unscrambling the genomic chaos of osteosarcomas reveals a recurrent gene fusion
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Unscrambling the genomic chaos of osteosarcomas reveals a recurrent gene fusion. Leonardo A. Meza-Zepeda, PhD Department of Tumor Biology Institute for Cancer Research The Norwegian Radium Hospital. Osteosarcomas, a genomic chaos? . Harald Rieder, Duesseldorf. Tumour panel. - PowerPoint PPT PresentationTRANSCRIPT
Unscrambling the genomic chaos of osteosarcomas reveals a recurrent
gene fusion
Leonardo A. Meza-Zepeda, PhDDepartment of Tumor BiologyInstitute for Cancer Research
The Norwegian Radium Hospital
The Norwegian Radium Hospital
Osteosarcomas, a genomic chaos?
Harald Rieder, Duesseldorf
The Norwegian Radium Hospital
Tumour panel19 Osteosarcoma cell lines (EuroBoNeT panel)• Well characterised preclinical model
• Genomics and phenotypic (Poster 107, Myklebost)
AimIdentify fusion genes in osteosarcomas, to improve the knowledge about pathways involved in osteosarcoma biology
Strategy• Sequence the transcriptome (RNA-Seq) to identify recurrent
fusion transcripts • Whole genome sequencing (DNA-Seq) to confirm the fusion
transcripts at the genomic level
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Sample Candidate fusion transcripts after
filtering
IOR/MOS 5
IOR/OS15 110
IOR/OS18 160
MHM 11
OSA 56
ZK-58 113
KPDN 56
MG-63 102
SaOS-S2 36
IOR/OS10 15
IOR/SARG 36
517 candidate fusion transcripts identified in 11 osteosarcoma cell lines
RNA SequencingSusanne Lorenz, PhD
The Norwegian Radium Hospital
Candidate fusion transcript validation
Candidate fusion transcript Found in RNA-Seq data
Samples tested by breakpoint PCR Transcript validation by Sanger
sequencingIOR/OS15 IOR/MOS MHM Norm bone Osteoblast
COL1A2-MMP14 IOR/OS15, IOR/OS18 + + + - - +
FUS-LGMN IOR/OS15, MG-63 + + - - - +
DHFR-PABPC1 IOR/OS15 + + - - - +
PTMA-NPM1 IOR/OS15, IOR/OS18 + + + - - +
ELOVL5-PMP22 IOR/OS15 + + - - - +
CDC5L-BTBD9 IOR/OS15 + - - - - +
RAI1-CLIC5 IOR/OS15 + - - - - +
TP53-PPRAD IOR/OS15 + - - - - +
MRPL39-MAML3 IOR/OS15 + - - - - +
PLXNA2-RUNX1 IOR/OS15 + - - - - +
EIF5A-HMGN2 IOR/OS15, MG-63, Saos2 + + + + + +
EEF1A1-VIM IOR/OS15, IOR/OS18, SARG + + + + + +
EIF2S1-RBM25 IOR/OS15 + - - - - +
COL1A1-HSP90AB1 IOR/OS15, MG-63 + - + - - -
SCO1-CLIC5 IOR/OS15 + + + + + -
CREB3L1-HNRPA IOR/OS15 - - - - - NA
PDZRN4-PKM2 IOR/OS15, IOR/OS18, MHM - - - - - NA
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Whole genome sequencing
WG Sequencing• IOR/OS15• MG-63• ZK-58• IOR/OS18
Large heterogeneity
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Comparison RNA-Seq and WGSFusion Identified in Type of translocationELOVL5-PMP22 IOR-OS15 inter-chromosomal CDC5L-BTBD9 IOR-OS15 inter-chromosomalRAI1-CLIC5 IOR-OS15 inter-chromosomalSCO1-CLIC5 IOR-OS15 inter-chromosomal MRPL39-MAML3 IOR-OS15 inter-chromosomal PLXNA2-RUNX1 IOR-OS15 inter-chromosomalEIF2S1-RBM25 IOR-OS15 inter-chromosomalTP53-PPRAD IOR-OS15 inter-chromosomalBAZ1A-NUMB IOR-OS15 intra-chromosomal MRPL39-NRIP1 IOR-OS15 intra-chromosomal CLIP4-EPHB4 MG63 intra-chromosomalDNER-ELL2 MG63 inter-chromosomalTP53-VAV1* MG63 inter-chromosomalTP53-EMR1* MG63 inter-chromosomalBNC2-MTAP MG63 intra-chromosomalHEATR7A-PARP10 MG63 intra-chromosomalCREBBP-TFAP4 ZK58 intra-chromosomalGDPD5-MAP6 ZK58 intra-chromosomal
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PMP22-ELOVL5 fusion
Fusion transcript5` 3`
5’ 3’exon1 exon2 exon3 exon4 exon5
5’ 3’exon1 exon2 exon3 exon4 65
PMP22
ELOVL5
exon6
7 exon8
1 2 43 25 43 5 76 8
full protein sequence
106 aa
out of 160 aa wtPhe
1 aa 326 aa
ATG (Start)
Exons
In frame fusion
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PMP22-ELOVL5 fusionPMP22
ELOVL5
3` adapter 5` adapter
Fusion transcript1 2 43 5 43 5 762 8
RACE confirmed structure of fusion gene
IOR/OS15 K. Szuhai, Leiden
PMP22ELOVL5
FISHValidation extended osteosarcoma panel
Osteosarcoma cell lines(4/21) IOR/OS15, IOR/MOS, MHM, U-2OS Ostesarcomas xenografts(1/20) OKTx Clinical samples (2/9) primary tumours
Recurrence of 14%
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PMP22 and ELOVL5
Mutations in Charcot-Marie-Tooth DiseaseAmino acids lost by the fusion with ELOVL5
PMP22: peripheral myelin protein 22• Component of myelin in the peripheral
nervous system• Associated with Charcot–Marie–Tooth diseaseOsteosarcomas• Overexpressed in a subset of tumour
ELOVL5: fatty acid elongase 5• plasma membrane protein; elongation of
long-chain polyunsaturated fatty acids
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• Identified a large number of candidate fusion transcripts in osteosarcomas
• Identified a recurrent fusion in osteosarcomas PMP22-ELOVL5
• Large number of genomic rearrangements and heterogeneity
• Discrepancies between genomic rearrangements and rearrangements at the RNA level
• Majority of fusion transcripts does not seem to be a direct consequence of the genomic chaos
Summary
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Acknowledgements
Dept. of Tumor Biology• Susanne Lorenz• Tale Barøy• Ola Myklebost
Leiden University Medical Center• Karoly Szuhai
Genomics Core Facility• Jinchang Sun• Jan-Christian Bryne
Bioinformatics Core Facility• Daniel VodákDept. Cancer Prevention
• Torfinn Nome• Gard O. S. Thomassen • Rolf Skotheim• Ragnhild A. Lothe
The Norwegian Radium Hospital
Transcriptome sequencing
Paired-end250-300 bp
2x100bp
Fused mRNA
Gene A Gene B
Gene junction
Fusion genes• Biology• Biomarkers
RNA-Seq • 11 osteosarcoma cell linesSusanne Lorenz, PhD