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

Recently, a polymorphic base in exon 13 of the BCR gene (exon b2 of the maj or breakpoint cluster region) has been identified in the eighth position be fore the junctional region of BCR-ABL cDNA. Cytosine replaces thymidine; th e corresponding triplets are AAT (T allele) and AAC (C allele), respectivel y, both coding for asparagine. Therefore, this polymorphism has no implicat ion in the primary structure of BCR and BCR-ABL proteins. However, since th e alteration is located close to the fusion region it may have a significan t influence on the annealing of PCR primers, probes for real time PCR, and antisense oligonucleotides. We have developed a RT-PCR-based screening meth od to easily identify polymorphic BCR and BCR-ABL alleles in CML patients a nd normal individuals in order to estimate their frequency. After amplifica tion from cDNA, a melting curve of a specific fluorogenic probe mapping to the 3' end of BCR exon b2 and spanning the polymorphism readily discriminat es between normal and polymorphic BCR and BCR-ABL alleles. This reporter pr obe 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 (LightCycle r technology). T and C alleles were discriminated by a melting temperature difference of the reporter probe of 3.2 K. We have investigated cDNAs deriv ed from leukocytes from seven cell lines and a total of 229 individuals: no rmal donors, n=15; BCR-ABL negative chronic myeloproliferative disorders, n =30; BCR-ABL negative acute leukemias, n=11; b2a2(BCR-ABL) positive CML, n = 93; and b3a2(BCR-ABL) positive CML, n=80, The frequency of the C allele w as 33.0% in BCR-ABL negative individuals, 30.6% in b2a2(BCR-ABL), and 23.8% in b3a2(BCR-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 b 2 of BCR or BCR-ABL alleles demonstrated this polymorphism. We conclude tha t 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 cri tical region in order to avoid underestimation of the number of BCR-ABL tra nscripts.