Variable breakpoints target PAX5 in patients with dicentric chromosomes: a model for the basis of unbalanced translocations in cancer

Abstract

The search for target genes involved in unbalanced acquired chromosomal abnormalities has been largely unsuccessful, because the breakpoints of these rearrangements are too variable. Here, we use the example of dicentric chromosomes in B cell precursor acute lymphoblastic leukemia to show that, despite this heterogeneity, single genes are targeted through a variety of mechanisms. FISH showed that, although they were heterogeneous, breakpoints on 9p resulted in the partial or complete deletion of PAX5. Molecular copy number counting further delineated the breakpoints and facilitated cloning with long-distance inverse PCR. This approach identified 5 fusion gene partners with PAX5: LOC392027 (7p12.1), SLCO1B3 (12p12), ASXL1 (20q11.1), KIF3B (20q11.21), and C20orf112 (20q11.1). In each predicted fusion protein, the DNA-binding paired domain of PAX5 was present. Using quantitative PCR, we demonstrated that both the deletion and gene fusion events resulted in the same underexpression of PAX5, which extended to the differential expression of the PAX5 target genes, EBF1, ALDH1A1, ATP9A, and FLT3. Further molecular analysis showed deletion and mutation of the homologous PAX5 allele, providing further support for the key role of PAX5. Here, we show that specific gene loci may be the target of heterogeneous translocation breakpoints in human cancer, acting through a variety of mechanisms. This approach indicates an application for the identification of cancer genes in solid tumours, where unbalanced chromosomal rearrangements are particularly prevalent and few genes have been identified. It can be extrapolated that this strategy will reveal that the same mechanisms operate in cancer pathogenesis in general.

Fig. 1.

FISH and sequence analysis of BCP-ALL patients with dicentric chromosomal abnormalities. (A) FISH data for chromosome 9 from patients with dicentric chromosomes. A partial idiogram of the short arm of chromosome 9 is shown on the left with the position of the PAX5-specific FISH clone shown in black. Each column shows an individual patient with the position and extent of the deleted region represented by the vertical black box. In several cases, it was not possible to refine the position of the deletion breakpoint (shown by the thin vertical line). This was due either to the lack of uniquely binding FISH clones in the polymorphic region of 9p11–13.1 or lack of material. (B) Representative MCC and sequence analysis of PAX5 disruption in dicentric chromosome translocations. The results from 3 rounds of MCC on patient 20–4551 are shown above, where each round further delineates the position of the breakpoint. The x and y axes show the genomic position and degree of copy number change, respectively. The size and position of the area with the copy number change is shown by the dashed box. Below is the sequence data from the same patient. The fusion sequence is shown between normal sequence for chromosome 9 and 20, where the chromosome 20 sequence is underlined. (C) Schematic of PAX5 genomic breakpoints and partner genes. The exons for PAX5 and the partner genes are shown by the white and gray boxes, respectively. The gene orientation is shown by the horizontal arrow. Below each genomic schematic is the corresponding predicted protein structure and domains. PD, paired; O, octapeptide; H, homeodomain-like; TA-A, transactivation, activating; I, inhibitory; HLH, helix–loop–helix; ETS, Ets; oATP, organic anion transporter polypeptide; KAZAL-ALC21, azal-type serine protease inhibitor; SMC, structural maintenance of chromosomes.

Fig. 2.

Quantitative PCR analysis of expression and copy number and sequence analysis of BCP-ALL patients with dicentric chromosomes. (A) Quantitative RT-PCR for PAX5 expression exons 1/2 and 4/5. The patient subgroup and fold change in expression are shown in the x and y axes, respectively. Patients with a dicentric chromosome resulting in either fusion or deletion of PAX5 [dic (fused/del)] and with a high-hyperdiploidy karyotype [HeH (retained)]. (B) Quantitative RT-PCR expression of PAX5 target genes. (C) Quantitative genomic PCR for exon 6 of PAX5. Based on a series of control patients (n = 180) with 2 normal copies of PAX5 a minimal ratio for a normal copy number was defined and is shown by the horizontal dashed line. Each patient is shown as a vertical bar on the x axis, where N1-N8 are patients with 2 copies of PAX5, 12-4443 and 20-10061, have homozygous, and 20-4887 has heterozygous loss of PAX5. (D) PAX5 mutation analysis in patients with dicentric chromosomes. The DHPLC traces are shown above for a normal and patient 20-4887, where the x and y axes show retention time and spectrophotometrical detection at 253 nM, respectively. Below are the sequence traces for 2 patients with mutations of PAX5. The box shows the mutation in each case. Patient 20-4887 contained a DelG1450 frame-shift mutation in exon 8, which translates to a truncated protein composed of the original 334 AA of PAX5 then a sequence of 19 AA followed by a premature stop. Patient 20–6789 harboured an insG525 frame-shift mutation in exon 2, which translates to a truncated protein with 25 intact AA followed by a sequence of 48 AA then a premature stop.