title: Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.
abstract: A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.
authors.affiliation: Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan; Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan.
authors.forename: Hiraku
authors.initials: H
authors.lastname: Takei
authors.affiliation: Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University Graduate School of Medicine, Tokyo, Japan.
authors.forename: Soji
authors.initials: S
authors.lastname: Morishita
authors.affiliation: Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University Graduate School of Medicine, Tokyo, Japan.
authors.forename: Marito
authors.initials: M
authors.lastname: Araki
authors.affiliation: Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Edahiro
authors.affiliation: Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Sunami
authors.affiliation: Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Hironaka
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Noda
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sekiguchi
authors.affiliation: Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tsuneda
authors.affiliation: Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University Graduate School of Medicine, Tokyo, Japan.
authors.forename: Akimichi
authors.initials: A
authors.lastname: Ohsaka
authors.affiliation: Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.
authors.forename: Norio
authors.initials: N
authors.lastname: Komatsu
mesh_terms: D005787:Gene Frequency / Q000235:genetics*; D006801:Humans; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D053628:Receptors, Thrombopoietin / Q000235:genetics*
chemical_list: D053628:Receptors, Thrombopoietin; C082218:MPL protein, human