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

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.

AberrantHRAStranscript processing underlies a distinctive phenotype within the RASopathy clinical spectrum

“…Because of its location, that is, a cis ‐acting exonic‐splicing silencer operating in concert with a downstream flanking intronic silencer sequence, the unusual 10‐nt long deletion is predicted to destabilize/relax the secondary structure of this region in the transcript, resulting in the constitutive retention of the mutated exon IDX. A disturbing effect on splicing has recently been reported for a different germline HRAS mutation, c.35_36GC>TG, identified in a subject with attenuated CS (Hartung et al., ). In this case, the dinucleotide change promoted high levels of exon 2 skipping, leading to a not functional, possibly rapidly degraded protein.…”

“…In this case, the dinucleotide change promoted high levels of exon 2 skipping, leading to a not functional, possibly rapidly degraded protein. In this context, the “mild” phenotype of the subject was hypothesized to be driven by the counterbalancing effect of enhanced function (c.35_36GC>TG is predicted to result in the oncogenic p.Gly12Val substitution) and dramatically reduced efficiency in proper transcript processing (retention of exon 2), resulting in a poorly expressed hyperactive protein (Hartung et al., ). In the present case, the peculiar type of mutation resulted in the combined effect of promoting retention of a mutated exon IDX characterized by an aberrant out‐of‐frame 52‐nucleotide‐long sequence escaping termination and having the same phase of the reading frame of the downstream exon 4, which encode the “canonical” C ‐terminal tail of the GTPase.…”

Costello syndrome: Clinical phenotype, genotype, and management guidelines

“…The p.G12V missense mutation is associated with severe cardiomyopathy and tachycardia, as well as respiratory distress resulting in early death (Aoki et al, ; Quezada & Gripp, ; Sol‐Church & Gripp, ). Detailed functional studies based on an unusual patient with a nonlethal phenotype due to a c.35_36GC>TG mutation (p.G12V) demonstrated the effect of alternative splicing on the phenotypic presentation (Hartung et al, ). The p.G13C mutation may be associated with a milder phenotype characterized by taller stature, absence of ulnar wrist deviation and a lower risk for malignant tumors or papillomata (Figure ; Sol‐Church & Gripp, ; Gripp et al, ).…”

Progressive myoclonus epilepsy in Gaucher Disease due to a new Gly–Gly mutation causing loss of an Exonic Splicing Enhancer

“…The c.363A > G mutation generates a long stretch of G’s, which could, in fact, be regarded as the generation of two GGG triplets (spaced by a G). Multiple triplet Gs in exons are known to inhibit splicing by recruiting splicing inhibitory proteins from the hnRNPF/H family [ 16 ], so this could be the possible mechanism involved in the missplicing of the patient’s GBA mRNA.…”