Variable Breakpoints Target PAX5 in Patients with Dicentric Chromosomes: A Model for the Basis of Unbalanced Translocations in Cancer
Authors: Strefford Jonathan C , An Qian , Wright Sarah L , Konn Zoe J , Matherson Elizabeth , Minto Lynne , Moorman Anthony V , Parker Helen , Hall Andrew , Irving Julie , Harrison Christine J 
Published: October 2019

Abstract
Although balanced translocations often result in the formation of chimaeric fusion genes, the analysis of unbalanced translocations has largely failed to identify target loci. This is due to the heterogeneity of the chromosomal breakpoints and the multiplicity of partner chromosomes. One such example is that of dicentric chromosomes found in patients with acute lymphoblastic leukaemia (ALL). We investigated patients with dic(7;9)(p11;p11~13) [n=13], dic(9;12)(p11~13;p13) [n=38] and dic(9;20)(p11~13;q11) [n=59] to determine the breakpoints of these translocations. FISH with probes for 9p showed that breakpoints were heterogeneous [n=54]. Apart from two cases with dic(9;20), all showed either deletion of the entire PAX5 gene [53.7%, n=29] or a breakpoint within the gene [42.6%, n=23]. For those cases with breakpoints within PAX5, FISH identified ETV6 as the partner in dic(9;12) cases (18/19 cases tested). Sequencing showed a novel breakpoint within intron 1 of ETV6 in a one case of dic(9;12). Molecular copy number counting (MCC) and long distance inverse PCR (LDI-PCR) identified five novel fusion sequences in which PAX5 partnered the LOC392027 (7p12.1), SLCO1B3 (12p12), ASXL1 (20q11.1), KIF3B (20q11.21) and C20orf112 (20q11.1) loci. The PAX5-ASXL1 and PAX5-KIF3B fusion sequences were in opposing orientation, the remaining three fusions were in the same orientation but out of frame, implicating loss of PAX5 function. To confirm the significance of PAX5 disruption, we demonstrated that both the deletion and gene fusion events resulted in the same under-expression of PAX5 exons 1–2 (p<0.04), and 4–5 (p<0.04) in the patients with dicentric chromosomes, in association with differential expression of the PAX5 target genes, EBF1 (p<0.001), ALDH1A1 (p<0.001), ATP9A (p<0.01) and FLT3 (p<0.006). Genomic quantitative PCR (gqPCR) confirmed the heterozygous loss of PAX5 [n=16] and showed homozygous deletions involving the homologous allele in two patients. Mutation analysis [n=20] identified two cases with a single base pair deletion of exon 8 and a single base pair insertion into exon 2, both of which would translate into a truncated PAX5 protein. This is further support that the formation of the dicentric chromosome provided leukemic potential by abrogating normal PAX5 function in these cases. The same analysis of chromosome 20 in 26 dic(9;20) cases highlighted ASXL1 as the potential target gene in 62% [n=16] patients with the dic(9;20) tested, manifesting as recurrent fusion events and deletions as shown for PAX5. Three additional fusion sequences were identified; involving ASXL1, FRG1B (20q11.1) and LOC149950 (20q11.21) fused to sequence centromeric of PAX5. Here, we have shown for the first time, that specific gene loci may be the target of heterogeneous breakpoints in human cancer, acting through a variety of mechanisms. Although several investigations have failed to identify the key molecular events in patients with dicentric chromosomes, we report PAX5 and ASXL1 as key target genes, as a consequence of their involvement in multiple fusion genes and by deletion. This approach has considerable application to the identification of cancer genes in solid tumours, where unbalanced chromosomal rearrangements are particularly prevalent while relatively few genes have been identified. In the absence of a dicentric chromosome, PAX5 is also targeted by interstitial deletions and copy number neutral LOH events, further supporting the importance of investigating the underlying molecular basis of unbalanced translocations. From the expansion of this approach into other tumour types, a large number of novel genes will surely emerge, expanding our understanding of carcinogenesis and ultimately leading to improved management of patients with cancer.