The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer

PLoS Genet. 2016 May 19;12(5):e1006039. doi: 10.1371/journal.pgen.1006039. eCollection 2016 May.

Abstract

Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.

Publication types

  • Case Reports

MeSH terms

  • Child
  • Codon / genetics
  • Costello Syndrome / genetics*
  • Costello Syndrome / pathology
  • Exons / genetics
  • Genotype
  • Germ-Line Mutation / genetics
  • Humans
  • Male
  • Neoplasms / genetics*
  • Neoplasms / pathology
  • Phenotype
  • Proto-Oncogene Proteins p21(ras) / genetics*
  • RNA Splice Sites / genetics
  • RNA Splicing / genetics

Substances

  • Codon
  • RNA Splice Sites
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)

Grant support

This work was supported by grants from The Danish Medical Research Council (FSS grant no.11-107174 and no. 4183-00244B http://ufm.dk/en/research-and-innovation/councils-and-commissions/the-danish-council-for-independent-research/the-council-1/the-danish-council-for-independent-research-medical-sciences/the-danish-council-for-independent-research-medical-sciences) to BSA, from The Novo Nordisk Foundation (Grant no.15430 - http://novonordiskfonden.dk/en) to BSA, and a clinical Scientist Development Award to DAS from the Doris Duke Charitable Foundation - http://www.ddcf.org/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.