SHORT REPORT A polymorphism in exon b2 of the major breakpoint cluster region (M-bcr) identified in chronic myeloid leukaemia patients ROSELY DE V. MEISSNER , 1 PAULA M. B. D IAS , 1 D IMAS T. C OVAS , 3 FANI J OB , 2 MA ´ RCIA L EITE 2 AND NANCE B. NARDI 1 1 Departamento de Gene ´tica, Universidade Federal do Rio Grande do Sul, Porto Alegre, 2 Hospital de Clı ´nicas de Porto Alegre, and 3 Centro Regional de Hemoterapia Hemocentro, Ribeira ˜o Preto, Brazil Received 6 April 1998; accepted for publication 3 July 1998 Summary. The BCR/ABL junctional region and the b3 exon from chronic myeloid leukaemia (CML) patients were sequenced. In all 21 samples analysed the junctional region, as well as the b3 exon of 8 b3a2 mRNA molecules, presented no differences to the already described sequences. However, we identified a polymorphic base in the b2 exon in two out of seven b3a2 samples, four out of 10 b2a2 samples and all four b3a2/b2a2 samples analysed. In the eighth position before the junctional region of BCR/ABL cDNA, a cytosine replaces thymine in these cases. The polymorphism described here could be a useful marker for the differentiation of normal and rearranged BCR alleles in heterozygotes. Keywords: chronic myeloid leukaemia, BCR/ABL, BCR exon polymorphism, RT-PCR, M-bcr sequencing. Chronic myeloid leukaemia (CML) is a clonal disease characterized by the presence of BCR/ABL hybrid gene in >90% of patients. Two principal types of mRNAs (b2a2 or b3a2), which differ by the presence of BCR exon 14 (b3) and can also be co-expressed by alternative splicing, are transcribed from the gene. Although the type of BCR/ABL mRNA in CML patients has been determined by RT-PCR in several studies, in most of them the kind of junction in the amplified product was determined through the electrophor- etic analysis of molecular weight of the fragments. In a smaller number of cases the PCR products were analysed by hybridization with oligomers specific for the junctions. None of these approaches seemed to be sensitive enough for the detection of small variations. Particularly in the case of hybridization, there have been reports of cross reactivity between oligomers specific for b2a2 and b3a2 junctions (Lion et al, 1991; Morgan et al, 1990), which is possibly due to the potential presented by single-strand exon b3 to form secondary loop structures (Mahon et al, 1995). To investi- gate possible differences on the hybrid mRNA produced by CML cells, which can be responsible for different leukaemic profiles, the BCR/ABL junction region as well as the b3 exon from CML patients were sequenced. MATERIALS AND METHODS Peripheral mononuclear cells from 21 patients with clini- cally and cytogenetically diagnosed CML, including patients in chronic or blast phase of the disease, were investigated. Total RNA was extracted from 5 × 10 6 to 1 × 10 7 cells with Trizol (Gibco BRL, Grand Island, N.Y., U.S.A.) and resuspend in 20 ml DEPC (diethylpyrocarbamate, Sigma, St Louis, Mo., U.S.A.) treated water. Subsequently 8 ml of RNA solution were converted to cDNA with a random hexamer primer with the use of First Strand cDNA Synthesis kit (Pharmacia Biotech, Sa ˜o Paulo, SP, Brazil) according to the manufacturer’s instructions. RT-PCR amplification was performed with primers located on ABL exon a2 (5 0 -CTCCACTGGCCA CAAAAT-3 0 , antisense) and on M-bcr exon b2 (5 0 -TTCA GAAGCTTCTCCCTG-3 0 , sense) as described by Lion et al (1991). The PCR products were separated by manual agarose purification and amplified during a second round of asymmetric PCR using the M-bcr exon b2 primer (5 0 - TTCAGAAGCTTCTCCCTGACATCCG-3 0 , sense) and ABL exon a2 primer (5 0 -CTCCACTGGCCACAAAATCATACAG-3 0 , antisense) in the proportion of 1:50 (Gyllesten & Erlich, 1988; Trka et al, 1995). These primers, as well as the first PCR primers, specify a 327 bp fragment in BCR/ABL rearranged cells with M-bcr exon b3 linked to ABL exon a2. The samples were heated at 54°C for 2 min initially and 40 s in each subsequent cycle to denature DNA. The primers were allowed to anneal at 60°C for 40 s and strand extension was carried out at 72°C for 1 min during 35 cycles of British Journal of Haematology , 1998, 103, 224–226 224  1998 Blackwell Science Ltd Correspondence: Dr Nance B. Nardi, Departamento de Gene ´tica, Universidade Federal do Rio Grande do Sul, C.P. 15053, 91. 501-970 Porto Alegre, RS, Brazil.