Abstract 397: Generation of Raf1 Mutant and Crispr-cas9 Corrected Isogenic iPSC-derived Cardiomyocytes to Model Hypertrophic Cardiomyopathy in Noonan Syndrome

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Fabrice Jaffré ◽  
Gang Wang ◽  
Amy Roberts ◽  
William Pu ◽  
Andreas Hahn ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Genome Editing ◽  
Noonan Syndrome ◽  
Molecular Mechanisms ◽  
Germ Line ◽  
Mapk Pathway ◽  
Heart Defects ◽  
Single Point ◽  
Cardiac Cells ◽  
Single Point Mutation

Background: Hypertrophic cardiomyopathy (HCM) is a major cause of death in infants and children. Noonan Syndrome (NS), an autosomal dominant RASopathy disorder, is characterized by multiple defects, including short stature, facial dysmorphia, and congenital heart defects that include HCM. RASopathies are caused by germ-line mutations that affect the canonical RAS-MAPK pathway. Indeed, 95% of NS patients with a mutation in Raf1 , a gene that plays an integral role in this signaling cascade, exhibit HCM. However, the molecular mechanisms that elicit HCM in these patients remain poorly understood. Objective: To generate human NS Raf1 induced-pluripotent stem cells (iPSCs), correct the mutation by genome editing and subsequently differentiate isogenic iPSC lines into cardiomyocytes to characterize the molecular and genetic basis of HCM in NS patients. Results: We generated iPSCs from skin fibroblasts obtained from a NS pediatric patient with a single point mutation in the Raf1 gene. Using electroporation of four episomal vectors containing the Yamanaka factors, we obtained several iPSC clones with normal karyotypes and strong expression of pluripotent markers (Nanog, Oct4, Lin28, Sox2) as detected by RT-qPCR and immunofluorescence. We next corrected the mutation in the NS Raf1 iPSCs using genome editing CRISPR-Cas9 nickase technology. Correction of the Raf1 mutation was determined at the clonal level by PCR followed by Restriction Fragment Length Polymorphism and confirmed by Sanger sequencing. In addition, by inducing a Cas9 nickase-dependent frame shift mutation we also generated an isogenic iPSC line where Raf1 gene was knockout (KO), as demonstrated at the RNA level by RT-qPCR and at the protein level by Western Blot. We next differentiated these multiple iPSC lines (mutant, corrected and KO) into isogenic beating cardiomyocytes, with more >98% of the cells positive for specific cardiomyocyte markers (α-actinin and cardiac TroponinT). Conclusion: We have successfully generated human NS Raf1 isogenic iPSC lines and corresponding cardiomyocytes. Currently, we are in progress of characterizing these cardiac cells to determine the molecular basis of NS-dependent HCM. Ultimately, our work should reveal new targets to treat HCM in NS patients.

scholarly journals Localization of the Kinesin-like Protein Xklp2 to Spindle Poles Requires a Leucine Zipper, a Microtubule-associated Protein, and Dynein

1998 ◽  
Vol 143 (3) ◽  
pp. 673-685 ◽  
Author(s):  
Torsten Wittmann ◽  
Haralabia Boleti ◽  
Claude Antony ◽  
Eric Karsenti ◽  
Isabelle Vernos
Keyword(s):  
Leucine Zipper ◽  
Molecular Mechanisms ◽  
Single Point ◽  
Spindle Pole ◽  
Single Point Mutation ◽  
Spindle Poles ◽  
Centrosome Separation ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Xklp2 is a plus end–directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49–59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein–dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends.

scholarly journals Metabonomics study of the effects of single copy mutant KRAS in the presence or absence of WT allele using human HCT116 isogenic cell lines

Metabolomics ◽  
2021 ◽  
Vol 17 (12) ◽  
Author(s):  
Dorna Varshavi ◽  
Dorsa Varshavi ◽  
Nicola McCarthy ◽  
Kirill Veselkov ◽  
Hector C. Keun ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Single Point ◽  
Hct116 Cell ◽  
Cellular Transformation ◽  
Single Copy ◽  
Driver Mutations ◽  
Glutathione Metabolism ◽  
Single Point Mutation

Abstract Introduction KRAS was one of the earliest human oncogenes to be described and is one of the most commonly mutated genes in different human cancers, including colorectal cancer. Despite KRAS mutants being known driver mutations, KRAS has proved difficult to target therapeutically, necessitating a comprehensive understanding of the molecular mechanisms underlying KRAS-driven cellular transformation. Objectives To investigate the metabolic signatures associated with single copy mutant KRAS in isogenic human colorectal cancer cells and to determine what metabolic pathways are affected. Methods Using NMR-based metabonomics, we compared wildtype (WT)-KRAS and mutant KRAS effects on cancer cell metabolism using metabolic profiling of the parental KRASG13D/+ HCT116 cell line and its isogenic, derivative cell lines KRAS+/– and KRASG13D/–. Results Mutation in the KRAS oncogene leads to a general metabolic remodelling to sustain growth and counter stress, including alterations in the metabolism of amino acids and enhanced glutathione biosynthesis. Additionally, we show that KRASG13D/+ and KRASG13D/− cells have a distinct metabolic profile characterized by dysregulation of TCA cycle, up-regulation of glycolysis and glutathione metabolism pathway as well as increased glutamine uptake and acetate utilization. Conclusions Our study showed the effect of a single point mutation in one KRAS allele and KRAS allele loss in an isogenic genetic background, hence avoiding confounding genetic factors. Metabolic differences among different KRAS mutations might play a role in their different responses to anticancer treatments and hence could be exploited as novel metabolic vulnerabilities to develop more effective therapies against oncogenic KRAS. Graphical abstract

scholarly journals Hypertrophic cardiomyopathy R403Q mutation in rabbit β-myosin reduces contractile function at the molecular and myofibrillar levels

2018 ◽  
Vol 115 (44) ◽  
pp. 11238-11243 ◽  
Author(s):  
Susan Lowey ◽  
Vera Bretton ◽  
Peteranne B. Joel ◽  
Kathleen M. Trybus ◽  
James Gulick ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Contractile Function ◽  
Isometric Force ◽  
Single Point ◽  
Rabbit Model ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Single Point Mutation ◽  
Loss Of Function ◽  
Transgenic Rabbits

In 1990, the Seidmans showed that a single point mutation, R403Q, in the human β-myosin heavy chain (MHC) of heart muscle caused a particularly malignant form of familial hypertrophic cardiomyopathy (HCM) [Geisterfer-Lowrance AA, et al. (1990) Cell 62:999–1006.]. Since then, more than 300 mutations in the β-MHC have been reported, and yet there remains a poor understanding of how a single missense mutation in the MYH7 gene can lead to heart disease. Previous studies with a transgenic mouse model showed that the myosin phenotype depended on whether the mutation was in an α- or β-MHC backbone. This led to the generation of a transgenic rabbit model with the R403Q mutation in a β-MHC backbone. We find that the in vitro motility of heterodimeric R403Q myosin is markedly reduced, whereas the actin-activated ATPase activity of R403Q subfragment-1 is about the same as myosin from a nontransgenic littermate. Single myofibrils isolated from the ventricles of R403Q transgenic rabbits and analyzed by atomic force microscopy showed reduced rates of force development and relaxation, and achieved a significantly lower steady-state level of isometric force compared with nontransgenic myofibrils. Myofibrils isolated from the soleus gave similar results. The force–velocity relationship determined for R403Q ventricular myofibrils showed a decrease in the velocity of shortening under load, resulting in a diminished power output. We conclude that independent of whether experiments are performed with isolated molecules or with ordered molecules in the native thick filament of a myofibril, there is a loss-of-function induced by the R403Q mutation in β-cardiac myosin.

Abstract 1: Abnormal Cardiac Differentiation Underlies Cardiac Hypertrophy in Noonan Syndrome With RAF1 Mutation

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Fabrice Jaffre ◽  
Gang Wang ◽  
Amy Roberts ◽  
William Pu ◽  
Andreas Hahn ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Noonan Syndrome ◽  
Germ Line ◽  
Mapk Pathway ◽  
Cardiac Differentiation ◽  
Subsequent Increase ◽  
Akt Activation ◽  
Stage Of Development ◽  
Hela Cell Lines ◽  
Induced Pluripotent

Noonan Syndrome (NS), an autosomal dominant RASopathy disorder, is caused by germ-line mutations that affect the canonical RAS-MAPK pathway. >95% of NS patients with an S257L mutation in Raf1 exhibit cardiac hypertrophy (CH) at a very young age. However, the molecular and developmental mechanisms that elicit CH in these patients remain unknown. Hence, we aimed at modeling CH by differentiating human NS Raf1 induced-pluripotent stem cells (iPSCs) towards the cardiomyocyte fate. We generated iPSCs from an NS pediatric patient with the S257L/+ mutation in the Raf1 gene and corrected the mutation using CRISPR-Cas9 double nickase system. Differentiation of the mutant NS line led to a significantly increased number of cardiomyocytes (CMs) as measured by FACS sorting at day 20, as compared to isogenic corrected cells (5.71 million ± 0.38 of cTNT+ cells vs 3.77±0.54, n=6, p<0.01). Differentiation of mutant iPSCs into mesodermal cells was normal; however, differentiation into cardiac progenitor cells was enhanced by the mutation, as demonstrated by the increased number of NKX2.5+ cells in the mutant culture at this stage of development. In addition, differentiated S257L/+ CMs had a significantly increased cell surface area compared to the control CMs (3,331 μm 2 ± 325 vs 1,638 μm 2 ± 97, n=6, p<0.01). To uncover the aberrant signaling pathways underlying these phenotypes, we generated RAF1 S257L/+ knockin and RAF1 null (KO) Hela cell lines using CRISPR-Cas9n technology. Interestingly, we found that ERK1/2 activity was enhanced in KO cells, both at baseline and in response to growth factor (GF) stimulation. At the opposite, ERK1/2 activity was reduced in mutant cells, suggesting that the RAF1 S257L/+ mutant actually suppresses ERK1/2 activation. Finally, we found that AKT activation was upregulated in S257L/+ cells under GF stimulation, due to decreased ERK1/2-dependent p70-S6 kinase activity and subsequent increase in mTORC2 activity (n=6). Similar experiments are underway in S257L/+, corrected and KO RAF1 iPSC-derived CMs. In conclusion, we propose that the S257L/+ mutation modulates development of CH in NS RAF1 patients through a reduction in ERK1/2 activity and overactivation of the AKT/mTOR signaling pathway, leading to both an increased number and size of CMs.

scholarly journals A severe clinical phenotype of Noonan syndrome with neonatal hypertrophic cardiomyopathy in the second case worldwide with RAF1 S259Y neomutation

2019 ◽  
Vol 101 ◽  
Author(s):  
Hager Jaouadi ◽  
Amel Ben Chehida ◽  
Lilia Kraoua ◽  
Heather C. Etchevers ◽  
Laurent Argiro ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Noonan Syndrome ◽  
De Novo ◽  
Mapk Pathway ◽  
Failure To Thrive ◽  
Clinical Severity ◽  
Facial Dysmorphism ◽  
Stunted Growth ◽  
Whole Exome ◽  
Tunisian Patient

AbstractNoonan syndrome and related disorders are a group of clinically and genetically heterogeneous conditions caused by mutations in genes of the RAS/MAPK pathway. Noonan syndrome causes multiple congenital anomalies, which are frequently accompanied by hypertrophic cardiomyopathy (HCM). We report here a Tunisian patient with a severe phenotype of Noonan syndrome including neonatal HCM, facial dysmorphism, severe failure to thrive, cutaneous abnormalities, pectus excavatum and severe stunted growth, who died in her eighth month of life. Using whole exome sequencing, we identified a de novo mutation in exon 7 of the RAF1 gene: c.776C > A (p.Ser259Tyr). This mutation affects a highly conserved serine residue, a main mediator of Raf-1 inhibition via phosphorylation. To our knowledge the c.776C > A mutation has been previously reported in only one case with prenatally diagnosed Noonan syndrome. Our study further supports the striking correlation of RAF1 mutations with HCM and highlights the clinical severity of Noonan syndrome associated with a RAF1 p.Ser259Tyr mutation.

scholarly journals Molecular mechanisms of activated protein C resistance. Properties of factor V isolated from an individual with homozygosity for the Arg506 to Gln mutation in the factor V gene

1996 ◽  
Vol 313 (2) ◽  
pp. 467-472 ◽  
Author(s):  
Cristina APARICIO ◽  
Björn DAHLBÄCK
Keyword(s):  
Risk Factor ◽  
Protein C ◽  
Molecular Mechanisms ◽  
Single Point ◽  
Activated Protein C ◽  
Factor Xa ◽  
Single Point Mutation ◽  
Factor V ◽  
Apc Resistance ◽  
Anticoagulant System

Resistance to activated protein C (APC), which is the most prevalent pathogenetic risk factor of thrombosis, is linked to a single point-mutation in the factor V (FV) gene, which predicts replacement of Arg (R) at position 506 with a Gln (Q). This mutation modifies one of three APC-cleavage sites in the heavy chain of activated FV (FVa), suggesting that mutated FVa (FVa:Q506) is at least partially resistant to APC-mediated degradation. To elucidate the molecular mechanisms of APC-resistance and to investigate the functional properties of FV in APC resistance, FV:Q506 was purified from an individual with homozygosity for the Arg to Gln mutation. Intact and activated FV:Q506 were demonstrated to convey APC resistance to FV-deficient plasma. Thrombin- or factor Xa-activated FV:Q506 were found to be approx. 10-fold less sensitive to APC-mediated degradation than normal FVa, at both high and low phospholipid concentrations. The degradation pattern observed on Western blotting suggested that FVa:Q506 was not cleaved at position 506. However, it was slowly cleaved at Arg306, which explains the partial APC sensitivity of FVa:Q506. FV is initially activated during clotting and then rapidly inactivated in a process which depends on the integrity of the protein C anticoagulant system. During clotting of APC-resistant plasma, FV:Q506 was activated in a normal fashion, but then only partially inactivated. In conclusion, the reduced sensitivity of FVa:Q506 to APC-mediated degradation is the molecular basis for the life-long hypercoagulable state which constitutes a risk factor for thrombosis in APC-resistant individuals.

scholarly journals Imatinib, a New Adjuvant Medical Treatment for Multifocal Villonodular Synovitis Associated to Noonan Syndrome: A Case Report and Literature Review

2022 ◽  
Vol 8 ◽  
Author(s):  
Romain Dalla-Torre ◽  
Vincent Crenn ◽  
Pierre Menu ◽  
Bertrand Isidor ◽  
Pascale Guillot ◽  
...  
Keyword(s):  
Noonan Syndrome ◽  
Technical Assistance ◽  
Kinase Inhibitors ◽  
Mapk Pathway ◽  
Heart Defects ◽  
Mitogen Activated Protein Kinase ◽  
Villonodular Synovitis ◽  
Multisystem Disorder ◽  
First Case

Noonan syndrome (NS) is an autosomal dominant multisystem disorder caused by the dysregulation of the Rat Sarcoma/Mitogen-activated protein kinase (RAS/MAPK) pathway and characterized by short stature, heart defects, pectus excavatum, webbed neck, learning disabilities, cryptorchidism, and facial dysmorphia. Villonodular synovitis is a joint disorder most common in young adults characterized by an abnormal proliferation of the synovial membrane. Multifocal Villonodular synovitis is a rare disease whose recurrent nature can make its management particularly difficult. Currently, there is no systemic therapy recommended in diffuse and recurrent forms, especially because of the fear of long-term side effects in patients, who are usually young. Yet, tyrosine kinase inhibitors seem promising to reduce the effects of an aberrant colony stimulating factor-1 (CSF-1) production at the origin of the synovial nodule proliferation. We present here the case of a 21-year-old woman with NS associated to diffuse multifocal villonodular synovitis (DMVS). Our clinical case provides therapeutic experience in this very rare association. Indeed, in association with surgery, the patient improved considerably: she had complete daily life autonomy, knee joint amplitudes of 100° in flexion and 0° in extension and was able to walk for 10 min without any technical assistance. To our knowledge, this is the first case of a patient suffering from DMVS associated with a Noonan syndrome treated with Glivec® (oral administration at a dosage of 340 mg/m2 in children, until disease regression) on a long-term basis.

Noonan Syndrome

2010 ◽  
Author(s):  
Ellen Wingbermüuhle ◽  
Ineke van der Burgt
Keyword(s):  
Congenital Heart Defects ◽  
Noonan Syndrome ◽  
Congenital Heart ◽  
Mapk Pathway ◽  
Heart Defects ◽  
Genetic Disorder ◽  
Tyrosine Phosphatase ◽  
Older Age ◽  
Diagnostic Process ◽  
Age Related

Noonan syndrome (NS) is a genetic disorder characterized by short stature, typical facial dysmorphology, and congenital heart defects. Noonan syndrome may occur on a sporadic basis or in a pattern consistent with autosomal dominant inheritance, with a predominance of maternal transmission (Noonan 1994). In approximately 50% of the patients with definite NS, a missense mutation is found in the PTPN11 gene on chromosome 12. PTPN11 is one of the genes of the Ras-MAPK pathway, a signal transduction cascade that has been studied extensively for its role in human oncogenesis. The signaling cascade regulates cell proliferation, differentiation, and survival. PTPN11 encodes the nonreceptor protein tyrosine phosphatase SHP-2. The mutations associated with NS result in a gain of function of SHP-2 (Tartaglia and Gelb 2005). Recently, activating mutations in other genes of the Ras-MAPK pathway (SOS1, KRAS, RAF1) were found as the causative dominant mutations in NS. These findings establish hyperactive Ras as a cause of the developmental abnormalities seen in NS (Schubbert et al. 2007). The diagnosis is made on clinical grounds, by observation of key features. Establishing the diagnosis can be very difficult, especially at an older age. There is great variability in expression, and mild expression is likely to be overlooked. Improvement of the phenotype occurs with increasing age. The age-related change of facial appearance can be subtle, especially at older age. Several scoring systems have been devised to guide the diagnostic process). The most recent scoring system was developed in 1994 (Van der Burgt et al. 1994). The incidence of NS is estimated to be between 1:1,000 and 1:2,500 live births (Mendez and Opitz 1985). Further details on the various medical aspects of NS (e.g., congenital heart defects, skeletal and urethrogenital abnormalities, growth delay) can be found in Van der Burgt (2007). A number of conditions have phenotypes strikingly similar to NS. The first is Turner syndrome (45, X0), a well-known chromosomal abnormality in girls. A group of distinct syndromes with partially overlapping phenotypes also exist in which causative mutations are also found in genes of the RAS-MAPK pathway.

scholarly journals Inherited Disorders of the Ras-MAPK Pathway

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. SCI-41-SCI-41
Author(s):  
Christian P. Kratz ◽  
Martin Zenker
Keyword(s):  
Cancer Risk ◽  
Noonan Syndrome ◽  
Conflicts Of Interest ◽  
Mapk Pathway ◽  
Heart Defects ◽  
Mapk Signaling ◽  
Juvenile Myelomonocytic Leukemia ◽  
Costello Syndrome ◽  
Inherited Disorders ◽  
Congenital Diseases

Abstract RASopathies are a group of rare congenital diseases in which dysregulated signaling through the RAS-MAPK signaling cases is the critical pathogenetic mechanism. This definition excludes postnatally acquired conditions (e.g. RAS-MAPK driven neoplasms) and PIK3-AKT pathway related disorders as well as conditions with only ancillary RAS pathway involvement (e.g. KAT6B-, RAP1A/B-related disorders). The definition, however, includes the following categories: (1) Noonan syndrome and related disorders, specifically Noonan syndrome (NS), NS with multiple lentigines, NS-like disorder with loose anagen hair, CBL syndrome, cardiofaciocutaneous syndrome, and Costello syndrome); (2) Neurofibromatosis type 1 and the related disorders Neurofibromatosis-Noonan syndrome and Legius syndrome; (3) Mosaic RASopathies including a rapidly growing group of mainly (neuro)cutaneous disorders with "oncogenic" mutations in a somatic mosaic state; (4) RAS-MAPK pathway dysregulation without a NS-like phenotype including non-syndromic intellectual disability due to SYNGAP1mutations and capillary malformation-arteriovenous malformation/Parkes-Weber syndrome; (5) RAS-MAPK pathway defects without overactivation such as metachondromatosis. Germline mutations leading to Ras-MAPK dysregulation typically lead to a characteristic pattern of craniofacial anomalies, heart defects, shorts stature, and variable neurodevelopmental deficits as seen in NS and other RASopathies of category 1. Confirmed genes leading to RASopathies from this category include BRAF,CBL, HRAS, KRAS, MAP2K1, MAP2K1, NRAS, PTPN11, RAF1, RIT1, SHOC2, SOS1, LZTR1, and PPP1CB. Newer genes include SOS2, MRAS, RRAS, and RASA2. For some individual RASopathy disease entities, specific genotype associations exist, for others, this correlation is not tight. The cancer risk in many patients with RASopathies is only moderately increased, however, for a subgroup of patients the cancer risk is very high. These include patients with Costello syndrome (HRAS) who develop rhabdomyosarcoma, neuroblastoma and bladder cancer, patients with NF1 who develop juvenile myelomonocytic leukemia (JMML), neurofibroma/-fibrosarcoma, and brain tumors, and patients and with CBL syndrome who develop JMML. New clinical studies explore the use of RAS-MAPK Pathway inhibitors in this unique population. Various animal and in vitro models have been described, which (partially) recapitulate the human RASopathy phenotype and phenotypic rescue by manipulating RAS-MAPK signal flow has been demonstrated. Disclosures No relevant conflicts of interest to declare.

Activating MRAS mutations cause Noonan syndrome associated with hypertrophic cardiomyopathy

2019 ◽  
Vol 29 (11) ◽  
pp. 1772-1783 ◽  
Author(s):  
Marialetizia Motta ◽  
Lena Sagi-Dain ◽  
Oliver H F Krumbach ◽  
Andreas Hahn ◽  
Amir Peleg ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Noonan Syndrome ◽  
De Novo ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Ras Signaling ◽  
Genetic Syndromes ◽  
Activating Mutations ◽  
High Level ◽  
Functional Analyses

Abstract The RASopathies are a group of genetic syndromes caused by upregulated RAS signaling. Noonan syndrome (NS), the most common entity among the RASopathies, is characterized mainly by short stature, cardiac anomalies and distinctive facial features. Mutations in multiple RAS-MAPK pathway-related genes have been associated with NS and related phenotypes. We describe two unrelated patients presenting with hypertrophic cardiomyopathy (HCM) and dysmorphic features suggestive of NS. One of them died in the neonatal period because of cardiac failure. Targeted sequencing revealed de novo MRAS variants, c.203C &gt; T (p.Thr68Ile) and c.67G &gt; C (p.Gly23Arg) as causative events. MRAS has only recently been related to NS based on the observation of two unrelated affected individuals with de novo variants involving the same codons here found mutated. Gly23 and Thr68 are highly conserved residues, and the corresponding codons are known hotspots for RASopathy-associated mutations in other RAS proteins. Functional analyses documented high level of activation of MRAS mutants due to impaired GTPase activity, which was associated with constitutive plasma membrane targeting, prolonged localization in non-raft microdomains, enhanced binding to PPP1CB and SHOC2 protein, and variably increased MAPK and PI3K-AKT activation. This report provides additional evidence that a narrow spectrum of activating mutations in MRAS represents another rare cause of NS, and that MRAS has to be counted among the RASopathy genes predisposing to HCM. Moreover, our findings further emphasize the relevance of the MRAS-SHOC2-PPP1CB axis in the control of MAPK signaling, and the contribution of both MAPK and PI3K-AKT pathways in MRAS functional upregulation.

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