Detection of the Janus kinase 2 V617F mutation using molecular methods

Dissertation (MSc)--University of Pretoria, 2016.In 2005, a mutation located at exon 14 of the Janus Kinase gene on chromosome 9 was discovered in patients with Polycythaenia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF). The mutation (JAK2 V617F/G1849T) causes valine to be substituted by phenylalanine at codon 617. As a result the World Health Organisation (WHO) revised the diagnostic criteria of myeloproliferative neoplasms (MPN) in 2008 to include the detection of the JAK2 V617F mutation as a major diagnostic criterion for PV, ET and PMF. Molecular assays with high sensitivity and specificity should be offered by diagnostic laboratories for this purpose. To comply with these requirements, commercial and in-house assays that offer different sensitivity and specificity levels have been developed. In addition to the performance characteristics of diagnostic assays used to detect the JAK2 V617F mutation, associated cost remains an important factor to consider when selecting the assay that is best suited to a particular laboratory. Commercial diagnostic kits are expensive which led to the development of more cost-effective in-house assays for use in routine diagnostic laboratories. The purpose of this study was to develop real time polymerase chain reaction (PCR) assays for the detection of the JAK2 V617F mutation that could be used in diagnostic laboratories. Primers and locked nucleic acid (LNA) probes were designed. DNA was extracted from 60 fresh peripheral blood specimens. Allele specific PCR (AS-PCR) was performed on 59 DNA samples while direct sequencing and real time PCR assays were performed on all 60 samples. Performance of the different molecular assays was compared. In addition, the analytical sensitivity of the LNA real time PCR assay was determined by processing specimens comprising serial dilution of homozygous mutant in wild type DNA. Allele specific PCR identified 26 of the 59 as positive for the JAK2 V617F mutation. The LNA real time PCR assay and direct sequencing both showed 24 of 60 samples to harbour the mutation. Diagnostic sensitivity and specificity of the developed real time PCR assay and sequencing assay was 88% and 100%, respectively. There was 100% agreement between the real time PCR and sequencing assays. Agreement between real time PCR and AS-PCR was calculated to be 94% with kappa value of 0.89. The developed real time PCR assay showed acceptable performance when assessed against the comparative AS-PCR method. In addition, analytical sensitivity of 0.1% was demonstrated for this assay. The findings confirm the suitability of the developed LNA probe real time PCR assay to detect the JAK2 V617F mutation in a clinical laboratory.HaematologyMScUnrestricte