Transcriptome-guided characterization of genomic rearrangements in a breast cancer cell line

Abstract

We have identified new genomic alterations in the breast cancer cell line HCC1954, using high-throughput transcriptome sequencing. With 120 Mb of cDNA sequences, we were able to identify genomic rearrangement events leading to fusions or truncations of genes including MRE11 and NSD1, genes already implicated in oncogenesis, and 7 rearrangements involving other additional genes. This approach demonstrates that high-throughput transcriptome sequencing is an effective strategy for the characterization of genomic rearrangements in cancers.

Fig. 1.

Schematic diagram of chimeric transcript detection and validation.

Fig. 2.

SKY analysis of HCC1954 karyotype and FISH confirmation of interchromosomal translocations. (A) SKY picture of HCC1954 genome. (M) chromosome is too complicated to be assigned. (B and C) FISH using BACs adjacent to the break point CTC-1286C20 (5q35.3, labeled in red) and RP11–17E16 (8q24.21, labeled in green) generated fusion signals (yellow) in HCC1954. (D and E) FISH using BACs adjacent to the break point RP11–72M5 (8q24.12, labeled in red) and RP11–12M21 (2q22.1, labeled in green) generated fusion signals in HCC1954 (yellow). The red arrow in C indicates a possible event of additional duplication of 5q35.3 region.

Fig. 3.

Schematic diagram of genomic rearrangement events captured by 454 transcriptome sequencing. Upper shows wild-type structures and Lower shows the rearranged structure. Red thick arrows are chimeric cDNAs captured by 454 reads. Untranslated exons are shown in gray bars, whereas translated exons are shown in colored bars. (A) Translocation between chromosomes 9 and 18 created an in-frame chimeric product proposed to be composed of 120 aa from 5′ terminus of PDCD1LG2 and 172 aa from 3′ terminus of C18orf10. (B) Transcription of the chimeric transcript involving NSD1 continued for another 134 bp on chromosome 8 before poly(A) tail was added to the mRNA. Translation of the chimeric protein contained 1,265 aa from the 5′end of NSD1 plus 19 aa from the intergenic region on chromosome 8 before stopped by an in-frame stop codon marked by an asterisk. (C) An ≈15-Mb genomic fragment was flipped as shown by the orange arrow. The PHF20L1 gene is truncated by a stop codon marked by an asterisk.

Fig. 4.

Detailed analysis of t(4;11)(q32;q21). (A) Local genomic features of chromosomes involved. The interchromosome translocation between chromosomes 11q21 and 4q32 truncates MRE11A at its DNA binding domain. Chimeric cDNAs span exon 9, 10 and 11 (brown bars) of MRE11A and intergenic sequences on chromosome 4 as shown by thick red arrows. A 9- to 10-kb genomic fragment containing the break junction was amplified with primers on chromosome 11 and chromosome 4 as shown in light blue arrows. Consensus splice acceptor sequences used for transcription of the chimeric cDNA on chromosome 4 are shown. LINE repeats are shown in shaded gray bars. The transcription orientation of MRE11A gene is marked by a black arrow. An in-frame stop codon in the chimeric 454 cDNA is marked by an asterisk. Coverage of 454 reads (shown by shaded pink areas) mapped to the 9- to 10-kb fragment was determined from an assembly output graph from CLC Bio. Coverage of the genome at the break junction is between 500× and 3,200×. (B) Final fine assembly of the break junction of t(4;11)(q32;q21) by mapping and assembly of 454 sequences on the 9- to 10-kb genomic fragment.