Comprehensive Characterization of a Novel Intronic Pseudo-Exon Inserted within an e14/a2 BCR-ABL Rearrangement in a Patient with Chronic Myeloid Leukemia

Abstract

We identified a novel breakpoint cluster region-ABL rearrangement in a chronic myeloid leukemia (CML) patient. The e14/a2 (b3/a2) type BCR-ABL mRNA incorporated a 42-nucleotide intronic insertion of ABL intron Ib between BCR exon e14 and ABL exon a2. As we hypothesized that the rearrangement between BCR and ABL genes occurred near the inserted sequence and because of the relative small size of BCR intron 14 , we determined the BCR-ABL breakpoint at the genomic DNA level. Using a PCR-based method, this analysis revealed that i) BCR intron 14 brought a potential lariat branch point and the polypyrimidine tract , ii) the BCR-ABL breakpoint created a chimeric acceptor site , and iii) the inserted sequence of ABL intron Ib carried at its 3 end a well-conserved donor splice site. Therefore , the inserted sequence was flanked by canonical consensus splice sites and recognized as a pseudo-exon (as shown by splice site prediction and exon finder software). Moreover, the insertion did not disrupt the reading frame between BCR and ABL and did not produce a premature stop codon. Instead , this novel BCR-ABL chimeric transcript encoded a functional oncoprotein with an in-frame insertion of 15 new amino acids. Philadelphia chromosome (Ph1) results from the reciprocal t(9;22)(q34;q11) translocation that fuses the BCR and ABL genes into a BCR-ABL chimeric gene, which encodes a constitutively activated tyrosine kinase oncoprotein.1 Breakpoints in ABL generally occur upstream of exon a2 (rarely exon a3). Depending on the location of the breakpoint in the BCR gene, three main types of BCR-ABL transcripts can be produced.2 In chronic myeloid leukemia (CML), most of the breakpoints in BCR occur in the 5.8-kb major breakpoint cluster region and result in the fusion at the mRNA level of BCR exon e13 (b2) or e14 (b3) to ABL exon a2, known as the e13/a2 or e14/a2 rearrangements. In Ph1-positive acute lymphoblastic leukemia, breakpoints in BCR are generally located in the minor major breakpoint cluster region and result in the e1/a2 BCR-ABL rearrangement. A third breakpoint in BCR (-BCR) is observed in typical CML or neutrophilic leukemia and leads to the e19/a2 rearrangement. Finally, e6/a2 and e8/a2 BCR-ABL rearrangements are uncommonly observed in typical CML. Thus, in the great majority of CML cases, BCR-ABL rearrangements result in the joining of BCR exons e13 or e14 to ABL exon a2.We report here a case of CML with an e14/a2 fusion mRNA containing a 42-bp sequence from ABL intron Ib inserted between BCR exon e14 and ABL exon a2. The insertion was characterized by sequencing at the mRNA level. The location of the BCR-ABL breakpoint was determined on genomic DNA, and the consensus donor and acceptor splice sites as well as a potential lariat branch point were identified. We demonstrated that, in this BCR-ABL rearrangement, the consensus splice sites were brought by BCR intron 14 (branch point and polypyrimiSupported by grants from the Ligue Contre le Cancer.

BCR‐ABL1 kinase domain mutations: Methodology and clinical evaluation

“…RNA preparation is an extremely important step prior to any procedure. For a detailed protocol see a recent previous publication [74].…”

“…It is also important to establish the correct type of fusion transcript, (qualitative test) (i.e., major, minor, or rare variants). In our laboratory, any sample with less than 10,000 ABL1 molecules (quantitative test) is generally rejected, re‐extracted, and retested [74]. Also, a minimum of 50 BCR‐ABL1 molecules by RT‐qPCR are required to perform a mutation analysis test on any sample (25 molecules for direct sequencing analysis and 50 molecules for pyrosequencing, see below).…”

“…The volume of cDNA required per reaction is 2.5 μl (containing a pre‐determined minimum of 10,000 ABL1 molecules as a measure of cDNA quality [74].…”

“…It is important to establish the fusion subtype for each patient, in order to use the appropriate primer set [74]. In details, for the major BCR‐ABL1 transcript type (referred as b2a2 and b3a2 or e13a2 and e14a2), the first round PCR amplifies from exon13 of the BCR gene to exon 10 of the ABL1 gene (with an amplicon approximately 1,600 bp in size) using primers BCR/Ex13‐F and ABL1/Ex10‐R1.…”

Novel BCR-ABL1 fusion and leukemic mutations of SETBP1, PAX5, and TP53 detected by next generation sequencing in chronic myeloid leukemia

“…1 These transcripts are well-characterized and frequently detected by screening methods based on real-time quantitative RT-PCR (qRT-PCR), which is considered as the gold standard for the diagnostic and follow-up examination of Ph positive leukemia due to its superior sensitivity and cost-effectiveness. 2 However, new and rare forms of BCR-ABL1 fusions have also been discovered and these novel fusion gene variants including those with a masked or undetermined Ph chromosome karyotyping, often cannot be identified by routine RT-PCR methods, [3][4][5][6][7][8][9][10][11][12][13][14][15] presenting a big challenge for conventional diagnostic approaches.…”