Department of Medicine III, University Hospital, LMU Munich, Munich, Germany; German Cancer Consortium (DKTK), partner site Munich; and; German Cancer Research Center (DKFZ), Heidelberg, Germany.
Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Munich, Germany.
Haematologica. 2022 Jan 1;107(1):100-111. doi: 10.3324/haematol.2021.278436.
Identification of fusion genes in clinical routine is mostly based on cytogenetics and targeted molecular genetics, such as metaphase karyotyping, fluorescence in situ hybridization and reverse-transcriptase polymerase chain reaction. 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 RNAsequencing compared to standard diagnostic techniques, we analyzed 806 RNA-sequencing samples from patients with acute myeloid leukemia using two state-of-the-art software tools, namely Arriba and FusionCatcher. RNA-sequencing detected 90% of fusion events that were reported by routine with high evidence, while samples in which RNA-sequencing 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-sequencing. 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-sequencing 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 mechanisms of leukemogenesis in acute megakaryoblastic leukemia. Our findings demonstrate that RNA-sequencing has a strong potential to improve the systematic detection of fusion genes in clinical applications and provides a valuable tool for fusion discovery.
在临床常规中，融合基因的鉴定主要基于细胞遗传学和靶向分子遗传学，例如中期核型分析、荧光原位杂交和逆转录聚合酶链反应。然而，随着每个样本的处理时间和成本降低，测序技术在临床常规中变得越来越重要。为了评估 RNA 测序在融合基因检测方面与标准诊断技术的性能相比，我们使用两种最先进的软件工具，即 Arriba 和 FusionCatcher，分析了 806 例急性髓系白血病患者的 RNA 测序样本。RNA 测序以高证据水平检测到 90%的融合事件，而在 RNA 测序未能检测到融合基因的样本中，整体的序列覆盖度较低且不均匀。基于已知和未知融合事件的特性，我们开发了一种工作流程，其中包含集成的筛选策略，用于通过 RNA 测序鉴定稳健的融合基因候选物。通过这种方式，我们在 26 例未通过常规方法报告的病例中检测到已知的复发性融合事件，并在 3 例中发现常规方法和 RNA 测序之间已知融合事件证据存在差异。此外，我们还鉴定出 157 个融合基因作为新的稳健候选物，与 ChimerDB 或 Mitelman 数据库的条目进行比较，在 14 例中发现融合基因的新复发性。最后，我们检测到由 inv(21)(q11.2;q21.1)引起的新型复发性融合基因 NRIP1-MIR99AHG 在 9 例患者（1.1%）和由 inv(21)(q21.3;q22.3)引起的 LTN1-MX1 在 2 例患者（0.25%）中。我们证明 NRIP1-MIR99AHG 导致 MIR99AHG 的 3' 区域过表达和三顺反子 miRNA 簇 miR-99a/let-7c/miR-125b-2 的破坏。有趣的是，MIR99AHG 的上调和位于 MIR99AHG 基因座的 miRNA 簇的失调是急性巨核细胞白血病中白血病发生的已知机制。我们的研究结果表明，RNA 测序具有在临床应用中提高融合基因系统检测的强大潜力，并为融合发现提供了有价值的工具。
