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

Anne-Mette Hartung, Jeff Swensen, Inaki E Uriz, Karen Kristjansdottir, Ulrika S S Petersen, Jeanne Mari V Bang, Barbara Guerra, Henriette Skovgaard Andersen, Steven F Dobrowolski, John C Carey, Ping Yu, Cecily Vaughn, Amy Calhoun, Martin R Larsen, Lars Dyrskjøt, David A Stevenson, Brage S Andresen

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Resumé

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.

OriginalsprogEngelsk
TidsskriftP L o S Genetics
Vol/bind12
Udgave nummer5
Sider (fra-til)e1006039
ISSN1553-7390
DOI
StatusUdgivet - 2016

Fingeraftryk

Costello Syndrome
exons
phenotype
mutation
cancer
Mutation
neoplasms
Neoplasms
oligonucleotides
protein
Protein Splicing
proto-oncogenes
mutants
RNA Splice Sites
vulnerability
Mutant Proteins
lethal genes
codons
germ cells
proteins

Citer dette

Hartung, Anne-Mette ; Swensen, Jeff ; Uriz, Inaki E ; Kristjansdottir, Karen ; Petersen, Ulrika S S ; Bang, Jeanne Mari V ; Guerra, Barbara ; Andersen, Henriette Skovgaard ; Dobrowolski, Steven F ; Carey, John C ; Yu, Ping ; Vaughn, Cecily ; Calhoun, Amy ; Larsen, Martin R ; Dyrskjøt, Lars ; Stevenson, David A ; Andresen, Brage S. / The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer. I: P L o S Genetics. 2016 ; Bind 12, Nr. 5. s. e1006039.
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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.",
author = "Anne-Mette Hartung and Jeff Swensen and Uriz, {Inaki E} and Karen Kristjansdottir and Petersen, {Ulrika S S} and Bang, {Jeanne Mari V} and Barbara Guerra and Andersen, {Henriette Skovgaard} and Dobrowolski, {Steven F} and Carey, {John C} and Ping Yu and Cecily Vaughn and Amy Calhoun and Larsen, {Martin R} and Lars Dyrskj{\o}t and Stevenson, {David A} and Andresen, {Brage S}",
year = "2016",
doi = "10.1371/journal.pgen.1006039",
language = "English",
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journal = "P L o S Genetics",
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Hartung, A-M, Swensen, J, Uriz, IE, Kristjansdottir, K, Petersen, USS, Bang, JMV, Guerra, B, Andersen, HS, Dobrowolski, SF, Carey, JC, Yu, P, Vaughn, C, Calhoun, A, Larsen, MR, Dyrskjøt, L, Stevenson, DA & Andresen, BS 2016, 'The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer', P L o S Genetics, bind 12, nr. 5, s. e1006039. https://doi.org/10.1371/journal.pgen.1006039

The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer. / Hartung, Anne-Mette; Swensen, Jeff; Uriz, Inaki E; Kristjansdottir, Karen; Petersen, Ulrika S S; Bang, Jeanne Mari V; Guerra, Barbara; Andersen, Henriette Skovgaard; Dobrowolski, Steven F; Carey, John C; Yu, Ping; Vaughn, Cecily; Calhoun, Amy; Larsen, Martin R; Dyrskjøt, Lars; Stevenson, David A; Andresen, Brage S.

I: P L o S Genetics, Bind 12, Nr. 5, 2016, s. e1006039.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer

AU - Hartung, Anne-Mette

AU - Swensen, Jeff

AU - Uriz, Inaki E

AU - Kristjansdottir, Karen

AU - Petersen, Ulrika S S

AU - Bang, Jeanne Mari V

AU - Guerra, Barbara

AU - Andersen, Henriette Skovgaard

AU - Dobrowolski, Steven F

AU - Carey, John C

AU - Yu, Ping

AU - Vaughn, Cecily

AU - Calhoun, Amy

AU - Larsen, Martin R

AU - Dyrskjøt, Lars

AU - Stevenson, David A

AU - Andresen, Brage S

PY - 2016

Y1 - 2016

N2 - 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.

AB - 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.

U2 - 10.1371/journal.pgen.1006039

DO - 10.1371/journal.pgen.1006039

M3 - Journal article

C2 - 27195699

VL - 12

SP - e1006039

JO - P L o S Genetics

JF - P L o S Genetics

SN - 1553-7390

IS - 5

ER -