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.

IFN-γ enhances the therapeutic efficacy of MSCs-derived exosome via miR-126-3p in diabetic wound healing by targeting SPRED1.

BACKGROUND AND AIMS: The traditional treatment of diabetic wounds is unsatisfactory. Exosomes isolated from bone marrow mesenchymal stem cells (BMSCs) promote the healing of diabetic wounds. However, whether the exosomes secreted by interferon (IFN)-γ-pretreated BMSCs have an enhanced therapeutic effect on diabetic wound healing and the relevant mechanisms remain unclear. METHODS: In this study, we isolated exosomes from the corresponding supernatants of BMSCs with (IExos) or without IFN-γ treatment (NExos). Human umbilical vein endothelial cells (HUVECs) were used to investigate the proliferation, migration, and tube formation under different treatments in vitro. Diabetic mice were induced by intraperitoneal administration of streptozotocin, and a circular full-thickness dermal defect was then made on the back of each mouse, followed by a multisite subcutaneous injection of phosphate buffered saline or exosomes. Hematoxylin-eosin (H&E) staining, Masson's trichrome staining, and histological analysis were performed to assess the speed and quality of wound healing. RESULTS: NExos treatment accelerated the healing of diabetic wounds by promoting angiogenesis in vivo and in vitro, and IExos exhibited superior therapeutic efficiency. MicroRNA (miR)-126-3p was significantly increased in IExos, and exosomal miR-126-3p promoted angiogenesis and diabetic wound healing via its transfer to HUVECs. miR-126-3p regulates SPRED1 by directly targeting the 3'-UTR. Mechanistically, IFN-γ-pretreated BMSCs secreted miR-126-3p-enriched exosomes, which enhanced the function of HUVECs and promoted angiogenesis via the SPRED1/Ras/Erk pathway. CONCLUSION: Exosomal miR-126-3p secreted from IFN-γ-pretreated BMSCs exhibited higher therapeutic efficacy than NExos in diabetic wound healing by promoting angiogenesis via the SPRED1/Ras/Erk axis.

Long noncoding RNA LOC646029 functions as a ceRNA to suppress ovarian cancer progression through the miR-627-3p/SPRED1 axis.

Long noncoding RNAs (lncRNAs) play a crucial regulatory role in the development and progression of multiple cancers. However, the potential mechanism by which lncRNAs affect the recurrence and metastasis of ovarian cancer remains unclear. In the current study, the lncRNA LOC646029 was markedly downregulated in metastatic ovarian tumors compared with primary tumors. Gain- and loss-of-function assays demonstrated that LOC646029 inhibits the proliferation, invasiveness, and metastasis of ovarian cancer cells in vivo and in vitro. Moreover, the downregulation of LOC646029 in metastatic ovarian tumors was strongly correlated with poor prognosis. Mechanistically, LOC646029 served as a miR-627-3p sponge to promote the expression of Sprouty-related EVH1 domain-containing protein 1, which is necessary for suppressing tumor metastasis and inhibiting KRAS signaling. Collectively, our results demonstrated that LOC646029 is involved in the progression and metastasis of ovarian cancer, which may be a potential prognostic biomarker.

Cardiac angiogenesis enhances by activating Mir-126 and related target proteins in type 2 diabetic rats: Rescue combination effect of Sodium butyrate and voluntary exercise therapy.

Objective: type 2 diabetes, metabolic disorder, is one of the main risk factors for cardiovascular disease, leading to angiogenesis injury. The present study wanted to discover the effect of sodium butyrate (NaB) and voluntary exercise, alone or together, on miR-126 and related proteins in rats with type 2 diabetes. Methods: thirty-five male Wistar rats (200-250 g) were randomly divided into five groups: control, diabetes, diabetes-NaB, diabetes-exercise, and diabetes-NaB-exercise. Type 2 diabetes was induced by intraperitoneal injection of streptozotocin (35 mg/kg) and high-fat diet. The rats were then administrated NaB (200 mg/kg. ip) or were subjected to voluntary exercise, or combined NaB and voluntary exercise for 8 weeks. MiR-126 expression in the cardiac tissue was determined by real-time PCR, and the SPRED-1 and RAF proteins expression levels were measured by western blot. Results: NaB and voluntary exercise up-regulated cardiac miR-126 and RAF expression levels and down-regulated SPRED-1 in cardiac tissue of type 2 diabetic rats. Moreover, the combination of NaB and voluntary exercise amplified their effects on those parameters. Both NaB and voluntary exercise or together markedly modulated serum glucose and HbA1c. Conclusion: The present findings demonstrated that NaB combined with exercise could improve cardiac angiogenesis by increasing miR-126 and affecting related proteins. Thus, NaB together with voluntary exercise might be a promising intervention for the treatment and prevention of type 2 diabetes.

KIT Gene Mutation Causing Piebaldism Associated with Multiple Café Au-Lait Like Macules and Freckling: Delineating a Cause of this Coexistence.

Piebaldism is a rare genetic disorder of congenital leukoderma caused by mutation in KIT proto-oncogene receptor tyrosine kinase. We present a 10-year-old boy with congenital depigmented macules suggestive of piebaldism associated with café au lait macules and skin fold freckling complicating the diagnosis. A diagnosis of piebaldism was made via exome sequencing that showed a pathogenic variant of KIT gene with no pathogenic variants of NF1 or SPRED1 gene. Our current understanding of the KIT tyrosine kinase function may provide a better explanation into this phenotypic coexistence and does not necessarily represent an overlap with Neurofibromatosis type 1.

Circ_TEX2 Functions as a Tumor Suppressor in Hepatoma via miR-96-5p/SPRED1 Axis.

Circular RNAs (circRNAs) have been shown to have a vital effect on hepatoma progression. The purpose of this study was to explore the function and mechanism of circRNA testis expressed 2 (circ_TEX2, circ_0004913) in hepatoma pathogenesis. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect circ_TEX2, miR-96-5p, and sprouty-related EVH1 domain containing 1 (SPRED1) expression. Western blot analyzed the proliferating cell nuclear antigen (PCNA), SPRED1, and the apoptosis-related protein levels. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), and colony formation assays were used to test cell proliferation. Cell migration and invasion were analyzed by transwell assay, and cell apoptosis was detected by flow cytometry. Dual-luciferase reporter assay was done to analyze the target relationship between miR-96-5p and circ_TEX2 or SPRED1. The effects of circ_TEX2 on tumor growth in vivo were verified by xenograft model experiment and immunohistochemistry assay. The levels of circ_TEX2 and SPRED1 were down-regulated in hepatoma tissues and cells, and miR-96-5p expression was up-regulated. Overexpression of circ_TEX2 could inhibit the proliferation, migration, and invasion and boost cell apoptosis of hepatoma cells. Circ_TEX2 affected SPRED1 expression by sponging miR-96-5p. The overexpression of miR-96-5p could overturn the influence of circ_TEX2 up-regulation on malignant behaviors of hepatoma cells, and reduced SPRED1 expression could reverse the function of miR-96-5p knockdown on hepatoma cell malignant behaviors. Circ_TEX2 could suppress the growth of xenograft tumors in vivo. Our study demonstrates the tumor-suppressive role of circ_TEX2 in hepatoma through miR-96-5p/SPRED1 axis, suggesting that strategies directed toward restoring the production of circ_TEX2 might have a therapeutic value for hepatoma treatment.

SPOCK2 and SPRED1 function downstream of EZH2 to impede the malignant progression of lung adenocarcinoma in vitro and in vivo.

Enhancer of zeste homolog 2 (EZH2) is an important epigenetic regulator, and is associated with the malignant progression of lung cancer. However, the mechanisms of EZH2 on lung adenocarcinoma (LUAD) remain unclear. The relationship between EZH2 and SPOCK2 or SPRED1 was confirmed by dual-luciferase reporter assay. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were analyzed to examine the expression of SPOCK2 and SPRED1 and their prognostic values of LUAD. The effects of SPOCK2 and SPRED1 on the biological characters of LUAD cells were identified on functional assays in vitro and in vivo. Our results showed that EZH2 suppressed the expression and transcriptional activity of SPOCK2 and SPRED1, and these effects were reversed by the EZH2 inhibitor, Tazemetostat. SPOCK2 and SPRED1 were expressed at low levels in LUAD patients, and a high expression level of SPOCK2 or SPRED1 predicted better survival. Moreover, overexpression of SPOCK2 or SPRED1 could inhibit tumoral proliferation, migration ratio, and invasion activity in vitro as well as retard tumor growth in vivo. However, EZH2 elevation could rescue these impacts and accelerate LUAD progression. Our findings reveal that SPOCK2 and SPRED1 are epigenetically suppressed by EZH2 and may act as novel regulators to inhibit the proliferation, migration, and invasion of LUAD cells.

Legius syndrome combined with central precocious puberty: a case report and literature review / 中华神经科杂志

Objective:To investigate the clinical phenotype and genotypic characteristics of Legius syndrome.Methods:The clinical data of a child with precocious puberty and scattered café-au-lait macules admitted to Department of Neurology of the Children′s Hospital Affiliated to Zhengzhou University in July 2021 were retrospectively analyzed. Trio-whole exome sequencing (trio-WES) was used for genetic analysis to confirm the molecular diagnosis of the family. The relevant literature was reviewed to summarize the clinical characteristics of the disease.Results:The proband was a 10-year and 9-month-old girl, presenting with more than 5 café-au-lait macules with diameter>5 mm on the face and trunk, freckles in the axillary, without Lisch tubercles of iris and tumor signs of neurofibromatosis type 1, diagnosed as central precocious puberty at the age of 8. trio-WES results of the family revealed a spontaneous heterozygous nonsense mutation c.751(exon7) C>T in SPRED1 gene, causing a nonsense mutation in the amino acid sequence p.Arg251Ter (p. Ter251 *). Literature review showed a total of 88 pathogenic mutations were reported in SPRED1 gene, including frameshift mutations (41/88), nonsense mutations (31/88), splice mutations (7/88), missense mutations (6/88), and others (3/88), and no mutational hotspots were found. Clinical phenotype was as follows:>5 café-au-lait macules accounted for 92.8% (168/181), armpit and inguinal freckles 43.5% (73/168), macrocephaly 21.4% (31/145), learning disability 18.0% (30/166), psychomotor retardation 13.8% (22/159), lipoma (adult) 13.7% (21/153), Noonan facial sign 12.1% (21/173), and tumor phenotype of neurofibromatosis type 1 was not reported. Conclusions:The central precocious puberty phenotype of Legius syndrome was not reported in China. The clinical phenotype of Legius syndrome was mild, with a large variation, but without neurofibromatosis type 1 tumor phenotype. Genetic testing can be beneficial for early diagnosis of Legius syndrome.

Long noncoding RNA LOC646029 functions as a ceRNA to suppress ovarian cancer progression through the miR-627-3p/SPRED1 axis / 医学前沿

Long noncoding RNAs (lncRNAs) play a crucial regulatory role in the development and progression of multiple cancers. However, the potential mechanism by which lncRNAs affect the recurrence and metastasis of ovarian cancer remains unclear. In the current study, the lncRNA LOC646029 was markedly downregulated in metastatic ovarian tumors compared with primary tumors. Gain- and loss-of-function assays demonstrated that LOC646029 inhibits the proliferation, invasiveness, and metastasis of ovarian cancer cells in vivo and in vitro. Moreover, the downregulation of LOC646029 in metastatic ovarian tumors was strongly correlated with poor prognosis. Mechanistically, LOC646029 served as a miR-627-3p sponge to promote the expression of Sprouty-related EVH1 domain-containing protein 1, which is necessary for suppressing tumor metastasis and inhibiting KRAS signaling. Collectively, our results demonstrated that LOC646029 is involved in the progression and metastasis of ovarian cancer, which may be a potential prognostic biomarker.

Mechanistic Insights into the Long-range Allosteric Regulation of KRAS Via Neurofibromatosis Type 1 (NF1) Scaffold Upon SPRED1 Loading.

Allosteric regulation is the most direct and efficient way of regulating protein function, wherein proteins transmit the perturbations at one site to another distinct functional site. Deciphering the mechanism of allosteric regulation is of vital importance for the comprehension of both physiological and pathological events in vivo as well as the rational allosteric drug design. However, it remains challenging to elucidate dominant allosteric signal transduction pathways, especially for large and multi-component protein machineries where long-range allosteric regulation exits. One of the quintessential examples having long-range allosteric regulation is the ternary complex, SPRED1-RAS-neurofibromin type 1 (NF1, a RAS GTPase-activating protein), in which SPRED1 facilitates RAS-GTP hydrolysis by interacting with NF1 at a distal, allosteric site from the RAS binding site. To address the underlying mechanism, we performed extensive Gaussian accelerated molecular dynamics simulations and Markov state model analysis of KRAS-NF1 complex in the presence and absence of SPRED1. Our findings suggested that SPRED1 loading allosterically enhanced KRAS-NF1 binding, but hindered conformational transformation of the NF1 catalytic center for RAS hydrolysis. Moreover, we unveiled the possible allosteric pathways upon SPRED1 binding through difference contact network analysis. This study not only provided an in-depth mechanistic insight into the allosteric regulation of KRAS by SPRED1, but also shed light on the investigation of long-range allosteric regulation among complex macromolecular systems.

Methylation of SPRED1: A New Target in Acute Myeloid Leukemia.

Sprouty-related, EVH1 domain-containing protein 1 (SPRED1) has been identified as a novel tumor suppressor gene in acute myeloid leukemia (AML). Previous studies showed that SPRED1 methylation levels were significantly increased in AML patients, making it an interesting candidate for further investigations. To confirm the association of SPRED1 methylation, clinical parameters, and known molecular prognosticators and to identify the impact of methylation level on treatment outcome, we conducted this study in a larger cohort of 75 AML patients. Significantly increased methylation levels of SPRED1 were detected at four of ten CpG units by quantitative high-resolution mass spectrometry-based approach (MassARRAY) in AML patients. Whereas overall survival (OS) and relapse-free survival (RFS) showed no statistical difference between hypermethylation and hypomethylation subgroups, the relationship between methylation level and treatment response was indicated in paired samples from pre- and post-induction. To determine the possible mechanism of SPRED1 methylation in AML, we performed in vitro experiments using THP-1 cells, as the latter showed the highest methylation level (determined by utilizing bisulfite modification) among the three AML cell lines we tested. When treated with 5-AZA and lentivirus transfection, upregulated SPRED1 expression, decreased cell proliferation, increased cell differentiation and apoptosis, and inactivated phosphorylated extracellular signal-regulated kinase (p-ERK) were detected in THP-1 cells. These results show that demethylation of SPRED1 can inhibit the proliferation of AML cells and promote their differentiation and apoptosis, possibly by the ERK pathway. The hypermethylation of SPRED1 is a potential therapeutic target for AML.

Up-regulated miR-204-5p promoted the migration, invasion, and angiogenesis of endothelial progenitor cells to enhance the thrombolysis of rats with deep venous thrombosis by targeting SPRED1.

Deep venous thrombosis (DVT) endangers human health. Endothelial progenitor cells (EPCs) were proven to promote thrombolysis and miR-204-5p was discovered to be low-expressed in DVT patients. This study concentrated on exploring whether miR-204-5p had a regulatory effect on EPCs and DVT. Concretely, the expression of miR-204-5p in DVT patients' blood was detected by qRT-PCR. The target of miR-204-5p was predicted by bioinformatics and verified by dual-luciferase reporter assay. After rat EPCs were isolated, identified, and transfected with miR-204-5p agomiR, antagomiR, or SPRED1 plasmids, the viability, migration, invasion, and tube formation of EPCs were detected by MTT, wound healing, Transwell, and tube formation assays, respectively. MiR-204-5p, SPRED1, p-PI3K, PI3K, p-AKT, AKT, VEGFA, and Ang1 expressions in EPCs were measured by qRT-PCR or Western blot. EPCs transfected with miR-204-5p overexpression lentivirus plasmid were injected into the DVT rat model. The histopathology of the thrombus and the homing of EPCs to thrombus in the DVT rats were observed by hematoxylin-eosin staining and confocal microscopy, respectively. We found that miR-204-5p was low-expressed in DVT patients and SPRED1 was a target gene of miR-204-5p. MiR-204-5p agomiR promoted the viability, migration, invasion, and tube formation of EPCs, the levels of VEGFA and Ang1 and the activation of PI3K/AKT pathway in EPCs, while miR-204-5p antagomiR and SPRED1 worked oppositely. SPRED1 reversed the effect of miR-204-5p agomiR on EPCs. Up-regulated miR-204-5p inhibited thrombosis and promoted EPCs homing to thrombus in DVT rats. Collectively, up-regulated miR-204-5p enhanced the angiogenesis of EPCs and thrombolysis in DVT rats by targeting SPRED1.

Updated Approach to Patients with Multiple Café au Lait Macules.

Café au lait macules (CALMs) are a normal and frequent finding in the general population, but multiple CALMs raise the possibility of an underlying neurocutaneous disease like neurofibromatosis type I. Certain features of CALMs like number, size, shape, and distribution are important in identifying children at higher risk of having a neurocutaneous disorder or another genetic disorder. Genetic testing can be especially helpful in establishing a diagnosis in atypical presentations, or when the child is young and other features of the disease aside from CALMs have not manifested.

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.

MEK inhibition ameliorates social behavior phenotypes in a Spred1 knockout mouse model for RASopathy disorders.

BACKGROUND: RASopathies are a group of disorders that result from mutations in genes coding for proteins involved in regulating the Ras-MAPK signaling pathway, and have an increased incidence of autism spectrum disorder (ASD). Legius syndrome is a rare RASopathy caused by loss-of-function mutations in the SPRED1 gene. The patient phenotype is similar to, but milder than, Neurofibromatosis type 1-another RASopathy caused by loss-of-function mutations in the NF1 gene. RASopathies exhibit increased activation of Ras-MAPK signaling and commonly manifest with cognitive impairments and ASD. Here, we investigated if a Spred1-/- mouse model for Legius syndrome recapitulates ASD-like symptoms, and whether targeting the Ras-MAPK pathway has therapeutic potential in this RASopathy mouse model. METHODS: We investigated social and communicative behaviors in Spred1-/- mice and probed therapeutic mechanisms underlying the observed behavioral phenotypes by pharmacological targeting of the Ras-MAPK pathway with the MEK inhibitor PD325901. RESULTS: Spred1-/- mice have robust increases in social dominance in the automated tube test and reduced adult ultrasonic vocalizations during social communication. Neonatal ultrasonic vocalization was also altered, with significant differences in spectral properties. Spred1-/- mice also exhibit impaired nesting behavior. Acute MEK inhibitor treatment in adulthood with PD325901 reversed the enhanced social dominance in Spred1-/- mice to normal levels, and improved nesting behavior in adult Spred1-/- mice. LIMITATIONS: This study used an acute treatment protocol to administer the drug. It is not known what the effects of longer-term treatment would be on behavior. Further studies titrating the lowest dose of this drug that is required to alter Spred1-/- social behavior are still required. Finally, our findings are in a homozygous mouse model, whereas patients carry heterozygous mutations. These factors should be considered before any translational conclusions are drawn. CONCLUSIONS: These results demonstrate for the first time that social behavior phenotypes in a mouse model for RASopathies (Spred1-/-) can be acutely reversed. This highlights a key role for Ras-MAPK dysregulation in mediating social behavior phenotypes in mouse models for ASD, suggesting that proper regulation of Ras-MAPK signaling is important for social behavior.

miR-126-3p contributes to sorafenib resistance in hepatocellular carcinoma via downregulating SPRED1.

BACKGROUND: Sorafenib can prolong the survival of patients with advanced hepatocellular carcinoma (HCC). However, drug resistance remains the main obstacle to improving its efficiency. This study aimed to explore the likely molecular mechanism of sorafenib resistance. METHODS: Differentially expressed microRNAs (miRNAs) related to sorafenib response were analyzed with the Limma package in R software. The expression levels of miR-126-3p and sprouty-related EVH1 domain-containing protein 1 (SPRED1) in HCC cells were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell viability and proliferation were detected with Cell Counting Kit-8 (CCK-8), EdU proliferation, and clone formation assays. Transwell assays were performed to measure cell migration and invasion. TargetScan, MicroRNA Target Prediction Database (miRDB), and StarBase v2.0 were used to predict the targets of miR-126-3p. SPRED1 was confirmed as a target gene of miR-126-3p by dual-luciferase reporter assay and Western blotting. Finally, the in vivo anti-tumor effect of LV-miR-126-3p inhibitor combined with sorafenib was evaluated via subcutaneous tumor models. RESULTS: HCC cells with high expression of miR-126-3p exhibited increased resistance to sorafenib. The results of bioinformatics analysis and the dual-luciferase reporter assay showed that miR-126-3p directly targeted SPRED1. The sensitivity of HCC cells to sorafenib was markedly enhanced by SPRED1 upregulation. Gain- and loss-of function experiments verified that miR-126-3p induced sorafenib resistance in HCC through downregulating SPRED1. Furthermore, the inhibition of miR-126-3p markedly increased the effectiveness of sorafenib against HCC in vivo. Mechanistically, our results suggested that miR-126-3p promoted sorafenib resistance via targeting SPRED1 and activating the ERK signaling pathway. CONCLUSIONS: Our study demonstrates that regulating the miR-126-3p/SPRED1 axis might be a promising strategy for enhancing the antitumor effect of sorafenib in the treatment of HCC.

Impaired instrumental learning in Spred1-/- mice, a model for a rare RASopathy.

RASopathies are neuro-cardio-facio-cutaneous disorders stemming from mutations in genes regulating the RAS-MAPK pathway. Legius syndrome is a rare RASopathy disorder caused by mutations in the SPRED1 gene. SPRED1 protein negatively regulates activation of Ras by inhibiting RAS/RAF and by its interaction with neurofibromin, a Ras GTPase-activating protein (RAS-GAP). Cognitive impairments have been reported in Legius syndrome as well as in other RASopathy disorders. Modelling these cognitive deficits in a Spred1 mouse model for Legius syndrome has demonstrated spatial learning and memory deficits, but other cognitive domains remained unexplored. Here, we attempted to utilize a cognitive touchscreen battery to investigate if Spred1-/- mice exhibit deficits in other cognitive domains. We show that Spred1-/- mice had heterogeneous performance in instrumental operant learning, with a large subgroup (n = 9/20) failing to reach the standard criterion on touchscreen operant pretraining, precluding further cognitive testing. To examine whether targeting the RAS-MAPK signalling pathway could rescue these cognitive impairments, Spred1-/- mice were acutely treated with the clinically relevant mitogen-activated protein kinase (MEK) inhibitor PD325901. However, MEK inhibition did not improve their instrumental learning. We conclude that Spred1-/- mice can model severe cognitive impairments that cannot be reversed in adulthood.

Lisch nodules and iris mammillations in two siblings with familial legius syndrome.

Legius syndrome is characterized by numerous café-au-lait macules and intertriginous freckling, but typically lacks the distinctive tumor manifestations of neurofibromatosis type 1. We report two siblings with Legius syndrome and Lisch nodules illustrating the importance of eye surveillance in these patients.

Simultaneous Detection of NF1, SPRED1, LZTR1, and NF2 Gene Mutations by Targeted NGS in an Italian Cohort of Suspected NF1 Patients.

Neurofibromatosis type 1 (NF1) displays overlapping phenotypes with other neurocutaneous diseases such as Legius Syndrome. Here, we present results obtained using a next generation sequencing (NGS) panel including NF1, NF2, SPRED1, SMARCB1, and LZTR1 genes on Ion Torrent. Together with NGS, the Multiplex Ligation-Dependent Probe Amplification Analysis (MLPA) method was performed to rule out large deletions/duplications in NF1 gene; we validated the MLPA/NGS approach using Sanger sequencing on DNA or RNA of both positive and negative samples. In our cohort, a pathogenic variant was found in 175 patients; the pathogenic variant was observed in NF1 gene in 168 cases. A SPRED1 pathogenic variant was also found in one child and in a one year old boy, both NF2 and LZTR1 pathogenic variants were observed; in addition, we identified five LZTR1 pathogenic variants in three children and two adults. Six NF1 pathogenic variants, that the NGS analysis failed to identify, were detected on RNA by Sanger. NGS allows the identification of novel mutations in five genes in the same sequencing run, permitting unambiguous recognition of disorders with overlapping phenotypes with NF1 and facilitating genetic counseling and a personalized follow-up.

Role of SPRED1 in keratinocyte proliferation in psoriasis.

Psoriasis is a recurrent inflammatory skin disease, affecting approximately 2% of the population. Previous studies have demonstrated that psoriatic dermal mesenchymal stem cells (DMSC) stimulated keratinocyte (KC) proliferation and that psoriasis exhibited missense SPRED1 mutations. To further investigate the molecular mechanism by which psoriatic DMSC stimulate KC proliferation, and the role of missense SPRED1 mutations in psoriasis, we assessed expression levels of miRNA, and both mRNA and protein of SPRED1 in normal human epidermal keratinocyte cells (NHEK) cocultured with either psoriatic or control DMSC. Expression levels of miRNA and mRNA were determined by RNA sequencing. Expression levels of spred1 protein were assessed using western blot analysis. Moreover, the variation in SPRED1 was also examined by whole-genome sequencing in 665 psoriatic patients, and verified by Sanger sequencing. Our results showed that coculture of NHEK with psoriatic DMSC induced 32 differentially expressed miRNA, in which expression levels of miR-1 increased approximately 16-fold over control DMSC-treated NHEK (P < 0.05). Likewise, expression levels of miR-21-3p increased over twofold (P < 0.05). Moreover, coculture of NHEK with psoriatic DMSC induced marked increase in expression levels of mRNA for MAPK3, CDC25B and CDC25C, while decreasing expression levels of SPRED1 mRNA and protein in comparison with control DMSC treatment (P < 0.05 for all between cocultured with control and psoriatic DMSC). Furthermore, psoriasis displayed non-synonymous mutation of SPRED1 enriched in exon 7: c.A881T:p.Y294F (chr15:38351210). These results suggest that dysregulation and mutations of SPRED1 may participate in the pathogenesis of psoriasis, including epidermal hyperproliferation.