Development of a fluorescent multiplex RT-PCR assay for detection of fusion-gene transcripts

The formation of fusion genes by chromosome rearrangement is a feature of many cancers and haematological neoplasms. Their expression is tumour-specific, so detection of fusion transcript is not only a valuable diagnostic tool but may also identify prognostic sub-types.The Northern Genetics Service employs RT-PCR for the detection / confirmation of gene fusion products from patient RNA samples for many common childhood solid tumours, i.e. Ewing’s sarcoma, rhabdomyosarcoma and synovial sarcoma. Current testing involves extraction of RNA and RT-PCR; control and target regions are amplified in separate reactions and analysed by agarose gel electrophoresis.The proposed project will establish a fluorescent RT-PCR based assay which will, compared to the current approach, increase sensitivity and enable multiplexing of primer pairs that detect specific gene fusions for a control fragment to determine if the PCR reaction has failed or not. The project will involve selection of appropriate control targets and the redesign of primers for the RT-PCR of EWSR1-FLI1 fusion in Ewing’s Sarcoma initially as proof-of-principle. Once this method has been established it will be applied to other fusion gene targets such as the EWSR1-ERG fusion in Ewing’s Sarcoma, the PAX3-FOX01 and PAX7-FOX01 in alveolar rhabdomyosarcoma and SS18-SSX1 and SS18-SSX2 in synovial sarcoma. The assay will be designed with a view to fully integrate the process into laboratory services including fragment capillary electrophoresis and Sanger sequencing services. Additionally, this work will include the generation of an SOP detailing the joint molecular/cytogenetics provision of a Gene Fusion Service.