title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors.forename: Anne-Mette
authors.initials: AM
authors.lastname: Hartung
authors.affiliation: Caris Life Sciences, Phoenix, Arizona, United States of America.|Department of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.|ARUP Laboratories, Salt Lake City, Utah, United States of America.
authors.forename: Jeff
authors.identifier: http://orcid.org/0000-0002-8204-4220
authors.initials: J
authors.lastname: Swensen
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors.forename: Inaki E
authors.initials: IE
authors.lastname: Uriz
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors.forename: Morten
authors.identifier: http://orcid.org/0000-0003-4183-0679
authors.initials: M
authors.lastname: Lapin
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors.forename: Karen
authors.initials: K
authors.lastname: Kristjansdottir
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors.forename: Ulrika S S
authors.initials: US
authors.lastname: Petersen
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors.forename: Jeanne Mari V
authors.initials: JM
authors.lastname: Bang
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors.forename: Barbara
authors.initials: B
authors.lastname: Guerra
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors.forename: Henriette Skovgaard
authors.initials: HS
authors.lastname: Andersen
authors.affiliation: Department of Pathology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Steven F
authors.initials: SF
authors.lastname: Dobrowolski
authors.affiliation: Department of Pediatrics, University of Utah, Salt Lake City, Utah, United States of America.
authors.forename: John C
authors.initials: JC
authors.lastname: Carey
authors.affiliation: ARUP Laboratories, Salt Lake City, Utah, United States of America.
authors.forename: Ping
authors.initials: P
authors.lastname: Yu
authors.affiliation: ARUP Laboratories, Salt Lake City, Utah, United States of America.
authors.forename: Cecily
authors.identifier: http://orcid.org/0000-0003-4289-6346
authors.initials: C
authors.lastname: Vaughn
authors.affiliation: Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, United States of America.
authors.forename: Amy
authors.initials: A
authors.lastname: Calhoun
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
authors.affiliation: Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark.
authors.forename: Lars
authors.initials: L
authors.lastname: Dyrskjøt
authors.affiliation: Division of Medical Genetics, Stanford University, Stanford, California, United States of America.
authors.forename: David A
authors.initials: DA
authors.lastname: Stevenson
authors.affiliation: Department of Biochemistry and Molecular Biology and The Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors.forename: Brage S
authors.initials: BS
authors.lastname: Andresen
mesh_terms: D002648:Child; D003062:Codon / Q000235:genetics; D056685:Costello Syndrome / Q000235:genetics* / Q000473:pathology; D005091:Exons / Q000235:genetics; D005838:Genotype; D018095:Germ-Line Mutation / Q000235:genetics; D006801:Humans; D008297:Male; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D010641:Phenotype; D000090063:Proto-Oncogene Mas; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics
chemical_list: D003062:Codon; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D022821:RNA Splice Sites; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)