You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer}
Article:
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.
Introduction:
The Harvey rat sarcoma viral proto-oncogene homolog( HRAS) was the first human proto-oncogene to be identified[1]. The HRAS protein is a GTPase, which mediates signal transduction from growth factor receptors important for cellular proliferation, growth and survival. Somatic mutations in HRAS are present in many cancers and the vast majority of mutations affect codons 12 and 13( c. 34-39) leading to a constitutively active protein. The c. 35G>T mutation( p. Gly12Val) in HRAS was the first mutation in a proto-oncogene that was implicated in cancer[2, 3] and it is the second most frequently reported HRAS mutation in human cancers( Cosmic database: http://cancer. sanger. ac. uk/cosmic/gene/overview? ln=HRAS). The p. Gly12Val mutant has the lowest GTPase activity[4] and highest transformation potential among HRAS mutants[5, 6]. Germline mutations in HRAS cause Costello syndrome( CS)( MIM: 218040), which is a congenital disease characterized by postnatal growth retardation, short stature, tumor predisposition, developmental delay, and abnormalities of the heart( cardiomyopathy), skin and skeletal muscles[7]. The vast majority( 75%) of CS is caused by heterozygous c. 34G>A( p. Gly12Ser) activating mutations in HRAS[8]. Heterozygous c. 35G>C( p. Gly12Ala) and c. 37G>T( p. Gly13Cys) HRAS mutations are also frequent in CS patients, making up 10% and 7% of alleles, respectively[8]. Whereas these mutations result in a relatively homogenous phenotype, a particularly severe, early lethal form of CS has been observed in a few patients with the less frequently observed p. Gly12Val( encoded by c. 35G>T, c. 35_36GC>TT or c. 35_36GC>TA), p. Gly12Asp( c. 35G>A) or p. Gly12Cys( c. 34G>T) mutations[9–12]. These severe CS phenotypes are consistent with the higher transforming potential of the p. Gly12Val, p. Gly12Asp and p. Gly12Cys mutant proteins[5] and are also reflected in the higher frequencies of these mutations in cancer( Cosmic). So far it has therefore been believed that the relative frequency of the different HRAS mutations in cancer and CS simply reflects differences in the oncogenic potential of the encoded proteins and differences in the rate they occur by spontaneous mutations. Recently, it was, however, demonstrated that a phenomenon termed “selfish selection” may be an important factor determining the mutation spectrum observed in CS. Selfish selection both offers an explanation for the puzzling fact that CS has an extreme paternal bias in origin and occurs with a frequency two—three orders of magnitude higher than expected from the background mutation rate[8]. This is explainable by the fact that HRAS mutations offer different selection advantages in male germ cells depending on their oncogenic potential, and that their proportion increases with paternal age dependent on the selective advantage. Exonic mutations may, however, also have other effects than the expected changes in the protein, which can be predicted based on the resulting change from one codon to another according to the genetic code. A so called “splicing code”[13], which predicts that exonic mutations may have effects by impacting splicing regulatory elements, is beginning to emerge as an important cause of human disease, including cancer. This was elegantly demonstrated in a study by Supek and co-workers, who showed that synonymous mutations frequently act as driver mutations in cancer by altering exonic splicing regulatory motifs[14]. In particular, it was demonstrated that cancer-associated synonymous mutations frequently create exonic splicing enhancers( ESEs) and destroy exonic splicing silencers( ESSs) which regulate oncogene splicing in tumors. Additionally, expression and activity of splicing regulatory factors, like the SR-proteins and the hnRNP proteins are often dysregulated in cancer leading to aberrant splicing of oncogenes and tumor suppressor genes. A prominent example is the SRSF2 splicing regulatory factor, which is frequently mutated to a dominant negative form in myelogenic diseases[15]. Other important splicing regulatory factors such as SRSF1, SRSF3 and SRSF6 have been described as potent oncogenes[16–18], underscoring the importance of dysregulated splicing in cancer. Here we demonstrate for the first time that HRAS exon 2 is a vulnerable exon, which is dependent on binding of splicing regulatory proteins to ESEs in order to be correctly included in the HRAS mRNA. Importantly, we show that a mutation in the codon for Glycine 12( c. 34-39) can abrogate formation of constitutively active p. Gly12Val HRAS in a CS patient by mediating pronounced exon 2 skipping. Moreover, we show that this vulnerability of HRAS exon 2 splicing can be exploited by employing splice switching oligonucleotides( SSOs) to induce HRAS exon 2 skipping. This provides proof of principle for a new mechanism for knocking out oncogenic HRAS, which may be used therapeutically to treat cancer.
Discussion:
We show that a particular mutation, c. 35_36GC>TG, which encodes the prototypical oncogenic, constitutively active p. G12V HRAS protein, causes exon 2 skipping in an individual with CS with an attenuated clinical phenotype. This key finding demonstrates, in vivo, in a patient that exon 2 skipping leads limited production of the constitutively active oncogenic HRAS, thereby attenuating clinical symptoms. Simultaneously, this points to a previously unrecognized “Achilles heel” of the HRAS gene, namely that exon 2 is weakly defined due to a suboptimal 3’splice site. It’s inclusion in the mRNA is therefore dependent on binding of splicing stimulatory proteins, like SRSF2 to ESEs, and that binding of splicing inhibitory proteins, like hnRNPF/H to ESSs is avoided. Thus HRAS exon 2 inclusion can be affected by mutations altering the balance between ESEs and ESSs and this could in turn also result in cell type specific differences in splicing efficiency dependent on the relative levels/activities of splicing regulatory proteins, like SRSF2 or hnRNPF/H in the relevant tissue. Consistent with this, we show that mutations in codon 12 and 13( c. 34-39) impact exon 2 inclusion differently, and that HRAS exon 2 splicing efficiency is different( e. g. T24 and HepG2 cells). Thus, our results illustrate that the oncogenic effect of different mutations in HRAS may be determined also by their effect on exon 2 splicing efficiency. This adds an additional layer to the complex interpretation of the molecular consequences of mutations in HRAS exon 2. We posit that a delicate balance exists between the mutability of the different nucleotides, the resulting efficiency of exon 2 inclusion, and the oncogenic effect of the encoded mutant protein. We postulate that the observed frequencies of the various mutations in codon 12 and 13 in CS and cancers are a result of this balance. This has clear implications for our understanding of the correlation between genotype and phenotype in diseases caused by HRAS mutations and highlights the general importance of the “splicing code”[13], by providing a striking example on how exonic mutations, like c. 35_36GC>TG, can affect splicing and have dramatically different effects than those predicted based solely on the genetic code. Finally, we show that this previously unknown weakness of the HRAS gene points to a new mechanism for knocking out oncogenic HRAS by employing SSOs that block binding of the required splicing regulatory factors, resulting in exon 2 skipping and decreased growth of cancer cells. SSOs targeting HRAS exon 2 splicing may represent a new therapeutic approach either when used alone or in combination with other therapies. In contrast to traditional drugs, SSOs are highly specific for a single gene and there are currently promising clinical trials employing SSOs for treating human disease. SSOs that are able to inhibit tumorigenesis in vivo by altering splicing of genes, like Bcl-X, STAT3 and MDM4 have been reported[35–37]. A particularly appealing characteristic of SSOs is that delivery of several different SSOs targeting different cancer genes, like those mentioned above, could be performed simultaneously using the same delivery method. In this way multiple oncogenic mechanisms could be targeted in a single approach. Our data suggest that SSO-based therapy targeting HRAS could also be included in such a future strategy.
HRAS was the first human proto-oncogene reported and p . Gly12Val in codon 12 the first oncogenic mutation described . Somatic mutations in HRAS are important drivers in cancer , and germline mutations cause Costello syndrome . Until now , it has been believed that the severity of mutations located in the HRAS codon 12/13 mutational hot spot sequence is exclusively determined by the activity of the encoded proteins . Here we show that mutations in exon 2 of the HRAS proto-oncogene can have a previously unrecognized effect on splicing efficiency and thereby determine HRAS activity . We report a patient with Costello syndrome ( CS ) , who despite having the classical , severe , oncogenic p . Gly12Val mutation in HRAS has a mild clinical phenotype . We show that this is due to his specific sequence change , c . 35_36GC>TG , encoding p . Gly12Val , which disrupts an ESE fundamental for efficient splicing of exon 2 . We have explored this in detail and were able to show that HRAS exon 2 is a weak exon , which is dependent on the balance between positive and negative splicing regulatory factors , like SRSF2 and hnRNPF/H in order to be properly included . We show that different mutations in the mutational hot spot in HRAS codon 12 and 13 ( c . 34-39 ) affect splicing of HRAS differently , suggesting that this mechanism may also influence their occurrence in CS and their oncogenic potential in somatic cancers . Finally , we show that blocking access to the fundamental ESE , located in HRAS c . 34-39 , using a splice switching oligonucleotide ( SSOs ) , causes exon 2 skipping , abolishes production of functional HRAS protein and halts proliferation of cancer cells . This shows a previously unknown weakness of the HRAS proto-oncogene , namely that exon 2 is weakly defined and therefore is a suitable target for SSO-based therapy , thereby pointing towards a potential new direction for therapeutic targeting of RAS in anti-cancer treatment .