Unfortunately, a multicenter study reported existence of significant discrepancies in the results of JAK2 V617F mutant allele burden quantitation when blinded samples were tested across some of the most specialized health institutes, which delineated the importance of using well-defined, accurate standards to refine JAK2 quantitative assays. There have been great efforts to improve the accuracy of detecting and quantifying JAK2 V617F mutation, such as allele-specific loop-mediated isothermal amplification, addition of a wild-type JAK2 blocker (any one of a non-extendible dideoxy oligonucleotide, a locked nucleic acid, and a peptide nucleic acid) to limit amplification to be mutant-specific in a quantitative PCR (q-PCR) reaction, and restriction fragment nested AS-PCR. Among these diagnostic techniques, AS-PCR seems to be the most commonly used method and allows detection of an allele burden as low as 1% however, the obtained results are usually ambiguous. These studies highlight the importance of precise determination and quantification of JAK2 V617F mutant allele burden in afflicted individuals.Ī large number of diagnostic techniques have been applied in the detection of JAK2 V617F mutation in MPNs, including direct sequencing, pyrosequencing, denaturing high performance liquid chromatography, restriction enzyme digestion, melting curve analysis, amplification-refractory mutation system, and allele-specific polymerase chain reaction (AS-PCR). For example, MPN patients with higher allele burden of JAK2 V617F are more likely to suffer from major thrombotic events. Previous studies have demonstrated that JAK2 V617F homozygosity could drive phenotypic switch from ET to PV in mice models, and the higher allele burden in PV than in ET as seen in clinical studies is associated with unique disease phenotypes even within a specific MPN subtype. In particular, allele burden of JAK2 V617F carries important pathogenetic and clinical significances in MPNs. In regard to classical MPNs, mutations in JAK2 include JAK2 V617F hotspot mutation and exon 12 mutations. Mutations in any one of the mutually exclusive driver genes result in constitutive activation of downstream signaling cascades, which in turn leads to clonal proliferation of hematopoietic precursors and excessive production of terminally differentiated, fully functional blood cells. A hallmark of the genetic background of MPNs that constitutes the diagnostic criteria of World Health Organization (WHO) classification is MPN-restricted driver mutations, including those in Janus kinase 2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia virus (MPL). MPNs include three major clinical entities, i.e., polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). BACKGROUNDĬlassical myeloproliferative neoplasms (MPNs) are multipotent hematopoietic stem cell disorders characterized by excess production of various blood cells. The disclosure relates to a method of quantifying a mutant allele burden of a target gene by using a recombinant plasmid pair as a standard. The instant application contains a Sequence Listing which has been filed electronically in ASCII format.
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