Uncovering genomic complexity of PAX5 internal tandem duplication using long-read and short-read sequencing

PAX5 internal tandem duplication (PAX5-ITD) is associated with poor outcomes in childhood B-ALL. Its precise genomic architecture remains unclear. Here we report the analysis of 26 PAX5-ITD cases using long- and short-read sequencing. We observe an inverse correlation between the size of the duplicated region (median 23.8 kb) and the number of gained copies (median 6×). Consistent copy number gains between DNA and RNA indicate high splicing fidelity of the ITD allele. All investigated PAX5-ITDs encode in-frame transcripts, with the creation of neo-splice sites when truncated exons are involved. Analysis of secondary mutations in the ITD region establishes a multi-step etiology for PAX5-ITD formation. Concordant copy number and structural events between diagnosis and relapse samples suggest that PAX5-ITD is invariant during clonal evolution through therapy. Collectively, our findings highlight the genomic complexity of PAX5-ITD and provide a foundation for functional investigation and precision targeting.