Peptide Nucleic Acid (PNA)-Clamping Competitive PCR: A New Tool for the High Sensitive Detection of JAK2V617F Mutation in Myeloproliferative Neoplasms (MPNs)

Myeloproliferative neoplasms (MPNs) are a group of diseases characterized by clonal expansion of single or multiple lineages of myeloid subset (i.e. granulocytic, erythroid, megakaryocytic and mast cell). Since 2008, the WHO included the detection of JAK2 mutations (the common V617F and the less common exon 12 mutations), into the diagnostic criteria of MPNs. The specific pathogenic implication of JAK2 mutations in MPNs is still under investigation. Preliminary data emerging from the treatment of Idiopathic Myelofibrosis patients with JAK2 inhibitors have shown only clinically significant benefits (improvement of splenomegaly and constitutional symptoms) but not a clear evidence of disease-modifying activity. Several techniques (e.g. ASO-PCR, ARMS-PCR, Direct sequencing, HRM, DHPLC, etc) have been used to detect the JAK2 V617F mutation, but all of them were low sensitive and time-consuming. We developed a PNA-clamping competitive PCR assay able to detect JAK2 V617F mutation with a very high level of sensitivity and specificity.

In order to promote the selective amplification of the JAK2 V617F mutant allele, a specific PNA oligonucleotide, full matching with the wild-type (wt) sequence of the portion of JAK2 gene containing the V617F mutation, was designed to compete with the reverse primer used in the PCR assay. It was experimentally demonstrated that a PNA concentration of 6 μmol/L occurred for the complete clamping of 100 ng of wt genomic DNA used as template for the PCR reaction. The sensitivity of the assay was determined by a serial dilution (100% through 0.01%) of genomic DNA containing JAK2 V617F mutation, obtained from HEL cell line, with wt DNA obtained from healthy donors. The specificity of PCR products was assessed by sequencing in both forward and reverse directions. The thermal dissociation profile of PNA/DNA and DNA/DNA duplexes was studied by monitoring the Enthalpy as function of the temperature (range 20–100°C), with an heating/cooling rate of 1°C/min.

PNA-clamping competitive PCR was able to detect JAK2 V617F mutation with a sensitivity of 0.01%. Thermodynamic studies clearly showed that the melting temperature (Tm) of fully matched PNA/DNA duplex is always higher than Tm of the corresponding DNA/DNA duplex. The enthalpy values for the hybrid PNA/DNA are always greater than the corresponding DNA/DNA duplex and a single mismatch has an energetic cost higher in a PNA/DNA than in DNA/DNA duplex. This energetic cost is more evident in the melting temperature with a ΔTm of 16°C and 7°C for the PNA/DNA and DNA/DNA duplex, respectively. In the PCR assay the PNA complementary to the JAK2 wild type sequence strongly compete with the primer resulting in a complete knock out of the wild type allele. In a blind screening of samples obtained from 308 MPN patients, PNA clamping competitive PCR was able to detect JAK2 V617F mutation in 61.4% cases of Polycythemia Vera, 54.4% of Essential Thrombocythemia, 57.9% of Idiopathic Myelofibrosis and in 21.4% of Ph negative-MPNs, respectively. In 40 unselected patients, PNA clamping PCR results were confirmed by other techniques such as ASO-PCR, ARMS-PCR and direct sequencing.

The PNA-clamping competitive PCR assay could be used as convenient, high-sensitive and reliable diagnostic test for detection of JAK2 V617F mutation both on genomic DNA and cDNA samples. As suggested from the thermodynamic studies, a similar technique could be developed for the detection of any known single point mutation, widely contributing to the improvement of molecular diagnostic tests usable in clinical practice.

No relevant conflicts of interest to declare.