Identification and Characterization of Novel Fusion Genes with Potential Clinical Applications in Mexican Children with Acute Lymphoblastic Leukemia

Abstract

Acute lymphoblastic leukemia is the most common type of childhood cancer worldwide. Mexico City has one of the highest incidences and mortality rates of this cancer. It has previously been recognized that chromosomal translocations are important in cancer etiology. Specific fusion genes have been considered as important treatment targets in childhood acute lymphoblastic leukemia (ALL). The present research aimed at the identification and characterization of novel fusion genes with potential clinical implications in Mexican children with acute lymphoblastic leukemia. The RNA-sequencing approach was used. Four fusion genes not previously reported were identified: CREBBP-SRGAP2B, DNAH14-IKZF1, ETV6-SNUPN, ETV6-NUFIP1. Although a fusion gene is not sufficient to cause leukemia, it could be involved in the pathogenesis of the disease. Notably, these new translocations were found in genes encoding for hematopoietic transcription factors which are known to play an important role in leukemogenesis and disease prognosis such as IKZF1, CREBBP, and ETV6. In addition, they may have an impact on the prognosis of Mexican pediatric patients with ALL, with the potential to be included in the current risk stratification schemes or used as therapeutic targets.

Keywords: RNA sequencing; chromosomal translocation; fusion gene; genetic variations; leukemogenesis; risk stratification.

Figure 1

Characterization of the CREBBP-SRGAP2B gene fusion. (A) 100 bp marker, PCR of CREBBP-SRGAP2B using H₂0 as negative control (lane1), DNA genomic (lane 2), and cDNA from total RNA isolated from 74MO sample (lane 3). (B) Schematic diagram showing the structure of CREBBP-SRGAP2B fusion, exon 1 of the CREBBP gene is fused in frame with exon 3 of the SRGAP2B gene. The symbol “lightning” indicates the breakpoint region. A CREBBP-SRGAP2B fusion sequence is shown below, with a schematic derived by the cloning and sequencing of the RT-PCR product.

Figure 2

Detection of the DNAH14-IKZF1 fusion sequence A. Genomic PCR products were obtained from original genomic DNA samples. We used primer sets at the intronic breakpoint between exon 36 of DNAH14 with exon 4 of IKZF1 (Table S1). (A) 100 bp marker, PCR of DNAH14-IKZF1 with genomic DNA of DNAH14-IKZF1-negative sample 74MO (lane 1), genomic DNA of 179 sample (lane 2), and H₂O as negative control (lane 3). (B) Schematic representation of the DNAH14-IKZF1 fusion. Exon 29, 33, and 34 of the DNAH14 gene and exon 3 and 4 of the IKZF1 gene could be fused together, synthesizing different fusion transcripts. The symbol “lightning” indicates the breakpoint region. Note: Only the intronic breakpoint between exon 36 of DNAH14 with exon 4 of IKZF1 was confirmed by cloning the sequencing of the PCR product.

Figure 3

Identification of the ETV6 gene fusion transcript. For ETV6 fusion confirmation, the original RNA sample was used. Primers were designed to amplify ETV6-NUFIP1 with the two regions of 118 bp and 269 bp named internal and external, respectively. For ETV6-SNUPN, two regions, 112 bp-internal and 339 bp-external, were also amplified (Table S1). (A) 100 bp marker, RT-PCR of ETV6-NUFIP1 with primers ETV6-NUFIP1-int (lane 1), with primers ETV6-NUFIP1-ext (lane 3). RT-PCR with primers ETV6-SNUPN-int (lane 5), ETV6-SNUPN-ext (lane 7), and negative control (H₂O) (lanes 2, 4, 6, and 8). (B) Schematic representation of the localization of the breakpoints within ETV6 and NUFIP1 and the corresponding result of sequencing where exon 1 of the ETV6 gene and exon 6 of the NUFIP1 gene are fused together in this transcript. (C) Schematic representation of ETV6-SNUPN where exon 2 of the ETV6 gene and exon 2 of the SNUPN gene are fused. The symbol “lightning” indicates the breakpoint region.

Figure 4

Identification of the EP300-ZNF384 fusion. For the confirmation of EP300-ZNF384 fusion, the original RNA sample was used. Primers were designed to amplify two regions of 106 bp and 250 bp, named internal and external, respectively (Table S1). (A) 100 bp marker, RT-PCR of EP300-ZNF384 with primers EP300-ZNF384-ext (lane 2), with primers EP300-ZNF384-int (lane 4), and negative control (H₂O) (lanes 1 and 3). (B) Schematic representation of the localization of the breakpoints within ZNF384 and EP300 and the corresponding result of sequencing where exon 6 of the EP300 gene and exon 3 of the ZNF384 gene are fused in this transcript. The symbol “lightning” indicates the breakpoint region.