Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K, Sonoda Y, Fujimoto T, Misawa S
Department of Internal Medicine III, Kyoto Prefectural University of Medicine, Japan.
Leukemia. 1996 Dec;10(12):1911-8.
We analyzed mRNA expression of the flt3 gene in 30 patients with acute myeloid leukemia (AML) and 50 with acute lymphoblastic leukemia (ALL). Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of flt3 was observed in 61 patients; 22 (73%) with AML and 39 (78%) with ALL. Among these, five patients with AML (one M2, two M4, and two M5) showed unexpected longer transcripts with a primer combination which could amplify the transmembrane (TM) domain through the juxtamembrane (JM) domain. For those patients who expressed flt3 mRNA, the extracellular domain of the flt3 gene was also examined by RT-PCR, but no length abnormality was seen in this region. We further analyzed the TM domain through the second tyrosine kinase domain by genomic amplifications. The five patients who showed aberrant flt3 transcripts exhibited abnormal longer PCR products in addition to the germline products at a region corresponding to the JM through the first TK (TK1) domains. Sequence analyses of the abnormal RT-PCR products demonstrated that partial sequences were tandemly duplicated. Because all these altered transcripts were in-frame, deduced protein products could be expected. Sequence analyses of the genomic DNA revealed that three of the five patients showed a simple internal duplication within exon 11; one had an internal duplication (26 bp) with a 4-bp insertion; and in the fifth patient, a 136-bp sequence from the 3' part of exon 11 to intron 11 and the first 16-bp sequence of exon 12 were each duplicated with 1-bp insertion. In order to confirm the tumor specificity of these alterations, DNA samples obtained at complete remission were also analyzed in the three patients harboring an flt3 duplication, but no abnormal PCR product other than germline was detected in any of the samples. Our results suggest that an internal tandem duplication at the JM/TK1 domains of the flt3 gene is a somatic change detected preferentially in AML, possibly containing a monocytic component.
我们分析了30例急性髓系白血病（AML）患者和50例急性淋巴细胞白血病（ALL）患者中flt3基因的mRNA表达。使用逆转录聚合酶链反应（RT-PCR），在61例患者中观察到flt3表达；22例（73%）AML患者和39例（78%）ALL患者。其中，5例AML患者（1例M2、2例M4和2例M5）使用可通过近膜（JM）结构域扩增跨膜（TM）结构域的引物组合时，显示出意外的更长转录本。对于那些表达flt3 mRNA的患者，还通过RT-PCR检测了flt3基因的胞外结构域，但该区域未发现长度异常。我们通过基因组扩增进一步分析了从第二个酪氨酸激酶结构域到TM结构域。这5例显示flt3转录本异常的患者，在对应于通过第一个酪氨酸激酶（TK1）结构域的JM区域，除了种系产物外，还表现出异常的更长PCR产物。对异常RT-PCR产物的序列分析表明，部分序列串联重复。由于所有这些改变的转录本都是框内的，因此可以预期推导的蛋白质产物。基因组DNA的序列分析表明，5例患者中有3例在外显子11内出现简单的内部重复；1例有内部重复（26 bp）并伴有4 bp插入；在第5例患者中，从外显子11的3'部分到内含子11的136 bp序列以及外显子12的前16 bp序列各自重复并伴有1 bp插入。为了确认这些改变的肿瘤特异性，还对3例存在flt3重复的患者在完全缓解时获得的DNA样本进行了分析，但在任何样本中均未检测到除种系以外的异常PCR产物。我们的结果表明，flt3基因的JM/TK1结构域处的内部串联重复是一种体细胞变化，在AML中优先检测到，可能包含单核细胞成分。
