Interlaboratory development and validation of a HRM method applied to the detection of JAK2 exon 12 mutations in polycythemia vera patients.
BACKGROUND
Myeloproliferative disorders are characterized by clonal expansion of normal mature blood cells. Acquired mutations giving rise to constitutive activation of the JAK2 tyrosine kinase has been shown to be present in the majority of patients. Since the demonstration that the V617F mutation in the exon 14 of the JAK2 gene is present in about 90% of patients with Polycythemia Vera (PV), the detection of this mutation has become a key tool for the diagnosis of these patients. More recently, additional mutations in the exon 12 of the JAK2 gene have been described in 5 to 10% of the patients with erythrocytosis. According to the updated WHO criteria the presence of these mutations should be looked for in PV patients with no JAK2 V617F mutation. Reliable and accurate methods dedicated to the detection of these highly variable mutations are therefore necessary.


RESULTS
For these reasons we have defined the conditions of a High Resolution DNA Melting curve analysis (HRM) method able to detect JAK2 exon 12 mutations. After having validated that the method was able to detect mutated patients, we have verified that it gave reproducible results in repeated experiments, on DNA extracted from either total blood or purified granulocytes. This HRM assay was further validated using 8 samples bearing different mutant sequences in 4 different laboratories, on 3 different instruments.


CONCLUSIONS
The assay we have developed is thus a valid method, adapted to routine detection of JAK2 exon 12 mutations with highly reproducible results.
Ugo
Valerie
V
CHU BREST, Laboratoire d'Hematologie, Brest, France.
Tondeur
Sylvie
S
Menot
Marie-Laurence
ML
Bonnin
Nadine
N
Le Gac
Gerald
G
Tonetti
Carole
C
Mansat-De Mas
Veronique
V
Lecucq
Lydie
L
Kiladjian
Jean-Jacques
JJ
Chomienne
Christine
C
Dosquet
Christine
C
Parquet
Nathalie
N
Darnige
Luc
L
Porneuf
Marc
M
Escoffre-Barbe
Martine
M
Giraudier
Stephane
S
Delabesse
Eric
E
Cassinat
Bruno
B
D001483:Base Sequence; D017931:DNA Primers; D005091:Exons; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D007753:Laboratories / Q000592:standards*; D009154:Mutation; D011087:Polycythemia Vera / Q000235:genetics*; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D053614:Janus Kinase 2