摘要
目的探讨寡核苷酸芯片在儿童急性淋巴细胞性白血病(ALL)融合基因检测中的应用。方法针对儿童ALL常见的5种融合基因:TEL/AML1、E2A/PBX1、BCR/ABLp190、BCR/ABLp210、MLL/AF4,设计特异的融合基因片段为芯片探针,合成后用点样仪点片制成寡核苷酸芯片。提取疾病初期白血病患儿的骨髓/外周血标本的总RNA,进行多重巢式逆转录聚合酶链反应(RT—PCR)、荧光标记并与芯片杂交,以检测白血病细胞中特异融合基因。结果该芯片可以从患儿骨髓样本RNA中准确检测到阳性内参序列、特异的融合基因以及融合位点。结论采用寡核苷酸芯片方法,可快速、同时筛选出5种染色体结构畸变产生的融合基因及融合位点,为ALL患儿危险分层、治疗选择、预后判断提供重要依据。此种方法应用于筛查初治白血病患儿融合基因表达时优缺点并存,具有一定临床实用价值。
Objective To evaluate the possibility of applying oligonucleotide microarrays for detection of the fusion genes in childhood acute lymphoblastic leukemia (ALL). Methods To detect five types of fusion genes emerging frequently in childhood ALL including TEL/AML1, E2A/PBX1, BCR/ ABLpl90, BCR/ABLp210, MLL/AF4, probes were designed, synthesized and spotted on the chemicalmaterial-coated-glass plates in array. The total RNAs were extracted from patients' bone marrow or peripheral blood cells at the beginning of diagnosis, analyzed by multiplex nested reverse-transcription-polymerase chain reaction (RT-PCR), and labeled by fluorescein. The products of RT-PCR were hybridized with microarray in order to detect specific types of fusion genes in leukemia cells. Results Distinctive hybridization signals were obtained for internal positive control and specific types of fusion genes. TEL/AML1 gene was found positive in 2 of the 36 cases, E2A/PBX1 gene in 3, BCR./ABLp190 gene in 2, BCR./ ABLp210 gene in one, and MLL/AF4 gene in one. The results of the microarray and RT-PCR were consistent. Conclusion The microarray-based assay could screen 5 types of chromosome structural aberrations and the splice variants at the same time. It can provide reliable and helpful information for patient stratification, evaluation of therapeutic effects and prediction of prognosis in childhood ALL, although there are both advantages and disadvantages in applying this new method.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2008年第3期198-202,共5页
Chinese Journal of Pediatrics
关键词
寡核苷酸类
基因融合
白血病
逆转录聚合酶链反应
Oligonucleotides
Gene fusion
Leukemia
Reverse transcriptase polymerase chain reaction