SHOC2 Mutations Research Articles

Noonan Syndrome Like with Loose Anagen-1 due to SHOC2 Mutation—A Report of Two Cases from India

Noonan Syndrome Like with Loose Anagen-1 due to SHOC2 Mutation—A Report of Two Cases from India

S3465 Protein-Losing Enteropathy Associated With SHOC2 Gene Mutation in Noonan-Like Syndrome

Introduction: Protein-losing enteropathy (PLE) is the abnormal loss of protein through the gastrointestinal tract and has been associated with many diseases. The group of disorders including Noonan syndrome and Noonan-like syndromes (NS) consist of various dysmorphic features and congenital abnormalities. Multiple gene mutations paired with diverse phenotypic presentations have made identifying relationships between genotype and phenotype challenging, particularly with PLE in NS. In rare cases, PLE has been reported with NS, but few genetic associations have been made. Our case describes a possible connection between PLE and the SHOC2 gene mutation. Case Description/Methods: A 21-year-old female with a history of NS presented to our outpatient clinic with lower extremity edema and diarrhea with four watery bowel movements per day for 3 months. Labs revealed albumin of 2.4 g/dL, creatinine of 0.35 mg/dL, and potassium of 3.1 mEq/L. Acute hepatitis panel and anti-tissue transglutaminase were negative. Stool studies were positive for Clostridioides difficile and she was treated with oral vancomycin. She had an unremarkable esophagogastroduodenoscopy and colonoscopy. Computed tomography enterography suggested altered lymphatic drainage, and a lymphangiogram showed abdominal collateral lymphatics and iliac lymphangiectasia. Overall, these findings were consistent with PLE. A high protein diet and medium-chain triglyceride supplementation were initiated, and she had normalization of serum albumin levels. Genetic analysis showed a SHOC2 gene mutation. Discussion: PLE consists of loss of serum proteins through the digestive tract, which may initially present as hypoalbuminemia in the absence of liver disease. This disorder is important to recognize due to the association between low albumin and increased mortality risk. Lymphatic disease presenting with lymphedema, chylothorax, and lymphangiectasia is well known in NS and may play a role in PLE development. PLE has been tied to the mutation of protein tyrosine phosphatase non-receptor type 11, but to our knowledge, has not been reported with SHOC2 mutation. Because little is understood about PLE in NS, there is no standard treatment. However, supplementation with medium-chain triglycerides and periodic albumin infusions has proven effective. The association between PLE and NS may be more common than acknowledged. This case identifies SHOC2 mutation with PLE and encourages consideration of PLE in NS in hopes of decreasing mortality and increasing quality of life.

Behavioral phenotype of Noonan-like syndrome with loose anagen hair

IntroductionNoonan-like syndrome with loose anagen hair (NSLH MIM 607721) is associated to mutations in PTPN11, RAF1, BRAF and SHOC2 genes.ObjectivesHere, we report behavioral phenotype of a child suspected to have NSLH.MethodsA 2-years-old Tunisian child harboring severe pulmonic valvular stenosis was referred to our genetic counselling for genetic assessment. Medical dysmorphology, cytogenetic analysis as well as genetic exploration of RAS-MAPK pathway genes were conducted.ResultsThe child had short stature and ectodermal features including ichthyotic skin and thin-soft nails. He has specific hair appearance associated to NS features. In fact, he had a small nasal tip, thick lips and sticking-out rotated ears. He harbored typical nasal voice and loose anagen hair with ungrowing thin hair, sparse and pale scalp hair and eyebrows. He showed cognitive deficits with mental retardation and hyperactive behavior. Considered as having NSLH, cytogenetic analysis revealed a 46,XY formula, but molecular screening of PTPN11, RAF1, BRAF, RIT1 and SHOC2 genes was negative.Conclusions Mutations within the RAS‐MAPK signaling pathway affect neurophysiologic activity in brain regions underlying attention and executive functions. Children with rasopathies demonstrated higher rates of attention deficit-hyperactivity (ADHD) and autism spectrum disorders. However, no studies have examined specifically the aspects of behavioral attention in the various types of Rasopathies. A recent study demonstrated that ADHD seems to be higher in children with NSLH and SHOC2 mutation, which is the case of our patient. We suggest that assessment of inattentive and hyperactivity symptoms in children should consider Rasopathies with specific molecular screening.DisclosureNo significant relationships.

Proteins of the ubiquitin system in the Shoc2 ‐ ERK1/2 signaling axis and Noonan‐like syndrome with loose anagen hair (NSLAH) RASopathy

The ERK1/2 signaling pathway is critical in organismal development and tissue morphogenesis. Deregulation of this pathway leads to congenital abnormalities with severe developmental dysmorphisms, i.e. RASopathies. The core ERK1/2 cascade relies on scaffold proteins such as Shoc2 to guide and fine‐tune its signals. Mutations in shoc2 lead to the development of the pathology termed Noonan‐like Syndrome with Loose Anagen Hair (NSLAH) RASopathy. However, the mechanisms underlying the functions of Shoc2 and its contributions to disease progression remain unclear.We found that Shoc2 assembles an elegant multi‐component complex that incorporates several proteins of the ubiquitin system. To fine‐tune the amplitude of ERK1/2 signal transmitted via the complex, Shoc2 tethers the E3 ligase HUWE1, the (AAA+) ATPases, PSMC5 and VCP/p97, and the deubiquitinating enzyme, USP7. All of these enzymes are integral to the intricate feedback mechanism.Our recent studies demonstrated that ERK1/2 pathway activation triggers the interaction of Shoc2 with the ubiquitin‐specific protease USP7. We identified that in the Shoc2 module, USP7 functions as a molecular “switch” that controls the E3 ligase HUWE1 and the HUWE1‐induced regulatory feedback loop. We also found that disruption of Shoc2‐USP7 binding leads to aberrant activation of the Shoc2‐ERK1/2 axis. The zebrafish vertebrate model was then used to show that Shoc2 congenital mutations affecting Shoc2 interaction with USP7 lead to aberrant Shoc2 ubiquitination and signal transmission. Thus, our studies reveal a role for USP7 in the pathogenic mechanisms underlying NSLAH extending our understanding of how ubiquitin‐specific proteases regulate intracellular signaling.In summary, our studies are the first to demonstrate that the Shoc2 scaffold employs multi‐protein enzymatic machinery to govern the amplitude of Shoc2‐ERK1/2 signals. We also uncover novel molecular mechanisms underlying the pathogenesis of Noonan‐like syndrome with loose anagen hair. Overall, these studies significantly advance our understanding of the mechanisms by which non‐enzymatic scaffolds regulate the specificity and dynamics of the ERK1/2 signaling networks.

AACE2021-A-1102: Growth Hormone Deficiency in Patient with De Novo Mutation in SHOC2 Causing Noonan Syndrome-Like Disorder with Loose Anagen Hair, and Castleman Syndrome

Clinical manifestations of Noonan syndrome (NS) include short stature, distinctive facial features, webbed/wide neck, cryptorchidism, and congenital heart disease, notably pulmonic stenosis. NS is also recognised to predispose to malignancy – particularly hematological disease. Almost half of all NS cases are due to PTPN11 mutation. Other common mutations include SOS1 and RAF. Several rare mutations and NS subtypes are recognised. All of the known genes encode proteins within the RAS-Mitogen Activated Protein Kinase pathway.

The role of USP7 in the Shoc2-ERK1/2 signaling axis and Noonan-like syndrome with loose anagen hair.

The ERK1/2 (also known as MAPK3 and MAPK1, respectively) signaling pathway is critical in organismal development and tissue morphogenesis. Deregulation of this pathway leads to congenital abnormalities with severe developmental dysmorphisms. The core ERK1/2 cascade relies on scaffold proteins, such as Shoc2 to guide and fine-tune its signals. Mutations in SHOC2 lead to the development of the pathology termed Noonan-like Syndrome with Loose Anagen Hair (NSLAH). However, the mechanisms underlying the functions of Shoc2 and its contributions to disease progression remain unclear. Here, we show that ERK1/2 pathway activation triggers the interaction of Shoc2 with the ubiquitin-specific protease USP7. We reveal that, in the Shoc2 module, USP7 functions as a molecular 'switch' that controls the E3 ligase HUWE1 and the HUWE1-induced regulatory feedback loop. We also demonstrate that disruption of Shoc2-USP7 binding leads to aberrant activation of the Shoc2-ERK1/2 axis. Importantly, our studies reveal a possible role for USP7 in the pathogenic mechanisms underlying NSLAH, thereby extending our understanding of how ubiquitin-specific proteases regulate intracellular signaling.

Genotype-cardiac phenotype correlations in a large single-center cohort of patients affected by RASopathies: Clinical implications and literature review.

Congenital heart disease (CHD) and hypertrophic cardiomyopathy (HCM) are common features in patients affected by RASopathies. The aim of this study was to assess genotype- phenotype correlations, focusing on the cardiac features and outcomes of interventions for cardiac conditions, in a single-center cohort of 116 patients with molecularly confirmed diagnosis of RASopathy, and compare these findings with previously published data. All enrolled patients underwent a comprehensive echocardiographic examination. Relevant information was also retrospectively collected through the analysis of clinical records. As expected, significant associations were found between PTPN11 mutations and pulmonary stenosis (both valvular and supravalvular) and pulmonary valve dysplasia, and between SOS1 mutations and valvular defects. Similarly, HRAS mutations were significantly associated with HCM. Potential associations between less prevalent mutations and cardiac defects were also observed, including RIT1 mutations and HCM, SOS2 mutations and septal defects, and SHOC2 mutations and septal and valve abnormalities. Patients with PTPN11 mutations were the most likely to require both a primary treatment (transcatheter or surgical) and surgical reintervention. Other cardiac anomalies less reported until recently in this population, such as isolated functional and structural mitral valve diseases, as well as a sigmoid-shaped interventricular septum in the absence of HCM, were also reported. In conclusion, our study confirms previous data but also provides new insights on cardiac involvement in RASopathies. Further research concerning genotype/phenotype associations in RASopathies could lead to a more rational approach to surgery and the consideration of drug therapy in patients at higher risk due to age, severity, anatomy, and comorbidities.

Abstract PR05: Selective contribution of the SHOC2 phosphatase complex to ERK pathway dynamics highlights its potential as a therapeutic target

Abstract The ERK signaling pathway is hyperactivated in the majority of human cancers. However, ERK signaling mediates myriad physiologic responses and on-target toxicity has severely limited the clinical efficacy of current ERK pathway inhibitors. Despite the key role of RAF kinases in physiology and disease, we still lack a complete understanding of their regulation. Here we show that dephosphorylation of the conserved ‘S259’ RAF inhibitory site is a key activating step mediated by the SHOC2-MRAS-PP1 complex. Gain-of-function mutations in SHOC2, MRAS, PP1 and CRAF (clustering around S259) found in Noonan syndrome independently validate the important role of the SHOC2 complex in RAF-ERK pathway regulation. We have used CRISPR/cas9 to knock out SHOC2 and the 3 RAF isoforms in colorectal DLD-1 cancer cells. Our data show that SHOC2 is required for EGF-induced dephosphorylation of S365 BRAF, 14-3-3 and MEK dissociation from BRAF and RAF dimerization. However, whereas SHOC2 function is essential for a rapid transient phase of ERK activation in response to EGF stimulation, it is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and N-region phosphorylation of CRAF. Redundancy makes SHOC2 dispensable for ERK activation and proliferation in 2D whereas KRAS mutant cells rely on SHOC2 for ERK signaling under anchorage-independent/3D conditions, which thus presents a therapeutic opportunity. This study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for how targeting the SHOC2 regulatory node would allow for selective ERK pathway inhibition, likely to provide improved therapeutic margins in the clinic. This abstract is also being presented as Poster A32. Citation Format: Isabel Boned del Rio, Lucy C. Young, Sibel Sari, Greg G. Jones, Amandeep Bhamra, Silvia Surinova, Pablo Rodriguez-Viciana. Selective contribution of the SHOC2 phosphatase complex to ERK pathway dynamics highlights its potential as a therapeutic target [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr PR05.

Multilayer regulatory mechanisms of ERK1/2 signals transmitted through the Shoc2 scaffolding complex

Signaling scaffolds guide the flow of information and the spatial organization of the enzymes within the ERK1/2 signaling pathway. However, mechanisms that control assembly and dynamics within scaffolding complexes as well as mechanisms regulating the cellular distribution of these complexes remain largely unknown. Our studies unravel a novel, multi‐level paradigm in which allosteric modifications alter the ability of the scaffold protein Shoc2 to actively accelerate transmission of ERK1/2 signals. Shoc2 accelerates ERK1/2 signaling by tethering to close proximity essential proteins of the pathway, Ras and RAF‐1. Germ‐line mutations in Shoc2 affect spatial distribution of Shoc2 or its ability to assemble the signaling complex and cause Noonan‐like syndrome, a congenital disorder that presents with a wide spectrum of developmental abnormalities.Our lab studies mechanisms that fine‐tune ERK1/2 signals transmitted through the Shoc2 complex. We found that Shoc2 assembles an elegant multi‐protein complex that incorporates a number of proteins of the ubiquitin system. To fine‐tune amplitude of ERK1/2 signal transmitted via the complex, Shoc2 tethers the E3 ligase HUWE1, the (AAA+) ATPases, PSMC5 and VCP/p97 as well as the deubiquitinating enzyme, USP7. All of these enzymes are integral to the intricate feedback mechanism by which ubiquitination controls amplitude of the Shoc2‐ERK1/2 signals.Our studies show that HUWE1 ubiquitinates Shoc2 and RAF‐1, while ATPases modulate ubiquitination of Shoc2 and RAF‐1 through remodeling of the complex. We used fluorescence microscopy and biochemical analysis to demonstrate that PSMC5 and VCP/p97 are involved in recruitment of the Shoc2 scaffolding complexes to endosomes for complex remodeling. Importantly, we found that in the context of the Shoc2 complex, USP7 functions as a “molecular switch” that uses its enzymatic activity to trigger the “turn‐off” of the ERK1/2 signals transmitted via the scaffolding complex. We found that congenital Shoc2 mutations affect mechanisms controlling Shoc2 ubiquitination leading to aberrant signal transmission.In summary, our studies are the first to demonstrate that the Shoc2 scaffold employs multi‐protein enzymatic machinery to govern the amplitude of Shoc2‐ERK1/2 signals. We also show that mislocalization of the Shoc2 complexes affects remodeling of the scaffolding module and may lead to the developmental pathology. Overall, these studies significantly advance our understanding of the mechanisms by which non‐enzymatic scaffolds regulate specificity and dynamics of the ERK1/2 signaling networks.Support or Funding InformationNational Cancer Institute (R00CA126161 to EG), National Institute of General Medical Sciences (GM113087 to EG), American Cancer Society (RSG‐14‐172‐01‐CSM to EG)

The FBXW7-SHOC2-Raptor Axis Controls the Cross-Talks between the RAS-ERK and mTORC1 Signaling Pathways

FBXW7 is a tumor suppressive E3 ligase, whereas RAS-ERK and mechanistic target of rapamycin kinase(mTORC1) are two major oncogenic pathways. Whether and how FBXW7 regulates these two oncogenic pathways are unknown. Here, we showed thatSHOC2, a RAS activator, is a FBXW7 substrate.Growth stimuli trigger SHOC2 phosphorylation on Thr507 by the mitogen-activated protein kinase (MAPK) signal, which facilitates FBXW7 binding for ubiquitylation and degradation. FBXW7-mediated SHOC2 degradation terminates the RAS-MAPK signals and inhibits proliferation. Furthermore, SHOC2 selectively binds to Raptor to competitively inhibit the Raptor-mTOR binding to inactivate mTORC1 andinduce autophagy, whereas Raptor binding of SHOC2 inhibits the SHOC2-RAS binding to block the MAPK pathway and proliferation. Finally, SHOC2 is overexpressed in pancreatic cancer, which correlated with poor patient survival. SHOC2 mutations were found in lung cancer tissues with gain-of-function activity. Collectively, the SHOC2-Raptor interaction triggers negative cross-talk between RAS-ERK and mTORC1 pathways, whereas FBXW7 regulates both pathways by targeting SHOC2 for ubiquitylation and degradation.

Clinical and mutation profile of pediatric patients with RASopathy-associated hypertrophic cardiomyopathy: results from a Chinese cohort

BackgroundThe RASopathies are a class of developmental disorders caused by germline mutations in the RAS-mitogen-activated protein kinase (MAPK) pathway. Hypertrophic cardiomyopathy (HCM) has been frequently described in children with RASopathy, but only a minority of patients have received formal genotyping. The purpose of this study was to evaluate the genetic basis and clinical outcome of pediatric patients with RASopathy-associated HCM.MethodsWe retrospectively reviewed the mutation spectrum and clinical outcome of all the patients with RASopathy derived from 168 pediatric HCM cases referred to our institution between January 2012 and July 2018.ResultsA heterozygous missense mutation in one of known RASopathy genes was identified in 46 unrelated children with HCM. Mutations in the PTPN11 gene were the most prevalent (19/46); this was followed by mutations in RAF1 (11/46), KRAS (5/46), RIT1 (4/46), BRAF (3/46), SOS1 (2/46), HRAS (1/46), and SHOC2 (1/46). Moreover, two compound heterozygous missense mutations in the LZTR1 gene were identified in one patient with the Noonan syndrome phenotype and HCM. The median age at the diagnosis of HCM was 3.0 months (range 0 months to 8.1 years). Twenty-one of the patients had significant left ventricular outflow tract obstruction and 32 had concomitant congenital heart disease. Three patients with a mutation in exon 13 of the PTPN11 gene died of cardiac failure at the ages of 3.0, 3.5, and 6.0 months. The remaining 44 patients were alive after an average follow-up time of 3.9 years (0.5 to 17.1 years, median 2.9 years) from the initial diagnosis of HCM, including 5 patients with spontaneous regression of their cardiac hypertrophy.ConclusionsRASopathy-associated HCM is a heterogeneous genetic condition characterized by early-onset cardiac hypertrophy and a high prevalence of co-existing congenital heart disease, which is most frequently related to specific mutations in the PTPN11 gene. Rapidly progressive HCM, resulting in an early death, is uncommon in RASopathy patients except those with specific mutations in exon 13 of the PTPN11 gene.

Recurrent Erythema Nodosum in a Child with a <i>SHOC2</i> Gene Mutation

We report the case of a 6-year-old male who developed recurrent erythema nodosum (EN) at the age of 3 years. The patient exhibited hypertelorism, low-set ears, micrognathia, moderate intellectual disability, thin long fingers, loose anagen hair, and prominent palmoplantar wrinkles. A heterozygous single nucleotide variant in the SHOC2 gene (c.4 A > G, p.S2G) was identified. Patients with a SHOC2 mutation exhibit a unique combination of ectodermal abnormalities including darkly pigmented skin and loose anagen hair. This report is the first to describe EN in a patient with SHOC2 mutation, and to examine the patient's hair using scanning electron microscopy. We hypothesize that the RAS/MAPK pathway is associated with the pathogenesis of cutaneous lesions in patients with SHOC2 mutations via autoinflammation and disturbance of epithelial stem cells.

SHOC2–MRAS–PP1 complex positively regulates RAF activity and contributes to Noonan syndrome pathogenesis

Dephosphorylation of the inhibitory "S259" site on RAF kinases (S259 on CRAF, S365 on BRAF) plays a key role in RAF activation. The MRAS GTPase, a close relative of RAS oncoproteins, interacts with SHOC2 and protein phosphatase 1 (PP1) to form a heterotrimeric holoenzyme that dephosphorylates this S259 RAF site. MRAS and SHOC2 function as PP1 regulatory subunits providing the complex with striking specificity against RAF. MRAS also functions as a targeting subunit as membrane localization is required for efficient RAF dephosphorylation and ERK pathway regulation in cells. SHOC2's predicted structure shows remarkable similarities to the A subunit of PP2A, suggesting a case of convergent structural evolution with the PP2A heterotrimer. We have identified multiple regions in SHOC2 involved in complex formation as well as residues in MRAS switch I and the interswitch region that help account for MRAS's unique effector specificity for SHOC2-PP1. MRAS, SHOC2, and PPP1CB are mutated in Noonan syndrome, and we show that syndromic mutations invariably promote complex formation with each other, but not necessarily with other interactors. Thus, Noonan syndrome in individuals with SHOC2, MRAS, or PPPC1B mutations is likely driven at the biochemical level by enhanced ternary complex formation and highlights the crucial role of this phosphatase holoenzyme in RAF S259 dephosphorylation, ERK pathway dynamics, and normal human development.

Hematopoietic and neural crest defects in zebrafish shoc2 mutants: a novel vertebrate model for Noonan-like syndrome.

The extracellular signal-related kinase 1 and 2 (ERK1/2) pathway is a highly conserved signaling cascade with numerous essential functions in development. The scaffold protein Shoc2 amplifies the activity of the ERK1/2 pathway and is an essential modulator of a variety of signaling inputs. Germline mutations in Shoc2 are associated with the human developmental disease known as the Noonan-like syndrome with loose anagen hair. Clinical manifestations of this disease include congenital heart defects, developmental delays, distinctive facial abnormalities, reduced growth and cognitive deficits along with hair anomalies. The many molecular details of pathogenesis of the Noonan-like syndrome and related developmental disorders, cumulatively called RASopathies, remain poorly understood. Mouse knockouts for Shoc2 are embryonic lethal, emphasizing the need for additional animal models to study the role of Shoc2 in embryonic development. Here, we characterize a zebrafish shoc2 mutant, and show that Shoc2 is essential for development, and that its loss is detrimental for the development of the neural crest and for hematopoiesis. The zebrafish model of the Noonan-like syndrome described here provides a novel system for the study of structure-function analyses and for genetic screens in a tractable vertebrate system.

Cleft palate and hypopituitarism in a patient with Noonan‐like syndrome with loose anagen hair‐1

Noonan syndrome (NS), the most common of the RASopathies, is a developmental disorder caused by heterozygous germline mutations in genes encoding proteins in the RAS-MAPK signaling pathway. Noonan-like syndrome with loose anagen hair (NSLH, including NSLH1, OMIM #607721 and NSLH2, OMIM #617506) is characterized by typical features of NS with additional findings of macrocephaly, loose anagen hair, growth hormone deficiency in some, and a higher incidence of intellectual disability. All NSLH1 reported cases to date have had an SHOC2 c.4A>G, p.Ser2Gly mutation; NSLH2 cases have been reported with a PPP1CB c.146G>C, p.Pro49Arg mutation, or c.166G>C, p.Ala56Pro mutation. True cleft palate does not appear to have been previously reported in individuals with NS or with NSLH. While some patients with NS have had growth hormone deficiency (GHD), other endocrine abnormalities are only rarely documented. We present a female patient with NSLH1 who was born with a posterior cleft palate, micrognathia, and mild hypotonia. Other findings in her childhood and young adulthood years include hearing loss, strabismus, and hypopituitarism with growth hormone, thyroid stimulating hormone (TSH), and gonadotropin deficiencies. The SHOC2 mutation may be responsible for this patient's additional features of cleft palate and hypopituitarism.

Ocular findings in Noonan syndrome: a retrospective cohort study of 105 patients

The aim of this retrospective study is to describe ocular findings in a large Noonan syndrome cohort and to detect associations between ocular features and genetic mutations that were not found in earlier studies. We collected ophthalmological and genetic data of 105 patients (median age, 12 years; range, 0–60 years) clinically diagnosed as Noonan syndrome. The ocular findings were linked to the genotypes. All patients with Noonan syndrome showed multiple abnormalities in the categories of vision and refraction, external ocular features, ocular alignment and motility, anterior ocular segment, and posterior ocular segment. In total, 50 patients have NS due to a mutation in PTPN11. Permanent visual impairment (bilateral best-corrected visual acuity < 0.3) was found in 7 patients, including patients with a mutation in RAF1, SHOC2, and KRAS. Keratoconus was found in 2 PTPN11 positive patients, and prominent corneal nerves were observed in a patient with a SOS1 mutation.Conclusions: This study shows an overview of ocular abnormalities in Noonan syndrome, including permanent visual impairment caused by binocular optic nerve abnormalities and nystagmus. Delay in ophthalmological diagnosis is still present, also in patients with visual impairment. All Noonan syndrome patients should have a complete ophthalmological examination at the time of diagnosis.What is Known:• Although we discover more pathogenic mutations in patients with Noonan syndrome, Noonan syndrome still is a clinical diagnosis• Ocular features of Noonan syndrome are characterized by developmental anomalies of the eyelids and associated with other ocular abnormalities in childhood (including refractive errors, strabismus and amblyopia).What is New:• There seems to be a delay in the ophthalmological diagnosis and awareness of the broad variety ofophthalmological features including refractive errors and visual impairment in Noonan syndrome is needed. All children should have a full ophthalmological examination at the time of diagnosis.• Permanent visual impairment (best-corrected visual acuity < 0.3) is found in patients with mutations in RAF1, SHOC2, and KRAS and the cause is probably a developmental disorder of the optic nerves.

Craniosynostosis in patients with RASopathies: Accumulating clinical evidence for expanding the phenotype

RASopathies are phenotypically overlapping genetic disorders caused by dysregulation of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway. RASopathies include Noonan syndrome, cardio-facio-cutaneous (CFC) syndrome, Costello syndrome, Neurofibromatosis type 1, Legius syndrome, Noonan syndrome with multiple lentigines, Noonan-like syndrome, hereditary gingival fibromatosis, and capillary malformation/arteriovenous malformation syndrome. Recently, six patients with craniosynostosis and Noonan syndrome involving KRAS mutations were described in a review, and a patient with craniosynostosis and Noonan syndrome involving a SHOC2 mutation has also been reported. Here, we describe patients with craniosynostosis and Noonan syndrome due to de novo mutations in PTPN11 and patients with craniosynostosis and CFC syndrome due to de novo mutations in BRAF or KRAS. All of these patients had cranial deformities in addition to the typical phenotypes of CFC syndrome and Noonan syndrome. In RASopathy, patients with cranial deformities, further assessments may be necessary to look for craniosynostosis. Future studies should attempt to elucidate the pathogenic mechanism responsible for craniosynostosis mediated by the RAS/MAPK signaling pathway.

237 - Prediction of Disease Progression Using Mutational Screening in Patients with Myelodysplastic Syndrome

The RAS-MAPK syndromes are a group of clinically and genetically related disorders caused by dysregulation of the RAS-MAPK pathway. A member of this group of disorders, Noonan syndrome (NS), is associated with several different genes within the RAS-MAPK pathway. To date, mutations in PTPN11, SOS1, KRAS, RAF1 and SHOC2 are known to cause NS and a small group of patients harbour mutations in BRAF, MEK1 or NRAS. The majority of the mutations are predicted to cause an up-regulation of the pathway; hence they are gain-of-function mutations. Despite recent advances in gene identification in NS, the genetic aetiology is still unknown in about ¼ of patients.To investigate the contribution of gene dosage imbalances of RAS-MAPK-related genes to the pathogenesis of NS, a multiplex ligation-dependent probe amplification (MLPA) assay was developed. Two probe sets were designed for seven RAS-MAPK-syndrome-related candidate genes: PTPN11, SOS1, RAF1, KRAS, BRAF, MEK1 and MEK2. The probe sets were validated in 15 healthy control individuals and in glioma tumour cell lines. Subsequently, 44 NS patients negative for mutations in known NS-associated genes were screened using the two probe sets. The MLPA results for the patients revealed no gene dosage imbalances. In conclusion, the present results exclude copy number variation of PTPN11, SOS1, RAF1, KRAS, BRAF, MEK1 and MEK2 as a common pathogenic mechanism of NS. The validated and optimised RAS-MAPK probe sets presented here enable rapid high throughput screening of further patients with RAS-MAPK syndromes.

Shoc2 Mediates Hematopoietic Signals of the ERK1/2 Pathway

Activity of the extracellular‐signal‐regulated kinase 1/2 (ERK1/2) pathway plays a critical role in normal human development. Aberrant ERK1/2 signaling caused by germline mutations in genes of the Ras‐mediated ERK1/2 pathway results in developmental disorders cumulatively called ‘RASopathies’. Shoc2, the scaffold protein in the ERK1/2 pathway, accelerates ERK1/2 activity by bringing the signaling components of the pathway, Ras and Raf‐1, to close proximity. Noonan‐like RASopathy causing Shoc2 mutations emphasize the significance of ERK1/2 signaling transmitted through the Shoc2 scaffolding module. However, the physiological role of Shoc2 and the specificity of the signals transduced through the Shoc2 scaffolding complex remain largely unknown.In our work, we took advantage of the vertebrate zebrafish model that is ideally suited for developmental genetic studies due to its optical transparency and ex vivo development. To delineate the developmental ERK1/2 signals mediated through the Shoc2 complex, a translation‐blocking shoc2 morpholino (MO) was injected into 1–2 cell stage zebrafish embryos. The outcomes of the Shoc2 depletion were examined using biochemical methods such as Western blot analysis, RT‐PCR and whole embryo staining/imaging. We found that Shoc2 depletion in zebrafish embryos results in decreased ERK1/2 activity. Shoc2 MO‐injected embryos also possessed a larger yolk, kinked tail, and an enlarged hindbrain. Importantly, Shoc2 morphants displayed dramatically reduced numbers of circulating blood cells and loss of erythropoietic and myeloid lineages. To further understand the role of Shoc2 in differentiation of hematopoietic stem cells, we utilized CRISPR/Cas9 gene editing to generate the Shoc2 zebrafish knockout. 12 germ‐line transmitted alleles were recovered from the mutagenesis. All alleles had lesions in similar locations, causing frame‐shifts around amino acid 225 and introducing aberrant stop codons (TAA or TAG) prior to termination.Our data demonstrate that Shoc2 depletion affects both waves of hematopoiesis: primitive and definitive. Shoc2 CRISPR zebrafish knockout will now be utilized to examine quantitative changes in the hematopoiesis‐specific ERK1/2 transcriptional program.Support or Funding InformationFunding sources: National Institute of General Medical Siences, American Cancer Society, American Heart Association

Nutritional aspects of Noonan syndrome and Noonan-related disorders.

Rasopathies are a group of rare disorders characterized by neurocardiofaciocutaneous involvement, and caused by mutations in several genes of the RAS/MAPK pathway. In the present study, we characterized growth parameters, body composition, and nutritional aspects of children and adults (n = 62) affected by these disorders, mainly Noonan syndrome, using an indirect method-anthropometry-and a 24-hr recall questionnaire. The growth parameters in our cohort showed short stature, especially in individuals with RAF1 and SHOC2 mutations, lower obesity rates compared to the control population, and BMI scores highest in individuals with BRAF mutations and lowest in individuals with SHOC2. Body composition showed a compromise in the upper arm muscle circumference, with a statistically significant difference in the z-score of triceps skinfold (P = 0.0204) and upper arm fat area (P = 0.0388) between BRAF and SHOC2 groups and in the z-score of triceps skinfold between RAF1 and SHOC2 (P = 0.0218). The pattern of macronutrient consumption was similar to the control population. Our study is the first to address body composition in RASopathy individuals and the data indicate a compromise not only in adipose tissue, but also in muscle mass. Studies using different techniques, such as dual-energy X-ray absorptiometry or imaging studies, which give a more precise delineation of fat and non-fat mass, are required to confirm our results, ultimately causing an impact on management strategies. © 2016 Wiley Periodicals, Inc.