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 performed in 12 cases of Ewing sarcoma on the cDNA and/or genomic DNA breakpoint regions of a t(11;22)(q24;q12), which joins the EWS gene located on chromosome 22 with the FLI1 gene located on chromosome 11, in order to understand the molecular mechanism of this translocation. Reverse transcriptase-PCR on total tumor cell RNA from the examined cases showed five types of EWS-FLI1 chimeric product, resulting from various junctions between EWS exon 7 or 10 with FLI1 exon 5, 6, or 8. Sequencing of the genomic fusion junctions of EWS-FLI1 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 clustered within 100 bp and 300 bp, respectively. All the junctions were found to be flanked by various oligomers, among which a consensus sequence, 5'-AGAAAARDRR-3', was found near the breakpoints of both genes in four cases, suggesting that these oligomers may have a functional significance in the genesis of t(11;22). In addition to these oligomers, sequences highly homologous to Alu repeats and/or eukaryotic topoisomerase II cleavage sites were located near, or flanked, or even encompassed, the breakpoints in most of the cases examined. Thus, these sequences may also mediate DNA double-strand breakage and rejoining to generate the t(11;22). Genomic sequence analysis of both EWS-FLI1 and FLI1-EWS chimeric genes in three of the seven cases demonstrated a deletion and duplication of both EWS and FLI1 sequences in two cases and no gain or loss in one case. The present findings suggest that multiple 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 these translocations are asymmetric at the molecular level.
Obata
K
K
Laboratory of Cytogenetics, Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan.
Hiraga
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H
Nojima
T
T
Yoshida
M C
MC
Abe
S
S
D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D002648:Child; D002874:Chromosome Mapping; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics*; D002892:Chromosomes, Human, Pair 22 / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012512:Sarcoma, Ewing / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics; D014178:Translocation, Genetic / Q000235:genetics*
D004273:DNA, Neoplasm; C106031:EWS-FLI fusion protein; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D014157:Transcription Factors