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Background: Over 90% of Ewing?s sarcomas and peripheral primitive neuroectodermal tumors (pPNETs) are characterized by rearrangements of the EWSR1 gene and ETS- related genes. Identification of these rearrangements serve as vital diagnostic adjuncts in establishing a Ewing?s/pPNET diagnosis. In the current study, cytogenetic analysis of a pPNET arising in a 5-year-old female revealed a variant chromosomal translocation t(4;22)(q31;q12) as the sole anomaly. Molecular cytogenetic analysis confirmed a rearrangement of the EWSR1 locus. We hypothesized that this primary translocation generated a fusion gene involving a novel EWSR1 translocation partner. Our study objectives were to: 1) further localize the 4q31 breakpoint; 2) uncover the underlying involved gene; and, 3) establish molecular diagnostic assays capable of detecting this variant translocation for clinical purposes. Methods: A fluorescence in situ hybridization (FISH)-based positional cloning strategy using a series of bacterial artificial chromosome (BAC) probe combinations on abnormal interphase cells was employed for narrowing the 4q31 breakpoint. Following identification of FREM3 and SMARCA5 as candidate genes on 4q31, 5? RACE and RT-PCR studies were conducted using EWSR1, FREM and SMARCA5 primers followed by sequencing. Results: Metaphase FISH analysis revealed a single BAC probe covered the 4q31 breakpoint; the latter corresponding to the FREM3 and SMARCA5 gene loci. 5' RACE distinguished SMARCA5 as the EWSR1 partner gene and sequencing studies of the RT-PCR generated transcripts defined the fusion occurring between exons 4 and 7 of SMARCA5 and EWSR1 respectively. The resulting fusion transcript encodes a chimeric protein composed of the transcriptional activation and regulatory domains of EWSR1 (265 N- terminal AA) and the 878 C-terminal AA of SMARCA5 plus a unique AA (N, Asp) at the breakpoint. Conclusions: A novel Ewing?s/pPNET variant translocation, t(4;22)(q31;q12), results in fusion of a non-ETS gene family member, SMARCA5, with EWSR1. SMARCA5 encodes for hSNF2H, a SWI/SNF-related matrix-associated actin- dependent regulator of chromatin subfamily A member 5. To the best of our knowledge, this is the first description of a fusion between EWSR1 and a gene coding for a chromatin-reorganizing protein. FISH and RT-PCR assays developed in this study can serve as diagnostic adjuncts for this rare translocation event.
