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

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 3Ј end of BCR exon b2 and spanning the polymorphism readily discriminates between normal and polymorphic BCR and BCR-ABL alleles. This reporter probe is 3Ј labeled with fluorescein and placed next to 5Ј LC Red640-labeled anchor probes mapping to the 5Ј 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; b2a2

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program

“…24 On the BCR gene, Y (cytidine or thymidine) appears on the BCR primer at position 3188, according to the polymorphism recently described. 53 RNA and cDNA from cell lines and leukemia samples at diagnosis RNA and/or cDNA samples were prepared centrally by the FG network leaders (Table 1) and distributed on dry ice to members of their respective networks during phases I-IVa. FG transcriptpositive cell line RNA was commonly used (see Table 1 for specific cell lines), except for rare FG transcripts (PML-RARA bcr2 and bcr3 and CBFB-MYH11 type D and E) for which patient RNA was provided by the network leader.…”

Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results

“…Technologies based on hydrolysis and hybridization probes are both suitable and can give comparable results 41,42 provided that standardized and validated procedures are followed, as recommended by the EAC. 13 Assay design should take account of the polymorphic site in BCR exon 13,7,43,44 and also the fact that the small intron between ABL exons 2 and 3 can be amplified efficiently from genomic DNA, which can thereby distort results and decrease sensitivity. The probes and primers should be RNA specific and tested with genomic DNA.…”

Molecular profiling of chronic myeloid leukemia in eastern India

“…The BCR intronic polymorphism, however, occurs at the invariant A of a sequence that is the best match to the poorly conserved branchpoint. It has been proposed that the change results in reduced efficiency of splicing of intron 13 of BCR and BCR-ABL allele [5][6][7] and use of the acceptor site at the end of intron 14, leading to coexpression of both the transcripts.…”