Characterization of the different BCR-ABL transcripts with a single multiplex RT-PCR

Abstract

The diagnosis of chronic myeloid leukemia is based on detection of the Philadelphia (Ph) chromosome or the BCR-ABL gene. The junction present in the transcript may vary according to the reciprocal translocation t(9;22)(q34;11). Identification of the transcript (p190, p210 or p230) does not reveal the type of junction but this information is very important for classification of patients in clinical trials. Most identification kits do not explore p230 transcripts and are unable to determine exotic breakpoints. We have developed a clinical molecular diagnosis assay, able to identify all of the BCR-ABL transcripts and, by single assay, to characterize all of the possible transcript junctions. This technique is based on RT-PCR and PCR-capillary electrophoresis. For each patient sample, we performed RT-PCR with three different BCR primers each coupled to a specific different fluorochrome and a unique reverse ABL primer. Depending on the transcript, only one BCR primer was used for each RT-PCR. After capillary electrophoresis and fluorescence determination, we were able to identify both the transcript and its junction at the same time.

Figure 1

Molecular structure of BCR and c-ABL genes and their main translocated transcripts. Arrows indicate breakpoints in BCR and c-ABL. Asterisks show sense primers which are used for the detection of different transcripts (e1, Tet; b1, Fam; e19, Hex). The reverse primer is systematically a3.

Figure 2

Analysis of BCR-ABL transcripts in five different patients. The bottom panel of each result is the GS 500 size standard labeled with Tamara (75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, and approximately 500 bases). The top panel shows the fluorescent multiplex electropherogram of each sample analyzed. For each sample, we indicate the color, the band size of the predominant amplicon in bases, and the corresponding transcript.