Interlaboratory Development and Validation of a HRM Method Applied to the Detection of JAK2 Exon 12 Mutations in Polycythemia Vera Patients

Abstract

BackgroundMyeloproliferative 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.Methods/FindingsFor 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.ConclusionThe assay we have developed is thus a valid method, adapted to routine detection of JAK2 exon 12 mutations with highly reproducible results.

The JAK2 V617F mutational status and allele burden may be related with the risk of venous thromboembolic events in patients with Philadelphia-negative myeloproliferative neoplasms

“…groups followed by the same letter do not differ statistically at Pb0.05. * the presence of JAK2 exon 12 mutations were confirmed in 3 JAK2 V617F-negative patients [85].…”

Frequency and Molecular Characteristics of Calreticulin Gene <b><i>(CALR)</i></b> Mutations in Patients with <b><i>JAK2</i></b>-Negative Myeloproliferative Neoplasms

“…Assessment for the presence of the JAK2 V617F mutation was conducted with a tetra primer amplification refractory mutation system (described previously [1]) and confirmed by quantitative allele-specific RQ-PCR standardized by MPN&MPNr EuroNet [6]. JAK2 exon 12 and MPL exon 10 mutations were detected using high-resolution melting (HRM) analysis with previously reported assays [7,8]. The CALR mutation search was done with PCR and subsequent nonfluorescent capillary electrophoresis on QIAxcel (QIAGEN) and simultaneously with HRM on Rotorgene Q (QIAGEN), using the primer set published by Klampfl et al [4].…”

How I treat polycythemia vera

“…Exon 12 mutations can be demonstrated in granulocyte DNA by high-resolution melting analysis (sensitivity, 5-10%) [36][37][38] and standard sequencing (sensitivity, 15-20%), 39 but in some cases, the mutant allele burden is low enough to prevent detection. 16 In this patient, I stubbornly ordered the search for exon 12 mutations by nextgeneration sequencing; this approach was successful, but I admit that this is not standard procedure.…”