Abstract
BackgroundPh- myeloproliferative neoplasms (MPNs) represent a heterogeneous group of chronic diseases characterized by increased expansion of hematopoietic cells of the myeloid lineage. JAK2V617F, an activation mutation form of tyrosine kinase JAK2, is found in the majority of patients with MPNs. Studies have demonstrated that JAK2V617F can cause MPNs, and various methods have been developed to detect JAK2V617F for diagnostic purposes. However, a highly sensitive method is still needed for the earliest possible detection and for disease prevention and treatment.MethodsIn the present study, we developed a method dubbed restriction fragment nested allele-specific PCR (RFN-AS-PCR). The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the JAK2V617F mutation site, 2) digestion of the PCR products with restriction enzyme BsaXI which only cleaves the wild type allele, and 3) detection of JAK2V617F by allele-specific PCR with nested primers.ResultsWe tested the sensitivity of the method by using purified plasmid DNAs and blood cell DNAs containing known proportions of JAK2V617F. We were able to detect JAK2V617F with a sensitivity of 0.001%. We further analyzed blood cell DNA samples from 105 healthy donors with normal blood cell counts and found three JAK2V617F-positive cases, which would have remained undetected using a less sensitive method.ConclusionsWe have developed a highly sensitive method that will allow for detection of JAK2V617F at a very early stage. This method may have major implications in diagnosis and prevention of MPNs and related diseases.
Highlights
Ph- myeloproliferative neoplasms (MPNs) represent a heterogeneous group of chronic diseases characterized by increased expansion of hematopoietic cells of the myeloid lineage
When the JAK2V617F mutation rate is low, these methods often give weak and ambiguous signals because allele-specific PCR (AS-PCR) is not absolutely specific, and restriction enzymes cannot digest with 100% efficiency [29]
BsaXI removes from the wild type PCR product a 30 bp fragment containing the V617F mutation site, which effectively eliminates the chance that the full-length wild type product is re-generated in the subsequent PCR
Summary
Ph- myeloproliferative neoplasms (MPNs) represent a heterogeneous group of chronic diseases characterized by increased expansion of hematopoietic cells of the myeloid lineage. A highly sensitive method is still needed for the earliest possible detection and for disease prevention and treatment. Ph- myeloproliferative neoplasms (MPNs) represent a group of chronic conditions including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) [1,2]. Complications associated with MPNs include thrombosis, hemorrhage, myeloid metaplasia, and acute leukemia. These diseases cause strokes and heart attacks that are usually fatal. A more reliable and more sensitive method is still needed for the earliest possible detection of JAK2V617F, which will have major implications in diagnosis and prevention of MPNs
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