混合系白血病全长基因在AML-M4/M5患者中的突变检测

Mutational Detection of Full-length Mixed Lineage Leukemia Gene in Patients with De Novo AML-M4 and M5

急性髓系白血病(AML)-M4、M5常有11号染色体异常,定位于11q23的混合系白血病(MLL)基因异常所形成的MLL-PTD和MLL融合基因在该病的发病机理中具有重要作用。但在该类白血病绝大多数未能检测出染色体水平异常,是否存在MLL基因的其他突变?本研究对MLL全长基因的外显子进行了测序分析。选取25例初发核型正常的AML M4、M5患者(排除MLL融合基因和MLL-PTD及M4eo),进行了MLL全长基因的外显子PCR扩增直接测序分析,对发现的突变在基因组DNA水平进行验证。利用Mass Array方法在正常对照样本和AML扩大样本中对发现的点突变进行检测。结果发现,在MLL的RD、PHD、TAD和SET功能域中存在着高频的缺失、插入及多个点突变;同时对照研究正常样本,发现也存在上述的变异,且突变频率无统计学差别(P>0.05)。结论:检测到的MLL基因发生在mRNA水平的缺失、插入和多个点突变,分别是MLL在转录水平的选择性剪接和MLL的单核苷酸多态性,它们共同构成了MLL的遗传多态性;这些MLL基因遗传多态性可能与AML M4、M5的发病不直接相关,对于治疗、预后及其他生物学表型的意义还有待进一步探讨。

Abnormalities of chromosome 11 involving mixed lineage leukemia （MLL） on 11 q23 are often seen in acute myeloid leukemia （AML） -M5 or AML-M4. The fusion gene of MLL-PTD and MLL plays a critical role in the pathogenesis of these AML. However, rare chromosome abnormalities have been identified in this type of leukemia. To explore whether there were other MLL gene mutations at M4 and M5, in this study all of the MLL exons were sequenced at cDNA level. 25 patients with de novo AML-M4 or M5 with normal karyotypes excluding M4eo and MLL fusion gene or MLL-PTD were selected, the amplification and direct sequencing analysis of full length MLL gene exons were carded out, then the mutations found were verified at genomic DNA level. Furthermore, the point mutations were tested in normal samples and a larger group of AML patients using the platform of Mass Array. The results showed that high- frequency deletion/insertion and point mutations in RD, PI-ID, TAD and SET domains of MLL were found, while these alterations in normal samples and other subtypes of AML samples were also verified, and without significant difference （ P 〉 0.05 ）. It is concluded that a variety of deletions/insertions in MLL mRNA and point mutations are respectively alternative splicing of MLL gene at transcriptional level and single nucleotide polymorphism. These alternations together constituted genetic polymorphisms of MLL. Although these variations may not play a direct role in the molecular pathogenesis of AML-M4 or M5, their correlations to clinical treatment and prognosis need to be further explored.