Cancer Discov. 2015 Apr;5(4):380-95. doi: 10.1158/2159-8290.CD-14-0892. Epub 2015 Jan 30.

The genetics of splicing in neuroblastoma.

Chen J¹, Hackett CS², Zhang S³, Song YK⁴, Bell RJ⁵, Molinaro AM⁶, Quigley DA⁷, Balmain A⁸, Song JS⁹, Costello JF⁸, Gustafson WC¹⁰, Van Dyke T¹¹, Kwok PY¹², Khan J⁴, Weiss WA¹³.

Author information

1: Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, California. Department of Neurology, University of California, San Francisco, San Francisco, California. Department of Neurosurgery, University of California, San Francisco, San Francisco, California.
2: Department of Neurology, University of California, San Francisco, San Francisco, California. Department of Neurosurgery, University of California, San Francisco, San Francisco, California.
3: Program in Bioinformatics, Boston University, Boston, Massachusetts. Oncogenomics Section, Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland.
4: Oncogenomics Section, Pediatric Oncology Branch, National Cancer Institute, Bethesda, Maryland.
5: Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, California. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California.
6: Department of Neurology, University of California, San Francisco, San Francisco, California. Department of Neurosurgery, University of California, San Francisco, San Francisco, California. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California. Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, California.
7: Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California. Institute for Cancer Research, Oslo, Norway.
8: Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California.
9: Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, California. Department of Bioengineering, University of Illinois, Urbana-Champaign, Urbana, Illinois. Department of Physics, University of Illinois, Urbana-Champaign, Urbana, Illinois.
10: Department of Pediatrics, University of California, San Francisco, San Francisco, California.
11: Mouse Cancer Genetics Program, Center for Advanced Preclinical Research, National Cancer Institute, Frederick, Maryland.
12: Institute for Human Genetics, University of California, San Francisco, San Francisco, California. Department of Dermatology, University of California, San Francisco, San Francisco, California. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California.
13: Department of Neurology, University of California, San Francisco, San Francisco, California. Department of Neurosurgery, University of California, San Francisco, San Francisco, California. Department of Pediatrics, University of California, San Francisco, San Francisco, California. waweiss@gmail.com.

Abstract

Regulation of mRNA splicing, a critical and tightly regulated cellular function, underlies the majority of proteomic diversity and is frequently disrupted in disease. Using an integrative genomics approach, we combined both genomic data and exon-level transcriptome data in two somatic tissues (cerebella and peripheral ganglia) from a transgenic mouse model of neuroblastoma, a tumor that arises from the peripheral neural crest. Here, we describe splicing quantitative trait loci associated with differential splicing across the genome that we use to identify genes with previously unknown functions within the splicing pathway and to define de novo intronic splicing motifs that influence splicing from hundreds of bases away. Our results show that these splicing motifs represent sites for functional recurrent mutations and highlight novel candidate genes in human cancers, including childhood neuroblastoma.

SIGNIFICANCE:

Somatic mutations with predictable downstream effects are largely relegated to coding regions, which comprise less than 2% of the human genome. Using an unbiased in vivo analysis of a mouse model of neuroblastoma, we have identified intronic splicing motifs that translate into sites for recurrent somatic mutations in human cancers.

PMID:: 25637275
PMCID:: PMC4390477
DOI:: 10.1158/2159-8290.CD-14-0892

[Indexed for MEDLINE]

Free PMC Article

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Figure 4

Recurrent somatic mutations occur in intronic splicing motifs in neuroblastoma

Analysis of 40 neuroblastoma samples reveals enrichment of somatic mutations in splicing motifs in DYNLRB1 (A), CGB7 (B), and MOXD2P (C). Germline variants (GVs) and somatic mutations (SMs) were identified by MuTect. MAST was used to examine the sequence immediately surrounding both intronic GVs and intronic SMs for a match to any of the 22 splicing motifs (sequence-level match p < 0.0001). The sequence logo of the splicing motif is drawn linked to the physical genomic location of the somatic mutation (black line). The total height of each nucleotide position is the information content in bits and represents the level of conservation for that position. The height of each nucleotide letter represents the ratio that they are found to occupy that position. The gene structures of known isoforms are depicted in red with arrows indicating the direction of transcription. The reference sequence (black) is given directly beneath the sequence logo with the position and nucleotide of the mutant allele shown in red. Red silhouettes indicate the number of tumor samples with that particular mutation. A black silhouette indicates the allele was found in a normal sample.
A. Two neuroblastoma samples (5%) had a recurrent C>T mutation within DYNLRB1 at chr20:33,121,706. This mutation created a stronger match to a splicing motif by substituting a base found infrequently to a base more commonly associated with the motif.
B. Two tumor samples had a recurrent G>C mutation at chr19:49,558,080 within CGB7; another neuroblastoma germline sample was also found to be G/C heterozygous at the same position. The splicing motif matched here was the same as observed in the DYNLRB1 mutation, and when analyzed in the orientation of the corresponding transcript, this mutation effectively destroyed the match to the motif.
C. Two neuroblastoma samples were identified with distinct splicing motif mutations at chr7:141,944,631 (C>T) and chr7:141,943,454 (A>C) in MOXD2P. When analyzed in the orientation of the corresponding transcript, the mutation at chr7:141,944,631 destroyed a splicing motif match whereas the chr7:141,943,454 mutation resulted in creating a match to the splicing motif.

The genetics of splicing in neuroblastoma

Cancer Discov. ;5(4):380-395.