Katherine A. Rauen

Cardio-facio-cutaneous syndrome: clinical features, diagnosis, and management guidelines.

Cardio-facio-cutaneous syndrome (CFC) is one of the RASopathies that bears many clinical features in common with the other syndromes in this group, most notably Noonan syndrome and Costello syndrome. CFC is genetically heterogeneous and caused by gene mutations in the Ras/mitogen-activated protein kinase pathway. The major features of CFC include characteristic craniofacial dysmorphology, congenital heart disease, dermatologic abnormalities, growth retardation, and intellectual disability. It is essential that this condition be differentiated from other RASopathies, as a correct diagnosis is important for appropriate medical management and determining recurrence risk. Children and adults with CFC require multidisciplinary care from specialists, and the need for comprehensive management has been apparent to families and health care professionals caring for affected individuals. To address this need, CFC International, a nonprofit family support organization that provides a forum for information, support, and facilitation of research in basic medical and social issues affecting individuals with CFC, organized a consensus conference. Experts in multiple medical specialties provided clinical management guidelines for pediatricians and other care providers. These guidelines will assist in an accurate diagnosis of individuals with CFC, provide best practice recommendations, and facilitate long-term medical care.

Dysregulation of astrocyte extracellular signaling in Costello syndrome

Excessive HRAS signaling in Costello syndrome accelerates Ras-specific astroglial development and extracellular matrix accumulation, resulting in premature cortical circuit maturation. Perineuronal nets close the door on brain development Creating a brain is exceedingly complex, so mutations in many different genes can cause the process to go awry. Mutations in one sensitive pathway, Ras signaling, cause a family of neurodevelopmental disorders called RASopathies. Krencik et al. have used patient-derived iPSCs and mutant mice to demonstrate that the mutation in HRAS that causes one RASopathy—Costello syndrome—acts by accelerating astrocyte differentiation. The authors examined astroglial development from patient iPSCs in vitro and, similarly, astroglial development in vivo in mice carrying the same mutation as that found in Costello syndrome patients. The authors suggest that premature formation of perineuronal nets around neurons may close the experience-dependent development of the brain, producing the typical intellectual disability in Costello syndrome patients. Astrocytes produce an assortment of signals that promote neuronal maturation according to a precise developmental timeline. Is this orchestrated timing and signaling altered in human neurodevelopmental disorders? To address this question, the astroglial lineage was investigated in two model systems of a developmental disorder with intellectual disability caused by mutant Harvey rat sarcoma viral oncogene homolog (HRAS) termed Costello syndrome: mutant HRAS human induced pluripotent stem cells (iPSCs) and transgenic mice. Human iPSCs derived from patients with Costello syndrome differentiated to astroglia more rapidly in vitro than those derived from wild-type cell lines with normal HRAS, exhibited hyperplasia, and also generated an abundance of extracellular matrix remodeling factors and proteoglycans. Acute treatment with a farnesyl transferase inhibitor and knockdown of the transcription factor SNAI2 reduced expression of several proteoglycans in Costello syndrome iPSC-derived astrocytes. Similarly, mice in which mutant HRAS was expressed selectively in astrocytes exhibited experience-independent increased accumulation of perineuronal net proteoglycans in cortex, as well as increased parvalbumin expression in interneurons, when compared to wild-type mice. Our data indicate that astrocytes expressing mutant HRAS dysregulate cortical maturation during development as shown by abnormal extracellular matrix remodeling and implicate excessive astrocyte-to-neuron signaling as a possible drug target for treating mental impairment and enhancing neuroplasticity.

RASopathies: unraveling mechanisms with animal models

ABSTRACT RASopathies are developmental disorders caused by germline mutations in the Ras-MAPK pathway, and are characterized by a broad spectrum of functional and morphological abnormalities. The high incidence of these disorders (∼1/1000 births) motivates the development of systematic approaches for their efficient diagnosis and potential treatment. Recent advances in genome sequencing have greatly facilitated the genotyping and discovery of mutations in affected individuals, but establishing the causal relationships between molecules and disease phenotypes is non-trivial and presents both technical and conceptual challenges. Here, we discuss how these challenges could be addressed using genetically modified model organisms that have been instrumental in delineating the Ras-MAPK pathway and its roles during development. Focusing on studies in mice, zebrafish and Drosophila, we provide an up-to-date review of animal models of RASopathies at the molecular and functional level. We also discuss how increasingly sophisticated techniques of genetic engineering can be used to rigorously connect changes in specific components of the Ras-MAPK pathway with observed functional and morphological phenotypes. Establishing these connections is essential for advancing our understanding of RASopathies and for devising rational strategies for their management and treatment. Summary: Developmental disorders caused by germline mutations in the Ras-MAPK pathway are called RASopathies. Studies with animal models, including mice, zebrafish and Drosophila, continue to enhance our understanding of these diseases.

Expansion of the RASopathies

Purpose of ReviewThe Ras/mitogen-activated protein kinase (MAPK) pathway is essential in the regulation of cell cycle, differentiation, growth, cell senescence and apoptosis, all of which are critical to normal development. A class of neurodevelopmental disorders, RASopathies, is caused by germline mutations in genes of the Ras/MAPK pathway. Through the use of whole exome sequencing and targeted sequencing of selected genes in cohorts of panel-negative RASopathy patients, several new genes have been identified.Recent FindingsNew genes have been identified and include RIT1, SOS2, RASA2, RRAS and SYNGAP1, that likely represent new, albeit rare, causative RASopathy genes. In addition, A2ML1, LZTR1, MYST4, SPRY1 and MAP3K8 may represent new rare genes for RASopathies, but, additional functional studies regarding the mutations are warranted. In addition, recent reports have demonstrated that chromosomal copy number variation in regions encompassing Ras/MAPK pathway genes may be a novel pathogenetic mechanism expanding the RASopathies.SummaryThe identification of potential new genes and chromosomal copy number variation being associated with the RASopathies is very exciting and broadens our understanding of the biology of Ras signaling and the RASopathies.

Recent developments in neurofibromatoses and RASopathies: Management, diagnosis and current and future therapeutic avenues

Neurofibromatosis type 1 (NF1) was the first RASopathy and is now one of many RASopathies that are caused by germline mutations in genes that encode components of the Ras/mitogen‐activated protein kinase (MAPK) pathway. Their common underlying pathogenetic etiology causes significant overlap in phenotypic features which includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, GI and ocular abnormalities, and a predisposition to cancer. The proceedings from the symposium “Recent Developments in Neurofibromatoses (NF) and RASopathies: Management, Diagnosis and Current and Future Therapeutic Avenues” chronicle this timely and topical clinical translational research symposium. The overarching goal was to bring together clinicians, basic scientists, physician‐scientists, advocate leaders, trainees, students and individuals with Ras pathway syndromes to discuss the most state‐of‐the‐art basic science and clinical issues in an effort to spark collaborations directed towards the best practices and therapies for individuals with RASopathies.

Autonomous and Non-autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes

Summary Germline mutations in BRAF cause cardio-facio-cutaneous syndrome (CFCS), whereby 40% of patients develop hypertrophic cardiomyopathy (HCM). As the role of the RAS/MAPK pathway in HCM pathogenesis is unclear, we generated a human induced pluripotent stem cell (hiPSC) model for CFCS from three patients with activating BRAF mutations. By cell sorting for SIRPα and CD90, we generated a method to examine hiPSC-derived cell type-specific phenotypes and cellular interactions underpinning HCM. BRAF-mutant SIRPα+/CD90− cardiomyocytes displayed cellular hypertrophy, pro-hypertrophic gene expression, and intrinsic calcium-handling defects. BRAF-mutant SIRPα−/CD90+ cells, which were fibroblast-like, exhibited a pro-fibrotic phenotype and partially modulated cardiomyocyte hypertrophy through transforming growth factor β (TGFβ) paracrine signaling. Inhibition of TGFβ or RAS/MAPK signaling rescued the hypertrophic phenotype. Thus, cell autonomous and non-autonomous defects underlie HCM due to BRAF mutations. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies.

The Fourth International Symposium on Genetic Disorders of the Ras/MAPK pathway

The RASopathies are a group of disorders due to variations of genes associated with the Ras/MAPK pathway. Some of the RASopathies include neurofibromatosis type 1 (NF1), Noonan syndrome, Noonan syndrome with multiple lentigines, cardiofaciocutaneous (CFC) syndrome, Costello syndrome, Legius syndrome, and capillary malformation–arteriovenous malformation (CM‐AVM) syndrome. In combination, the RASopathies are a frequent group of genetic disorders. This report summarizes the proceedings of the 4th International Symposium on Genetic Disorders of the Ras/MAPK pathway and highlights gaps in the field.

The third international meeting on genetic disorders in the RAS/MAPK pathway: Towards a therapeutic approach

“The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Towards a Therapeutic Approach” was held at the Renaissance Orlando at SeaWorld Hotel (August 2–4, 2013). Seventy‐one physicians and scientists attended the meeting, and parallel meetings were held by patient advocacy groups (CFC International, Costello Syndrome Family Network, NF Network and Noonan Syndrome Foundation). Parent and patient advocates opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes, to identification of therapeutic targets in the RAS/MAPK pathway, to preclinical drug development and testing, to clinical trials. These proceedings will review the major points of discussion.

Patient-derived iPSCs show premature neural differentiation and neuron type-specific phenotypes relevant to neurodevelopment

Ras/MAPK pathway signaling is a major participant in neurodevelopment, and evidence suggests that BRAF, a key Ras signal mediator, influences human behavior. We studied the role of the mutation BRAFQ257R, the most common cause of cardiofaciocutaneous syndrome (CFC), in an induced pluripotent stem cell (iPSC)-derived model of human neurodevelopment. In iPSC-derived neuronal cultures from CFC subjects, we observed decreased p-AKT and p-ERK1/2 compared to controls, as well as a depleted neural progenitor pool and rapid neuronal maturation. Pharmacological PI3K/AKT pathway manipulation recapitulated cellular phenotypes in control cells and attenuated them in CFC cells. CFC cultures displayed altered cellular subtype ratios and increased intrinsic excitability. Moreover, in CFC cells, Ras/MAPK pathway activation and morphological abnormalities exhibited cell subtype-specific differences. Our results highlight the importance of exploring specific cellular subtypes and of using iPSC models to reveal relevant human-specific neurodevelopmental events.

Age and ASD symptoms in Costello syndrome

To the Editor, Costello syndrome (CS), a RASopathy caused by a mutation in the HRAS gene in the ras/MAPK pathway, has previously been associated with increased risk of autism spectrum disorder (ASD) (Adviento et al., 2014; Alfieri et al., 2014; Garg et al., 2017). However, authors of a recent paper, Age Related Differences in Prevalence of Autism Spectrum Disorder Symptoms in Children and Adolescents with Costello Syndrome (Schwartz et al., 2017) found no ASD symptoms in CS subjects over age 4. In their study, Schwartz et al. found significantly elevated rates of ASD only in children 4 years old and younger in their sample of 14 CS subjects, and ultimately concluded that “CS individuals may appear to fall on the autism spectrum in early childhood due to severe feeding and orthopedic problems that improve by age four, suggesting many of these children may eventually emerge out of an ASD presentation.” We noticed several elements of the design of Schwartz et al.’s study that may have influenced its results and the authors’ conclusions. First, as the authors point out, their sample size is 14 individuals (seven in their young 4 years group and seven in the older group). As the previously reported frequency of notable ASD symptoms in CS patients is approximately 26–44% (Adviento et al., 2014; Alfieri et al., 2014), only a small number of individuals ( 4 to 6) would be expected to have clinically relevant ASD traits (consistent with their observed 5/14) and the likelihood of falling by chance into only one of two age groups is not negligible. Neither their result of 5/7 in the young group nor 0/7 in the old group are significantly different than the expected 2.5/7 in each group if ASD were evenly distributed by age (p> .1). Second, the authors used different questionnaires to assess ASD in the younger and older groups, thus confounding age with the method of ASD-assessment used. Third, Schwartz et al. used a lenient threshold in the Modified Checklist for Autism in Toddlers (M-CHAT) questionnaire for their younger age group, and as they report, only two CS individuals met the more conservative threshold—in which case there would also be no significant difference by age group. They used a standard threshold for Social Communication Questionnaire (SCQ) in the older group, similar to the previous studies of CS and ASD. However, the version of the SCQ this study (and previous studies) used is the “Lifetime” version, and asks whether an individual has “ever” displayed a behavior. Thus, it may not be appropriate as a metric sensitive to changes with age. With these caveats in mind, we wanted to test whether our CS data (Adviento et al., 2014) showed any relationship between parent-reported ASD symptoms and age. We re-examined CS data from Autism Traits in RASopathies (Adviento et al., 2014), as well as data collected by our lab since then, to determine if we saw the same trend as Schwartz et al. We used the SCQ to assess ASD traits as a binary metric, observing a cutoff score of 15 (as did Schwartz et al. for the older age group), and the Social Responsiveness Scale (SRS) to measure symptoms of ASD on a continuous scale. We used these questionnaires to assess ASD traits in CS subjects younger than the minimum ages that have been verified, and to ensure that this did not influence our results, we ran analyses with and without young subjects and found no significant difference in our results. After omitting subjects with more than 50% of questions on either survey unanswered and those without age and total score recorded, we had a final sample of 53 CS subjects with SCQ scores and 47 CS subjects with SRS scores, aged 1–32 years. We found no correlation between age and SRS score (Figure 1a). When examining the SCQ data most similar to