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Splicing mutation analysis reveals previously unrecognized pathways in lymph node-invasive breast cancer.

Dorman SN, Viner C, Rogan PK - Sci Rep (2014)

Bottom Line: Of the 5,206 putative mutations identified, exon skipping, leaky or cryptic splicing was confirmed for 988 variants.Pathway enrichment analysis of the mutated genes revealed mutations in 9 NCAM1-related pathways, which were significantly increased in samples with evidence of lymph node metastasis, but not in lymph node-negative tumours.We suggest that comprehensive reporting of DNA sequencing data should include non-trivial splicing analyses to avoid missing clinically-significant deleterious splicing mutations, which may reveal novel mutated pathways present in genetic disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Western Ontario, London, Ontario, N6A 5C1, Canada.

ABSTRACT
Somatic mutations reported in large-scale breast cancer (BC) sequencing studies primarily consist of protein coding mutations. mRNA splicing mutation analyses have been limited in scope, despite their prevalence in Mendelian genetic disorders. We predicted splicing mutations in 442 BC tumour and matched normal exomes from The Cancer Genome Atlas Consortium (TCGA). These splicing defects were validated by abnormal expression changes in these tumours. Of the 5,206 putative mutations identified, exon skipping, leaky or cryptic splicing was confirmed for 988 variants. Pathway enrichment analysis of the mutated genes revealed mutations in 9 NCAM1-related pathways, which were significantly increased in samples with evidence of lymph node metastasis, but not in lymph node-negative tumours. We suggest that comprehensive reporting of DNA sequencing data should include non-trivial splicing analyses to avoid missing clinically-significant deleterious splicing mutations, which may reveal novel mutated pathways present in genetic disorders.

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Related in: MedlinePlus

Cancer genes with validated cryptic splicing.mRNA of ABL1, CBFB, GATA3 and PALB2, which each have validated cryptic splicing mutations confirmed using tumour-matched RNA-Seq data. Full gene lengths are displayed with vertical black bars outlining exon boundaries. The location of the cryptic variant is denoted by the red V, and the variant consequence is highlighted by white (wild type), dark grey (exonic deletion), and red (frameshift mutation). Conserved domains and protein interactions are labeled by the yellow and blue horizontal bars. In ABL1, the catalytic and C-terminal F-actin binding domains are disrupted. In PALB2, the region that interacts with BRCA2 is truncated. In the GATA3 aberrant transcript, the second zinc finger domain and a conserved motif crucial for DNA binding and protein function are affected by the altered reading frame.
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f1: Cancer genes with validated cryptic splicing.mRNA of ABL1, CBFB, GATA3 and PALB2, which each have validated cryptic splicing mutations confirmed using tumour-matched RNA-Seq data. Full gene lengths are displayed with vertical black bars outlining exon boundaries. The location of the cryptic variant is denoted by the red V, and the variant consequence is highlighted by white (wild type), dark grey (exonic deletion), and red (frameshift mutation). Conserved domains and protein interactions are labeled by the yellow and blue horizontal bars. In ABL1, the catalytic and C-terminal F-actin binding domains are disrupted. In PALB2, the region that interacts with BRCA2 is truncated. In the GATA3 aberrant transcript, the second zinc finger domain and a conserved motif crucial for DNA binding and protein function are affected by the altered reading frame.

Mentions: Predicted cryptic splicing mutations were confirmed based on the presence of unique junction-spanning reads corresponding the ectopically spliced isoforms in GATA3, PALB2, CBFB, ABL1, C2CD2L, ENSA, NASP, NOP9, and TFE3 (Supplementary Fig. S2). Four of these genes have been linked to tumourigenesis: ABL1, an oncogene, GATA3 and PALB2, which are associated with familial breast cancer2627, and CBFB has been recently implicated in breast cancer by TCGA5 and others12. These cryptic splicing mutations lead to short exonic deletions that alter the reading frame, and likely affect the activity of the gene products (Fig. 1). The GATA3 cryptic isoform is the only detectable transcript in the majority of controls, although it is substantially more abundant in the tumour sample (Supplementary Fig. S3).


Splicing mutation analysis reveals previously unrecognized pathways in lymph node-invasive breast cancer.

Dorman SN, Viner C, Rogan PK - Sci Rep (2014)

Cancer genes with validated cryptic splicing.mRNA of ABL1, CBFB, GATA3 and PALB2, which each have validated cryptic splicing mutations confirmed using tumour-matched RNA-Seq data. Full gene lengths are displayed with vertical black bars outlining exon boundaries. The location of the cryptic variant is denoted by the red V, and the variant consequence is highlighted by white (wild type), dark grey (exonic deletion), and red (frameshift mutation). Conserved domains and protein interactions are labeled by the yellow and blue horizontal bars. In ABL1, the catalytic and C-terminal F-actin binding domains are disrupted. In PALB2, the region that interacts with BRCA2 is truncated. In the GATA3 aberrant transcript, the second zinc finger domain and a conserved motif crucial for DNA binding and protein function are affected by the altered reading frame.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231324&req=5

f1: Cancer genes with validated cryptic splicing.mRNA of ABL1, CBFB, GATA3 and PALB2, which each have validated cryptic splicing mutations confirmed using tumour-matched RNA-Seq data. Full gene lengths are displayed with vertical black bars outlining exon boundaries. The location of the cryptic variant is denoted by the red V, and the variant consequence is highlighted by white (wild type), dark grey (exonic deletion), and red (frameshift mutation). Conserved domains and protein interactions are labeled by the yellow and blue horizontal bars. In ABL1, the catalytic and C-terminal F-actin binding domains are disrupted. In PALB2, the region that interacts with BRCA2 is truncated. In the GATA3 aberrant transcript, the second zinc finger domain and a conserved motif crucial for DNA binding and protein function are affected by the altered reading frame.
Mentions: Predicted cryptic splicing mutations were confirmed based on the presence of unique junction-spanning reads corresponding the ectopically spliced isoforms in GATA3, PALB2, CBFB, ABL1, C2CD2L, ENSA, NASP, NOP9, and TFE3 (Supplementary Fig. S2). Four of these genes have been linked to tumourigenesis: ABL1, an oncogene, GATA3 and PALB2, which are associated with familial breast cancer2627, and CBFB has been recently implicated in breast cancer by TCGA5 and others12. These cryptic splicing mutations lead to short exonic deletions that alter the reading frame, and likely affect the activity of the gene products (Fig. 1). The GATA3 cryptic isoform is the only detectable transcript in the majority of controls, although it is substantially more abundant in the tumour sample (Supplementary Fig. S3).

Bottom Line: Of the 5,206 putative mutations identified, exon skipping, leaky or cryptic splicing was confirmed for 988 variants.Pathway enrichment analysis of the mutated genes revealed mutations in 9 NCAM1-related pathways, which were significantly increased in samples with evidence of lymph node metastasis, but not in lymph node-negative tumours.We suggest that comprehensive reporting of DNA sequencing data should include non-trivial splicing analyses to avoid missing clinically-significant deleterious splicing mutations, which may reveal novel mutated pathways present in genetic disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Western Ontario, London, Ontario, N6A 5C1, Canada.

ABSTRACT
Somatic mutations reported in large-scale breast cancer (BC) sequencing studies primarily consist of protein coding mutations. mRNA splicing mutation analyses have been limited in scope, despite their prevalence in Mendelian genetic disorders. We predicted splicing mutations in 442 BC tumour and matched normal exomes from The Cancer Genome Atlas Consortium (TCGA). These splicing defects were validated by abnormal expression changes in these tumours. Of the 5,206 putative mutations identified, exon skipping, leaky or cryptic splicing was confirmed for 988 variants. Pathway enrichment analysis of the mutated genes revealed mutations in 9 NCAM1-related pathways, which were significantly increased in samples with evidence of lymph node metastasis, but not in lymph node-negative tumours. We suggest that comprehensive reporting of DNA sequencing data should include non-trivial splicing analyses to avoid missing clinically-significant deleterious splicing mutations, which may reveal novel mutated pathways present in genetic disorders.

Show MeSH
Related in: MedlinePlus