Molecular characterization of the genomic breakpoint junction in a t(11;22) translocation in Ewing sarcoma

Polymerase chain reaction (PCR)-based nucleotide sequence analysis was perf ormed in 12 cases of Ewing sarcoma on the cDNA and/or genomic DNA breakpoin t regions of a t(11;22)(q24;q12), which joins the EWS gene located on chrom osome 22 with the FL11 gene located on chromosome 11, in order to understan d the molecular mechanism of this translocation. Reverse transcriptase-PCR on total tumor cell RNA from the examined cases showed five types of EWS-FL 11 chimeric product, resulting from various junctions between EWS exon 7 or 10 with FL11 exon 5, 6, or 8. Sequencing of the genomic fusion junctions o f EWS-FL11 in seven cases showing three types of the chimeric cDNA products revealed that most of the breakpoint junctions shared common nucleotide(s) from both genes, and that the breakpoints in EWS introns 7 and 10 clustere d within 100 bp and 300 bp, respectively. All the junctions were found to b e flanked by various oligomers, among which a consensus sequence, 5'-AGAAAA RDRR-3', was found near the breakpoints of both genes in four cases, sugges ting that these oligomers may have a functional significance in the genesis of t(11;22). In addition to these oligomers, sequences highly homologous t o Alu repeats and/or eukaryotic topoisomerase 11 cleavage sites were locate d near, or flanked, or even encompassed, the breakpoints in most of the cas es examined. Thus, these sequences may also mediate DNA double-strand break age and rejoining to generate the t(11;22). Genomic sequence analysis of bo th EWS-FL11 and FL11-EWS chimeric genes in three of the seven cases demonst rated a deletion and duplication of both EWS and FL11 sequences in two case s and no gain or loss in one case. The present findings suggest that multip le mechanisms may be operative for the break and rejoining of the fragments of chromosomes 11 and 22 in the genesis of t(11;22), and that some of thes e translocations are asymmetric at the molecular level. Genes Chromosomes C ancer 25:6-15, 1999. (C) 1999 Wiley-Liss, Inc.