Panhandle PCR for cDNA: a rapid method for isolation of MLL fusion transcripts involving unknown partner genes.
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.
Megonigal
M D
MD
Division of Oncology, Joseph Stokes, Jr., Research Institute, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, 19104, USA.
Rappaport
E F
EF
Wilson
R B
RB
Jones
D H
DH
Whitlock
J A
JA
Ortega
J A
JA
Slater
D J
DJ
Nowell
P C
PC
Felix
C A
CA
D000483:Alleles; D017398:Alternative Splicing / Q000235:genetics; D002648:Child; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005091:Exons / Q000235:genetics; D011495:Histone-Lysine N-Methyltransferase; D006801:Humans; D007223:Infant; D007621:Karyotyping; D008297:Male; D008969:Molecular Sequence Data; D051788:Myeloid-Lymphoid Leukemia Protein; D009363:Neoplasm Proteins; D009690:Nucleic Acid Conformation; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D010445:Peptide Elongation Factors; D016133:Polymerase Chain Reaction / Q000379:methods*; D011519:Proto-Oncogenes; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D015203:Reproducibility of Results; D018232:Rhabdomyosarcoma, Alveolar / Q000235:genetics; D012512:Sarcoma, Ewing / Q000235:genetics; D013698:Templates, Genetic; D014157:Transcription Factors / Q000235:genetics; D035602:Transcriptional Elongation Factors; D014178:Translocation, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C090862:ELL protein, human; C497422:KMT2A protein, human; C092513:MLLT10 protein, human; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D051788:Myeloid-Lymphoid Leukemia Protein; D011495:Histone-Lysine N-Methyltransferase