A comprehensive analysis of breakpoint cluster region-abelson fusion oncogene splice variants in chronic myeloid leukemia and their correlation with disease biology

Abstract

Background: BCR-ABL fusion oncogene is a hallmark of Chronic Myeloid Leukemia (CML). It results due to translocation between chromosome 22 and chromosome 9 [t (9; 22)(q34; q11)]. It gives rise to translation of a 210 KDa chimeric protein (p210), leading to enhanced tyrosine kinase activity and activation of leukemogenic pathways, ultimately causing onset of CML. In case of CML, the classic fusions are b2a2 or b3a2, fusing exon 13 (b2) or exon 14 (b3) of BCR, respectively, to exon 2 (a2) of ABL. The type of BCR-ABL transcripts are thought to be have different prognosis and hence useful in clinical decision-making. The frequencies of different fusion oncogenes associated with leukemia can vary in different ethnic groups and geographical regions due to interplay of genetic variation in different ethnic populations, diverse environmental factors and living style. Moreover, earlier relevant studies from our region were carried out in small subset of patients. Therefore, objective of this study was to find out frequencies of different BCR-ABL splice variants in larger subset of CML patients.

Methods: A nested reverse transcriptase polymerase chain reaction (RT-PCR) was established to detect BCR-ABL splice variants in 130 CML patients. Sensitivity of RT-PCR and ability to detect BCR-ABL fusion gene in least possible time was studied.

Results: BCR-ABL detection using our optimized RT-PCR protocol could be completed in 8 hours, starting from RNA extraction to Gel electrophoresis. Sensitivity of RT-PCR assay was of the order of 10(-6). Out of 130 Pakistani patients, 83 (63.84%) expressed b3a2 while 47 (36.15%) expressed b2a2 transcript.

Conclusion: Our RT-PCR was proved to be very quick to detect BCR-ABL fusion oncogene in CML patients within one working day. Because of its sensitivity, it can be used to monitor complete molecular response in CML. BCR-ABL RT-PCR and BCR-ABL splice variants frequency in our study differs from other ethnic groups. It shows that ethnic and geographical differences exist in BCR-ABL splice variant frequency, which may have a profound effect on disease biology as well as implications in prognosis and clinical management of BCR-ABL positive leukemias.

Keywords: b2a2; b3a2; breakpoint cluster region-abelson; breakpoint cluster region-abelson alternative splicing.

Figure 1

Formaldehyde gel showing bands of ribonucleic acid from three different samples of chronic myeloid leukemia patients

Figure 2

Agarose gel showing two types of characteristics breakpoint cluster region-abelson transcripts b2a2 (lanes 1, 4) and b3a2 (lanes 2, 3, 6), 234 bp and 305 bp respectively (M: 50 bp deoxyribonucleic acid ladder, NC: Negative control)

Figure 3

Amplification of breakpoint cluster region-abelson (BCR-ABL) fusion transcript b3a2 (305 bp) from serial dilutions of complementary deoxyribonucleic acid (cDNA) (10⁻⁰–10⁻⁷) showing sensitivity of nested polymerase chain reaction for the detection of BCR-ABL in chronic myeloid leukemia (M; 50 bp DNA ladder)