Megonigal M D, Rappaport E F, Wilson R B, Jones D H, Whitlock J A, Ortega J A, Slater D J, Nowell P C, Felix C A
Division of Oncology, Joseph Stokes, Jr., Research Institute, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, 19104, USA.
Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9597-602. doi: 10.1073/pnas.150241797.
Identifying translocations of the MLL gene at chromosome band 11q23 is important for the characterization and treatment of leukemia. However, cytogenetic analysis does not always find the translocations and the many partner genes of MLL make molecular detection difficult. We developed cDNA panhandle PCR to identify der(11) transcripts regardless of the partner gene. By reverse transcribing first-strand cDNAs with oligonucleotides containing coding sequence from the 5' MLL breakpoint cluster region at the 5' ends and random hexamers at the 3' ends, known MLL sequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with the fusion point of the chimeric transcript in the loop and the use of MLL primers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normal MLL allele in the cell line MV4-11. cDNA panhandle PCR then was used to identify the fusion transcripts in two cases of treatment-related acute myeloid leukemia where the karyotypes were normal and the partner genes unknown. cDNA panhandle PCR revealed a fusion of MLL with AF-10 in one case and a fusion of MLL with ELL in the other. Alternatively spliced transcripts and exon scrambling were detectable by the method. Leukemias with normal karyotypes may contain cryptic translocations of MLL with a variety of partner genes. cDNA panhandle PCR is useful for identifying MLL translocations and determining unknown partner sequences in the fusion transcripts.
识别位于染色体带11q23的MLL基因易位对于白血病的特征描述和治疗很重要。然而，细胞遗传学分析并不总能发现这些易位，而且MLL的众多伙伴基因使得分子检测变得困难。我们开发了cDNA锅柄PCR来识别der(11)转录本，而不考虑伙伴基因。通过用5'端含有来自5'MLL断点簇区域编码序列且3'端含有随机六聚体的寡核苷酸逆转录第一链cDNA，已知的MLL序列被连接到未知的伙伴序列上。这使得能够形成茎环模板，嵌合转录本的融合点在环中，并在双向PCR中使用MLL引物。通过检测细胞系MV4-11中已知的融合转录本和正常MLL等位基因的转录本对该检测方法进行了验证。然后，cDNA锅柄PCR被用于识别两例治疗相关急性髓系白血病中的融合转录本，这两例患者的核型正常且伙伴基因未知。cDNA锅柄PCR在一例中揭示了MLL与AF-10的融合，在另一例中揭示了MLL与ELL的融合。该方法还可检测到选择性剪接的转录本和外显子重排。核型正常的白血病可能含有MLL与多种伙伴基因的隐匿性易位。cDNA锅柄PCR对于识别MLL易位和确定融合转录本中的未知伙伴序列很有用。
