Chasseriau Jacques, Rivet Jérôme, Bilan Frédéric, Chomel Jean-Claude, Guilhot François, Bourmeyster Nicolas, Kitzis Alain
Laboratoire de Génétique Cellulaire et Moléculaire, UPRES EA2622, Poitiers Cedex, France.
J Mol Diagn. 2004 Nov;6(4):343-7. doi: 10.1016/S1525-1578(10)60530-2.
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.
慢性髓性白血病的诊断基于费城(Ph)染色体或BCR-ABL基因的检测。转录本中存在的连接可能因相互易位t(9;22)(q34;11)而有所不同。转录本(p190、p210或p230)的鉴定并不能揭示连接的类型,但该信息对于临床试验中患者的分类非常重要。大多数鉴定试剂盒不检测p230转录本,也无法确定异常断点。我们开发了一种临床分子诊断检测方法,能够识别所有BCR-ABL转录本,并通过单次检测对所有可能的转录本连接进行表征。该技术基于逆转录聚合酶链反应(RT-PCR)和PCR毛细管电泳。对于每个患者样本,我们使用三种不同的BCR引物进行RT-PCR,每种引物与特定的不同荧光染料以及唯一的反向ABL引物偶联。根据转录本的不同,每次RT-PCR仅使用一种BCR引物。经过毛细管电泳和荧光测定后,我们能够同时识别转录本及其连接。