Structural analysis of the genome of breast cancer cell line ZR-75-30 identifies twelve expressed fusion genes

Abstract

Background: It has recently emerged that common epithelial cancers such as breast cancers have fusion genes like those in leukaemias. In a representative breast cancer cell line, ZR-75-30, we searched for fusion genes, by analysing genome rearrangements.

Results: We first analysed rearrangements of the ZR-75-30 genome, to around 10kb resolution, by molecular cytogenetic approaches, combining array painting and array CGH. We then compared this map with genomic junctions determined by paired-end sequencing. Most of the breakpoints found by array painting and array CGH were identified in the paired end sequencing-55% of the unamplified breakpoints and 97% of the amplified breakpoints (as these are represented by more sequence reads). From this analysis we identified 9 expressed fusion genes: APPBP2-PHF20L1, BCAS3-HOXB9, COL14A1-SKAP1, TAOK1-PCGF2, TIAM1-NRIP1, TIMM23-ARHGAP32, TRPS1-LASP1, USP32-CCDC49 and ZMYM4-OPRD1. We also determined the genomic junctions of a further three expressed fusion genes that had been described by others, BCAS3-ERBB2, DDX5-DEPDC6/DEPTOR and PLEC1-ENPP2. Of this total of 12 expressed fusion genes, 9 were in the coamplification. Due to the sensitivity of the technologies used, we estimate these 12 fusion genes to be around two-thirds of the true total. Many of the fusions seem likely to be driver mutations. For example, PHF20L1, BCAS3, TAOK1, PCGF2, and TRPS1 are fused in other breast cancers. HOXB9 and PHF20L1 are members of gene families that are fused in other neoplasms. Several of the other genes are relevant to cancer-in addition to ERBB2, SKAP1 is an adaptor for Src, DEPTOR regulates the mTOR pathway and NRIP1 is an estrogen-receptor coregulator.

Conclusions: This is the first structural analysis of a breast cancer genome that combines classical molecular cytogenetic approaches with sequencing. Paired-end sequencing was able to detect almost all breakpoints, where there was adequate read depth. It supports the view that gene breakage and gene fusion are important classes of mutation in breast cancer, with a typical breast cancer expressing many fusion genes.

Figure 1

Chromosome rearrangements observed in the genome of breast cancer cell line ZR-75-30. A. Genome-wide Circos plot of structural variation in the ZR-75-30 genome. An ideogram of a normal karyotype is shown around the outside. Copy number variation is represented by the blue line, shown inside the ideogram. Chromosome rearrangements are depicted with green (interchromosomal) and purple (intrachromosomal) lines. All 125 structural variants shown have been independently validated by PCR (red lines) or by matching to a copy number step on a SNP6 array. B. Structural variation in the complex 8;17 amplicon. Colours as in A. C. Copy number variation in the 8;17 amplicon of ZR-75-30. Ideograms of chromosomes 8 and 17 are shown, with regions containing amplified segments highlighted with a red box and expanded below. Copy number changes, as measured by paired-end sequencing, illustrate the complexity of the amplification. An example set of ten confirmed rearrangement junctions are shown with green (interchromosomal) and purple (intrachromosomal) lines. Genome positions are based on Hg19.

Figure 2

Schematic representation of gene fusions and the expressed fusion transcripts in the breast cancer cell line ZR75-30 (not to scale). A. Fusions in the 8;17 amplicon. B. Structure of fusion transcripts detected by Robinson et al. [15]. C. Fusions at single copy breaks. Relevant exons are represented as numbered boxes, the transcription start site (AUG) is indicated with a black arrow and the breakpoint is indicated with a zig-zag line at the approximate chromosomal position (based on the UCSC Genome Browser, hg19). The (sequenced) expressed fusion transcripts and (where applicable) alternative splice products are shown below as black boxes joined by a dotted line. Exons depicted in grey are expected to be expressed, but were not sequenced. For numbering of exons see Additional file 1.