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Panhandle PCR strategy to amplify MLL genomic breakpoints in treatment-related leukemias
M D Megonigal1, E F Rappaport, D H Jones
1Division of Oncology, Jr. Research Institute, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA 19104-4318, USA.
Abstract:
Panhandle PCR amplifies genomic DNA with known 5' and unknown 3' sequences from a template with an intrastrand loop schematically shaped like a pan with a handle. We used panhandle PCR to clone MLL genomic breakpoints in two pediatric treatment-related leukemias. The karyotype in a case of treatment-related acute lymphoblastic leukemia showed the t(4;11)(q21;q23). Panhandle PCR amplified the translocation breakpoint at position 2158 in intron 6 in the 5' MLL breakpoint cluster region (bcr). The karyotype in a case of treatment-related acute myeloid leukemia was normal, but Southern blot analysis showed a single MLL gene rearrangement. Panhandle PCR amplified the breakpoint at position 1493 in MLL intron 6. Screening of somatic cell hybrid and radiation hybrid DNAs by PCR and reverse transcriptase-PCR analysis of the leukemic cells indicated that panhandle PCR identified a fusion of MLL intron 6 with a previously uncharacterized sequence in MLL intron 1, consistent with a partial duplication. In both cases, the breakpoints in the MLL bcr were in Alu repeats, and there were Alu repeats in proximity to the breakpoints in the partner DNAs, suggesting that Alu sequences were relevant to these rearrangements. This study shows that panhandle PCR is an effective method for cloning MLL genomic breakpoints in treatment-related leukemias. Analysis of additional pediatric cases will determine whether breakpoint distribution deviates from the predilection for 3' distribution in the bcr that has been found in adult cases.
Insights
Panhandle PCR effectively cloned mixed lineage leukemia (MLL) gene breakpoints in pediatric leukemias. This method identified novel MLL gene fusions and highlighted the role of Alu repeats in these rearrangements.
Area of Science:
- Genetics
- Molecular Biology
- Oncology
Background:
- Treatment-related leukemias can involve complex genomic rearrangements.
- The mixed lineage leukemia (MLL) gene is frequently implicated in leukemogenesis.
Purpose of the Study:
- To utilize panhandle PCR for cloning MLL gene genomic breakpoints in pediatric treatment-related leukemias.
- To investigate the nature of MLL gene rearrangements and identify potential contributing sequences.
Main Methods:
- Panhandle PCR was employed to amplify MLL genomic breakpoints from DNA of pediatric leukemia patients.
- Karyotyping, Southern blot analysis, and PCR-based screening of hybrid DNAs were performed.
Main Results:
- Panhandle PCR successfully cloned MLL breakpoints in two pediatric treatment-related leukemias, including one with a normal karyotype.
- A novel MLL gene fusion involving MLL intron 6 and a previously uncharacterized sequence in MLL intron 1 was identified, suggesting a partial duplication.
- Breakpoints within the MLL breakpoint cluster region (bcr) and partner DNA sequences were found in proximity to Alu repeats, implicating them in the rearrangements.
Conclusions:
- Panhandle PCR is an effective technique for cloning MLL genomic breakpoints in treatment-related leukemias.
- Alu sequences may play a significant role in mediating MLL gene rearrangements.
- Further studies on pediatric cases are needed to compare breakpoint distribution with adult cases.