Cytogenetic and molecular genetic investigations show that breakpoint sites of recurring structural chromosome abnormalities mark the location of cancer-related genes. The molecular characterisation of these recurring chromosome abnormalities in the leukaemias has provided powerful and sensitive diagnostic and treatment monitoring assays, and has refined prognostic stratification. For example, the t(4;11)/AF4-MLL or t(9;22)/BCR-ABL1 fusion genes are now known to confer a poor prognosis in pre-B-ALL, indicating the need for more intensive treatment. Such genetic markers are not only crucial for monitoring treatment responses and relapse, but provide targets for more effective and less toxic therapies such as Imatinib (Glivec) for chronic myeloid leukaemia (CML). However, in spite of these advances, the genetic cause of almost 40% of human ALL cases remains unknown.
We have targeted the breakpoints of a novel t(5;10)(q22;q24) translocation found as the sole cytogenetic abnormality in the leukemic cells of a 59 year old male with ALL. Positional cloning using fluorescent in situ hybridization (FISH), sequencing, in silico analyses, PCR analyses and Southern hybridization studies of leukemic DNA, have mapped the 10q24 breakpoint to within intron 2 of UniGene Hs.568769. The unique transcriptional activity of Hs.568769 has been confirmed by RACE and Northern blot studies, and interestingly overlaps 5'DNTT, the gene encoding terminal deoxynucleotidyltransferase (TdT), a key diagnostic marker for leukemia. On chromosomes 5, FISH studies map the breakpoint centromeric of the APC gene at 5q22.1, identifying 10 candidate genes for disruption by the t(5;10).
We have recently developed a red/green 10q24 breakapart FISH probe to begin screening a larger cohort of acute leukaemia patients for similar genetic aberrations. Current studies are focused on isolating the 10q24 breakpoint junction sequence using custom designed PCR protocols. Preliminary sequence analysis has identified chromosome 15 sequence attached to 10q24 indicating that this may be a complex rearrangement. Reverse transcription PCR analyses of the Hs.568769 transcript and DNTT in normal purified haematopoietic tissues and 10 leukemic cell lines is presently in progress to characterize the normal activity of Hs.568769 compared with that of DNTT and to determine if Hs.568769 is an alternate transcript of DNTT as suggested by the latest build (36.2) of the Human Genome Project. UniGene ‘Hs.568769’ has been retired and the full length cDNA transcript of Hs.568769 is now clustered with the DNTT transcripts. If Hs.568769 is an alternative transcript of DNTT, then this is the first reported case of DNTT disruption in leukemia.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA