A very mild phenotype in six individuals of a three-generation family with the novel HRAS variant c.176C > G p.(Ala59Gly): Emergence of a new HRAS-related RASopathy distinct from Costello syndrome.

Costello syndrome is a clinically recognizable, severe neurodevelopmental disorder caused by heterozygous activating variants in HRAS. The vast majority of affected patients share recurring variants affecting HRAS codons 12 and 13 and a relatively uniform phenotype. Here, we report the unique and attenuated phenotype of six individuals of an extended family affected by the HRAS variant c.176C>T p.(Ala59Gly), which, to our knowledge, has never been reported as a germline variant in patients so far. HRAS Alanine 59 has been previously functionally investigated as an oncogenic hotspot and the p.Ala59Gly substitution was shown to impair intrinsic GTP hydrolysis. All six individuals we report share a phenotype of ectodermal anomalies and mild features suggestive of a RASopathy, reminiscent of patients with Noonan syndrome-like disorder with loose anagen hair. All six are of normal intelligence, none have a history of failure to thrive or malignancy, and they have no known cardiac or neurologic pathologies. Our report adds to the previous reports of patients with rare variants affecting amino acids located in the SWITCH II/G3 region of HRAS and suggests a consistent, attenuated phenotype distinct from classical Costello syndrome. We propose the definition of a new distinct HRAS-related RASopathy for patients carrying HRAS variants affecting codons 58, 59, 60.

Severe neuroglycopenic symptoms due to nonketotic hypoglycemia in children with cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous syndrome (CFC) (OMIM 115150) is a congenital disease caused by constitutive activation of the Raf/MEK/ERK signaling cascade. Unlike aspects of morphological anomalies, metabolic functions related to the disease have garnered little attention. We present severe neuroglycopenic symptoms due to nonketotic hypoglycemia in two children with CFC (Case 1, a 4-year-old male with c.389A > G heterozygous variant in MAP2K1; Case 2, a 3-year-old male with c.770A > G heterozygous variant in BRAF). Case 1 exhibited a nonketotic hypoglycemic coma and clustered left-hemispheric convulsions despite receiving infusion therapy, leading to severe sequelae with choreoathetosis. Brain magnetic resonance imaging of Case 1 showed T2-elongation with restricted diffusion on the bilateral basal ganglia and thalamus, with the dominance of the right putamen. Case 2 presented a prolonged generalized seizure as an initial clinical symptom but fully recovered. The presence of growth hormone and cortisol deficiency was ruled out in both cases. Blood spots acylcarnitine profiles excluded the co-occurrence of mitochondrial HMG-CoA synthase deficiency and HMG-CoA lyase deficiency. These cases demonstrate the potential vulnerability to nonketotic hypoglycemia, especially during lipid shortages. As children with CFC frequently have difficulties feeding, we suggest great attention should be paid to the potential risk of severe nonketotic hypoglycemia.

Fibrous dysplasia in cardio-facio-cutaneous syndrome: A case report and review of literature.

Cardio-facio-cutaneous (CFC) syndrome (OMIM #:115150, 615278, 615279, 615280) is a rare genetic condition caused by variants in the RAS/mitogen-activated protein kinase (MAPK) signal transduction pathway. Up to 75% of cases are caused by mutations in the BRAF gene, whereas KRAS gene mutation has only been reported in <2% of cases. CFC syndrome is characterized by cardiac abnormalities, distinctive craniofacial dysmorphism, and various cutaneous abnormalities. Musculoskeletal and orthopedic manifestations are also prevalent in patients with CFC syndrome, among which the most common are skeletal deformities and joint laxities. Dysplastic bone disorders, on the other hand, have not been reported in CFC syndrome before. We report on a case of symmetrical polyostotic fibrous dysplasia (FD) in a patient with CFC syndrome with the KRAS(NM_004985.5):c.57G>C; p.Leu19Phe variant. The FDs were incidentally picked up, and patient was conservatively managed and remained asymptomatic on follow-up. The same variant was reported previously in a patient with Oculoectodermal Syndrome (OES), who developed polyostotic non-ossifying fibroma (NOF). This case explores FD as a possible new clinical feature of CFC syndrome, and when linked to the historical case of OES, explores whether the KRAS(NM_004985.5):c.57G>C; p.Leu19Phe mutation may potentially contribute to the development of dysplastic bone lesions in patients with this particular mutation.

TRAPγ-CDG shows asymmetric glycosylation and an effect on processing of proteins required in higher organisms.

Newly synthesised glycoproteins enter the rough endoplasmic reticulum through a translocation pore. The translocon associated protein (TRAP) complex is located close to the pore. In a patient with a homozygous start codon variant in TRAPγ (SSR3), absence of TRAPγ causes disruption of the TRAP complex, impairs protein translocation into the endoplasmic reticulum and affects transport, for example, into the brush-border membrane. Furthermore, we observed an unbalanced non-occupancy of N-glycosylation sites. The major clinical features are intrauterine growth retardation, facial dysmorphism, congenital diarrhoea, failure to thrive, pulmonary disease and severe psychomotor disability.

Ras/MAPK dysregulation in development causes a skeletal myopathy in an activating BrafL597V mouse model for cardio-facio-cutaneous syndrome.

BACKGROUND: Cardio-facio-cutaneous (CFC) syndrome is a human multiple congenital anomaly syndrome that is caused by activating heterozygous mutations in either BRAF, MEK1, or MEK2, three protein kinases of the Ras/mitogen-activated protein kinase (MAPK) pathway. CFC belongs to a group of syndromes known as RASopathies. Skeletal muscle hypotonia is a ubiquitous phenotype of RASopathies, especially in CFC syndrome. To better understand the underlying mechanisms for the skeletal myopathy in CFC, a mouse model with an activating BrafL597V allele was utilized. RESULTS: The activating BrafL597V allele resulted in phenotypic alterations in skeletal muscle characterized by a reduction in fiber size which leads to a reduction in muscle size which are functionally weaker. MAPK pathway activation caused inhibition of myofiber differentiation during embryonic myogenesis and global transcriptional dysregulation of developmental pathways. Inhibition in differentiation can be rescued by MEK inhibition. CONCLUSIONS: A skeletal myopathy was identified in the CFC BrafL597V mouse validating the use of models to study the effect of Ras/MAPK dysregulation on skeletal myogenesis. RASopathies present a novel opportunity to identify new paradigms of myogenesis and further our understanding of Ras in development. Rescue of the phenotype by inhibitors may help advance the development of therapeutic options for RASopathy patients.

Clinical and molecular spectra of BRAF-associated RASopathy.

Noonan syndrome (NS) and cardio-facio-cutaneous (CFC) syndrome are the most common subtypes of RASopathy. As an effector of Ras, BRAF is one of the molecules responsible for RASopathy. We investigated the phenotypic and genotypic features of 26 patients with BRAF-associated RASopathy. The clinical diagnoses were CFC (n = 21, 80.8%), NS (n = 3, 11.5%), NS/CFC (n = 1, 3.8%), and undefined syndromic intellectual disability (ID) (n = 1, 3.8%). The mostly shared phenotypes were ID (90.5%), cutaneous manifestations (84.6%), congenital heart defects (76.9%), short stature (76.9%), and dysmorphic features such as short neck (65.4%) and low-set ears (65.4%). Importantly, moderate to severe ID (57.1%) and epilepsy (26.9%) were noted. Eighteen different missense mutations were found, including a novel mutation, p.Phe498Tyr. p.Gln257Arg (n = 9, 34.6%) was the most common mutation, and the mutations were clustered in the cysteine-rich domain or protein kinase domain. A review of previously reported cases along with our findings revealed the existence of multiple sub-phenotypes of RASopathy within a single genotype, indicating that BRAF-associated RASopathy is not variant-specific. Our study further delineated the diverse and expanded clinical phenotypes of BRAF-associated RASopathy with their molecular genetic characteristics.

Familial cardio-facio-cutaneous syndrome: Vertical transmission of the BRAF p.G464R pathogenic variant and review of the literature.

Cardio-facio-cutaneous syndrome (CFC) is one of the RASopathies and is caused by germline mutations that activate the Ras/mitogen-activated protein kinase (MAPK) pathway. CFC is due to heterozygous germline mutations in protein kinases BRAF, MEK1, or MEK2 and rarely in KRAS, a small GTPase. CFC is a multiple congenital anomaly disorder in which individuals may have craniofacial dysmorphia, heart issues, skin and hair anomalies, and delayed development. Pathogenic variants for CFC syndrome are usually considered de novo because vertical transmission has only been reported with MEK2 and KRAS. The index case was a 3-year-old male with features consistent with the clinical diagnosis of CFC. Sequencing revealed a previously reported heterozygous likely pathogenic variant BRAF p.G464R. Upon detailed family history, the index case's pregnant mother was noted to have similar features to her son. Targeted familial testing of the BRAF pathogenic variant was performed on the mother, confirming her diagnosis. Prenatal genetic testing for the fetus was declined, but postnatal molecular testing of the index case's sister was positive for the familial BRAF p.G464R variant. Functional analysis of the variant demonstrated increased kinase activity. We report the first identified vertically transmitted functional BRAF pathogenic variant. Our findings emphasize the importance of obtaining a comprehensive evaluation of family members and that activating pathogenic variants within the canonical MAPK cascade mediated by BRAF are compatible with human reproduction.

Expanding the genotypic and phenotypic spectrum in a diverse cohort of 104 individuals with Wiedemann-Steiner syndrome.

Wiedemann-Steiner syndrome (WSS) is an autosomal dominant disorder caused by monoallelic variants in KMT2A and characterized by intellectual disability and hypertrichosis. We performed a retrospective, multicenter, observational study of 104 individuals with WSS from five continents to characterize the clinical and molecular spectrum of WSS in diverse populations, to identify physical features that may be more prevalent in White versus Black Indigenous People of Color individuals, to delineate genotype-phenotype correlations, to define developmental milestones, to describe the syndrome through adulthood, and to examine clinicians' differential diagnoses. Sixty-nine of the 82 variants (84%) observed in the study were not previously reported in the literature. Common clinical features identified in the cohort included: developmental delay or intellectual disability (97%), constipation (63.8%), failure to thrive (67.7%), feeding difficulties (66.3%), hypertrichosis cubiti (57%), short stature (57.8%), and vertebral anomalies (46.9%). The median ages at walking and first words were 20 months and 18 months, respectively. Hypotonia was associated with loss of function (LoF) variants, and seizures were associated with non-LoF variants. This study identifies genotype-phenotype correlations as well as race-facial feature associations in an ethnically diverse cohort, and accurately defines developmental trajectories, medical comorbidities, and long-term outcomes in individuals with WSS.

Expanding the clinical spectrum in trichohepatoenteric syndrome.

Trichohepatoenteric syndrome (THES) is a very rare autosomal recessive genetic disorder, which is characterized by intractable diarrhea during infancy, dysmorphic features, immunodeficiency, and a failure to thrive. There are still significant difficulties for patients and clinicians in terms of the management of THES, even though its molecular basis has been uncovered in the last decade. In this article, we have presented two cases relating to siblings that have been diagnosed with the condition. Concerning one of the patients, we described a novel variation (c.2114 + 5G > A) in the TTC37 gene and a mild clinical course; meanwhile, the other one was clinically diagnosed with THES at 17 years of age, but they had seizures and died suddenly. These cases expand the spectrum of clinical findings in relation to THES.

Congenital heart defects in Noonan syndrome: Diagnosis, management, and treatment.

Noonan syndrome is a pleomorphic genetic disorder, in which a high percentage of affected individuals have cardiovascular involvement, most prevalently various forms of congenital heart disease (i.e., pulmonary valve stenosis, septal defects, left-sided lesions, and complex forms with multiple anomalies). Care includes attentiveness to several comorbidities, some directly impacting cardiac management (bleeding diatheses and lymphatic anomalies). More than 50% of patients with Noonan syndrome harbor PTPN11 pathogenic variation, which results in hyperactivation of RAS/mitogen-activated protein kinase signaling. Several other disease genes with similar biological effects have been uncovered for NS and phenotypically related disorders, collectively called the RASopathies. Molecular diagnosis with gene resequencing panels is now widely available, but phenotype variability and in some cases, subtlety, continues to make identification of Noonan syndrome difficult. Until genetic testing becomes universal for patients with congenital heart disease, alertness to Noonan syndrome's broad clinical presentations remains crucial. Genotype-phenotype associations for Noonan syndrome enable better prognostication for affected patients when a molecular diagnosis is established. We still lack Noonan syndrome-specific treatment; however, newly developed anticancer RAS pathway inhibitors could fill that gap if safety and efficacy can be established for indications such as pulmonary valve stenosis.

Extending the phenotypic spectrum of Bohring-Opitz syndrome: Mild case confirmed by functional studies.

Bohring-Opitz syndrome (BOS) has been described as a clinically recognizable genetic syndrome since 1999. Clinical diagnostic criteria were established in 2011 and include microcephaly, trigonocephaly, distinctive craniofacial dysmorphic features, facial nevus flammeus, failure to thrive, and severe developmental delays. The same year, different de novo heterozygous nonsense mutations in the ASXL1 were found in affected individuals. Since then, several cases have been reported confirming the association between this chromatin remodeling gene and BOS. Most affected individuals die in early childhood because of unexplained bradycardia, obstructive apnea, or pulmonary infections. Those that survive usually cannot walk independently and are nonverbal. Some have had success using walkers and braces in late childhood. While few are able to speak, many have been able to express basic needs using communication devices as well as gestures with associated basic vocalizations. In this article, we present a mild case of BOS with a de novo pathogenic mutation c.1720-2A>G (p.I574VfsX22) in ASXL1 detected on whole-exome sequencing and confirmed by functional analysis of the messenger RNA splicing pattern on the patient's fibroblasts. She has typical dysmorphic features and is able to run and walk independently as well as to communicate with basic sign language.

Neck masses due to internal jugular vein phlebectasia: Frequency in Menkes disease and literature review of 85 pediatric subjects.

Classic Menkes disease is a rare X-linked recessive disorder of copper metabolism caused by pathogenic variants in the copper transporter gene, ATP7A. Untreated affected individuals suffer failure to thrive and neurodevelopmental delays that begin at 6-8 weeks of age and progress inexorably to death, often within 3 years. Subcutaneous injections of Copper Histidinate (US Food and Drug Administration IND #34,166, Orphan product designation #12-3663) are associated with improved survival and neurological outcomes, especially when commenced within a month of birth. We previously identified internal jugular vein phlebectasia (IJP) in four Menkes disease subjects. This feature and other connective tissue abnormalities appear to be consequences of deficient activity of lysyl oxidase, a copper-dependent enzyme. Here, we report results from a prospective study of IJP based on 178 neck ultrasounds in 66 Menkes subjects obtained between November 2007 and March 2018. Nine patients met the criterion for IJP (one or more cross-sectional area measurements exceeding 2.2 cm2 ) and five subjects had clinically apparent neck masses that enlarged over time. Our prospective results suggest that IJP occurs in approximately 14% (9/66) of Menkes disease patients and appears to be clinically benign with no specific medical or surgical actionability. We surveyed the medical literature for prior reports of IJP in pediatric subjects and identified 85 individuals and reviewed the distribution of this abnormality by gender, sidedness, and underlying etiology. Taken together, Menkes disease accounts for 16% (15/94) of all reported IJP individuals. Neck masses from IJP represent underappreciated abnormalities in Menkes disease.

Advancing RAS/RASopathy therapies: An NCI-sponsored intramural and extramural collaboration for the study of RASopathies.

RASopathies caused by germline pathogenic variants in genes that encode RAS pathway proteins. These disorders include neurofibromatosis type 1 (NF1), Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFC), and Costello syndrome (CS), and others. RASopathies are characterized by heterogenous manifestations, including congenital heart disease, failure to thrive, and increased risk of cancers. Previous work led by the NCI Pediatric Oncology Branch has altered the natural course of one of the key manifestations of the RASopathy NF1. Through the conduct of a longitudinal cohort study and early phase clinical trials, the MEK inhibitor selumetinib was identified as the first active therapy for the NF1-related peripheral nerve sheath tumors called plexiform neurofibromas (PNs). As a result, selumetinib was granted breakthrough therapy designation by the FDA for the treatment of PN. Other RASopathy manifestations may also benefit from RAS targeted therapies. The overall goal of Advancing RAS/RASopathy Therapies (ART), a new NCI initiative, is to develop effective therapies and prevention strategies for the clinical manifestations of the non-NF1 RASopathies and for tumors characterized by somatic RAS mutations. This report reflects discussions from a February 2019 initiation meeting for this project, which had broad international collaboration from basic and clinical researchers and patient advocates.

Cardiofaciocutaneous syndrome with KRAS gene mutation presenting as chylopericardium.

A 12-year-old female patient with cardiofaciocutaneous syndrome in the presence of a KRAS gene mutation had episodes of pericardial effusion on ultrasound, later confirmed to be chylopericardium, which resolved after a lymphangiography. We discussed herein the pathophysiological background of this rare case and the efficacy of lymphangiography in the treatment of chylopericardium.

NRAS associated RASopathy and embryonal rhabdomyosarcoma.

RASopathies are a group of phenotypically overlapping disorders that arise from dysregulation of the RAS/MAPK pathway. These disorders include Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome, and neurofibromatosis-Type 1. While somatic mutations in the three human Ras genes (KRAS, HRAS, and NRAS) are a common finding in a variety of cancers, germline mutations in each of the these genes cause developmental RASopathy phenotypes with mutations in specific genes typically correlating with specific phenotypes. We present the case of a germline heterozygous NRAS mutation producing a severe phenotype involving embryonal rhabdomyosarcoma, severe intellectual disability, and numerous melanocytic nevi in addition to more typical manifestations of Noonan syndrome. Additionally, the specific p.G12R NRAS mutation in this case is a common somatic mutation in cancer cells, and analysis of previously reported NRAS-RASopathy cases suggests that mutations at traditionally oncogenic codons are associated with elevated cancer risk not present with mutations at other sites.

Short stature and growth hormone deficiency in a subset of patients with Potocki-Lupski syndrome: Expanding the phenotype of PTLS.

Potocki-Lupski Syndrome (PTLS, MIM 610883), or duplication of chromosome 17p11.2, is a clinically recognizable condition characterized by infantile hypotonia, failure to thrive, developmental delay, intellectual disability, and congenital anomalies. Short stature, classified as greater than two standard deviations below the mean, has not previously been considered a major feature of PTLS. Retrospective chart review on a cohort of 37 individuals with PTLS was performed to investigate the etiology of short stature. Relevant data included anthropometric measurements, insulin growth factor-1 (IGF-1), insulin-like growth factor binding protein 3 (IGFBP-3), growth hormone (GH) stimulation testing, blood glucose levels, brain MRI, and bone age. Approximately 25% (9/37) of individuals with PTLS had short stature. Growth hormone deficiency (GHD) was definitively identified in two individuals. These two PTLS patients with growth hormone deficiency, as well as three others with short stature and no documented GHD, received growth hormone and obtained improvement in linear growth. One individual was identified to have pituitary abnormalities on MRI and had complications of hypoglycemia due to unrecognized GHD. Individuals with PTLS can benefit from undergoing evaluation for GHD should they present with short stature or hypoglycemia. Early identification of GHD could facilitate potential therapeutic benefit for individuals with PTLS, including linear growth, musculoskeletal, and in cases of hypoglycemia, potentially cognitive development as well.

Genetic landscape of RASopathies in Chinese: Three decades' experience in Hong Kong.

RASopathies are a group of genetic disorders due to dysregulation of the RAS-MAPK signaling pathway, which is important in regulating cell growth, proliferation, and differentiation. These include Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML), cardiofaciocutaneous (CFC) syndrome, and Costello syndrome (CS), clinical manifestations include growth retardation, developmental delay, cardiac defects, and specific dysmorphic features. There were abundant publications describing the genotype and phenotype from the Western populations. However, detailed study of RASopathies in Chinese population is lacking. We present here the largest cohort of RASopathies ever reported in Chinese populations, detailing the mutation spectrum and clinical phenotypes of these patients. The Clinical Genetic Service, Department of Health, and Queen Mary Hospital are tertiary referral centers for genetic disorders in Hong Kong. We retrospectively reviewed all the genetically confirmed cases of RASopathies, including NS, NSML, CFC syndrome, and CS, over the past 29 years (from 1989 to 2017). Analyses of the mutation spectrum and clinical phenotypes were performed. One hundred and ninety-one ethnic Chinese patients with genetically confirmed RASopathies were identified, including 148 patients with NS, 23 NSML, 12 CFC syndrome, and eight CS. We found a lower incidence of hypertrophic cardiomyopathy in individuals with NSML (27.3%), and NS caused by RAF1 mutations (62.5%). Another significant finding was for those NS patients with myeloproliferative disorder, the mutations fall within Exon 3 of PTPN11 but not only restricted to the well-known hotspots, that is, p.Asp61 and p.Thr731, which suggested that re-evaluation of the current tumor surveillance recommendation maybe warranted.

Activated Braf induces esophageal dilation and gastric epithelial hyperplasia in mice.

Germline mutations in BRAF are a major cause of cardio-facio-cutaneous (CFC) syndrome, which is characterized by heart defects, characteristic craniofacial dysmorphology and dermatologic abnormalities. Patients with CFC syndrome also commonly show gastrointestinal dysfunction, including feeding and swallowing difficulties and gastroesophageal reflux. We have previously found that knock-in mice expressing a Braf Q241R mutation exhibit CFC syndrome-related phenotypes, such as growth retardation, craniofacial dysmorphisms, congenital heart defects and learning deficits. However, it remains unclear whether BrafQ241R/+ mice exhibit gastrointestinal dysfunction. Here, we report that BrafQ241R/+ mice have neonatal feeding difficulties and esophageal dilation. The esophagus tissues from BrafQ241R/+ mice displayed incomplete replacement of smooth muscle with skeletal muscle and decreased contraction. Furthermore, the BrafQ241R/+ mice showed hyperkeratosis and a thickened muscle layer in the forestomach. Treatment with MEK inhibitors ameliorated the growth retardation, esophageal dilation, hyperkeratosis and thickened muscle layer in the forestomach in BrafQ241R/+ mice. The esophageal dilation with aberrant skeletal-smooth muscle boundary in BrafQ241R/+ mice were recovered after treatment with the histone H3K27 demethylase inhibitor GSK-J4. Our results provide clues to elucidate the pathogenesis and possible treatment of gastrointestinal dysfunction and failure to thrive in patients with CFC syndrome.

Leucine-485 deletion variant of BRAF may exhibit the severe end of the clinical spectrum of CFC syndrome.

The genotype-phenotype correlation in BRAF variant in cardio-facio-cutaneous (CFC) syndrome is not clearly defined. Here we report a case with a severe clinical phenotype and a novel BRAF variant, p.Leu485del. The present case showed severe intellectual disability, impaired awareness, hyperekplexia, involuntary movements, early onset refractory seizures, and delayed myelination on brain magnetic resonance imaging as well as a polycystic and dysplastic kidney, which are previously unreported anomalies in CFC or RAS/mitogen-activated protein kinase syndromes related to BRAF variant. CFC syndrome, especially caused by BRAF variant, should be included in the differential diagnosis of patients with developmental and epileptic encephalopathies and hyperekplexia. Furthermore, we need to keep in mind that missense variants or the deletion of Leucine-485 may be associated with severe symptoms.

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.