Identification of gene fusion transcripts by transcriptome sequencing in BRCA1-mutated breast cancers and cell lines.
BACKGROUND
Gene fusions arising from chromosomal translocations have been implicated in cancer. However, the role of gene fusions in BRCA1-related breast cancers is not well understood. Mutations in BRCA1 are associated with an increased risk for breast cancer (up to 80% lifetime risk) and ovarian cancer (up to 50%). We sought to identify putative gene fusions in the transcriptomes of these cancers using high-throughput RNA sequencing (RNA-Seq).


METHODS
We used Illumina sequencing technology to sequence the transcriptomes of five BRCA1-mutated breast cancer cell lines, three BRCA1-mutated primary tumors, two secretory breast cancer primary tumors and one non-tumorigenic breast epithelial cell line. Using a bioinformatics approach, our initial attempt at discovering putative gene fusions relied on analyzing single-end reads and identifying reads that aligned across exons of two different genes. Subsequently, latter samples were sequenced with paired-end reads and at longer cycles (producing longer reads). We then refined our approach by identifying misaligned paired reads, which may flank a putative gene fusion junction.


RESULTS
As a proof of concept, we were able to identify two previously characterized gene fusions in our samples using both single-end and paired-end approaches. In addition, we identified three novel in-frame fusions, but none were recurrent. Two of the candidates, WWC1-ADRBK2 in HCC3153 cell line and ADNP-C20orf132 in a primary tumor, were confirmed by Sanger sequencing and RT-PCR. RNA-Seq expression profiling of these two fusions showed a distinct overexpression of the 3' partner genes, suggesting that its expression may be under the control of the 5' partner gene's regulatory elements.


CONCLUSIONS
In this study, we used both single-end and paired-end sequencing strategies to discover gene fusions in breast cancer transcriptomes with BRCA1 mutations. We found that the use of paired-end reads is an effective tool for transcriptome profiling of gene fusions. Our findings suggest that while gene fusions are present in some BRCA1-mutated breast cancers, they are infrequent and not recurrent. However, private fusions may still be valuable as potential patient-specific biomarkers for diagnosis and treatment.
Department of Human Genetics, McGill University, Montreal, Quebec, H3A 1B1, Canada.
Kevin C H
KC
Ha
Emilie
E
Lalonde
Lili
L
Li
Luca
L
Cavallone
Rachael
R
Natrajan
Maryou B
MB
Lambros
Costas
C
Mitsopoulos
Jarle
J
Hakas
Iwanka
I
Kozarewa
Kerry
K
Fenwick
Chris J
CJ
Lord
Alan
A
Ashworth
Anne
A
Vincent-Salomon
Mark
M
Basik
Jorge S
JS
Reis-Filho
Jacek
J
Majewski
William D
WD
Foulkes
D019313:BRCA1 Protein / Q000235:genetics*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D056905:Chromosome Breakpoints; D056915:DNA Copy Number Variations / Q000235:genetics; D005240:Feasibility Studies; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D050939:Gene Fusion / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*
D019313:BRCA1 Protein; D012333:RNA, Messenger