New real-time PCR-based method for the joint genotyping of JAK2 (V617F) with inherited thrombophilic F5 and F2 mutations.
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.


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 20microl 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.
Martínez-Serra
J
J
Servicio de Hematología, Hospital Universitario de Son Dureta, Palma de Mallorca, C/Andrea Doria, n degrees . 55, 07014, Spain. jorgej.martinez@ssib.es
Maffiotte
E
E
Gutierrez
A
A
Durán
M A
MA
Amat
J C
JC
Besalduch
J
J
D017931:DNA Primers; D005165:Factor V / Q000235:genetics*; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D008722:Methods; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction / Q000379:methods*; D011516:Prothrombin / Q000235:genetics*; D012189:Retrospective Studies; D019851:Thrombophilia / Q000235:genetics*
D017931:DNA Primers; D005165:Factor V; D011516:Prothrombin; D053614:Janus Kinase 2