IDENTIFICATION OF COMPLEX GENOMIC BREAKPOINT JUNCTIONS IN THE T(9-11)MLL-AF9 FUSION GENE IN ACUTE-LEUKEMIA

The MLL gene at chromosome 11, band q23, is involved in translocations with as many as 40 different chromosomal bands. Virtually all breakpo ints occur within an 8.3 kb BamH1 fragment and result in 5' MLL fused to partner genes in a 5'-3' orientation. The translocation t(9;11)(p22 ;q23), which results in the fusion of MLL to AF9, is the most common o f the 11q23 chromosomal abnormalities observed in de novo acute myeloi d leukemia (AML), in therapy related leukemia (t-AML), and rarely in a cute lymphoblastic leukemia (ALL). We have studied 24 patients with a t(9;11) and an MLL rearrangement, including 19 patients with AML, four with t-AML, and one with ALL. To understand the mechanisms of this il legitimate recombination, we cloned and sequenced the t(9;11) transloc ation breakpoint junctions on both derivative chromosomes from one AML patient and from the Mono Mac 6 (MM6) cell line, which was derived fr om a patient with AML. Two different complex junctions were noted. In the AML patient, both chromosome 11 and 9 breaks were staggered, occur red in Alu DNA sequences, and resulted in a 331 bp duplication. In the MM6 cell line, breaks in chromosomes 11 and 9 were also staggered, bu t, in contrast to the finding in the AML patient, the breaks did not i nvolve Alu DNA sequences and resulted in a 664 bp deletion at the brea kpoints. Using reverse transcriptase (RT-) PCR, we analyzed 11 patient samples, including the two just described, for MLL-AF9 fusions. The f usion occurred in six of seven AML patients, two of two t-AML patients , one patient with ALL, and in the MM6 cell line. Interestingly, all o f the breaks within the AFP gene in AML patients occurred in the centr al AF9 exon, called Site A by others, whereas in the single ALL patien t the breakpoint mapped to a more 3' region of the AF9 gene. Our data, when combined with those of others, suggest that the fusion point wit hin the AF9 gene, and thus the amount of AF9 material included in the MLL-AF9 fusion gene product, may influence the phenotype of the result ing leukemia. This further supports the proposal that the MLL transloc ation partner genes play a critical role in the leukemogenic process. (C) 1997 Wiley-Liss, Inc.