Site-specific translocation and evidence of postnatal origin of the t(1;19) E2A-PBX1 fusion in childhood acute lymphoblastic leukemia

Abstract

The t(1;19) translocation yields a fusion between E2A and PBX1 genes and occurs in 5% of acute lymphoblastic leukemia in children and adults. We used chromosomal translocations and Ig heavy chain (IGH)/T cell antigen receptor (TCR) rearrangements to develop an understanding of the etiology and natural history of this subtype of leukemia. We sequenced the genomic fusion between E2A and PBX1 in 22 preB acute lymphoblastic leukemias and two cell lines. The prenatal origin of the leukemia was assessed in 15 pediatric patients by screening for the clonotypic E2A-PBX1 translocation in neonatal blood spots, or Guthrie cards, obtained from the children at the time of birth. Two patients were determined to be weakly positive for the fusion at the time of birth, in contrast to previously studied childhood leukemia fusions, t(12;21), t(8;21), and t(4;11), which were predominantly prenatal. The presence of extensive N-nucleotides at the point of fusion in the E2A-PBX1 translocation as well as specific characteristics of the IGH/TCR rearrangements provided additional evidence for a postnatal, preB cell origin. Intriguingly, 16 of 24 breakpoints on the 3.2-kb E2A intron 14 were located within 5 bp, providing evidence for a site-specific recombination mechanism. Breakpoints on the 232-kb PBX1 intron 1 were more dispersed but highly clustered proximal to exon 2. In sum, the translocation breakpoints displayed evidence of unique temporal, ontological, and mechanistic formation than the previously analyzed pediatric leukemia translocation breakpoints and emphasize the need to differentiate cytogenetic and molecular subgroups for studies of leukemia causality.

Figures

Fig 1.

Inverse PCR scheme. Locations of PCR primers and restriction sites around intron 13 of the E2A gene. G, BglII; X, XbaI; B, BamH1; A, location of invA and invA-1 primers; B, location of invB and invB-1 primers. InvB-2, 3, 4, and 5 are located just to the right of invB-1. C corresponds to invC and invC-1; D corresponds to invD and invD-1, respectively. InvC-2, 3, 4, and 5 are the complement of invB-5, 4, 3, and 2, respectively.

Fig 2.

Locations of E2A-PBX1 breakpoints and V(D)J recombinase sites. Exon sequences are indicated with boxes, and intronic sequences are indicated with horizontal lines. Patient breakpoints are identified with hash marks above the introns, and putative cryptic RSS are indicated below the introns. V(D)J sites are shown with an up arrow if they are on the sense strand and a down arrow if antisense. Eighteen breakpoints within a 12-bp segment of E2A are shown as a tree-like structure, and brackets above the introns (with a *) show the clusters indicated in Results.

Fig 3.

Fine structure of cluster region on E2A. Eighteen forward E2A-PBX1 breakpoints are indicated with lines and labels above the sequence segment of E2A intron 13, and six reciprocal (i.e., PBX1-E2A) breakpoints are indicated below the segment. The vertical lines delimit the E2A sequence to the left of the line for the E2A-PBX1 and delimit the E2A sequence to the right of the line in the case of the reciprocal. Six E2A-PBX1 translocations are designated at a single base, at which two E2A breakpoints from previously sequenced E2A-HLF+ patients were located, as shown with a dashed line (B, ref. 23). An additional E2A-HLF breakpoint is located 1 bp prior at A (24). Candidate RSS heptamers are boxed; nonamers are not shown for clarity.

Fig 4.

Fine structure of E2A-PBX1 genomic fusions. Patient number is displayed with the E2A and PBX1 sequences proximal to the fusions and N-nucleotides that were present between the E2A and PBX1 sequence (lowercase letters). The antisense strand of the reciprocal fusions (R) are displayed. Cell line RCH had two segments of N-nucleotides along with a tandem duplication of 30 nucleotides of PBX1 sequences shown in between, derived from sequence 7,398 bp 3′ of the breakpoint in reverse orientation. The “T” is a microhomology nucleotide (derived from E2A or PBX1, not an N-nucleotide). The reciprocal partner fusion to 913 is in italics, indicating a fusion of E2A to RODH intron 2.

Fig 5.

Guthrie card analysis of patient #295. The second-round PCR products are shown for the dilution series of diagnostic patient DNA (lanes 1–4, 1:10 dilution series, starting at 50 ng/μl patient DNA in lane 1). Guthrie card segments for control (C) cards are shown with segments from patient #295 (P) and a no-DNA blank (B). A single-lane positive shown was sequenced and determined to match the sequence from the patient. Results were similar for patient #58.