Breakpoint analysis for discovering novel cancer gene rearrangements.

<p>Schematic depiction of the approach and workflow, demonstrated by example of the rediscovery of a known gene fusion, <i>SET/NUP214</i>, in the T-ALL cell line LOUCY. Various publicly-available and in-house exon microarray and high-density CGH/SNP array experiments were analyzed. RNA breakpoint analysis (RBA) identifies significant transitions in exon expression level, which may reflect elevated expression of exons distal (3′ partner) or proximal (5′ partner) to a gene fusion junction. To identify such transitions a “walking” Student's t-test was applied, comparing expression levels of proximal and distal exons. Candidate rearrangements were subsequently filtered for those disrupting genes of the Cancer Gene Census, with directional orientation (i.e. being the 5′ or 3′ partner) consistent with known rearrangements of that gene. RBA candidates were further filtered using a Bonferroni correction to adjust for multiple t-tests. DNA breakpoint analysis (DBA) screens for intragenic DNA copy number transitions, which may reflect unbalanced chromosomal rearrangements leading to the formation of gene fusions. The fused lasso method (FDR 1%) followed by a copy number smoothing algorithm was applied to identify CNAs. Copy number transitions were filtered for those disrupting any annotated gene and then further filtered for those disrupting genes of the Cancer Gene Census. We included only candidate breakpoints where the directional orientation of the copy number transition was consistent with known rearrangements of that gene. Several candidates were then validated using molecular and cytogenetic approaches. The average numbers of candidate rearrangements per cancer sample are depicted along the left and right panels at various stages of the workflow.