Identification of e19a2 BCR–ABL fusions (μ-BCR breakpoints) at the DNA level by ligation-mediated PCR

TO THE EDITOR

The Philadelphia chromosome (Ph) derives from a balanced translocation between chromosomes 9 and 22, and results in a BCR–ABL fusion gene. In the vast majority of Ph+ leukemias, breakpoints in the ABL gene are distributed over a 200 kb breakpoint region between exons 1b and a2, while breakpoints in the BCR gene are clustered in three well-defined regions.1 The major breakpoint cluster region (M-BCR) is a 2.9 kb chromosomal region between exons 13 and 15.1 The breakpoint can be detected in more than 95% of chronic myeloid leukemia (CML) and can lead to two types of mRNA molecules (e13a2 or e14a2, formerly assigned as b2a2 and b3a2), both encoding a p210BCR–ABL fusion protein. The minor breakpoint cluster region (m-BCR) spans a 55 kb intronic sequence between the two alternative exons 1 and 2. The corresponding e1a2 fusion gene transcript encodes the p190BCR–ABL fusion protein and is frequently associated with Ph+ acute lymphoblastic leukemia. The micro-breakpoint cluster region (μ-BCR) spans a 1 kb sequence which is located in intron 19 of the BCR gene. The corresponding fusion gene transcript e19a2 results in a 230 kDa protein. The e19a2 fusion gene was initially described to be associated with neutrophilic-CML (N-CML), a mild Ph+ myeloproliferative disease with only rarely progression, but in later reports e19a2 fusion gene transcripts were also found in classical CML and in acute myeloid leukemia (AML).2, 3

Here, we report in a new case of CML with an e19a2 transcript the fusion at the DNA level between BCR and ABL genes by using ligation-mediated PCR (LM-PCR). Additionally, the genomic μ-BCR–ABL breakpoints in an e19a2-positive AML and the AR230 cell line were identified.

In January 1999, a 69-year-old female was referred to the hospital with a leucocytosis of 130 × 109/l and immature granulocyte precursors. A bone marrow (BM) aspirate showed an increased cellularity with a myeloid/erythroid ratio of 10. Cytogenetic analysis revealed three pathological clones: 46,XX,t(9;22)(9q34;22q11)[10]/47,XX,idem,+der(22)t(9;22)[2]/46,XX,der(9)t(9;22),idic(22)(22pter → 22q11::9q34::9q34::22q11 → 22pter)[2], supporting the diagnosis of CML in acceleration. An e19a2 fusion gene was identified by RT-PCR analysis using a BCR exon 19 primer (Table 1) and two previously described ABL exon a3 primers.1 She was treated with hydroxyurea (HU) and showed a hematologic response. In May 2002, her BM showed 11% blasts. Gleevec® was started, but soon discontinued because of side effects. HU was restarted, but in December 2002 the patient deceased due to disease progression.

Table 1 Sequences (5′–3′ sequence) of the primers used in this study

To determine the DNA breakpoint region of the μ-BCR–ABL rearrangement in this CML as well as in a previously reported e19a2-positive AML and the e19a2-positive AR230 cell line, an LM-PCR was performed.3, 4, 5 DNA (1 μg) was digested with 30 U blunt end restriction enzymes (DraI, PvuII, HincII, and StuI). Ten restriction sites were found in the germline BCR sequence between exons 18 and 21 (Figure 1a). To both ends of the restriction fragments, 25 μ M of adaptor DNA was ligated.5 The ligation products were subjected to two PCRs, essentially performed as described before.5 Briefly, a 50 μl reaction contained 1 μl of ligation products, 10 pmol of AP1 primer and BCR–LM1 primer (Table 1), 10 mM dNTP (Amersham Biosciences Corp., Piscataway, NJ, USA), 2 U rTth (Applied Biosystems, Foster City, CA, USA), and 1.5 mM Mg(OAc)2. Subsequently, a second round PCR was performed using 1 μl of the first-round PCR product and 10 pmol of the internal AP2 and BCR–LM2 primers (Table 1). The first-round LM-PCR identified germline PCR products of 253, 1992, 1679, and 1317 bp in DraI, PvuII, HincII, and StuI digests, respectively, while the second-round LM-PCR gave germline PCR products of 206, 1945, 1632, and 1270 bp, respectively. In the AR230 cell line, an additional non-germline PCR product of 1014 bp was identified after the second PCR of HincII digested and amplified DNA, while nested LM-PCR of the CML patient and the AML patient revealed additional PCR products of 1469 and 1086 bp, respectively, in PvuII digested DNA (Figure 1b). By using specific primers (Table 1), direct sequencing of the gel-extracted bands (Zymoclean gel DNA recovery kit, Zymo Research, CA, USA) identified all the three genomic μ-BCR-ABL fusion sites (Table 2). The presence of μ-BCR–ABL rearrangements was confirmed by PCR analysis performed on undigested genomic DNA using μ-BCR- and ABL-specific primers (Table 1). At the fusion site of BCR and ABL intronic sequences, no insertions or duplications of nucleotides were found.

Figure 1
figure1

(a) Schematic representation of the BCR gene. In total, 10 restriction sites of DraI (D), PvuII (P), HincII (H), and StuI (S) in the BCR gene between exons 18 and 21 are shown. The bold arrows show the location of the μ-BCR DNA breakpoints in the AML patient, the CML patient, and the AR230 cell line (Genbank accession code: U07000). (b) Agarose gel electroforesis of amplified DNA after nested LM-PCR. Results of germline controls (gc), the AML patient, the CML patient, and the AR230 cell line are shown. Germline bands of 1945 and 1632 bp were found in PvuII and HincII digested DNA, respectively. Additional bands (encircled) differing in size from the germline control were seen in PvuII digested DNA of the AML patient (1086 bp) and the CML patient (1469 bp), and in HincII digested DNA of the AR230 cell line (1014 bp). *100 bp ladder; **λ/PstI DNA ladder. (c) Schematic representation of the ABL gene showing the approximate location of the breakpoint regions in the first intron of the ABL gene. A, B and C indicate breakpoint regions, respectively, 30 kb±5, 100 kb±13, and 135 kb±8 downstream from exon 1b, involved in M-BCR rearrangements.8 D indicates the ABL breakpoint region involved in the three μ-BCR rearrangements, which is located 162±12 kb downstream from exon 1b. The bold arrows show the precise location of the ABL DNA breakpoints in the AR230 cell line, the CML patient and the AML patient (Genbank accession code: U07563).

Table 2 μ-BCR–ABL DNA breakpoint sequences for the three e19a2 junctions amplified by LM-PCR

The three μ-BCR breaks occurred in a 393 bp stretch of intron 19. In the CML patient, almost the entire sequence of intron 19 was preserved, with only six nucleotides deleted. The BCR breakpoint in AR230 was also located in the downstream part of intron 19, while in the AML patient the break occurred more towards the middle of intron 19 (Figure 1a). Breakpoints in the ABL gene were distributed over 24 kb at the 3′end of the first ABL intron (Figure 1c). In the CML patient, the ABL breakpoint occurred within an Alu element, and in the AR230 cell line Alu repeats were located within 400 bp at both sides of the breakpoint. No repeats were found in the μ-BCR breakpoint region.

This is the first study in which LM-PCR was used to identify the exact DNA breakpoints in the ABL and BCR genes in e19a2 junctions. The low incidence of e19a2 junctions may partly be caused by the relatively short length of intron 19 (1 kb) as compared with m-BCR (55 kb) and M-BCR (2.9 kb). Currently, primary factors that determine the preferential breakage sites in the BCR and ABL genes are unknown. It has been proposed that high densities of repetitive DNA, such as Alu elements, could provide hot spots for homologous recombination and mediate chromosomal translocation. The characterization of genomic DNA breakpoints in CML with BCR–ABL translocations has been reported.6, 7 In some cases, the BCR gene preferentially recombined with Alu elements; however, no unique pattern explained all DNA breaks. In our study, the μ-BCR recombined with regions close to or within Alu elements of the ABL gene in two out of three cases.

Conflicting data are reported with regard to the breakpoint sites in the ABL gene in CML. Nonrandom chromosomal breakpoints within the first intron of ABL have been suggested, with three breakpoint cluster regions downstream from exon 1b.8 However, others reported equally dispersed breaks over intron 1b without any clustering.9 The ABL breaks in our three e19a2 junctions were scattered over a 24 kb region at the 3′ part of intron 1b. During transcription exon 1a is probably spliced out, resulting in the BCR exon 19/ABL exon a2 mRNA junction.

Transcription of the p230BCR–ABL gene in the great majority of patients with N-CML is extremely low: the level of p230BCR–ABL transcripts in untreated N-CML patients was similar to the level of p210BCR–ABL transcripts in typical CML patients in cytogenetic remission after interferon-α treatment.10 This low transcription level of the p230BCR–ABL gene in most patients may hamper the use of RT-PCR at diagnosis and particularly during follow-up, which can be overcome by using genomic DNA breakpoint fusion sites as PCR targets. However, because the DNA breakpoint fusion regions, particularly of the ABL gene, are scattered over large regions and differ in each patient, special methods are needed for identification of these DNA breakpoint fusion sites. In our study, we used an LM-PCR to identify the e19a2 fusion at the DNA level.

Patient-specific DNA breakpoint fusion sites may be attractive PCR targets for monitoring of minimal residual disease (MRD) by real-time quantitative PCR using an allele-specific reverse primer positioned at the breakpoint area of the fusion genes, in combination with a μ-BCR-specific forward primer and probe. DNA targets for disease monitoring have several advantages over RNA targets, including low degradation rate, easy quantification (as only one target per cell is present), and stability throughout the disease course.

In conclusion, using LM-PCR, we identified DNA sequences of three e19a2 junctions which may potentially be used as patient-specific DNA targets for monitoring of MRD. Although Alu elements might be involved in some μ-BCR–ABL rearrangements, further studies on a larger series of e19a2-positive leukemias are needed to better understand the mechanisms by which μ-BCR–ABL DNA breaks are mediated.

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Acknowledgements

We are grateful to Professor Dr N Blanckaert for the continuous support, to Annemarie Wijkhuijs and Daniëlle Jacobs for their technical assistance and to Marieke Comans-Bitter for preparation of the figures. We thank Dr Hiroshi Wada who kindly donated the AR230 cell line. This work was supported by a grant of the IGA MZ CR: NC/7560-3.

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Correspondence to J J M van Dongen.

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Boeckx, N., Jansen, M., Haskovec, C. et al. Identification of e19a2 BCR–ABL fusions (μ-BCR breakpoints) at the DNA level by ligation-mediated PCR. Leukemia 19, 1292–1295 (2005). https://doi.org/10.1038/sj.leu.2403761

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