Nanopore Targeted Sequencing for Rapid Gene Mutations Detection in Acute Myeloid Leukemia

Genes (Basel). 2019 Dec 9;10(12):1026. doi: 10.3390/genes10121026.

Abstract

Acute myeloid leukemia (AML) clinical settings cannot do without molecular testing to confirm or rule out predictive biomarkers for prognostic stratification, in order to initiate or withhold targeted therapy. Next generation sequencing offers the advantage of the simultaneous investigation of numerous genes, but these methods remain expensive and time consuming. In this context, we present a nanopore-based assay for rapid (24 h) sequencing of six genes (NPM1, FLT3, CEBPA, TP53, IDH1 and IDH2) that are recurrently mutated in AML. The study included 22 AML patients at diagnosis; all data were compared with the results of S5 sequencing, and discordant variants were validated by Sanger sequencing. Nanopore approach showed substantial advantages in terms of speed and low cost. Furthermore, the ability to generate long reads allows a more accurate detection of longer FLT3 internal tandem duplications and phasing double CEBPA mutations. In conclusion, we propose a cheap, rapid workflow that can potentially enable all basic molecular biology laboratories to perform detailed targeted gene sequencing analysis in AML patients, in order to define their prognosis and the appropriate treatment.

Keywords: FLT3 internal tandem duplications; acute myeloid leukemia; biallelic CEBPA mutations; mutational analysis; nanopore targeted sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Alleles
  • CCAAT-Enhancer-Binding Proteins / genetics
  • Female
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Isocitrate Dehydrogenase / genetics
  • Leukemia, Myeloid, Acute / genetics*
  • Male
  • Mutation
  • Nanopores
  • Nuclear Proteins / genetics
  • Nucleophosmin
  • Prognosis
  • Sequence Analysis, DNA / methods
  • Tumor Suppressor Protein p53 / genetics
  • fms-Like Tyrosine Kinase 3 / genetics

Substances

  • CCAAT-Enhancer-Binding Proteins
  • CEBPA protein, human
  • NPM1 protein, human
  • Nuclear Proteins
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • Nucleophosmin
  • IDH2 protein, human
  • Isocitrate Dehydrogenase
  • IDH1 protein, human
  • FLT3 protein, human
  • fms-Like Tyrosine Kinase 3