Technical Notes
PML/RARa translocation t (15; 17) for diagnosis
Clinical Significance:
PML/RARa is the molecular equivalent of t (15; 17) that is highly characteristic of acute promyelocytic leukemia (AML-M3), about 95% of acute promyelocytic leukemias harbor this translocation (1). It is important to identify leukemias harboring PML/RARa translocation because they have distinct clinical features and therapeutic management options (2).The translocation involves the PML gene on chromosome 15 and the Retinoic acid receptor alpha (RARa) gene on chromosome 17. The altered retinoic acid receptor structure renders it less sensitive to the effects of retinoic acid, resulting in uncontrolled cell proliferation and failure of promyelocytes to differentiate. The defect can be overcome by treating with high doses of retinoic acid thus underlining the importance of sensitive detection of this translocation (3).
The mRNA for PML/RARa transcript can be sensitively identified by a Reverse Transcriptase PCR assay (4, 5).
Methodology:
Patient RNA would be isolated, purified, and converted to cDNA by the enzyme reverse transcriptase. The cDNA would be detected by a nested PCR reaction. An additional RT-PCR amplification directed at RARa gene segment would be performed as a control for sample RNA and reverse transcription quality. PCR products would be analyzed by electrophoresis and UV transillumination of ethidium bromide-stained gels.The assay has been validated by a positive control reaction using RNA of confirmed AML-M3 patients.
References:
1) Melnick A., Licht J.D. (1999) Blood 93, 3167.2) Grignani F., Fagioli M., Alcalay M. et al (1994) Blood 83, 10.
3) Miller W.H. Jr., Kakizuka A., Frankel S.R. et al (1992) Proc. Natl. Acad. Sci. USA, 89, 2694.
4) Fukutani H., Naoe T., Ohno R. et al (195) Leukemia 9, 588.
5) Hussey C.E., Lyon E., Millson A. et al (1999) Am. J. Clin. Pathol. 112, 256.