Whole transcriptome sequencing of Philadelphia positive acute lymphoblastic leukemia (ALL) by RNA-seq: an exhaustive overview of novel point mutations, gene expression and alternative splicing profiles

Background: Although the pathogenesis of BCR-ABL1+ ALL is mainly related to the expression of BCR-ABL1, additional genetic lesions are supposed to be involved in its development and progression. Aim: In order to define the full repertoire of leukemia-related mutations, changes in expression profiles and alternative splicing (AS) events, the leukemia transcriptome of a BCR-ABL1+ ALL patient at diagnosis and relapse was sequenced using a Whole Transcriptome Sequencing (RNA-Seq) approach. Methods: Poly(A) RNA from blast cells was used to prepare cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained sequence reads were mapped to the human genome reference sequence (UCSC hg18) to identify single nucleotide variants (SNVs). Reads that showed no match were mapped to a dataset of all possible splice junctions created in silico to identify AS events. The number of reads corresponding to RNA from known exons was also estimated and a normalized measure of gene expression level (RPKM) was computed. Results: RNA-seq analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples, respectively, detecting transcripts from 62% and 64% of human annotated genes. Low RPKM estimates (0.01 <RPKM< 10) were computed for the great majority of genes (78% at diagnosis and 73% at relapse), whereas a moderate expression (10 <RPKM< 100) was observed for 20% − 24% of active genes and only 2% − 3% of transcripts had high RPKM values (100 <RPKM< 8000). Moreover, 4,334 and 3,651 primary ALL and relapse isoforms with at least one AS event were identified. An average of 1.5 and 1.3 AS per isoform was estimated. The well-known alternatively spliced IKZF1 gene was also detected. Finally, 2,011 and 2,103 SNVs were found at diagnosis and relapse respectively, about 94% of which have been already reported in the dbSNP. As potential ALL-related mutations, 124 and 114 not annotated SNVs were found at diagnosis and relapse, respectively. Of these, 43 affected both samples, while 81 and 71 resulted private variants. The analysis was focused on the coding sequences of annotated genes, finding 12 non-synonymous changes: 1 affecting the PLXNB2 gene on both samples, 6 affecting genes involved in metabolic processes (PDE4DIP, EIF2S3, DPEP1, ZC3H12D, TMEM46) and transport (MVP) at diagnosis and 3 affecting genes involved in cell cycle regulation (CDC2L1) and catalytic activity (CTSZ, CXorf21) at relapse. Furthermore, the T315I mutation in the Bcr-Abl kinase domain was also identified. Conclusions: Discovery of novel missense mutations, as well as exhaustive alternative splicing and gene expression profiles were achieved for the first time for a BCR-ABL1+ positive ALL patient demonstrating that RNA-Seq is a suitable approach for identifying a wide spectrum of genetic alterations.