RNA-sequencing analysis in B-cell acute lymphoblastic leukemia reveals aberrant gene expression and splicing alterations
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Background: B-cell acute lymphoblastic leukemia (B-ALL) is a neoplasm of immature lymphoid progenitors and is the leading cause of cancer-related death in children. The majority of B-ALL cases are characterized by recurring structural chromosomal rearrangements that are crucial for triggering leukemogenesis, but do not explain all incidences of disease. Therefore, other molecular mechanisms, such as alternative splicing and epigenetic regulation may alter expression of transcripts that are associated with the development of B-ALL. To determine differentially expressed and spliced RNA transcripts in precursor B-cell acute lymphoblastic leukemia patients a high throughput RNA-seq analysis was performed. Methods: Eight B-ALL patients and eight healthy donors were analyzed by RNA-seq analysis. Statistical testing was performed in edgeR. Each annotated gene was mapped to its corresponding gene object in the Ingenuity KB. Analysis of RNA-seq data for splicing alterations in B-ALL patients and healthy donors was performed with custom Perl script. Results: Using edgeR analysis, 3877 DE genes between B-ALL patients and healthy donors based on TMM (trimmed mean of M-values) normalization method and false discovery rate, FDR less than 0.01, logarithmically transformed fold changes, logFC greater than 2) were identified. IPA revealed abnormal activation of ERBB2, TGFB1 and IL2 transcriptional factors that are crucial for maintaining proliferation and survival potential of leukemic 26 cells. B-ALL specific isoforms were observed for genes with roles in important canonical signaling pathways, such as oxidative phosphorylation and mitochondrial dysfunction. A mechanistic study with the Nalm 6 cell line revealed that some of these gene isoforms significantly change their expression upon 5-Aza treatment, suggesting that they may be epigenetically regulated in B-ALL. Conclusion: Our data provide new insights and perspectives on the regulation of the transcriptome in B-ALL. In addition, we identified transcript isoforms and pathways that may play key roles in the pathogenesis of B-ALL. These results further our understanding of the transcriptional regulation associated with B-ALL development and will contribute to the development of novel strategies aimed towards improving diagnosis and managing patients with B-ALL. Keywords: B-ALL, RNA-sequencing, differential gene expression, alternative splicing.