title: Simultaneous detection of mutations and copy number variation of NPM1 in the acute myeloid leukemia using multiplex ligation-dependent probe amplification.
abstract: The NPM1 gene encodes nucleophosmin, a protein involved in multiple cell functions and carcinogenesis. Mutation of the NPM1 gene, causing delocalization of the protein, is the most frequent genetic lesion in acute myeloid leukemia (AML); it is considered a founder event in AML pathogenesis and serves as a favorable prognostic marker. Moreover, in solid tumors and some leukemia cell lines, overexpression of the NPM1 gene is commonly observed. Therefore, the purpose of this study was to develop a new method for the detection of NPM1 mutations and the simultaneous analysis of copy number alterations (CNAs), which may underlie NPM1 gene expression deregulation. To address both of the issues, we applied a strategy based on multiplex ligation-dependent probe amplification (MLPA). A designed NPM1mut+ assay enables the detection of three of the most frequent NPM1 mutations: A, B and D. The accuracy of the assay was tested using a group of 83 samples from Polish patients with AML and other blood-proliferative disorders. To verify the results, we employed traditional Sanger sequencing and next-generation transcriptome sequencing. With the use of the NPM1mut+ assay, we detected mutations A, D and B in 14, 1 and 0 of the analyzed samples, respectively. All of these mutations were confirmed by complementary sequencing approaches, proving the 100% specificity and sensitivity of the proposed test. The performed sequencing analysis allowed the identification of two additional rare mutations (I and ZE). All of the mutations were identified exclusively in AML cases, accounting for 25% of those cases. We did not observe any CNAs (amplifications) of the NPM1 gene in the studied samples, either with or without the mutation. The presented method is simple, reliable and cost-effective. It can be easily introduced into clinical practice or developed to target both less-frequent mutations in the NPM1 gene and other cancer-related genes.
authors.affiliation: European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan, Poland. Electronic address: m-marcinkowska@o2.pl.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Marcinkowska-Swojak
authors.affiliation: European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan, Poland; Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan, Poland. Electronic address: luizahan@ibch.poznan.pl.
authors.forename: Luiza
authors.initials: L
authors.lastname: Handschuh
authors.affiliation: European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan, Poland; Institute of Computing Science, Poznan University of Technology, Piotrowo 2, 60-965 Poznan, Poland. Electronic address: Pawel.Wojciechowski@cs.put.poznan.pl.
authors.forename: Pawel
authors.initials: P
authors.lastname: Wojciechowski
authors.affiliation: European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan, Poland. Electronic address: mgoralsk@ibch.poznan.pl.
authors.forename: Michal
authors.initials: M
authors.lastname: Goralski
authors.affiliation: European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan, Poland. Electronic address: kamil.tomaszewsky@gmail.com.
authors.forename: Kamil
authors.initials: K
authors.lastname: Tomaszewski
authors.affiliation: Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan, Poland. Electronic address: maciej.kazmierczak@onet.eu.
authors.forename: Maciej
authors.initials: M
authors.lastname: Kazmierczak
authors.affiliation: Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan, Poland. Electronic address: krzysztof.lewandowski@skpp.edu.pl.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Lewandowski
authors.affiliation: Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569 Poznan, Poland. Electronic address: mak7@pro.onet.pl.
authors.forename: Mieczyslaw
authors.initials: M
authors.lastname: Komarnicki
authors.affiliation: European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan, Poland; Institute of Computing Science, Poznan University of Technology, Piotrowo 2, 60-965 Poznan, Poland. Electronic address: jblazewicz@cs.put.poznan.pl.
authors.forename: Jacek
authors.initials: J
authors.lastname: Blazewicz
authors.affiliation: European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan, Poland; Institute of Computing Science, Poznan University of Technology, Piotrowo 2, 60-965 Poznan, Poland. Electronic address: marekf@ibch.poznan.pl.
authors.forename: Marek
authors.initials: M
authors.lastname: Figlerowicz
authors.affiliation: European Center of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan, Poland. Electronic address: kozlowp@yahoo.com.
authors.forename: Piotr
authors.initials: P
authors.lastname: Kozlowski
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D045744:Cell Line, Tumor; D056915:DNA Copy Number Variations; D005260:Female; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D000090243:Nucleophosmin; D011044:Poland; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D055815:Young Adult
chemical_list: C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D000090243:Nucleophosmin
keywords: Acute myeloid leukemia; Copy number alterations; Multiplex ligation-dependent probe amplification; Mutation detection; NPM1 gene; Sequencing