Clinical profiling of MRD48 and functional characterization of two novel pathogenic RAC1 variants.

RAC1 is a member of the Rac/Rho GTPase subfamily within the RAS superfamily of small GTP-binding proteins, comprising 3 paralogs playing a critical role in actin cytoskeleton remodeling, cell migration, proliferation and differentiation. De novo missense variants in RAC1 are associated with a rare neurodevelopmental disorder (MRD48) characterized by DD/ID and brain abnormalities coupled with a wide range of additional features. Structural and functional studies have documented either a dominant negative or constitutively active behavior for a subset of mutations. Here, we describe two individuals with previously unreported de novo missense RAC1 variants. We functionally demonstrate their pathogenicity proving a gain-of-function (GoF) effect for both. By reviewing the clinical features of these two individuals and the previously published MRD48 subjects, we further delineate the clinical profile of the disorder, confirming its phenotypic variability. Moreover, we compare the main features of MRD48 with the neurodevelopmental disease caused by GoF variants in the paralog RAC3, highlighting similarities and differences. Finally, we review all previously reported variants in RAC proteins and in the closely related CDC42, providing an updated overview of the spectrum and hotspots of pathogenic variants affecting these functionally related GTPases.

Dominant ARF3 variants disrupt Golgi integrity and cause a neurodevelopmental disorder recapitulated in zebrafish.

Vesicle biogenesis, trafficking and signaling via Endoplasmic reticulum-Golgi network support essential developmental processes and their disruption lead to neurodevelopmental disorders and neurodegeneration. We report that de novo missense variants in ARF3, encoding a small GTPase regulating Golgi dynamics, cause a developmental disease in humans impairing nervous system and skeletal formation. Microcephaly-associated ARF3 variants affect residues within the guanine nucleotide binding pocket and variably perturb protein stability and GTP/GDP binding. Functional analysis demonstrates variably disruptive consequences of ARF3 variants on Golgi morphology, vesicles assembly and trafficking. Disease modeling in zebrafish validates further the dominant behavior of the mutants and their differential impact on brain and body plan formation, recapitulating the variable disease expression. In-depth in vivo analyses traces back impaired neural precursors' proliferation and planar cell polarity-dependent cell movements as the earliest detectable effects. Our findings document a key role of ARF3 in Golgi function and demonstrate its pleiotropic impact on development.

Radiosensitivity in patients affected by ARPC1B deficiency: a new disease trait?

Actin-related protein 2/3 complex subunit 1B (ARPC1B) deficiency is a recently described inborn error of immunity (IEI) presenting with combined immunodeficiency and characterized by recurrent infections and thrombocytopenia. Manifestations of immune dysregulation, including colitis, vasculitis, and severe dermatitis, associated with eosinophilia, hyper-IgA, and hyper-IgE are also described in ARPC1B-deficient patients. To date, hematopoietic stem cell transplantation seems to be the only curative option for patients. ARPC1B is part of the actin-related protein 2/3 complex (Arp2/3) and cooperates with the Wiskott-Aldrich syndrome protein (WASp) in the regulation of the actin cytoskeleton remodeling and in driving double-strand break clustering for homology-directed repair. In this study, we aimed to investigate radiosensitivity (RS) in ARPC1B-deficient patients to assess whether it can be considered an additional disease trait. First, we performed trio-based next-generation-sequencing studies to obtain the ARPC1B molecular diagnosis in our index case characterized by increased RS, and then we confirmed, using three different methods, an increment of radiosensitivity in all enrolled ARPC1B-deficient patients. In particular, higher levels of chromatid-type aberrations and γH2AX foci, with an increased number of cells arrested in the G2/M-phase of the cell cycle, were found in patients' cells after ionizing radiation exposition and radiomimetic bleomycin treatment. Overall, our data suggest increased radiosensitivity as an additional trait in ARPC1B deficiency and support the necessity to investigate this feature in ARPC1B patients as well as in other IEI with cytoskeleton defects to address specific clinical follow-up and optimize therapeutic interventions.

Expanding the molecular spectrum of pathogenic SHOC2 variants underlying Mazzanti syndrome.

We previously molecularly and clinically characterized Mazzanti syndrome, a RASopathy related to Noonan syndrome that is mostly caused by a single recurrent missense variant (c.4A > G, p.Ser2Gly) in SHOC2, which encodes a leucine-rich repeat-containing protein facilitating signal flow through the RAS-mitogen-associated protein kinase (MAPK) pathway. We also documented that the pathogenic p.Ser2Gly substitution causes upregulation of MAPK signaling and constitutive targeting of SHOC2 to the plasma membrane due to the introduction of an N-myristoylation recognition motif. The almost invariant occurrence of the pathogenic c.4A > G missense change in SHOC2 is mirrored by a relatively homogeneous clinical phenotype of Mazzanti syndrome. Here, we provide new data on the clinical spectrum and molecular diversity of this disorder and functionally characterize new pathogenic variants. The clinical phenotype of six unrelated individuals carrying novel disease-causing SHOC2 variants is delineated, and public and newly collected clinical data are utilized to profile the disorder. In silico, in vitro and in vivo characterization of the newly identified variants provides evidence that the consequences of these missense changes on SHOC2 functional behavior differ from what had been observed for the canonical p.Ser2Gly change but converge toward an enhanced activation of the RAS-MAPK pathway. Our findings expand the molecular spectrum of pathogenic SHOC2 variants, provide a more accurate picture of the phenotypic expression associated with variants in this gene and definitively establish a gain-of-function behavior as the mechanism of disease.

Prevalence of bladder cancer in Costello syndrome: New insights to drive clinical decision-making.

Costello syndrome (CS) is a rare disorder affecting development and growth characterized by cancer predisposition and caused by mutations in HRAS proto-oncogene. Somatic HRAS mutations drive bladder carcinogenesis. The aim of this study was to analyze prevalence and histological characterization of bladder cancer (BC) in a cohort of patients with CS to help clinicians plan effective management strategies. This study included 13 patients above 10 years of age with molecular diagnosis of CS. Screening cystoscopies (31 total procedures) were performed to exclude BC. Any lesion was analyzed through cold-cup biopsy or trans-urethral resection of the bladder. According to histology, patients were followed-up with urinalysis and abdominal ultrasound yearly, and cystoscopies every 12-24 months. During study enrollment, bladder lesions (often multifocal) were detected in 11/13 patients. Histological analysis documented premalignant lesions in 90% of cystoscopies performed, epithelial dysplasia in 71%, and papillary urothelial neoplasm of low-malignant potential in 19%. BC G1/low grade (Ta) were removed in 10%. Overall, 76% of patients showed a bladder lesion at first cystoscopy. The present findings document that individuals with CS aged 10 years and older have high prevalence of bladder lesions (premalignant/malignant), highlighting the importance of personalized screening protocols.

SPRED2 loss-of-function causes a recessive Noonan syndrome-like phenotype.

Upregulated signal flow through RAS and the mitogen-associated protein kinase (MAPK) cascade is the unifying mechanistic theme of the RASopathies, a family of disorders affecting development and growth. Pathogenic variants in more than 20 genes have been causally linked to RASopathies, the majority having a dominant role in promoting enhanced signaling. Here, we report that SPRED2 loss of function is causally linked to a recessive phenotype evocative of Noonan syndrome. Homozygosity for three different variants-c.187C>T (p.Arg63∗), c.299T>C (p.Leu100Pro), and c.1142_1143delTT (p.Leu381Hisfs∗95)-were identified in four subjects from three families. All variants severely affected protein stability, causing accelerated degradation, and variably perturbed SPRED2 functional behavior. When overexpressed in cells, all variants were unable to negatively modulate EGF-promoted RAF1, MEK, and ERK phosphorylation, and time-course experiments in primary fibroblasts (p.Leu100Pro and p.Leu381Hisfs∗95) documented an increased and prolonged activation of the MAPK cascade in response to EGF stimulation. Morpholino-mediated knockdown of spred2a and spred2b in zebrafish induced defects in convergence and extension cell movements indicating upregulated RAS-MAPK signaling, which were rescued by expressing wild-type SPRED2 but not the SPRED2Leu381Hisfs∗95 protein. The clinical phenotype of the four affected individuals included developmental delay, intellectual disability, cardiac defects, short stature, skeletal anomalies, and a typical facial gestalt as major features, without the occurrence of the distinctive skin signs characterizing Legius syndrome. These features, in part, characterize the phenotype of Spred2-/- mice. Our findings identify the second recessive form of Noonan syndrome and document pleiotropic consequences of SPRED2 loss of function in development.

Co-Occurring Heterozygous CNOT3 and SMAD6 Truncating Variants: Unusual Presentation and Refinement of the IDDSADF Phenotype.

Objective, the application of genomic sequencing in clinical practice has allowed us to appreciate the contribution of co-occurring pathogenic variants to complex and unclassified clinical phenotypes. Besides the clinical relevance, these findings have provided evidence of previously unrecognized functional links between genes in the context of developmental processes and physiology. Patients and Methods, a 5-year-old patient showing an unclassified phenotype characterized by developmental delay, speech delay, peculiar behavioral features, facial dysmorphism and severe cardiopathy was analyzed by trio-based whole exome sequencing (WES) analysis to identify the genomic events underlying the condition. Results, two co-occurring heterozygous truncating variants in CNOT3 and SMAD6 were identified. Heterozygous loss-of-function variants in CNOT3, encoding a subunit of the CCR4-NOT protein complex, have recently been reported to cause a syndromic condition known as intellectual developmental disorder with speech delay, autism and dysmorphic facies (IDDSADF). Enrichment of rare/private variants in the SMAD6 gene, encoding a protein negatively controlling transforming growth factor ß/bone morphogenetic protein (TGFB/BMP) signaling, has been described in association with a wide spectrum of congenital heart defects. We dissected the contribution of individual variants to the complex clinical manifestations and profiled a previously unappreciated set of facial features and signs characterizing IDDSADF. Conclusions, two concomitant truncating variants in CNOT3 and SMAD6 are the cause of the combination of features documented in the patient resulting in the unique multisystem neurodevelopmental condition. These findings provide evidence for a functional link between the CCR4-NOT complex and TGFB/BMP signaling in processes controlling cardiac development. Finally, the present revision provides evidence that IDDSADF is characterized by a distinctive facial gestalt.

Broadening the phenotypic spectrum of Beta3GalT6-associated phenotypes.

Biallelic mutations in B3GALT6, coding for a galactosyltransferase involved in the synthesis of glycosaminoglycans (GAGs), have been associated with various clinical conditions, causing spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMDJL1 or SEMDJL Beighton type), Al-Gazali syndrome (ALGAZ), and a severe progeroid form of Ehlers-Danlos syndrome (EDSSPD2). In the 2017 Ehlers-Danlos syndrome (EDS) classification, Beta3GalT6-related disorders were grouped in the spondylodysplastic EDSs together with spondylodysplastic EDSs due to B4GALT7 and SLC39A13 mutations. Herein, we describe a patient with a previously unreported homozygous pathogenic B3GALT6 variant resulting in a complex phenotype more severe than spondyloepimetaphyseal dysplasia with joint laxity type 1, and having dural ectasia and aortic dilation as additionally associated features, further broadening the phenotypic spectrum of the Beta3GalT6-related syndromes. We also document the utility of repeating sequencing in patients with uninformative exomes, particularly when performed by using "first generations" enrichment capture methods.

When to test fetuses for RASopathies? Proposition from a systematic analysis of 352 multicenter cases and a postnatal cohort.

PURPOSE: Recent studies have identified suggestive prenatal features of RASopathies (e.g., increased nuchal translucency [NT], cystic hygroma [CH], hydrops, effusions, congenital heart diseases [CHD], polyhydramnios, renal anomalies). Our objective is to clarify indications for RASopathy prenatal testing. We compare genotype distributions between pre- and postnatal populations and propose genotype-phenotype correlations. METHODS: Three hundred fifty-two chromosomal microarray-negative cases sent for prenatal RASopathy testing between 2012 and 2019 were collected. For most, 11 RASopathy genes were tested. Postnatal cohorts (25 patients with available prenatal information and 108 institutional database genotypes) and the NSeuroNet database were used for genotypic comparisons. RESULTS: The overall diagnostic yield was 14% (50/352), with rates >20% for effusions, hydrops, and CHD. Diagnostic yield was significantly improved in presence of hypertrophic cardiomyopathy (HCM), persistent or associated CH, any suggestive finding combined with renal anomaly or polyhydramnios, or ≥2 ultrasound findings. Largest prenatal contributors of pathogenic variants were PTPN11 (30%), RIT1 (16%), RAF1 (14%), and HRAS (12%), which considerably differ from their prevalence in postnatal populations. HRAS, LZTR1, and RAF1 variants correlated with hydrops/effusions, and RIT1 with prenatal onset HCM. CONCLUSION: After normal chromosomal microarray, RASopathies should be considered when any ultrasound finding of lymphatic dysplasia or suggestive CHD is found alone or in association.

SPEN haploinsufficiency causes a neurodevelopmental disorder overlapping proximal 1p36 deletion syndrome with an episignature of X chromosomes in females.

Deletion 1p36 (del1p36) syndrome is the most common human disorder resulting from a terminal autosomal deletion. This condition is molecularly and clinically heterogeneous. Deletions involving two non-overlapping regions, known as the distal (telomeric) and proximal (centromeric) critical regions, are sufficient to cause the majority of the recurrent clinical features, although with different facial features and dysmorphisms. SPEN encodes a transcriptional repressor commonly deleted in proximal del1p36 syndrome and is located centromeric to the proximal 1p36 critical region. Here, we used clinical data from 34 individuals with truncating variants in SPEN to define a neurodevelopmental disorder presenting with features that overlap considerably with those of proximal del1p36 syndrome. The clinical profile of this disease includes developmental delay/intellectual disability, autism spectrum disorder, anxiety, aggressive behavior, attention deficit disorder, hypotonia, brain and spine anomalies, congenital heart defects, high/narrow palate, facial dysmorphisms, and obesity/increased BMI, especially in females. SPEN also emerges as a relevant gene for del1p36 syndrome by co-expression analyses. Finally, we show that haploinsufficiency of SPEN is associated with a distinctive DNA methylation episignature of the X chromosome in affected females, providing further evidence of a specific contribution of the protein to the epigenetic control of this chromosome, and a paradigm of an X chromosome-specific episignature that classifies syndromic traits. We conclude that SPEN is required for multiple developmental processes and SPEN haploinsufficiency is a major contributor to a disorder associated with deletions centromeric to the previously established 1p36 critical regions.

Variants of SOS2 are a rare cause of Noonan syndrome with particular predisposition for lymphatic complications.

RASopathies are caused by variants in genes encoding components or modulators of the RAS/MAPK signaling pathway. Noonan syndrome is the most common entity among this group of disorders and is characterized by heart defects, short stature, variable developmental delay, and typical facial features. Heterozygous variants in SOS2, encoding a guanine nucleotide exchange factor for RAS, have recently been identified in patients with Noonan syndrome. The number of published cases with SOS2-related Noonan syndrome is still limited and little is known about genotype-phenotype correlations. We collected previously unpublished clinical and genotype data from 17 individuals carrying a disease-causing SOS2 variant. Most individuals had one of the previously reported dominant pathogenic variants; only four had novel changes at the established hotspots for variants that affect protein function. The overall phenotype of the 17 patients fits well into the spectrum of Noonan syndrome and is most similar to the phenotype observed in patients with SOS1-related Noonan syndrome, with ectodermal anomalies as common features and short stature and learning disabilities as relatively infrequent findings compared to the average Noonan syndrome phenotype. The spectrum of heart defects in SOS2-related Noonan syndrome was consistent with the known spectrum of cardiac anomalies in RASopathies, but no specific heart defect was particularly predominating. Notably, lymphatic anomalies were extraordinarily frequent, affecting more than half of the patients. We therefore conclude that SOS2-related Noonan syndrome is associated with a particularly high risk of lymphatic complications that may have a significant impact on morbidity and quality of life.

The clinical significance of A2ML1 variants in Noonan syndrome has to be reconsidered.

The RASopathies are a group of clinically and genetically heterogeneous developmental disorders caused by dysregulation of the RAS/MAPK signalling pathway. Variants in several components and regulators of this pathway have been identified as the pathogenetic cause. In 2015, missense variants in A2ML1 were reported in three unrelated families with clinical diagnosis of Noonan syndrome (NS) and a zebrafish model was presented showing heart and craniofacial defects similar to those caused by a NS-associated Shp2 variant. However, a causal role of A2ML1 variants in NS has not been confirmed since. Herein, we report on 15 individuals who underwent screening of RASopathy-associated genes and were found to carry rare variants in A2ML1, including variants previously proposed to be causative for NS. In cases where parental DNA was available, the respective A2ML1 variant was found to be inherited from an unaffected parent. Seven index patients carrying an A2ML1 variant presented with an alternate disease-causing genetic aberration. These findings underscore that current evidence is insufficient to support a causal relation between variants in A2ML1 and NS, questioning the inclusion of A2ML1 screening in diagnostic RASopathy testing.

De Novo VPS4A Mutations Cause Multisystem Disease with Abnormal Neurodevelopment.

The endosomal sorting complexes required for transport (ESCRTs) are essential for multiple membrane modeling and membrane-independent cellular processes. Here we describe six unrelated individuals with de novo missense variants affecting the ATPase domain of VPS4A, a critical enzyme regulating ESCRT function. Probands had structural brain abnormalities, severe neurodevelopmental delay, cataracts, growth impairment, and anemia. In cultured cells, overexpression of VPS4A mutants caused enlarged endosomal vacuoles resembling those induced by expression of known dominant-negative ATPase-defective forms of VPS4A. Proband-derived fibroblasts had enlarged endosomal structures with abnormal accumulation of the ESCRT protein IST1 on the limiting membrane. VPS4A function was also required for normal endosomal morphology and IST1 localization in iPSC-derived human neurons. Mutations affected other ESCRT-dependent cellular processes, including regulation of centrosome number, primary cilium morphology, nuclear membrane morphology, chromosome segregation, mitotic spindle formation, and cell cycle progression. We thus characterize a distinct multisystem disorder caused by mutations affecting VPS4A and demonstrate that its normal function is required for multiple human developmental and cellular processes.

Enhanced MAPK1 Function Causes a Neurodevelopmental Disorder within the RASopathy Clinical Spectrum.

Signal transduction through the RAF-MEK-ERK pathway, the first described mitogen-associated protein kinase (MAPK) cascade, mediates multiple cellular processes and participates in early and late developmental programs. Aberrant signaling through this cascade contributes to oncogenesis and underlies the RASopathies, a family of cancer-prone disorders. Here, we report that de novo missense variants in MAPK1, encoding the mitogen-activated protein kinase 1 (i.e., extracellular signal-regulated protein kinase 2, ERK2), cause a neurodevelopmental disease within the RASopathy phenotypic spectrum, reminiscent of Noonan syndrome in some subjects. Pathogenic variants promote increased phosphorylation of the kinase, which enhances translocation to the nucleus and boosts MAPK signaling in vitro and in vivo. Two variant classes are identified, one of which directly disrupts binding to MKP3, a dual-specificity protein phosphatase negatively regulating ERK function. Importantly, signal dysregulation driven by pathogenic MAPK1 variants is stimulus reliant and retains dependence on MEK activity. Our data support a model in which the identified pathogenic variants operate with counteracting effects on MAPK1 function by differentially impacting the ability of the kinase to interact with regulators and substrates, which likely explains the minor role of these variants as driver events contributing to oncogenesis. After nearly 20 years from the discovery of the first gene implicated in Noonan syndrome, PTPN11, the last tier of the MAPK cascade joins the group of genes mutated in RASopathies.

Refinement of the clinical and mutational spectrum of UBE2A deficiency syndrome.

UBE2A deficiency, that is, intellectual disability (ID) Nascimento type (MIM 300860), is an X-linked syndrome characterized by developmental delay, moderate to severe ID, seizures, dysmorphisms, skin anomalies, and urogenital malformations. Forty affected subjects have been reported thus far, with 31 cases having intragenic UBE2A variants. Here, we report on additional eight affected subjects from seven unrelated families who were found to be hemizygous for previously unreported UBE2A missense variants (p.Glu62Lys, p.Arg95Cys, p.Thr99Ala, and p.Arg135Trp) or small in-frame deletions (p.Val81_Ala83del, and p.Asp101del). A wide phenotypic spectrum was documented in these subjects, ranging from moderate ID associated with mild dysmorphisms to severe features including congenital heart defects (CHD), severe cognitive impairment, and pineal gland tumors. Four variants affected residues (Glu62, Arg95, Thr99 and Asp101) that contribute to stabilizing the structure of the E3 binding domain. The three-residue in-frame deletion, p.Val81_Ala83del, resulted from aberrant processing of the transcript. This variant and p.Arg135Trp mapped to regions of the protein located far from the E3 binding region, and caused variably accelerated protein degradation. By reviewing available clinical information, we revise the clinical and molecular profile of the disorder and document genotype-phenotype correlations. Pineal gland cysts/tumors, CHD and hypogammaglobulinemia emerge as recurrent features.

Pathogenic PTPN11 variants involving the poly-glutamine Gln255 -Gln256 -Gln257 stretch highlight the relevance of helix B in SHP2's functional regulation.

Germline PTPN11 mutations cause Noonan syndrome (NS), the most common disorder among RASopathies. PTPN11 encodes SHP2, a protein tyrosine-phosphatase controlling signaling through the RAS-MAPK and PI3K-AKT pathways. Generally, NS-causing PTPN11 mutations are missense changes destabilizing the inactive conformation of the protein or enhancing its binding to signaling partners. Here, we report on two PTPN11 variants resulting in the deletion or duplication of one of three adjacent glutamine residues (Gln255 -to-Gln257 ). While p.(Gln257dup) caused a typical NS phenotype in carriers of a first family, p.(Gln257del) had incomplete penetrance in a second family. Missense mutations involving Gln256 had previously been reported in NS. This poly-glutamine stretch is located on helix B of the PTP domain, a region involved in stabilizing SHP2 in its autoinhibited state. Molecular dynamics simulations predicted that changes affecting this motif perturb the SHP2's catalytically inactive conformation and/or substrate recognition. Biochemical data showed that duplication and deletion of Gln257 variably enhance SHP2's catalytic activity, while missense changes involving Gln256 affect substrate specificity. Expression of mutants in HEK293T cells documented their activating role on MAPK signaling, uncoupling catalytic activity and modulation of intracellular signaling. These findings further document the relevance of helix B in the regulation of SHP2's function.

Activating MRAS mutations cause Noonan syndrome associated with hypertrophic cardiomyopathy.

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 > T (p.Thr68Ile) and c.67G > 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.

A novel disorder involving dyshematopoiesis, inflammation, and HLH due to aberrant CDC42 function.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival.

Activating Mutations of RRAS2 Are a Rare Cause of Noonan Syndrome.

Aberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%-20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations.

Clinical and functional characterization of a novel RASopathy-causing SHOC2 mutation associated with prenatal-onset hypertrophic cardiomyopathy.

SHOC2 is a scaffold protein mediating RAS-promoted activation of mitogen-activated protein kinase (MAPK) signaling in response to extracellular stimuli. A recurrent activating mutation in SHOC2 (p.Ser2Gly) causes Mazzanti syndrome, a RASopathy characterized by features resembling Noonan syndrome and distinctive ectodermal abnormalities. A second mutation (p.Met173Ile) supposed to cause loss-of-function was more recently identified in two individuals with milder phenotypes. Here, we report on the third RASopathy-causing SHOC2 mutation (c.807_808delinsTT, p.Gln269_His270delinsHisTyr), which was found associated with prenatal-onset hypertrophic cardiomyopathy. Structural analyses indicated a possible impact of the mutation on the relative orientation of the two SHOC2's leucine-rich repeat domains. Functional studies provided evidence of its activating role, revealing enhanced binding of the mutant protein to MRAS and PPP1CB, and increased signaling through the MAPK cascade. Differing from SHOC2 S2G , SHOC2 Q269_H270delinsHY is not constitutively targeted to the plasma membrane. These data document that diverse mechanisms in SHOC2 functional dysregulation converge toward MAPK signaling upregulation.