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  • Biotechnical Methods Section BTS
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Biotechnical Methods Section (BTS)

Frequent polymorphism in BCR exon b2 identified in BCR-ABL positive and negative individuals using fluorescent hybridization probes

Leukemia volume 14, pages 2006–2010 (2000)Cite this article

Abstract

Recently, a polymorphic base in exon 13 of the BCR gene (exon b2 of the major breakpoint cluster region) has been identified in the eighth position before the junctional region of BCR-ABL cDNA. Cytosine replaces thymidine; the corresponding triplets are AAT (T allele) and AAC (C allele), respectively, both coding for asparagine. Therefore, this polymorphism has no implication in the primary structure of BCR and BCR-ABL proteins. However, since the alteration is located close to the fusion region it may have a significant influence on the annealing of PCR primers, probes for real time PCR, and antisense oligonucleotides. We have developed a RT-PCR-based screening method to easily identify polymorphic BCR and BCR-ABL alleles in CML patients and normal individuals in order to estimate their frequency. After amplification from cDNA, a melting curve of a specific fluorogenic probe mapping to the 3′ end of BCR exon b2 and spanning the polymorphism readily discriminates between normal and polymorphic BCR and BCR-ABL alleles. This reporter probe is 3′ labeled with fluorescein and placed next to 5′ LC Red640-labeled anchor probes mapping to the 5′ ends of BCR exon b3 or ABL exon a2 so that resonance energy transfer occurs when the probes are hybridized (LightCycler technology). T and C alleles were discriminated by a melting temperature difference of the reporter probe of 3.2 K. We have investigated cDNAs derived from leukocytes from seven cell lines and a total of 229 individuals: normal donors, n = 15; BCR-ABL negative chronic myeloproliferative disorders, n = 30; BCR-ABL negative acute leukemias, n = 11; b2a2BCR-ABL positive CML, n = 93; and b3a2BCR-ABL positive CML, n = 80. The frequency of the C allele was 33.0% in BCR-ABL negative individuals, 30.6% in b2a2BCR-ABL, and 23.8% in b3a2BCR-ABL positive CML. In CML patients, 27.7% of BCR-ABL and 27.2% of BCR alleles had the C allele (NS). In total, 132 of 458 (28.8%) exons b2 of BCR or BCR-ABL alleles demonstrated this polymorphism. We conclude that a thymidine/cytosine replacement occurs frequently in BCR exon b2. Probes for real time quantitative RT-PCR should be designed not to map to the critical region in order to avoid underestimation of the number of BCR-ABL transcripts.

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Acknowledgements

Our study was supported by grants from the Deutsche José-Carreras-Stiftung eV, the Tumorzentrum Mannheim/ Heidelberg, the Forschungsfonds der Fakultät für Klinische Medizin Mannheim, Germany, and by Roche Diagnostics, Mannheim, Germany. Cell lines BV173, K562, and HL60 were bought from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany, cDNA from cell lines KCL22, LAMA84, and KG1a was kindly provided by Dr Junia V Melo, Imperial College School of Medicine, London, UK.

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  1. III. Medizinische Universitätsklinik, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Wiesbadener Strasse 7–11, Mannheim, 68305, Germany

    S Saußele, A Weißer, MC Müller, M Emig, PLa Rosée, P Paschka, C Kuhn, A Willer, R Hehlmann & A Hochhaus

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Saußele, S., Weißer, A., Müller, M. et al. Frequent polymorphism in BCR exon b2 identified in BCR-ABL positive and negative individuals using fluorescent hybridization probes. Leukemia 14, 2006–2010 (2000). https://doi.org/10.1038/sj.leu.2401929

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