TRANSLOCATION IDENTIFICATION OF AN EWS-PSEUDOGENE USING DETECTION BY RT-PCR IN EWINGS-SARCOMA

The presence of a t(11;22)(q24;q12) translocation is one of the charac teristic features of the Ewing family of tumors. The detection of the fusion gene product by RT-PCR using primers at both sides of the break points has been advocated as a diagnostic tool. By applying this techn ique appropriate internal controls are required. We found that the use of normal non-rearranged EWS mRNA as an internal control for RNA qual ity may lead to conflicting data. We obtained PCR products of the expe cted size for the normal EWS mRNA in both RNA and DNA samples, suggest ing 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 jun ction and was 97% homologous to the EWS cDNA sequence. Similarly two s uch 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 f or the RNA quality in a RT-PCR based test for the presence of the tran slocation. (C) 1995 Academic Press. Inc.