Identification of an EWS-pseudogene using translocation detection by RT-PCR in Ewing's sarcoma.

The presence of a t(11;22)(q24;q12) translocation is one of the characteristic features of the Ewing family of tumors. The detection of the fusion gene product by RT-PCR using primers at both sides of the breakpoints has been advocated as a diagnostic tool. By applying this technique appropriate internal controls are required. We found that the use of normal non-rearranged EWS mRNA as an internal control for RNA quality may lead to conflicting data. We obtained PCR products of the expected size for the normal EWS mRNA in both RNA and DNA samples, suggesting, the existence of one or more EWS pseudogenes. A 109 bp sequence at the 5' end of this PCR-product contained a correctly spliced exon junction and was 97% homologous to the EWS cDNA sequence. Similarly two such junctions were found in a 346 bp sequence of the 3' end, which was 89% homologous. Hence EWS should not be used as an internal control for the RNA quality in a RT-PCR based test for the presence of the translocation.