Analysis of genomic breakpoints in p190 and p210 BCR–ABL indicate distinct mechanisms of formation

Abstract

We sought to understand the genesis of the t(9;22) by characterizing genomic breakpoints in chronic myeloid leukemia (CML) and BCR-ABL-positive acute lymphoblastic leukemia (ALL). BCR-ABL breakpoints were identified in p190 ALL (n ¼ 25), p210 ALL (n ¼ 25) and p210 CML (n ¼ 32); reciprocal breakpoints were identified in 54 cases. No evidence for significant clustering and no association with sequence motifs was found except for a breakpoint deficit in repeat regions within BCR for p210 cases. Comparison of reciprocal breakpoints, however, showed differences in the patterns of deletion/insertions between p190 and p210. To explore the possibility that recombinase-activating gene (RAG) activity might be involved in ALL, we performed extra-chromosomal recombination assays for cases with breakpoints close to potential cryptic recombination signal sequence (cRSS) sites. Of 13 ALL cases tested, 1/10 with p190 and 1/3 with p210 precisely recapitulated the forward BCR-ABL breakpoint and 1/10 with p190 precisely recapitulated the reciprocal breakpoint. In contrast, neither of the p210 CMLs tested showed functional cRSSs. Thus, although the t(9;22) does not arise from aberrant variable (V), joining (J) and diversity (D) (V(D)J) recombination, our data suggest that in a subset of ALL cases RAG might create one of the initiating double-strand breaks.

Natural course and biology of CML

“…The location of the BCR and ABL1 genomic breakpoints is highly variable [17], but the recombination usually involves fusion of intron 13 or 14 of BCR with a 140-kilobase (kb) region of ABL1 between exons 1b and 2 ( Fig. 1a) [17]. Regardless of the breakpoint location on the ABL1 gene, mRNA splicing gives rise to major BCR-ABL1 transcripts with e13a2 (BCR exon 13 and ABL1 exon 2) or e14a2 junctions.…”

“…The BCR-ABL1 fusion gene consists of the 5′ end of the BCR (breakpoint cluster region) gene and the 3′end of the ABL1 gene (also known as Abelson). The location of the BCR and ABL1 genomic breakpoints is highly variable [17], but the recombination usually involves fusion of intron 13 or 14 of BCR with a 140-kilobase (kb) region of ABL1 between exons 1b and 2 ( Fig. 1a) [17].…”

Developmental origins and impact of BCR-ABL1 fusion and IKZF1 deletions in monozygotic twins with Ph+ acute lymphoblastic leukemia

“…18 When PCR with the primer mix produced a PCR product, a split out PCR was run with the individual primers contained within the mix to amplify a band with BCR F and ABL1 R. The breakpoints were confirmed by re-amplification and sequencing in the patient samples with BCR F and a twin-and breakpointspecific ABL1 reverse primers (Figure 1). More specifically, the breakpoints were designated as a fusion between BCR intron 1 (nucleotide 2982232 in genomic sequence NT_011520) and ABL1 intron 1 (nucleotide 417579 in genomic sequence NT_035014) in twin 1A and between BCR intron 1 (nucleotide 2970354 in genomic sequence NT_011520) and ABL1 intron 1 (nucleotide 504164 in genomic sequence NT_035014) in twin 2A.…”

“…To cover the BCR and ABL1 regions, within which breakpoints can occur, 21 BCR forward primers and 20 ABL1 reverse primers were used in multiplex, combining each BCR forward primer with 4 mixes of 5 ABL1 reverse primers, as described elsewhere. 18 …”

In search of the original leukemic clone in chronic myeloid leukemia patients in complete molecular remission after stem cell transplantation or imatinib

“…23 The reaction used the Expand Long Template PCR System (Roche Applied Science) polymerase and Buffer 2 from the kit. The reaction conditions and thermocycling parameters were optimized to amplify templates of up to 12 kb.…”