spain b Servicio de Análisis Clínico, Hospital Universitario de Son Dureta, Palma de Mallorca, C/Andrea Doria, n°. 55, 07014, Spain c Unidad de Investigación, Hospital Universitario de Son Dureta, Institut Universitari d'Investigacions en Ciències de la Salut (IUNICS) Palma de Mallorca, C/Andrea Doria, n°. 55, 07014, Spain abstract article info Article history: Received 3 July 2009 Received in revised form 10 August 2009 Accepted 9 September 2009 Available online 22 September 2009 Keywords: Jak-2 V617F Thrombosis F5 Leiden F2 Real-time PCR FRET Background: During the last years the appearance of the acquired V617F mutation of the Janus Kinase 2 gene (JAK2) in patients suffering different thrombotic events has been described. We decided to develop a new and rapid multiplex real-time Polymerase Chain Reaction (PCR) in order to detect the V617F mutation together with the inherited prothrombotic mutations of factors F5 and F2. Design and methods: The method was carried out on the LightCycler 2.0 (Roche Diagnostics, Mannheim, Germany) and consisted in a first step of simultaneous amplification by real-time PCR of the three genes to be genotyped, in a 20 μl closed tube, using a primer pair together with the correspondent FRET-hybridization probes for each gene. Results: We assayed 41 samples in the multiplex PCR reaction, 19 were positive (46.34%) for V617F mutation. From the V617F positive samples we found 1 sample heterozygous for F2 (5.26%) and 1 sample heterozygous for F5 (5.26%), so a 10.52% of the samples tested combine V617F mutation with inherited thrombophilic mutations. Results were clear, rapid and reliable allowing a significant time saving. Conclusions: The technique presented in this manuscript is a new achievement in the field of the molecular diagnosis that combines the genotyping of F5 and F2 with the assessment of the JAK2 (V617F) mutation load. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The pathogenesis of the venous and arterial thrombosis with an annual incidence in developed countries of 0.1% is multifactorial and it includes inherited and acquired causes. Among the inherited factors directly associated to this disease, two main thrombosis-related polymorphisms have been implicated by its major incidence, the factor V Leiden (FV) and the mutation G20210A of the Factor 2 (F2) or prothrombin (PT) [1–3]. During the last years the appearance of the acquired V617F mutation of the Janus Kinase 2 gene (JAK2) as a new independent thrombotic risk factor has been described. Initially this mutation was only associated to the diagnosis of Philadelphia- negative Chronic Mieloproliferative Neoplasms (Ph-CMNs) where it is present in the majority of patients developing polycythemia vera (PV) and in half of those with essential thrombocythemia (ET) and/or idiopatic myelofibrosis (IM) as an acquired mutation implicated in its pathogenesis. However recently this acquired mutation has also been associated with thrombosis in special areas like portal and mesenteric venous thrombosis [4], splanchnic venous thrombosis and suprahe- patic veins (Bud–Chiari syndrome), in patients with or without overt signs of CMNs [4–6]. This evidence clearly suggests that further investigation is necessary in order to clarify the real implication of V617F as a new independent thrombotic risk factor, and its relationship with inherited thrombotic risk factors such as F5 and F2. For all these reasons the detection of V617F mutation has become an important new acquired element in order to recognize new independent risk factor to be analyzed in patients that have developed a thrombotic event. In this context and because genotyping of F5 an F2 is mandatory to be tested in patients who have suffered thrombosis, we decided to develop a new and rapid multiplex real-time Polymerase Chain Reaction (PCR) in order to detect in a 20 μl close tube the V617F mutation in association with the inherited mutations of genes encoding for prothrombotic factors F5 and F2. With this in mind we have focused the design of this new protocol following the methodology already published by our group based in the use of asymmetric real-time PCR reaction in order to efficiently amplify the fluorescence signalling derived from the probes used in a multiplex assay. The rapid and easy multiplex PCR described in this manuscript may have an important role in order to define the real implication of the V617F acquired mutation in the development of thrombotic Clinica Chimica Acta 410 (2009) 59–63 ⁎ Corresponding author. Servicio Hematología, Hospital Son Dureta, Palma de Mallorca, C/Andrea Doria n°. 55, 07014, Spain. Tel.: +34 971175155, +34 971175000; fax: +34 971175360. E-mail address: jorgej.martinez@ssib.es (J. Martínez-Serra). 1 Both authors contributed equally to this paper. 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.09.022 Contents lists available at ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim