Two novel e13a3 variants detected in CML patients and their DNA breakpoint analysis

Even in the era of tyrosine kinase inhibitor (TKI) therapy, the prognostic landscape of atypical transcripts in chronic myeloid leukemia (CML) remains poorly defined. In our preliminary work, we identified two atypical transcripts harboring distinct DNA breakpoints that ultimately translate into an identical truncated protein. To investigate the DNA breakpoints of the BCR::ABL fusion gene in two patients with novel e13a3-type CML and to clarify the underlying mechanism of their generation, we conducted a breakpoint analysis. Target DNA fragments were amplified via nested PCR using specific primers, followed by agarose gel purification and sequencing analysis. RT-PCR sequencing identified two distinct e13a3 transcript variants: an 819-bp insertion was detected between BCR exon 13 and ABL exon 3 in patient 1, whereas a 131-bp insertion was found in patient 2. DNA sequencing verified that the breakpoint regions were located in BCR intron 13 and ABL intron 2, which were fully consistent with the cDNA fusion sites. The intronic sequence insertion induced a frameshift, leading to identical premature translation termination at BCR intron 13 in both cases. This resulted in the complete deletion of the entire ABL region, generating a truncated protein with 20-amino acids at the C-terminus of BCR exon 13. This novel e13a3 transcript originates from DNA breakage. Both patients achieved remission after imatinib treatment; molecular docking simulation results show that e13a3 has a lower binding energy with imatinib than e13a2. This finding suggests that in the TKI era, more attention should be paid to the types of truncated proteins resulting from DNA breakpoints.