SPRED proteins and their roles in signal transduction, development, and malignancy.

The roles of SPRED proteins in signaling, development, and cancer are becoming increasingly recognized. SPRED proteins comprise an N-terminal EVH-1 domain, a central c-Kit-binding domain, and C-terminal SROUTY domain. They negatively regulate signaling from tyrosine kinases to the Ras-MAPK pathway. SPRED1 binds directly to both c-KIT and to the RasGAP, neurofibromin, whose function is completely dependent on this interaction. Loss-of-function mutations in SPRED1 occur in human cancers and cause the developmental disorder, Legius syndrome. Genetic ablation of SPRED genes in mice leads to behavioral problems, dwarfism, and multiple other phenotypes including increased risk of leukemia. In this review, we summarize and discuss biochemical, structural, and biological functions of these proteins including their roles in normal cell growth and differentiation and in human disease.

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.

Epilepsy in Legius syndrome: Coincidence or causation?

Legius syndrome is a rare genetic disorder, caused by heterozygous SPRED1 pathogenic variants, which shares phenotypic features with neurofibromatosis type 1 (NF1). Both conditions typically involve café-au-lait macules, axillary freckling, and macrocephaly; however, patients with NF1 are also at risk for tumors, such as optic nerve gliomas and neurofibromas. Seizure risk is known to be elevated in NF1, but there has been little study of this aspect of Legius syndrome. The reported epilepsy incidence is 3.3%-5%, well above the general population incidence of ~0.5%-1%, but the few reports in the literature have very little data regarding epilepsy phenotype. We identified two unrelated individuals, both with Legius syndrome and epilepsy, and performed thorough phenotyping. One individual's mother also had Legius syndrome and now-resolved childhood epilepsy, as well as reports of more distant relatives who also had multiple café-au-lait macules and seizures. Both probands had experienced childhood-onset focal seizures, with normal brain MRI. In one patient, EEG later showed apparently generalized epileptiform abnormalities. Based on the data from this small case series and literature review, seizure risk is increased in people with Legius syndrome, but the epilepsy prognosis appears to be generally good, with patients having either self-limited or pharmacoresponsive courses.

Novel causative variants in Legius syndrome: SPRED1 Genotype spectrum expansion.

Legius syndrome, commonly referred to as SPRED1-related neurofibromatosis type 1-like syndrome, is a rare autosomal dominant disorder characterized by café-au-lait macules, freckling, lipomas, macrocephaly, and heterogeneous neurodevelopmental manifestations, including a different degree of learning difficulties. Although a partial clinical overlap exists with neurofibromatosis type 1 (NF1), Legius syndrome is distinguished by its genetic etiology and the absence of neurofibromas, indicating an inherent lack of tumor risk. The SPRED1 gene encodes the Sprouty-related protein with an EVH1 domain 1 (SPRED1), a negative regulator of the RAS-MAPK signaling pathway with a crucial role in cellular growth and development. Despite various genetic variants and genomic deletions associated with Legius syndrome, the full genetic spectrum of this condition remains elusive. In this study, we investigated the underlying genetic etiology in a cohort of patients presenting with typical manifestations of Legius syndrome using a custom Next Generation Sequencing (NGS) panel and Multiplex Ligation-Dependent Probe Amplification (MLPA) for NF1 and SPRED1. We identified 12 novel SPRED1 damaging variants segregating with the phenotype in all families. These rare variants affect conserved residues of the protein and are predicted damaging according to in silico tools. No clear genotype-phenotype correlations could be observed in the current cohort and previously reported patients, underscoring the heterogeneous genotype spectrum of this condition. Our findings expand the understanding of SPRED1 variants causing Legius syndrome and underscore the importance of comprehensively characterizing the genetic landscape of this disorder. Despite the absence of clear genotype-phenotype correlations, elucidating the genetic etiology of Legius syndrome is pertinent for facilitating accurate diagnosis, genetic counseling, and therapeutic interventions.

Neurofibromatosis type 1: A comparison of the 1997 NIH and the 2021 revised diagnostic criteria in 75 children and adolescents.

PURPOSE: Examining a cohort of patients suspicious of neurofibromatosis type 1 (NF1) we compared the revised diagnostic criteria with the previous National Institutes of Health (NIH) diagnostic criteria. We asked whether the refinement improved distinguishing between NF1, Legius syndrome, and constitutional mismatch repair deficiency (CMMRD). METHODS: A database search in the hospital information system of the University Children's Hospital Augsburg between 2017 and 2020 ascertained patients with International Classification of Diseases-10 code Q85.0; their clinical phenotype was evaluated by retrospective chart review. RESULTS: A total of 75 patients were identified (median age 11.0 years [range 1.1-22.6 years]; 35 female). At first suspicion of NF1, 44 patients met the NIH criteria and 56 met the revised diagnostic criteria. In total, 12 patients were diagnosed with NF1 after performing molecular genetic testing. In 31 patients, only pigmentary findings were present, whereas nonpigmentary NF1 manifestations presented with time in 9 patients. In 1 patient a heterozygous variant of uncertain significance was identified in SPRED1. Requirements for CMMRD testing were fulfilled in another patient. A total of 3 patients presented with segmental clinical findings. Three additional patients did not meet the NIH criteria, 1 of them presented with 1 additional feature of CMMRD without fulfilling requirements for testing. CONCLUSION: In our pediatric cohort, the revised diagnostic criteria discovered more patients with proven NF1 than the NIH criteria.

Value of a café-au-lait macules screening clinic: Experience from The Hospital for Sick Children in Toronto.

BACKGROUND/OBJECTIVES: Café-au-lait macules (CALMs) are a characteristic feature of neurofibromatosis type 1 (NF1), but also occur in other genetic disorders. Differential diagnosis of CALMs remains challenging and can be stressful for families. We sought to examine the role of an established CALMs screening clinic in diagnosing CALMs-related disorders. METHOD: We retrospectively reviewed patients seen between July 2012 and January 2019 in a CALMs screening clinic at The Hospital for Sick Children, a tertiary pediatric hospital in Toronto, Canada. Pediatric patients were referred because of multiple CALMs or suspected NF1. Selection was based on a chronological referral sample with no exclusions. A pediatric dermatologist examined all patients for CALMs and NF1 manifestations. Genetic testing was offered to confirm a clinical diagnosis or when clinical findings were inconclusive. RESULTS: Three hundred patients, of which 152 (50.7%) were female and had a mean age of 5.6 ± 4.8 years were seen during the study period. NF1 was diagnosed in 76 (25.3%) patients, mosaic NF1 in 38 (12.7%) patients, and 8 (2.7%) patients received other genetic diagnoses. One hundred and twelve (37.3%) patients were diagnosed with isolated CALMs not associated with an underlying genetic disease. Furthermore, 36 (12%) of our patients did not have CALMs. CONCLUSIONS: The CALMs screening clinic aided in the early diagnosis of genetic disorders such as NF1 and distinguished CALMs from other hyperpigmented lesions. We encourage the adoption of this clinic model in referral centers to streamline and optimize care of patients with presumptive diagnosis of CALMs.

Legius Syndrome and its Relationship with Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is the most common disorder characterized by multiple café-au-lait macules. Most individuals with this autosomal dominant disorder also have other features, such as skinfold freckling, iris Lisch nodules and benign or malignant peripheral nerve sheath tumours. Legius syndrome is a less frequent autosomal dominant disorder with similar multiple café-au-lait macules and skinfold freckling. Legius syndrome is not characterized by an increased risk of tumours, and a correct diagnosis is important. In young children with a sporadic form of multiple café-au-lait macules with or without freckling and no other manifestations of NF1 these 2 conditions cannot be differentiated based on clinical examination. Molecular analysis of the NF1 and SPRED1 genes is usually needed to differentiate the 2 conditions. Other less frequent conditions with café-au-lait macules are Noonan syndrome with multiple lentigines, constitutional mismatch repair deficiency and McCune-Albright syndrome.

Proceedings of the fifth international RASopathies symposium: When development and cancer intersect.

This report summarizes and highlights the fifth International RASopathies Symposium: When Development and Cancer Intersect, held in Orlando, Florida in July 2017. The RASopathies comprise a recognizable pattern of malformation syndromes that are caused by germ line mutations in genes that encode components of the RAS/mitogen-activated protein kinase (MAPK) pathway. Because of their common underlying pathogenetic etiology, there is significant overlap in their phenotypic features, which includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, gastrointestinal and ocular abnormalities, neurological and neurocognitive issues, and a predisposition to cancer. The RAS pathway is a well-known oncogenic pathway that is commonly found to be activated in somatic malignancies. As in somatic cancers, the RASopathies can be caused by various pathogenetic mechanisms that ultimately impact or alter the normal function and regulation of the MAPK pathway. As such, the RASopathies represent an excellent model of study to explore the intersection of the effects of dysregulation and its consequence in both development and oncogenesis.

Choroidal abnormalities in café-au-lait syndromes: a new differential diagnostic tool?

The best known café-au-lait syndrome is neurofibromatosis type 1 (NF1). Legius syndrome (LS) is another, rarer syndrome with café-au-lait macules (CALMs). In young patients their clinical picture is often indistinguishable. We investigated the presence of choroidal abnormalities in syndromes with CALMs as a candidate tool for a more efficient diagnosis. Thirty-four patients with NF1 (14 with a truncating mutation, 14 with a non-truncating mutation and 6 with unknown mutation) and 11 patients with LS. All patients underwent an ophthalmological examination. Infrared images were performed. Choroidal nodules were diagnosed in 65% of the NF1 group. About 71% of NF1 patients with a truncating mutation and 50% of patients with a non-truncating mutation were found to have nodules. Choroidal nodules were seen in 18% of the LS patients, never more than one nodule/eye was detected in this group. Choroidal nodules are more abundantly present in NF1 genotypes with truncating mutations. In contrast, the number of choroidal nodules in LS is comparable with their presence in healthy individuals. Especially at an early age, when the clinical picture is incomplete, the detection of choroidal nodules is of diagnostic value, and helps in an appropriate genetic counselling and follow-up. These results support the suggestion to include choroidal nodules to the diagnostic criteria for NF1.

An Update on Neurofibromatosis Type 1: Not Just Café-au-Lait Spots, Freckling, and Neurofibromas. An Update. Part I. Dermatological Clinical Criteria Diagnostic of the Disease. / Neurofibromatosis tipo 1: más que manchas café con leche, efélides y neurofibromas. Parte I. Actualización sobre los criterios dermatológicos diagnósticos de la enfermedad.

Neurofibromatosis type 1 (NF1) is the most common neurocutaneous syndrome and probably the one best known to dermatologists, who are generally the first physicians to suspect its diagnosis. Although the genetic locus of NF1 was identified on chromosome 17 in 1987, diagnosis of the disease is still mainly based on clinical observations and the diagnostic criteria of the National Institute of Health, dating from 1988. Cutaneous manifestations are particularly important because café-au-lait spots, freckling on flexural areas, and cutaneous neurofibromas comprise 3 of the 7 clinical diagnostic criteria. However, café-au-lait spots and freckling can also be present in other diseases. These manifestations are therefore not pathognomonic and are insufficient for definitive diagnosis in the early years of life. NF1 is a multisystemic disease associated with a predisposition to cancer. A multidisciplinary follow-up is necessary and dermatologists play an important role.

High Incidence of Noonan Syndrome Features Including Short Stature and Pulmonic Stenosis in Patients carrying NF1 Missense Mutations Affecting p.Arg1809: Genotype-Phenotype Correlation.

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients.

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.

Nevus anemicus in neurofibromatosis type 1: a potential new diagnostic criterion.

BACKGROUND: Children with multiple café-au-lait macules (CALMs) may be followed for years before a second National Institutes of Health clinical criterion of neurofibromatosis type 1 (NF1) develops to confirm the diagnosis. OBJECTIVE: We sought to assess the prevalence of nevus anemicus (NA) in NF1 and its association with neuro-ophthalmologic complications. METHODS: This was a prospective multicenter case-control study of 210 consecutive patients with multiple CALMs. Patients with NF1 were matched for age, sex, and center with control subjects. We documented the number, location, and morphologic appearance of NA; dermatologic features of NF1; magnetic resonance imaging results; and family history. RESULTS: In all, 77 (51%) patients with NF1 had NA compared with 6 (2%) control subjects. NA was not detected in 26 patients with other genodermatoses associated with CALMs. Patients with NF1 and NA were younger than those without NA (median age: 17 years) (P = .002). NA was mostly localized to the upper anterior aspect of the chest. NA was not significantly linked with other clinical manifestations of NF1, including optic glioma and unidentified bright objects. LIMITATIONS: A potential referral bias associated with tertiary care centers is a limitation. CONCLUSIONS: NA appears to have a high prevalence and specificity in NF1 and might serve as a marker for NF1 in children with multiple CALMs.

Legius Syndrome and Inflammatory Bowel Disease: A Pediatric Case Report.

Legius syndrome (LS) is a rare and underrecognized disorder that is often misdiagnosed as neurofibromatosis type 1 (NF1). It is characterized by café-au-lait macules without the tumoral manifestations of NF1. We report the case of an 11-year-old patient with multiple café-au-lait macules and intertriginous freckling who was admitted for bloody stools, joint pain, and weight loss. His clinical and endoscopic findings were consistent with inflammatory bowel disease (IBD). He also met the clinical diagnostic criteria for NF1 but not for LS. Genetic testing played a pivotal role in the differential diagnosis and revealed a loss-of-function mutation in the SPRED1 gene, confirming the diagnosis of LS. This is the first reported case of a patient with IBD and LS. The subtle manifestations of LS make it an underdiagnosed disease, which reduces the likelihood of it being diagnosed in association with other diseases, such as IBD. There are, however, 10 published case reports linking IBD and NF1, and some pathophysiological mechanisms have been proposed. Continued reporting will help clarify the relationship between IBD and RASopathies such as NF1 and LS.

How common are ear, nose and throat disorders in children with Noonan syndrome and other RASopathies?

INTRODUCTION: Noonan syndrome and related conditions (RASopathies) are known to be associated with abnormalities in many organ systems. It is our impression that few otolaryngologists are familiar with the manifestations of these syndromes and we therefore reviewed our hospital's patient cohort to identify the prevalence of ear, nose and throat disorders in these children. METHODS: We cross-referenced various hospital department databases (otolaryngology, audiology, cardiology, haematology and genetics) to try to identify as many children with Noonan and other RASopathies as possible. We then performed a retrospective review of electronic patient records. RESULTS: We identified 67 children with Noonan, Costello, LEOPARD and other RASopathy syndromes. Around half have been seen in otolaryngology and audiology clinics. Otitis media with effusion requiring ventilation tubes occurred in 4% of children. 10% have suffered recurrent acute otitis media. 9% have a sensorineural hearing loss. 7% have undergone adenotonsillectomy for obstructive sleep apnoea. Airway anomalies and head and neck malformations occur but are rare. DISCUSSION: Children with Noonan and other RASopathies present commonly to otolaryngology and audiology clinics. The prevalence of sensorineural hearing loss is high and audiological screening is likely to be worthwhile. Surgeons should be aware that complications of surgery are common and can be very severe, especially in those with cardiac anomalies.

The RASopathies: from pathogenetics to therapeutics.

The RASopathies are a group of disorders caused by a germline mutation in one of the genes encoding a component of the RAS/MAPK pathway. These disorders, including neurofibromatosis type 1, Noonan syndrome, cardiofaciocutaneous syndrome, Costello syndrome and Legius syndrome, among others, have overlapping clinical features due to RAS/MAPK dysfunction. Although several of the RASopathies are very rare, collectively, these disorders are relatively common. In this Review, we discuss the pathogenesis of the RASopathy-associated genetic variants and the knowledge gained about RAS/MAPK signaling that resulted from studying RASopathies. We also describe the cell and animal models of the RASopathies and explore emerging RASopathy genes. Preclinical and clinical experiences with targeted agents as therapeutics for RASopathies are also discussed. Finally, we review how the recently developed drugs targeting RAS/MAPK-driven malignancies, such as inhibitors of RAS activation, direct RAS inhibitors and RAS/MAPK pathway inhibitors, might be leveraged for patients with RASopathies.

RASopathies: Dermatologists' viewpoints.

Ras/mitogen-activated protein kinase pathway dysregulation results in a group of disorders, collectively termed as RASopathies. Neurofibromatosis type 1, Noonan syndrome, Noonan syndrome with multiple lentigines, Noonan syndrome/loose anagen hair, Legius syndrome, Costello syndrome, cardio-facio-cutaneous syndrome and capillary malformation-arteriovenous malformation are the well-recognized RASopathies. These are characterized by multi-organ tumours and hamartomas. Some other features in common are facial dysmorphism, skeletal abnormalities, congenital heart disease, neurocognitive abnormalities and risk of various solid-organ and haematological malignancies. Some of the RASopathies are heterogeneous, caused by several gene mutations resulting in variations in phenotypes and severity ranging from mild to fatal. Significant phenotypic overlaps among different disorders, often makes it difficult to pinpoint a clinical diagnosis. Specific cutaneous manifestations are present in some of the RASopathies and are often the earliest clinical signs/symptoms. Hence, dermatologists contribute significantly as primary care physicians by identifying disorder-specific cutaneous lesions. However, diagnostic work-up and management of these disorders are often multidisciplinary. Confirmation of diagnosis is possible only by genetic mapping in each case. Genetic counseling of the patients and the affected families is an important component of the management. The aim of this review is description of cutaneous manifestations of RASopathies in the background of multi-system involvement to enable dermatologists a comprehensive and logical approach to work up and diagnose such patients in the absence of facility for specific molecular testing.

Central precocious puberty in a girl with LEGIUS syndrome: an accidental association?

BACKGROUND: Central precocious puberty is a condition characterized by precocious activation of the hypothalamic-pituitary-gonadal axis. It may be idiopathic or secondary to organic causes, including syndromes such as Neurofibromatosis type 1 (NF1). CASE PRESENTATION: We presented a girl of 6 years and 10 months with almost 11 café-au-lait skin macules, without other clinical or radiological signs typical of NF1, and with a central precocious puberty. Genetic analysis evidenced the new variant NM-152594.2:c.304delAp. (Thr102Argfs*19) in SPRED1 gene, which allowed to diagnose Legius syndrome. CONCLUSIONS: We report for the first time a case of central precocious puberty in a girl with Legius syndrome. The presence of central precocious puberty in a child with characteristic café-au-lait macules should suggest pediatricians to perform genetic analysis in order to reach a definitive diagnosis. Further studies on timing of puberty in patients with RASopathies are needed to better elucidate if this clinical association is casual or secondary to their clinical condition.

RASopathies: The musculoskeletal consequences and their etiology and pathogenesis.

The RASopathies comprise an ever-growing number of clinical syndromes resulting from germline mutations in components of the RAS/MAPK signaling pathway. While multiple organs and tissues may be affected by these mutations, this review will focus on how these mutations specifically impact the musculoskeletal system. Herein, we review the genetics and musculoskeletal phenotypes of these syndromes in humans. We discuss how mutations in the RASopathy syndromes have been studied in translational mouse models. Finally, we discuss how signaling molecules within the RAS/MAPK pathway are involved in normal and abnormal bone biology in the context of osteoblasts, osteoclasts and chondrocytes.

Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR.

Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.