title: Interlaboratory development and validation of a HRM method applied to the detection of JAK2 exon 12 mutations in polycythemia vera patients.
abstract: 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.
authors.affiliation: CHU BREST, Laboratoire d'Hematologie, Brest, France.
authors.forename: Valerie
authors.initials: V
authors.lastname: Ugo
authors.forename: Sylvie
authors.initials: S
authors.lastname: Tondeur
authors.forename: Marie-Laurence
authors.initials: ML
authors.lastname: Menot
authors.forename: Nadine
authors.initials: N
authors.lastname: Bonnin
authors.forename: Gerald
authors.initials: G
authors.lastname: Le Gac
authors.forename: Carole
authors.initials: C
authors.lastname: Tonetti
authors.forename: Veronique
authors.initials: V
authors.lastname: Mansat-De Mas
authors.forename: Lydie
authors.initials: L
authors.lastname: Lecucq
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Kiladjian
authors.forename: Christine
authors.initials: C
authors.lastname: Chomienne
authors.forename: Christine
authors.initials: C
authors.lastname: Dosquet
authors.forename: Nathalie
authors.initials: N
authors.lastname: Parquet
authors.forename: Luc
authors.initials: L
authors.lastname: Darnige
authors.forename: Marc
authors.initials: M
authors.lastname: Porneuf
authors.forename: Martine
authors.initials: M
authors.lastname: Escoffre-Barbe
authors.forename: Stephane
authors.initials: S
authors.lastname: Giraudier
authors.forename: Eric
authors.initials: E
authors.lastname: Delabesse
authors.forename: Bruno
authors.initials: B
authors.lastname: Cassinat
mesh_terms: 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
chemical_list: D017931:DNA Primers; D053614:Janus Kinase 2