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Kerbs, Paul; Vosberg, Sebastian; Krebs, Stefan; Graf, Alexander; Blum, Helmut; Swoboda, Anja; Batcha, Aarif M. N.; Mansmann, Ulrich ORCID logoORCID: https://orcid.org/0000-0002-9955-8906; Metzler, Dirk; Heckman, Caroline A.; Herold, Tobias and Greif, Philipp A. (2021): Fusion gene detection by RNA sequencing complements diagnostics of acute myeloid leukemia and identifies recurring NRIP1-MIR99AHG rearrangements. In: Haematologica

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Identification of fusion genes in clinical routine is mostly based on cytogenetics and targeted molecular genetics, such as metaphase karyotyping, FISH and RT-PCR. However, sequencing technologies are becoming more important in clinical routine as processing-time and costs per sample decrease. To evaluate the performance of fusion gene detection by RNA sequencing (RNAseq) compared to standard diagnostic techniques, we analyzed 806 RNA-seq samples from acute myeloid leukemia (AML) patients using two state-of-the-art software tools, namely Arriba and FusionCatcher. RNA-seq detected 90% of fusion events that were reported by routine with high evidence, while samples in which RNA-seq failed to detect fusion genes had overall lower and inhomogeneous sequence coverage. Based on properties of known and unknown fusion events, we developed a workflow with integrated filtering strategies for the identification of robust fusion gene candidates by RNA-seq. Thereby, we detected known recurrent fusion events in 26 cases that were not reported by routine and found discrepancies in evidence for known fusion events between routine and RNA-seq in three cases. Moreover, we identified 157 fusion genes as novel robust candidates and comparison to entries from ChimerDB or Mitelman Database showed novel recurrence of fusion genes in 14 cases. Finally, we detected the novel recurrent fusion gene NRIP1-MIR99AHG resulting from inv(21)(q11.2;q21.1) in nine patients (1.1%) and LTN1-MX1 resulting from inv(21)(q21.3;q22.3) in two patients (0.25%). We demonstrated that NRIP1-MIR99AHG results in overexpression of the 3' region of MIR99AHG and the disruption of the tricistronic miRNA cluster miR-99a/let-7c/miR-125b-2. Interestingly, upregulation of MIR99AHG and deregulation of the miRNA cluster, residing in the MIR99AHG locus, are known mechanism of leukemogenesis in acute megakaryoblastic leukemia. Our findings demonstrate that RNA-seq has a strong potential to improve the systematic detection of fusion genes in clinical applications and provides a valuable tool for fusion discovery.

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