Intellectual disability (ID) is a pathological condition characterized by limited intellectual functioning and adaptive behaviors. It affects 1–3% of the worldwide population, and no pharmacological therapies are currently available. More than 1000 genes have been found mutated in ID patients pointing out that, despite the common phenotype, the genetic bases are highly heterogeneous and apparently unrelated. Bibliomic analysis reveals that ID genes converge onto a few biological modules, including cytoskeleton dynamics, whose regulation depends on Rho GTPases transduction. Genetic variants exert their effects at different levels in a hierarchical arrangement, starting from the molecular level and moving toward higher levels of organization, i.e., cell compartment and functions, circuits, cognition, and behavior. Thus, cytoskeleton alterations that have an impact on cell processes such as neuronal migration, neuritogenesis, and synaptic plasticity rebound on the overall establishment of an effective network and consequently on the cognitive phenotype. Systems biology (SB) approaches are more focused on the overall interconnected network rather than on individual genes, thus encouraging the design of therapies that aim to correct common dysregulated biological processes. This review summarizes current knowledge about cytoskeleton control in neurons and its relevance for the ID pathogenesis, exploiting in silico modeling and translating the implications of those findings into biomedical research. ID is a heterogeneous group of neurodevelopmental disorders (NDDs), usually diagnosed before the age of 18, characterized by significant limitations in both intellectual functioning (IQ < 70) and adaptive behavior as expressed in conceptual, social, and practical adaptive skills ,3,4,5. BID is manifested as both syndromic and non-syndromic forms, depending on whether the disability is associated with other symptoms, and it is clinically classified referring to severity  and penetrance  . A phenoID mutations can be autosomal–recessive, autosomal–dominant (mostly de novo), or X-linked. The latter two are responsible for the higher prevalence of ID in males versus females.OPHN1 (oligophrenin 1), ARHGEF9 (Cdc42 guanine nucleotide exchange factor 9), FGD1 , RAC1 , and PAK3 (P21-activated kinase 3).The causes of ID are heterogeneous and still to be completely defined: it has been estimated that half of all cases are due to environmental factors such as intrauterine/neonatal insults  and postnatal risk factors such as severe malnutrition during infancy. The other half of the cases are associated with genetic variants, highly heterogeneous, and only partially identified. According to the SysID database , 1454 IDBased on a wealth of experimental data from animal models and cultured neurons, it is widely accepted that cognitive deficits in ID patients are linked to altered neuronal networking, impaired synaptic plasticity, and excitation/inhibition unbalance in the cerebral cortex and hippocampus, resulting in abnormal information processing ,9,10,11.As genome-sequencing technologies improve and become accessible, more ID-causing mutations will surely be identified in patients. However, our mechanistic understanding of ID pathophysiology continues to lag behind the pace of gene discovery.Considering the elevated number of risk genes and their heterogeneity, it is unlikely that each identified mutation represents an independent pathway that, when misregulated, causes a similar cognitive phenotype. On the contrary, it can be assumed that the identified mutations may converge to, or participate in, a limited number of core regulatory intracellular modules that are beginning to be identified, although they are not yet fully characterized. The dysfunction of different genes impacting the same process will result in analogous dysfunctions of the process itself. Thus, multiple genetic causes converge on a few common cellular outcomes and result in one overall phenotype. For this reason, an integrated approach that collects a large set of data but focuses on single biological processes is more suitable for furthering genetic diagnostics and developing treatment strategies to target shared pathways rather than single genes.Three key questions arise: (i) What are the common core regulatory mechanisms dysregulated in ID? (ii) What are the key proteins  and/or posttranslational modifications at the basis of the cell endophenotype resulting in ID? (iii) Do we have adequate tools to identify and study such hubs and biological processes?Integrative methods and data meta-analyses, protein::protein interaction (PPI) networks, and transcriptomics analysis coupled with gene ontology (GO) ,13 have CCNT2 (cyclin T2), CDK9 (cyclin-dependent kinase 9), and TAF2 (TATA-box binding protein associated factor 2), all encoding for transcription factors [A second approach used RNA-seq data derived from the blood of patients harboring mutations in the ID genes  factors . DiffereCDC42 (cell division cycle 42) and RAC1, two known cytoskeleton regulators, APP (amyloid β precursor protein), involved in proliferation, cell–cell adhesion, migration, and synaptogenesis, EP300 (E1A binding protein p300), important for genomic stability through chromatin regulation, TP53 (tumor protein p53), and GNB1 (G protein subunit β1).A third approach adopted a systematic analysis by building a highly stringent PPI network from genes previously related to ID and global developmental delay (GDD) in the Human Phenotype Ontology database . This anOverall, the SB approaches reported above identified the following wide biological processes on which ID genes converge A.EHMT1 (euchromatic histone lysine methyltransferase 1) and SWI/SNF chromatin remodeling complex were shown to have a role in Kleefstra syndrome and Coffin–Siris syndrome [KDM5C (lysine demethylase 5C), encoding an eraser enzyme for di-methylated and tri-methylated histone H3 lysine 4, account for 2% of X-linked ID (XLID) [DDX3X (DEAD-box helicase 3 X-linked), coding for an RNA helicase involved in post-transcriptional modifications, were found to be relevant in ID because of its fundamental role in neurite outgrowth and dendritic spine formation via modulation of RAC1 transcription [Many chromatin-modifying enzymes and other epigenetic regulators have been genetically associated with ID and other syndromes in which ID is one of the major clinical outcomes ,18. A casyndrome ,20, respD (XLID) ; mutatiocription . Interescription .EIF4E (eukaryotic translation initiation factor 4E), a regulator of MTOR (mechanistic target of rapamycin kinase) translation [PTEN (phosphatase and tensin homolog) and TSC1/2  results in overactive MTOR signaling, causing dendritic and axonal overgrowth, neuronal hypertrophy, and ASD-like behavioral patterns [CTNNB1 (catenin β1) and CHD8 (chromodomain helicase DNA binding protein 8), are associated with ID, as CTNNB1 is involved in synaptic function and its mutations are associated with deficits in intra-hemispheric connections, dendritic branching, long-term potentiation (LTP), and cognitive functions [BRAF , MAP2K1 , MAP2K2 , and KRAS  are associated with cardio-facio-cutaneous syndrome, in which ID is present in the majority of patients [12V expressed in both forebrain excitatory and inhibitory neurons cause decreased excitatory transmission, accompanied by reduced hippocampal LTP [WNT, MTOR, and MAPK signaling pathways have been shown to play a central role in brain development. Perturbations of these pathways have been implicated in multiple neuropsychiatric disorders, including autism spectrum disorder (ASD) and ID ,24,25. Mnslation , while lpatterns ,28. Simiunctions . MAPK papatients . The conmpal LTP .UBE3A (ubiquitin protein ligase E3A) have been associated with increased risk of ASD and ID through dysregulation of WNT and MTOR pathways [HUWE1 , which regulates both CTNNB1 and EIF4E, was associated to ID and ASD [CUL4B (cullin 4B) LOF mutations [Mutations in several ubiquitination system genes have been linked to ID. Gain and LOF mutations in pathways ,33; the  and ASD ,35; CUL4utations  cause deutations .SLC6A8  mutations, cause a mild to moderate ID phenotype [SLC6A8 pathogenic mutation [The contribution of metabolic dysregulations to ID is highly dependent on the period in which the defect becomes relevant . For example, alterations in oxidative phosphorylation during the prenatal period lead to abnormalities in brain formation . Creatinhenotype . A mutatmutation . Disordemutation , as seenmutation . Finallymutation .CACNA1A , coding the α-1A subunit of the P/Q-type voltage-dependent calcium channel, cause congenital ataxia and ID [Synaptic signaling pathways, such as the Rab and Arf pathways, are commonly altered in ID. Genome-wide weighted co-expression network analysis showed specific enrichment for synaptic functioning . Mutatioa and ID ,46. De na and ID . In 2019a and ID . Notablya and ID ,50.CDC42 and WASP, known regulators of actin filament polymerization and branching, have been identified as hubs in PPI networks based on differentially expressed genes in DS [The altered control of cytoskeleton dynamics within developing neurons is a core dysfunction in ID as well as in Down syndrome (DS), Rett syndrome, Fragile X syndrome (FXS), and ASD. es in DS . Schizopes in DS .Imbalance of actin cytoskeleton regulation has also been reported in FXS, a condition caused by an LOF mutation in FMR1  . TranscrOverall, strong evidence indicates that cytoskeleton dynamics isaffected by mutations detected in ID B and othDuring development, neurons migrate to find synaptic partners and establish the complexity of the neuronal wiring. Neurite extension and navigation are possible thanks to the formation of the growth cone, a sensory-motile structure at the tip of the growing axon directed by chemotaxis . The strMicrotubules’ and actin filaments’ polymerization result from the addition of α/β-tubulin and glomerular actin (G-actin), respectively ,66. The It is assumed that growth cones are already provided with all the proteins necessary for synaptogenesis during their searching for contacts . The proSynapses are not static formations. They undergo changes in postnatal life, while carrying out specific activities, e.g., learning, and in specific periods, e.g., synaptic pruning during adolescence. In particular, synaptic plasticity takes place in an activity-dependent manner: LTP is the result of strengthened synapses after high-frequency stimulation from the presynaptic terminal, while long-term depression (LTD) is a decrease in synaptic activity after low-frequency signals . For LTPAlterations in neurites and spine morphology, as well as in neuronal migration properties, have been consistently associated with ID and other NDDs that include ID as a main and recurrent phenotype . These dThis section illustrates in detail the key components of the signaling pathway responsible for the control of the dynamics of the actin cytoskeleton, focusing on the biochemical and cellular role of each protein and its links with neurological and cognitive deficits in human and animal models.RAC1: RAC1 is a key regulator of neurite elongation, axon migration, synaptic function, and synaptic plasticity, as it promotes neurite outgrowth [utgrowth , spine futgrowth , and cluutgrowth ,88, and utgrowth . Formatiutgrowth . Moreoveutgrowth , indicatRAC1 variants have been reported in patients with mild to severe ID [Seven de novo missense evere ID ,93. Amonevere ID .Rac1 in the ventricular zone of the telencephalon resulted in ventricles enlargement, impaired migration of median ganglionic eminence-derived interneurons, and impaired projection of commissural and corticothalamic axons. Interestingly, primary Rac1-deficient neurons had increased neurites formation and branching, indicating that RAC1 may be dispensable for neuritogenesis per se [In mice, the deletion of s per se .CDC42: CDC42 plays a critical role in neurite outgrowth [utgrowth , neuronautgrowth . It is autgrowth ,98.CDC42 de novo missense mutations have been reported in 13 unrelated patients showing several developmental abnormalities, including ID and dysmorphic facial features [C. elegans showed that these mutations result in proteins with altered activity and/or impaired target interactions, with some mutations acting as a gain of function and others acting as hypomorphs [CDC42, which has been described as a de novo mutation in one individual and inherited mutation in three related individuals, resulted in a hypomorphic allele associated with several developmental phenotypes, but not with ID [CDC42 mutations may reflect different functional consequences of the single mutations.Eight features ,101,102.Cdc42-KO mice die soon after birth and show a reduced cortical mass and a widespread loss of axonal tracts [CDC42 depletion in the postnatal brain have been assessed using flox/flox, Camk2a-CRECdc42 mice, in which CRE recombinase is expressed in cortical pyramidal neurons and hippocampus starting from P16-P19. These mice showed reduced spine density and LTP in the hippocampus, together with memory deficits [Brain-specific l tracts . The effdeficits .RHOA: Broadly speaking, the effects of RAC1 and CDC42 signaling on neurite outgrowth and dendritic spine formation are antagonized by RHOA signaling [ignaling ,90,104. ignaling , promoteignaling ,106,107,ignaling .KCTD13 (potassium channel tetramerization domain containing 13) and CUL3 (cullin 3), two genes linked to NDDs, are involved in RHOA ubiquitination [Kctd13-KO mice [Interestingly, tination ,110,111,-KO mice ,113. ThePAK1: Six PAK proteins have been identified in mammals. Based on their sequence homology, PAKs are classified into two groups, the first including PAK1, PAK2, and PAK3 and the second including PAK4, PAK5, and PAK6. All six PAKs are expressed in the nervous system with a different spatio-temporal pattern, with PAK1 and 3 being the most studied in the context of neuronal function [function . Active function ,116,117.Pak1-KO mice show impaired LTP at hippocampal CA1 synapses, reduced enrichment of F-actin at dendritic spines, and impaired NMDA-dependent ADF/cofilin phosphorylation [PAK1 plays a critical role in both synaptic function and axon migration. rylation . Both ovrylation .PAK1 have been associated with ID, macrocephaly, and seizures [In humans, gain of function missense variants in seizures ,121. Intseizures .PAK3: Differently from PAK1, which is activated by both RAC1 and CDC42, PAK3 is mainly activated by CDC42 [by CDC42 .PAK3 are associated with XLID [PAK3 variants responsible for severe ID and corpus callosum agenesis (G424R and K389N) were shown to suppress kinase activity, increase the interaction between PAK3 and the guanine exchange factor ARHGEF6 , and inhibit cell migration [Mutations of ith XLID ,125. Twoigration . Anotherigration .Pak3-KO mice have no apparent defects in the actin cytoskeleton, but showed impaired hippocampal LTP, together with learning and memory deficits [In mice, the latter variant impacts cognitive functions and adult hippocampal neurogenesis . Likewisdeficits .LIMK1: LIMK1 (LIM domain kinase 1) is a serine–threonine kinase that possesses two LIM domains, a PDZ domain, and a C-terminal kinase catalytic domain [c domain . LIMK1 ic domain . Dominanc domain , CDC42-ic domain .Limk1-KO mice showed reduced growth cone size and altered dendritic spine morphology [Neurons of rphology .LIMK1, results in Williams syndrome, a complex developmental disorder characterized by ID and impaired long-term memory [Limk1+/− mice also showed impaired long-term memory, together with reduced late-LTP in the hippocampus [In humans, heterozygous deletion of 27 genes, including m memory . Interespocampus , indicatpocampus ,135, altpocampus ,137.ROCK: ROCK (Rho-associated coiled-coil containing protein kinase) is activated by active RHOA [ROCK was identified in an ID patient [ive RHOA ,139. Twoive RHOA . ROCK acive RHOA ,142; on ive RHOA , and by ive RHOA ,145. A n patient .The pharmacological inhibition of actomyosin contractility inhibits actin retrograde flow and actin filaments’ severing, and promotes neurite outgrowth in the early stages of neuronal polarization ,148, indNotably, PAK1 inhibits MLCK , suggestCdk5-p35: CDK5 (cyclin-dependent kinase 5) is activated by binding with the specific protein partners CDK5R1  and CDK5R2  [ as p39) ,151. CDK as p39) ,153. CDK as p39) . CDK5 fu as p39)  or inhib as p39) ,157.Similarly, CDK5 activates RAC1 via phosphorylation of KALRN  to promote dendritic spine stabilization  or inhibCDK5 and p35 were identified: three intronic variations and four heterozygous variations in a cohort of 360 patients with non-syndromic ID, suggesting that these mutations and polymorphisms may contribute to ID phenotype [In a mutation screening, novel silent mutations in henotype .Small GTPases cycle between a GTP-bound active state and a GDP-bound inactive state. The most important regulators of small GTPases are GTPase-activating proteins (GAPs), which promote GTP hydrolysis, GEFs, which promote activation by inducing the release of GDP and the binding of GTP, and guanine dissociation inhibitors, which prevent GDP dissociation .OPHN1: OPHN1 is an F-actin binding protein ubiquitously expressed in the central nervous system in both glial cells and neurons, where it mainly localizes at the tip of growing neurites, growth cones, and dendritic spines [c spines ,165. It c spines ,166.OPHN1 cause syndromic XLID, in which ID is associated with epilepsy, ventriculomegaly, and cerebellar hypoplasia [In humans, LOF mutations in poplasia ,168,169.Ophn1-KO mice recapitulate some aspects of the human phenotype, such as social, behavioral, and cognitive impairments, as well as ventricular enlargement and susceptibility to seizures [Ophn1-KO mice show hyperexcitability of the hippocampal network, associated with a reduced number of hippocampal GABAergic interneurons, impaired dendritic spine maturation, and short-term synaptic plasticity [OPHN1-deficient human iPSCs showed decreased neurogenic potential and impaired neurite elongation [seizures ,170. At asticity ,170. Morongation .ARHGAP15: ARHGAP15 (Rho GTPase activating protein 15) is a RAC-specific GAP protein, expressed in both excitatory and inhibitory neurons of the adult hippocampus and cortex. It is a negative regulator of RAC1/RAC3 activity, and its loss results in the hyperactivation of the RAC1 pathway [ pathway . ARHGAP1 pathway .Arhgap15-KO mice showed altered neuritogenesis and synaptic density, resulting in increased spike frequency and bursts, accompanied by poor synchronization. Its loss mainly impacts interneuron-dependent inhibition. Adult Arhgap15-KO mice showed defective hippocampus-dependent functions such as working and associative memories [memories .In humans, the loss of ARHGAP15 has been reported in a rare variant of Mowat–Wilson disease, which is characterized by severe neurological deficits, severe ID, speech impairment, and ASD ,174.NOMA-GAP: ARHGAP33  is a multi-adaptor protein with GAP activity, and it is a major negative regulator of CDC42 [Noma-gap-KO mice showed hyperactivity of CDC42 and reduced complexity of dendritic arborization [of CDC42 . NOMA-GAof CDC42 . Noma-garization .TRIO: TRIO (trio Rho guanine nucleotide exchange factor) is a conserved Rho GTPase regulator that is highly expressed during brain development [Trio-KO has been shown to be embryonically lethal [Trio-KO and heterozygous mice show progressive defects in learning ability, sociability, and motor coordination [TRIO de novo mutations in several patients affected by NDDs in which ID appears as a prominent phenotype [elopment ,178. It elopment . In the y lethal , hippocadination . Whole-ehenotype .ARHGEF6 and ARHGEF7: ARHGEF6 and ARHGEF7  are GEFs of the Rho GTPases. ARHGEF6 has been shown to be specific for RAC1, activating and targeting it to membrane ruffles and focal adhesions [Arhgef6-KO mice showed a significant reduction in the activity of both RAC1 and CDC42, but only at the hippocampal level [dhesions . On the al level .Both proteins share an SH3 domain, a prerequisite for the binding with PAK1, PAK2, and PAK3 . SantiagBoth ARHGEF6 and ARHGEF7 present the Dbl homology and pleckstrin domains, which possess RhoGEF activity. Moreover, an ARHGEF7 transcriptional isoform presents a PDZ target at the C-terminal, functional to the binding with PDZ protein, e.g., SHANKs (SH3 and multiple ankyrin repeat domains), at the excitatory synaptic sites .ARHGEF6 (c.166-11T > C) [ARHGEF6, was then identified [Mutation screening of 119 patients with nonspecific ID revealed a T > C variant in the first intron of 11T > C) ,189, alt11T > C) . In addientified .Arhgef6-KO mice showed an increased dendritic length of hippocampal pyramidal neurons, reduced spine synapses, an overall reduction in early-phase LTP, and an increase in LTD, together with impaired spatial and complex learning and less behavioral control in mildly stressful situations, resembling the human ID phenotype, thus validating Arhgef6-KO mice as a proper ID animal model [al model .For what concerns ARHGEF7, the case of two siblings presenting generalized epilepsy and ID was reported, consequently to the 13q34 deletion. This genomic locus contains two protein-coding genes, SOX1 (SRY-box transcription factor 1) and ARHGEF7, thereby supporting the possible contribution of ARHGEF7 haploinsufficiency to the pathogenic phenotype .Arhgef7-KO mice showed embryonic lethality at E9.5; for this reason, the in vivo role of ARHGEF7 was investigated through heterozygous or cortex-specific KO mice [Arhgef7 heterozygous mice exhibited impaired social interactions [ KO mice ,193. Theractions .ARHGEF9: ARHGEF9 is a brain-specific GEF that specifically activates CDC42 [A receptors [es CDC42 . It regues CDC42 . GPHN cleceptors .Arhgef9-KO mice showed reduced GABAA receptor clusters at dendritic spines, enhanced LTP, increased levels of anxiety, and impaired spatial learning [learning .ARHGEF9 mutations cause an XLID syndrome associated with seizures and facial dysmorphism [In humans, morphism ,199,200.TIAM1: TIAM1 (TIAM Rac1 associated GEF 1) is a GEF protein highly expressed in the developing nervous system that activates RAC1 and, to a lesser extent, CDC42 and RHOA [and RHOA ,202. RACand RHOA ,204. Morand RHOA ,206. TIAand RHOA ,208; intand RHOA .TIAM1 activity seems to be exquisitely relevant for granule neurons of the dentate gyrus, as TIAM1 knock-down (KD) in these cells led to a decreased number of glutamatergic synapses expressing AMPA receptor and to an increased spine length, while no effect was observed upon TIAM1 KD in CA1 neurons .Tiam1-KO mice showed defective maintenance of the dendritic arborization and impaired stabilization of dendritic spines in the granule neurons [Consistent with this finding,  neurons . Strikin neurons . For thiThe effects of RAC1, CDC42, and RHOA on actin dynamics are mediated by actin-binding proteins (ABPs), which are classified according to their activity: actin depolymerization, such as ADF/cofilin, branching, such as ARP2/3, severing, such as GSN (gelsolin), bundling, such as fascin family proteins, and nucleotide exchanging, such as profilin family proteins .ADF/cofilin: LIMK1 inhibits ADF/cofilin proteins by phosphorylation at the Ser3 residue [ residue ,213.This protein family is composed of three isoforms: DSTN , CFL1 (cofilin 1), which is the most expressed in the central nervous system, and CFL2, which is specifically expressed in muscle tissue ,215. SinADF/cofilin binds to ADP-actin, increasing the depolymerization rate of the pointed end and causing the severing of actin filaments ,216. ThiSSH: Proteins of the Slingshot family  dephosphorylate ADF/cofilin at Ser3 [ at Ser3 , thereby at Ser3 .YWHAZ: YWHAZ  is an adaptor protein that affects actin dynamics via the stabilization of phospho-ADF/cofilin [/cofilin  and the /cofilin . Additio/cofilin , a compo/cofilin ,227,228.ζ-KO mice present reduced spine density, stressing the importance of this protein in the regulation of the actin cytoskeleton [14-3-3 skeleton .ARP2/3: The ARP2/3 complex is a heptameric complex formed by ACTR2 , ACTR3 , and ARPC1-5 (actin-related protein complex 1–5) [lex 1–5) . It bindlex 1–5) . The axolex 1–5) ,233. Thulex 1–5)  but alsolex 1–5) ,235.The postnatal loss of ARPC3 in forebrain excitatory neurons leads to progressive spine loss and defective LTP-induced spine volume expansion . MoreoveNPFs: The activity of nucleation-promoting factors (NPFs) is required to activate the nucleation and branching activity of the ARP2/3 complex. These factors include WASP (Wiskott–Aldrich syndrome protein), N-WASP , the WAVE regulatory complex (WRC) formed by WASF1 (WASP family member 1), CYFIP1 (cytoplasmic FMR1 interacting protein 1), ABI2 (abl interactor 2), NCKAP1 (NCK associated protein 1), and BRIK (BRICK1 subunit of SCAR/WAVE actin nucleating complex), or paralogues of these, and the WASH complex, formed by WASHC1-5 (WASH complex subunit 1–5) [nit 1–5) ,238. Thenit 1–5) ,240. Actnit 1–5) . Notablynit 1–5) .CYFIP1, were identified in patients with ASD and ID [CYPFI2 variants, most of which were shown to impact on WRC-mediated actin remodeling, have been reported in 37 ID patients [NCKAP1 variants predicted to be deleterious for protein function have been associated with ID [ABI2 is a candidate autosomal recessive ID gene [WASHC5 were shown to cause Ritscher–Schinzel/3C syndrome, a disorder characterized by several phenotypes, among which ID [Strong genetic evidence indicates that alterations in the NPFs-ARP2/3 signaling module may lead to ID: copy number variants of the chromosomal region 15q11-q13, encompassing D and ID , with seD and ID ,245,246;patients ,248; WASpatients ,250; NCK with ID ; ABI2 is ID gene ; de novowhich ID .GSN: GSN (gelsolin) acts by severing actin filaments and capping free barbed ends [bed ends . Its depbed ends . GSN is bed ends , presumabed ends , suggestFMN2: FMN2 (formin 2) is an ABP that belongs to the family of formin homology (FH) domain proteins. Since it is involved in the maturation of tip adhesion, it is essential for the generation of traction forces by filopodia and the stabilization of the growth cone [punctae along dendrites in neurons [wth cone . By bindwth cone  and punc neurons .FMN2 truncating mutations in two consanguineous families lead to decreased spine density and non-syndromic autosomal-recessive ID [Notably, ssive ID .Profilins: Profilin family proteins (PFN1-4) promote the conversion of ADP-actin into ATP-actin, thus providing the actin monomers necessary to sustain barbed end elongation [ongation . In striongation ,262,263.ongation , and it ongation .SHTN1: PAK1 phosphorylates SHTN1 (shootin 1), promoting its interaction with F-actin [ F-actin . SHTN1 p F-actin ,268.Interestingly, SHTN1 mRNA was found to be consistently down-regulated in blood samples of ID patients harboring mutations in the transcription factors CCNT2, CDK9, and TAF2 .A set of mutations identified in ID specifically affect spine development and morphological changes during maturation. In this section, we summarize the most relevant findings.KALRN: KALRN  is a GEF for RAC1 [Kalrn-KO mice showed decreased synaptic density in the apical dendrites of CA1 hippocampal neurons, along with learning deficits [for RAC1 . In micefor RAC1 . Kalrn-Kdeficits .PPP1R9A and PPP1R9B: PPP1R9A (neurabin -I) and PPP1R9B  show F-actin cross-linking and phosphatase activity. They are enriched in dendritic spines [c spines  and are c spines .Ppp1r9b-KO mice have a reduced number of filopodia and an increase in spine density [ density .ARHGEF2: Neurabin-I and Neurabin-II interact with the Rho GEF ARHGEF2 . This interaction selectively regulates Rho-dependent organization of F-actin in spines; ARHGEF2 is maintained inactive/sequestered through the interaction with microtubules and targeted to dendritic spines as a result of the interaction with Neurabin-I and Neurabin-II [rabin-II .ARHGEF2 mutation leads to reduced activity of the RHOA pathway. A homozygous frameshift mutation in the ARHGEF2 is associated with ID, midbrain–hindbrain malformation, and mild microcephaly in a consanguineous family of Kurdish–Turkish descent [ descent .SHANK: SHANK family proteins are higher-order organizing scaffold proteins of the PSD. They are known to interact with the ABP DBNL (drebrin like) in the PSD to promote the reorganization of actin after stimulation [mulation . SHANK pmulation . Indeed,mulation . SHANK3 mulation . A largemulation .SHANK3 is responsible for the clinical disorder [It has been proposed that in the 22q13 deletion syndrome, an NDD characterized by ID, the disruption of disorder ,284,285.CAMK2: The activity of CAMK2, which is stimulated upon the increase in intracellular calcium concentration, is essential for AMPA receptor delivery to the membrane of silent synapses and SYNGAP activation [tivation ,286. Whotivation .Microtubules are basic elements of the cytoskeleton and actively participate in most neurodevelopmental processes. Neurons depend on microtubule dynamics for cell division, axon guidance, intracellular trafficking, and synapse formation . They arADNP mutations are associated with ASD; ASPM, MCPH1, STIL, CDK5RAP2, CENPJ, PRUNE1, and KIF20 mutations are associated with microcephaly; TUBB2B mutations are associated with polymicrogyria; LIS1, DCX, and TUBA1A are linked to lissencephaly [Many microtubule-associated genes are linked to ID and to other NDDs in which ID appears as a prominent and recurrent phenotype: ncephaly .KIF1A, KIF4A, KIF5C, and KIF7: Kinesins are motor proteins that move along microtubules in an anterograde fashion, transporting their cargo towards microtubules’ plus end. KIF1A (kinesin family member 1A) is selectively expressed in neurons, and its partial or total depletion results in the disruption of axonal and dendritic transports [KIF1A was found in a patient with non-syndromic ID [KIF4A and KIF5C for which the causative role in ID is supported by evidence obtained using KO models [KIF2A and KIF5C were reported in patients with malformations of cortical development presenting ID [KIF7 were found in patients with ciliary disorders in which ID is part of the phenotype [ansports . A dominromic ID , and othromic ID . Next-geO models . Mutationting ID , and homhenotype .KIFBP: KIFBP (kinesin family binding protein) is a modulator of kinesins activity. Homozygous nonsense mutations of KIFBP are associated with Goldberg–Shprintzen syndrome, which is a form of syndromic ID [romic ID .CHAMP1: CHAMP1  is a zinc finger protein that regulates chromosome segregation during mitosis. De novo CHAMP1 mutations are associated with GDD and ID [D and ID ,307,308.CLIP1: CLIP1 (CAP-Gly Domain Containing Linker Protein 1) is a +TIP that regulates microtubule growth and bundling. Next-generation sequencing revealed an autosomal recessive form of ID associated with a nonsense variant in CLIP1 in an Iranian consanguineous family [s family .KATNAL1: KATNAL1  is one of the two major catalytic subunits of the microtubule-severing enzyme Katanin, together with KATNAL2. Three unrelated patients with heterozygous de novo deletions encompassing 13q12.3 presented moderate ID phenotype; since this region contains KATNAL1, this gene has been proposed as a candidate ID gene [ ID gene .MID1 and MID2: MID1 (midline 1) and MID2 are E3 ubiquitin ligases that have a role in microtubule stability and organization. MID1 mutations are associated with Opitz G/BBB syndrome, in which mild to severe ID can appear [MID2 missense mutation that disrupts its interaction with microtubules is associated with XLID [n appear . A MID2 ith XLID .CDKL5: CDKL5 (cyclin-dependent kinase-like 5) is a kinase protein essential for brain development. CDKL5 interacts with IQGAP1 (IQ motif containing GTPase activating protein 1), through which it forms a functional complex with its effectors RAC1 and CLIP1, MAPRE2 (microtubule-associated protein RP/EB family member 2), MAP1S (microtubule-associated protein 1S), ARHGEF2, and SHTN1 [CDKL5 mutations cause the so-called CDKL5 deficiency disorder in which severe ID is one of the most important clinical manifestations [nd SHTN1 ,314,315,stations .Astrocytes, oligodendrocytes, and microglia cells express a fair fraction of ID genes, including those involved in Rho GTPases signaling and cytoskeleton organization . For thiAstrocytes: It is well established that astrocytes play a role in learning and memory formation [ormation ,318. Forormation , while cormation .The actin cytoskeleton is involved in many aspects of astrocyte physiology and function, such as the plasticity of the perisynaptic astrocytic processes , small mOphn1-KO mice are less ramified and show altered migration and glial scar formation [Arntl-deficient astrocytes [Therefore, one might speculate that defects in cytoskeleton dynamics may lead to ID by affecting astrocyte functions. In line with this hypothesis, astrocytes of ormation . Moreovetrocytes .Ophn1-KO and Arntl-KO mice may elucidate the non-neuron autonomous contribution of these proteins.The generation of astrocyte-specific Oligodendrocytes: Oligodendrocytes, the myelin-forming cells of the central nervous system, are essential to establish neuronal networks with proper functions [PAK3 [Pak3-KO mice showed myelination defects of the axons of corpus callosum [PAK3 mutations.unctions ,329. Myeunctions ,331. Theunctions . This reunctions ,334. Myeunctions ,336 and ns [PAK3 ,338. PAKns [PAK3 ,340,341.Microglia: Microglia cells, the resident innate immune cells of the brain, are involved in the regulation of brain development by promoting both synaptic pruning and synapse formation [ormation . Interesormation . Since mormation ,344 and ‑simultaneously consider a large number of interacting proteins and link their relationships with emerging phenotypes‑elaborate hypotheses and design new experiments‑search for biomarkers and druggable targets for translational purposesSB approaches complement the wealth of experimental and mutational data derived from small-scale studies and high-throughput methods with computational and mathematical modeling to reconstruct the dynamic organization of a specific cellular regulatory network, a task that is often out of reach of molecular technologies. Moreover, computational models can be implemented with existing data to raise predictions of how the biological system would behave in particular conditions. Our ability to model a complex regulation of connected elements in silico has largely improved, allowing us to:SB studies are performed with bottom-up (dynamic models such as Boolean models) or top-down  procedures. A Boolean model was implemented to study the molecular dynamics underlying the behavior of the neuronal growth cone during axon growth and guidance. Interestingly, simulations performed with mutant networks suggest that many mutations underlying ID and ASD affect the motility of the growth cone and in particular the formation of filopodia and lamellipodia . To our Bottom-up pipelines start with a survey and manual curation of the scientific literature and databases looking for key genes, proteins, and interactions regulating the process under investigation, e.g., neurite extension, in order to derive a graphical depiction of the network in which nodes are genes/proteins and edges are the interactions among them. Here, we provide an example of a protein regulatory network for neurite elongation centered on GTPases A. The gr, we show the Boolean model corresponding to the network of There are different frameworks for mathematical/computational modeling , which differ in their trade-off in terms of biological complexity, scalability, or simulation reliability. Referring to Boolean models, there are only two mutually exclusive states for each node, the logic 1 and 0. The initial graphical map is rewritten converting the interactions between proteins/genes to Boolean functions by using the three logical operators: AND, OR, and NOT. As an example, in Table 1Simulations can be run by choosing the initial conditions to be used , i.e., selecting which node is set as active (1) or inactive (0). During the simulation, the nodes state can be either synchronous or asynchronous: in the synchronous type, the state is updated simultaneously, while in the asynchronous type, only one transition function—randomly chosen—is updated at each simulation.The simulation aims to identify the steady states reached by the system, named “attractors”, corresponding to particularly stable configurations of activity for each node of the network B. AttracThe simulation performed on the neurite elongation Boolean network using the exhaustive method and the synchronous type yielded 2155 attractors Nf1+/− mice (a model of NF1) were treated with both farnesyl-transferase and HMG-CoA reductase inhibitors to decrease RAS activity; the treatment was successful in rescuing spatial memory and attention deficits [Starting from the hypothesis that learning deficits in NF1 (neurofibromatosis 1) are caused by an excess of RAS activity and by the consequent increase in GABA-mediated inhibition, deficits ,386.(2)B and GABAA receptor antagonists; both treatments rescued memory in novel place and object recognition tests and contextual fear conditioning tasks [A receptor signaling is excitatory rather than inhibitory in Ts65Dn mice and DS patients, because of an increased hippocampal expression of the cation chloride cotransporter SLC12A2 (solute carrier family 12 member 2). Its inhibitor, bumetanide, a common diuretic, was able to restore synaptic plasticity and hippocampus-dependent memory in adult Ts65Dn mice [The observation that the hippocampal signaling through postsynaptic GABA receptors was significantly increased in Ts65Dn mice (a model of DS) prompted the testing of selective GABAng tasks ,388,389.5Dn mice . Recentl5Dn mice .(3)CREBBP (CREB-binding protein) is considered the most significant mutation in Rubinstein–Taybi syndrome, pharmacological strategies to enhance CREBBP-dependent gene expression were investigated. Crebbp+/− mice (a model of Rubinstein–Taybi syndrome) treated with either PDE4 inhibitor  or HDAC inhibitor  were rescued for long-term memory deficit [+/βGeoKmt2d mice (a model of Kabuki syndrome) were rescued by the treatment with HDAC inhibitors [As the mutation in  deficit ,393. Simhibitors .(4)TSC1 or TSC2 that form an MTOR-inhibiting complex can cause tuberous sclerosis by hyperactivating MTOR signaling. +/−Tsc2 and Tsc1 homozygous mutant mice (a model of tuberous sclerosis), were treated with rapamycin, rescuing spatial learning and context discrimination deficits together with neurological findings [Strada-KO model, an upstream inhibitor of MTORC1, in a rare NDD called Pretzel syndrome [Hyperactivity of MTOR signaling is observed in several neurodevelopmental disorders, the so-called “mTORopathies”; therefore, it is not surprising that MTOR inhibitors were extensively tested. Heterozygous mutations in either findings ,396. Rapsyndrome . Interessyndrome . This clsyndrome  and is ssyndrome .(5)−Fmr1−/ mice (a model of FXS) were used to study the GABAAergic deficit that underlies FXS; treatment with a mGluR5 antagonist rescued associative learning [A receptors in animal models [B receptor agonists, which, in patients, seemed to rescue behavioral functions [learning , as welll models  and GABAunctions . Additiounctions .For many years, ID and the other cognitive disorders were thought to be irreversible, especially in adults, and treatments were focused only on personalized educational plans and co-morbidity alleviation, except for a few syndromic forms of ID, for which specific treatments, like enzyme replacement therapy, are used and result in intellectual improvements . NonetheAlthough ID genetic heterogeneity sets a limit on individual treatment, these studies have the merit to demonstrate that ID is potentially responsive to therapeutic interventions. The identification of common disrupted molecular and cellular mechanisms will help in finding flexible therapies, targeting central nodes more than individual genes. One of these nodes is represented by the cytoskeleton under the control of Rho GTPases signaling.Several genes associated with ID code for proteins acting on microtubules’ formation and regulation . LikewisMicrotubule stabilizers were mostly studied in neurodegenerative disorders characterized by compromised MAPT  functions. According to the hypothesis that microtubule-stabilizing drugs could offset the loss of normal tau functions , taxane Cdkl5-deficient neurons rescuing morphological defects [Microtubule stabilization can be achieved also by targeting microtubule-binding proteins. The neurosteroid pregnenolone (PREG) was shown to induce CLIP1 active conformation and to restore its association with microtubules in  defects .ADNP haploinsufficiency results in increased tau phosphorylation and memory impairments in neurodegenerative diseases, including Alzheimer’s disease [ADNP mutations have been linked also to ASD and ID [Another solution was provided by small peptides able to act out the activity of microtubule-associated proteins. NAP (NAPVSIPQ), alias Davunetide, is a neuroprotective peptide snipped by ADNP (activity-dependent neuroprotector homeobox).  disease . ADNP muD and ID . In precD and ID . InteresD and ID ,414.+/-LIS1 mice  [Several other compounds are under investigation for their activity over microtubules and microtubule-related proteins, e.g., blood–brain barrier-penetrant heterocyclic molecules able to stabilize microtubules, and calpain inhibitors, which, by protecting the protein LIS1 (platelet-activating factor acetylhydrolase 1b regulatory subunit 1) against proteolysis, are able to recover retrograde transport and network formation in cephaly) ,416.Altered actin polymerization kinetics features most of the ID models presenting mutations on genes involved in actin-reorganizing signaling pathways. Yet, the phenomenon was characterized only in ASD and schizophrenia patients, in which a defective actin polymerization was observed ,56.Because of its ubiquitous expression, directly targeting actin raises many concerns. Conversely, a more appealing possibility is ABPs’ or upstream regulators’ targeting, which are largely brain-specific, e.g., CTTNBP2 (cortactin binding protein 2) , particuShank3-deficient mouse model [The validity of such an approach was demonstrated in a study on a se model . First, Actin dissociation/association rate can also be affected by the presence of other natural actin ligands, fungal and bacterial toxins, and cytotoxic macrolides derived from marine sponges. As for ABPs, actin ligands are classified according to their activities and roughly divided into actin stabilizers, e.g., phalloidin, jasplakinolide, and miurenamide A, and destabilizers, e.g., latrunculin, cytochalasin D, and kabiramide C. Both stabilizing and destabilizing agents are intended in their proper meaning only in vitro, while in vivo they are expected to affect the cytoskeleton dynamics only by delaying or accelerating them. These compounds have been largely used as a cell biology tool, like phalloidin in conjugation with fluorophores, overlooking their ability to compete with specific ABPs in a biomimetic mode. Trisoxazole macrolides, e.g., kabiramide C, are small biomimetic molecules promoting actin filament severing and capping by competing specifically with Gelsolin . AnalogoSince the reorganization of the cell cytoskeleton is an essential process during metastasis formation, this class of molecules has always been seen as a good candidate for anticancer drug development. However, they could be a starting point in the creation of new compounds that do not influence actin polymerization per se but selectively antagonize specific ABPs.Several forms of ID are caused by mutations that either increase or decrease Rho GTPases’ signaling. However, it is important to note that both hyper- and hypo-activation of these signaling pathways led to similar phenotypes. A good example is provided by the mouse model bearing RAC1 conditional inactivation at the hippocampal level, which resembles the phenotypic abnormalities reported for the FXS model, which in contrast shows a massive activation of RAC1 ,423. ThiSeveral compounds were characterized for their inhibitory activity toward RAC1, which is of particular interest in cancer research. Most of them act by interfering with RAC1-GEF PPI, such as NSC23766 and EHop-016 (which interfere with TRIO and TIAM1) ,425 and Ophn1-KO model of ID, which exhibits hyperactive RHOA signaling. In these mice, the administration of the ROCK/PKA inhibitor Fasudil rescued spine density, hippocampal hyperexcitability, ventricular enlargement, and behavioral abnormalities [A striking example of such a pharmacological treatment is provided by the malities ,429. Fasmalities .In other cases, the opposite approach is required, i.e., positive modulation of the GTPase activity. Hyper-activation of RAC1 through the inoculation of the bacterial toxin CNF1 has been shown to improve the behavioral phenotype of a mouse model of Rett syndrome and to reverse the astrocytic deficits, which were assumed to have an impact on dendritic maturation . CNF1 acCurrent treatments of ID are largely based on psychosocial measures, environmental enrichment, dedicated educational plans, and motor activity, while pharmacotherapies are lagging. Recent evidence suggests that some phenotypes associated with cognitive disabilities can be reversed, through either genetic approaches  or pharmOne obstacle is represented by the high genetic variability observed in ID. SB approaches and integrative tools are beginning to deconvolute and model the core biological processes responsible for the altered circuitry and synaptic dysfunctions in ID. One of these processes is the regulation of cytoskeleton dynamics, whose hubs are the small GTPases of the Rho class. A relevant outcome of our current knowledge is the possibility to leverage the wealth of experimental and mutational data to derive a computational model, through which we will learn more about the behavior of the system.Rho class GTPases are promising targets for pharmacological intervention and can be positively or negatively modulated by acting upstream, on the regulatory partners, or downstream, on actin stability. To achieve a brain-specific, GTPase-specific, modest, and controlled remodulation, a full characterization of the PPI between GTPases and their regulatory partners is required. Current knowledge led to the identification of several potentially valid compounds, some of which are well characterized for their biological activity, while others still need a proper characterization.Future efforts should focus on completing our knowledge about the cytoskeleton core regulatory network, considering that relevant elements might still be missing or have been overlooked. Proteomic analyses focused on the human neuronal cytoskeleton are needed, intending to identify novel druggable elements participating in the GTPase regulatory network or neurodevelopmental processes, i.e., neurite elongation, neuronal migration, and adhesion.Since most of the current knowledge is based on animal models, an area of strong interest is the generation and validation of cellular models of ID based on human neurons. Such models should recapitulate the cortical and hippocampal endophenotype of human ID and offer the possibility to examine the excitatory/inhibitory balance. In light of these observations, human iPSCs offer a valid model to identify new valuable readouts and to start screening compounds able to alleviate ID .


Epstein–Barr Virus (EBV) is a common virus that is readily controlled by a healthy immune system and rarely causes serious problems in infected people. However, patients with certain genetic defects of their immune system might have difficulties controlling EBV and often develop severe and life-threatening conditions, such as severe inflammation and malignancies. In this review, we provide a summary of inherited immune diseases that lead to a high susceptibility to EBV infection and discuss how this infection is associated with cancer development.TNFRSF9, CD27, CD70, CORO1A, CTPS1, ITK, MAGT1, RASGRP1, STK4, CARMIL2, SH2D1A, and XIAP affect the development, differentiation, and function of key factors involved in the immunity against EBV, leading to increased susceptibility to lymphoproliferative disease and lymphoma.Epstein–Barr Virus (EBV) is a ubiquitous virus affecting more than 90% of the world’s population. Upon infection, it establishes latency in B cells. It is a rather benign virus for immune-competent individuals, in whom infections usually go unnoticed. Nevertheless, EBV has been extensively associated with tumorigenesis. Patients suffering from certain inborn errors of immunity are at high risk of developing malignancies, while infection in the majority of immune-competent individuals does not seem to lead to immune dysregulation. Herein, we discuss how inborn mutations in  Both receptors are part of the TNFR superfamily and act as co-stimulatory receptors, increasing T cell proliferation, survival, cytokine production, and cytotoxicity. Unlike CD27, which is constitutively expressed by resting T cells, CD137 is induced after cell activation ,108. TheTNFRSF9 and were EBV viremic, only one sibling developed symptoms. Importantly, specific CD8+ T cell responses towards LCL were impaired in both kindreds. The symptomatic sibling further showed digenic mutations in the PIK3CD gene (causative of activated PI3 kinase delta syndrome) that might have further contributed to the EBV-related clinical phenotype [EBV-specific T cells from CD137-deficient patients presented lower interferon (IFN)-γ and perforin expression and showed impaired expansion in response to EBV-infected B cells compared to healthy cells. Similar results were also observed following CD137 blockage in T cells from healthy donors, highlighting its non-redundant role in the immune response against EBV ,63. Suschenotype .2+ release, while DAG induces NF-κB and MAPK/ERK pathways [Interleukin-2 inducible T cell kinase (ITK) is a member of the Tec family tyrosine kinases with a crucial role in mediating antigen receptor signaling in T cells. Following T cell receptor (TCR) engagement, the CD3 immunoreceptor tyrosine-based activation motifs (ITAMs) are phosphorylated by lymphocyte-specific protein tyrosine kinase (Lck). It allows zeta-chain-associated protein kinase 70 (Zap-70) to bind to phosphorylated ITAMs and subsequently to phosphorylate adapters of linker for activation of T cells (LAT) and the SH2 domain-containing leukocyte protein of 76kDa (SLP-76). ITK is recruited to the phosphorylated LAT/SLP-76 adapter complex, and together they activate phospholipase Cγ1 (PLCγ1). Activated PLCγ1 hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce the second messenger molecules inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces intracellular Capathways ,109.+ T cells, and abrogates development of NKT cells [+ T cells show delayed effector function upon activation, decreased proliferation, and intrinsic defects in degranulation. Interestingly, those defects could be rescued by increasing costimulatory signals, such as prolonged IL-2 stimulation or the addition of IL-12 [−/− mice with murine gammaherpesvirus-68 (MHV-68) leads to latent intestinal infection, which develops into lethal colitis [ITK is not indispensable for TCR downstream signaling, it rather acts as an amplifier. Therefore, although some processes are barely affected, the development and differentiation of T cells might follow abnormal paths. ITK deficiency leads to a skewed Th1 response in detriment of the Th2 response, favors Treg differentiation over Th17, induces development of “innate like” CD8KT cells ,115,116.of IL-12 . Intrape colitis .2+ flux upon TCR stimulation with anti-CD3 [T cells from ITK-deficient patients were also reported to have low or delayed Caanti-CD3 . Clinicaanti-CD3 .+ T cells leads to defective proliferation and cytotoxic function [Similar to ITK, the nucleotide exchange factor RAS guanyl-releasing protein 1 (RASGRP1) is a secondary TCR messenger. Following increased DAG production by PLCγ1, RASGRP1 is recruited to the membrane and activates the small G protein RAS that in turn activates the cascade of MAP kinase  . RASGRP1function ,105. Salfunction . CD27/CDfunction . Given tfunction .+: CD8+ T cell ratio, poor T cell proliferation, defective NK cell function, autoimmunity, and EBV-associated lymphoma. Six out of nine patients developed EBV-associate malignancies, among them two were diagnosed with diffuse large B cell lymphoma (DLBCL), two with HL, one with low-grade lymphoma, and one with polymorphic B cell lymphoma [RASGRP1-deficient patients commonly show recurrent infections, inverted CD4lymphoma ,104,105.CTPS1 is a key enzyme for de novo synthesis of CTP, a limiting nucleotide in cells, and therefore, essential for DNA replication. Resting T cells express rather low levels of CTPS1, but it is readily upregulated after TCR stimulation in accordance with its requirement for DNA synthesis and proliferation ,122,123.+ T cells, and EBV-induced LPD and lymphoma [2+ influx that was abrogated in MAGT1 deficiency. It was thought that Mg2+ was a second intracellular messenger of the TCR linked with PLCγ1 activation and subsequently Ca2+ influx upon TCR activation [2+ transport. It was shown that defects in MAGT1 cause glycosylation errors in specific subsets of glycoproteins, including NKG2D and CD70 expressed by immune cells [+ T cells plays a crucial role in killing EBV-infected and transformed cells, and its decreased expression makes it a perfect biomarker [+ T cell lymphopenia, hypogammaglobulinemia, and lymphadenopathy, the clinical phenotype of patients with MAGT1 deficiency includes high susceptibility to EBV-induced LPD and malignancies (14 of 37 reported patients). Again, HL was the most prevalent lymphoma (7 of 14 patients) (Mutations in the gene encoding the magnesium transporter protein 1 (MAGT1) are causative of XMEN  syndrome. This disease was initially described in 2011 in two males showing recurrent pulmonary infections, low CD4lymphoma . Followitivation ,125,126.ne cells . NKG2D eiomarker ,128,129.iomarker ,130. Henatients) . InteresCORO1A) belongs to a family of coronins that is highly expressed in leukocytes. They are actin-binding proteins, which regulate cytoskeletal remodeling in response to extracellular signals. They modulate processes such as migration, phagocytosis, and cell polarization [2+ mobilization [CORO1A.Coronin 1A  phenotype, most coronin 1A-deficient patients reported to date suffered from recurrent  infections, and an inability to control EBV, leading to EBV-associated LPD and lymphomas . Althoug synapse .Serine-threonine kinase 4 , is a key kinase involved in the signaling of the canonical and non-canonical Hippo pathway. In the canonical path, STK4 phosphorylates large tumor suppressor kinases (LATS1/2), which further activate the yes-associated protein (YAP) and transcriptional co-activator PDZ-binding motif (TAZ). Activated YAP and TAZ act as transcription factors and induce the expression of various genes controlling cell growth, proliferation, and differentiation . ThroughSTK4 with tumorigenesis in mice [STK4-deficient patients suffer from recurrent bacterial, fungal, and/or viral infections, including EBV-associated LPD, intermittent neutropenia, T and B cell lymphopenia, and increased risk of autoimmune diseases and lymphoma. Although around half of reported STK4-deficient patients have manifested with EBV-LPD and viremia, there is a further EBV independent risk of developing malignancies. Out of six lymphomas reported in five of the 28 patients, three were tested EBV-negative ,80. Seve in mice . Kim et  in mice . AdditioCapping protein regulator and myosin 1 linker 2  is a protein expressed in many cell types, including lymphoid tissue and the gastrointestinal tract. It controls actin polymerization; hence it regulates a variety of functions as cell polarization and migration . DespiteSH2D1A, which encodes the signaling lymphocyte activation molecular (SLAM)-associated protein (SAP) [+ T and NK cells increases their cytotoxic effect. However, in SAP-deficient cells, stimulation of those receptors showed an inhibitory effect [+ T cells were still able to kill other cell types, i.e., fibroblasts, monocytes, or dendritic cells [X-linked lymphoproliferative disease type 1 (XLP1) is caused by mutations in in (SAP) ,143,144.in (SAP) ,146. Engy effect ,148,149.ic cells ,150. Thi−/− mice infected with MHV-68 develop hypogammaglobulinemia and chronic inflammation with exacerbated proliferation of virus-specific CD8+ T cells and consequently increased tissue damage [+ T cells against B cells, play a pivotal role in the development of B cell lymphoma, rather than the ability of EBV to induce transformation [SAPe damage ,152. Simormation .+ T cells are unaffected but CD8+ T cells lacking XIAP show increased apoptosis followed stimulation [In X-linked inhibitor of apoptosis protein (XIAP)-deficient patients, cytotoxicity of NK and CD8mulation . XIAP pamulation ,155.Since EBV was firstly identified 60 years ago, the immunological sequelae of EBV infection in immunocompetent and immunocompromised individuals has to a large extent been revealed ,156. Its+ T cell dysfunction to various degrees  the uncontrolled EBV-infection, (2) the inability of the organism to control transformed cells independently of EBV, or most likely (3) a combination of both factors.As the primary target of EBV is the B cell, it is not surprising that lymphoproliferative diseases in patients with IEIs are usually of B cell origin. However, our review did not include the rare but equally important as well as often fatal clinical manifestations of T/NK cell proliferative diseases. Fujiwara and Nakamura provide an in-depth review of the unique characteristics of chronic active EBV infection in IEIs with EBV-positive T/NK cell LPDs in a recent special issue of this journal .The discovery of “new” IEIs have rapidly increased in the past years due to the increased application of next-generation sequences . Novel d


In the original article, the authors neglected to include the funder Collaborative Research Centre (CRC) 1181 (Erlangen), grant number TP-B08 N to authors Zuqin Yang and Susetta Finotto.In the original article, there was an error. The scientific meaning of the following sentence was unclear owing to poor syntax: “In asthma, are predominantly induced ILC2 producing type 2 cytokine IL-4, IL-5, IL-13, and IL-9”.Innate Immune Response in Asthma, Innate Lymphoid Cells (ILC2), Paragraph 1. The sentence should read: “In asthma, ILC2 producing type 2 cytokine IL-4, IL-5, IL-13, and IL-9 are predominantly induced”.A correction has been made to as IFI44L, MX1, ISG15, IRF and STAT1”.The same is true of the following sentence: “After RV16 infection, in Tregs were induced both in healthy control and asthmatic subjects with upregulated anti-viral gene expression, such Adaptive Immune Response in Asthma, Treg, Paragraph 2. The sentence should read: “After RV16 infection, Tregs were induced both in healthy control and asthmatic subjects with upregulated anti-viral gene expression, such as IFI44L, MX1, ISG15, IRF and STAT1”.A correction has been made to The authors apologize for these errors and state that they do not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Early determination of coronavirus disease 2019 (COVID-19) pneumonia from numerous suspected cases is critical for the early isolation and treatment of patients.The purpose of the study was to develop and validate a rapid screening model to predict early COVID-19 pneumonia from suspected cases using a random forest algorithm in China.A total of 914 initially suspected COVID-19 pneumonia in multiple centers were prospectively included. The computer-assisted embedding method was used to screen the variables. The random forest algorithm was adopted to build a rapid screening model based on the training set. The screening model was evaluated by the confusion matrix and receiver operating characteristic (ROC) analysis in the validation.The rapid screening model was set up based on 4 epidemiological features, 3 clinical manifestations, decreased white blood cell count and lymphocytes, and imaging changes on chest X-ray or computed tomography. The area under the ROC curve was 0.956, and the model had a sensitivity of 83.82% and a specificity of 89.57%. The confusion matrix revealed that the prospective screening model had an accuracy of 87.0% for predicting early COVID-19 pneumonia.Here, we developed and validated a rapid screening model that could predict early COVID-19 pneumonia with high sensitivity and specificity. The use of this model to screen for COVID-19 pneumonia have epidemiological and clinical significance. Since December 2019, the COVID-19 pneumonia outbreak has rapidly swept across China and beyond through human-to-human transmission.–7 recently showed that the initial symptoms of COVID-19 pneumonia are nonspecific and usually present as fever, cough, headache, vomiting, or diarrhea; additionally, some cases present without any symptoms. Laboratory results can include normal or reduced leukocyte levels and chest computed tomography (CT) findings appearing as patchy/punctate ground-glass opacities in single or multiple lung lobes. Currently, the real-time reverse transcription-polymerase chain reaction (RT-PCR) assessments are regarded as the gold standard for COVID-19 diagnosis.,10 Nevertheless, the COVID-19 specific IgM antibody is usually generated 3–5 days after onset, COVID-19 pneumonia cases initially presented negative via RT-PCR.,12 There were numerous suspected COVID-19 pneumonia cases in clinics during the COVID-19 infection pandemic season. In addition, a shortage of RT-PCR test kits and specimen sampling uncertainty could adversely impact early COVID-19 pneumonia diagnosis, lead to delayed patient isolation and treatment, and challenge COVID-19 pneumonia epidemiology and prognosis. Therefore, a rapid diagnostic model to screen high-risk or suspected COVID-19 pneumonia patients is urgently needed.Medical reportsIn the research, we aimed to develop and validate a rapid, computer-assisted screening model based on epidemiological data, clinical characteristics, and laboratory results and imaging examinations to detect early COVID-19 pneumonia from numerous suspected subjects. The random forest algorithm was adopted to build a rapid screening model. Model performance was assessed with the receiver operating characteristic curve (ROC) and areas under the curves (AUC). The predictive power of the prediction model was validated by the confusion matrix in the validation set. This is the first prospective screening model to predict early COVID-19 pneumonia, as well as its widespread application in other countries.22.1A total of 914 participants suspected of COVID-19 infection were enrolled from multiple medical institutions in Hangzhou, Shaoxing, Jinxing, Wenzhou, Ningbo, and Taizhou from January 17, 2020 to February 29, 2020. The sample size was calculated by G∗Power 3.1.9.2. The suspected or confirmed patients were diagnosed based on the 7th edition of the Chinese recommendations for the diagnosis and treatment of pneumonia caused by COVID-19. The purpose of the study was to develop and validate a rapid screening model to predict early COVID-19 pneumonia from suspected cases using a random forest algorithm in China.2.1.1COVID-19 pneumonia suspected case met any 1 of the epidemiological criteria and any 2 clinical presentation criteria. If there were no epidemiological factors, then the suspected patients should meet 3 of the clinical presentation criteria.Epidemiological factors: (1) a history of residence or travel in the outbreak area (Wuhan) or its nearby areas, communities with confirmed cases or other areas with persistent local transmission within 14 days; (2) a history of being in contact with confirmed COVID-19 infected patients (positive nucleic acid detection) within 14 days; (3) a history of being in contact with patients with fever or respiratory symptoms who had a history of residence or travel to the outbreak area (Wuhan) or its neighboring areas, communities with confirmed cases or other areas with persistent local transmission within 14 days; and (4) association with a cluster outbreak, defined as the confirmed COVID-19 pneumonia case in a place of work or a family or community, which was not in the outbreak area, within 14 days, along with other patients suffering from a fever or respiratory symptoms.Clinical presentations: (1) fever and/or respiratory symptoms; (2) typical chest imaging features of COVID-19 pneumonia, such as ground-glass opacities, pulmonary consolidation, and infiltrating shadows; and (3) normal or decreased white blood cells (WBC), or decreased lymphocytes in an early stage of the disease.2.1.2Confirmed COVID-19 pneumonia cases were defined as a suspected COVID-19 pneumonia case that had any 1 of the following criteria: (1) positive COVID-19 nucleic acid found using RT-PCR from sputum, blood samples, throat swab, or stool sample and (2) genetic DNA sequencing results of the samples were homologous with the known COVID-19.A total of 914 patients were prospectively recruited in the study. The exempt informed consent was approved because the patients would not be exposed to any risks, and the patients’ information was anonymously collected prior to analysis in this observational study. This study was reviewed and approved by the Ethics Committees of all the medical institutions.2.2Epidemiological factors and clinical manifestations were collected for each case. Age, sex, region of residence, body temperature, dry cough, fatigue, dyspnea, sputum, conjunctival congestion, nasal congestion, diarrhea or abdominal ache, dizziness or headache, nausea or vomiting, sore throat, muscle soreness, and comorbidities were recorded for all patients. Blood routine examination and C-reactive protein (CRP) were performed by standard laboratory methods. The X-ray examination followed the common chest protocol: the patients stood with their backs to the X-ray device and their chests pressed firmly against the plate, with their hands resting on the ILIUM, shoulders drooped, upper arms turned inward (pulling apart the shoulder blades), head tilted back slightly, and lower jaw resting on the upper edge of the plate. CT scanning followed the common chest protocol: the patients were placed in a supine position with their arms raised. The patients were instructed to hold their breath during the data acquisition, which included the whole lung volume. The overall scan time was 2 seconds, and the slice thickness for reconstruction was 1.25 mm. Throat swabs, sputum, stool, or blood samples were collected to test for COVID-19 nucleic acid using standardized RT-PCR test kits following the standard protocol. If an initial RT-PCR test was negative, 2 repeated tests were performed after 24 hours and COVID-19 specific IgM and IgG antibodies were negative after 7 days.2.3We included age, sex, comorbidities, epidemiological data, clinical symptoms, body temperature, WBC count, lymphocyte count, neutrophil count, and chest imaging findings to establish a novel diagnostic model for COVID-19 pneumonia based on the epidemiology in China. The epidemiological features and symptoms were considered binary variables and were scored as “1” if “yes” and “0” if “no.” The thoracic radiologic findings were simply classified as “normal,” “unilateral local patchy shadowing,” “bilateral multiple ground-glass opacity,” “bilateral diffuse ground-glass shadow with pulmonary consolidation,” and “other imaging alterations such as pulmonary nodule or pleural effusion,” and were scored as “0,” “0.5,” “1,” “2,” and “3,” respectively.The samples from the patients were classified into a COVID-19 pneumonia group with 361 individuals total and a non-COVID-19 pneumonia group with 553 individuals total, based on the RT-PCR outcomes, which were considered the gold standard; the patients were randomly divided into 2 groups: 80% for model development and 20% for model validation. The computer-assisted embedding method was used to screen the variables. The random forest algorithm was applied to build and validate the predictive model.2.4t test, and categorical variables are expressed as numbers and percentages and were compared using the chi-squared test. For multiple comparisons, a one-way analysis of variance was applied.All statistical analyses were performed using SPSS software , G∗Power (version 3.1.9.2), and Scientific Python 3.6 libraries (Scikit-Learn package). Continuous variables are expressed as the mean ± standard deviation and were compared using Student P < 0.05 was considered statistically significant.G∗Power was used for sample size, prior analysis was conducted, the effect size was 0.25, α err prob was 0.05, and power was 0.98. The computer-assisted embedding method was used for variable selection, and the algorithm of variable selection used in this study was logistic regression; the threshold of the variable selection was manually set to 0.85 based on the optimal detection principle. Collinearity diagnosis was performed to screen the variables, which were used for all subsequent analyses. In the training set, the random forest algorithm, an ensemble, supervised machine learning algorithm, was used to build a classifier (a predictive model based on a panel), and the importance of each variable was calculated. To evaluate the prediction accuracy of the screening model for determining COVID-19 pneumonia, the predictive model was evaluated by the confusion matrix, ROC, and AUC in the validation set. The cutoff value was defined as the value that allowed the maximum sensitivity and specificity values. All statistical tests were carried out in two-tailed ranges, and a probability level of 2.5A total of 935 patients were recruited in this study, including 21 excluded patients due to data missing, and 914 participants were eligible for the evaluation. Among them, 553 patients were excluded because of at least 2 negative results by RT-PCR, and the remaining 361 patients were diagnosed as having COVID-19 pneumonia with a positive COVID-19 detection using RT-PCR.P < 0.001). Of the confirmed COVID-19 pneumonia patients, 33.24% had a travel or residence history in the outbreak area (Wuhan) within 14 days, 25.76% had contact with patients with fever or respiratory symptoms, and 36.84% were associated with cluster outbreaks within their families or working places.The patient's characteristics are shown in Table P = 1.000). Reduced or normal WBC counts were found in 340 (94.18%) of the COVID-19 pneumonia patients, and the mean WBC count was 5.40 ± 2.52 (× 109/L) in the COVID-19 pneumonia group, which was remarkably lower than that in the non-COVID-19 pneumonia group . Moreover, reduced lymphocytes were also present in 193 (53.46%) patients; the lymphocytes in COVID-19 pneumonia patients were 1.25 ± 1.03 (×109/L), much lower than those in COVID-19 pneumonia patients . No significant difference was found between the 2 groups, in terms of the CRP level (P = 0.254). Almost all COVID-19 pneumonia patients (94.37%) had pulmonary radiological abnormalities, such as unilateral or bilateral patchy shadows, stiffening of the lungs, or ground-glass opacity on thoracic X-ray or CT.The most common symptoms of COVID-19 pneumonia were fever (48.48%), dry cough (43.77%), fatigue (27.15%), dyspnea (9.14%), and sputum (36.01%); however, fever was not a specific symptom, as it was also common in non-COVID-19 pneumonia patients  in Hubei Province, other areas with persistent local transmission, or a community with confirmed COVID-19 pneumonia cases within 14 days; contact with patients with fever or respiratory symptoms from the outbreak area (Wuhan), areas near of the outbreak area (Wuhan) in Hubei Province, other areas with persistent local transmission, or a community with confirmed cases within 14 days; association with a cluster COVID-19 pneumonia outbreak; exposure to wildlife animals; contact with patients with influenza A or influenza B, which were tested by standard kits; the presence of fever, dry cough, sputum, fatigue, dyspnea, conjunctival congestion, nasal congestion, dizziness or headache, nausea or vomiting, sore throat, and muscle soreness; laboratory tests including WBC, lymphocyte, and neutrophil cell counts and CRP levels; and radiological examination findings including chest X-ray or CT scanning.The logistic regression coefficients are shown in Supplementary Table 1, and the variable selection threshold was manually set to be an absolute value of 0.85 based on the optimal detection principle. The top 10 variables ranked by regression coefficient are shown in Fig. 2.7To identify crucial predictors for the probability of early COVID-19 pneumonia, a random forest model was trained on 80% of the patients using the top 10 variables. We established the importance ranking with the random forest algorithm. High ranks with variable importance are important for tree building and prediction. As shown in Fig. Model performance was assessed on the 20% validation set with ROC and AUC. As shown in Table The predictive power of the prediction model for COVID-19 pneumonia was also subsequently validated by the confusion matrix in the validation set Table . The ove3In this study, we analyzed and compared the epidemiological factors, clinical manifestations, and laboratory and imaging findings between COVID-19 pneumonia patients and suspected COVID-19 pneumonia patients, for whom COVID-19 pneumonia was excluded. We applied the computer-assisted embedding method and random forest algorithm to build and validate a predictive model for early COVID-19 pneumonia. The model included using 4 epidemiological features: travel or residence in the outbreak area (Wuhan); contact with patients with fever or respiratory symptoms from the outbreak area (Wuhan) within 14 days; contact with patients from other areas with persistent local transmission or communities with confirmed cases; and association with a cluster outbreak. The model also included 3 clinical manifestations: fatigue, dyspnea, and muscle soreness; decreased WBC and lymphocyte count; and imaging changes on chest X-ray or CT scanning. The diagnostic performance of this established model was excellent, with an AUC of 0.956. The confusion matrix showed that the overall predictive percentage was 87.4%. A proportion of patients gradually progressed to dyspnea, especially in patients with low immune functions. During treatment, the clinicians found that dyspnea was more likely to occur in populations with low immune functions than in those with normal immune functions. The onset of complications, such as arrhythmia, acute respiratory distress syndrome, and shock was often a sign of poor prognosis.–16Prior studies have shown that most COVID-19 pneumonia patients were characterized by non-specific clinical presentations, such as fever, cough, fatigue, myalgia, diarrhea, nausea, headache, and sore throat in the early stage of the disease. The most frequent imaging findings were patchy/punctate ground-glass opacities in a single lobe or multiple lobes of the lungs. In addition, abnormalities on chest CT scanning could reflect disease progress and severity. COVID-19 infection could also present with normal pulmonary imaging, particularly in the early stage, suggesting a necessity to combine epidemiology with clinical characteristics, laboratory tests, and imaging findings in the screening and diagnosis of COVID-19 pneumonia.Among the laboratory results, the most common laboratory abnormalities were leukopenia and lymphocytopenia. Previous study showed that hypoalbuminemia elevated CRP and lactate dehydrogenase, and decreased CD8 count could be found in some patients as well. RT-PCR has high specificity and sensitivity and has been widely used in determining different coronavirus infections; however, The RT-PCR has some disadvantages, such as time-consuming, test kit shortages, and specimen sampling issues. Furthermore, RT-PCR might show false-negative results when used with unstable kits or non-standardized sampling, and repeated tests are required for a number of patients with initial negative RT-PCR results. Moreover, COVID-19 specific IgM antibodies are usually generated 3–5 days after onset. Some COVID-19 pneumonia patients were diagnosed only based on clinical and imaging findings due to earlier negative testing results for viral RNA in the outbreak area. All of these presentations and disadvantages have made early COVID-19 pneumonia diagnosis challenging, especially during the pandemic, and have prevented timely isolation and early treatment. A rapid screening diagnostic model is urgently needed to distinguish highly suspicious patients in a large scale population to support epidemiologists and help clinicians make early treatment decisions to ultimately reduce patient mortality.RT-PCR can provide confirmation of the diagnosis of COVID-19 pneumonia. Moreover, compared with other prediction models, this model could more effectively identify interactions and nonlinear relationships between variables.We applied the random forest algorithm as a classification method that consists of multiple nodes of decision trees in establishing a concise and accurate diagnosis model. This method has clear advantages including a low chance of overfit, more robust noise reduction, faster training speed, and more prediction accuracy.There were 10 candidate variables selected by the computer-assisted embedding method in this study. We set the random forest parameters to “n_estimators = 40 and criterion = entropy” and calculated the importance of each variable according to the original random-forest algorithm. The rapid screening model was subsequently established based on 4 epidemiological features, 3 clinical characteristics, 2 laboratory test results, and radiographic imaging findings. The area under the ROC curve was 0.956 with a high sensitivity of 83.82% and a high specificity of 89.57%, and the confusion matrix achieved 87.0% accuracy for the screening model to predict early COVID-19 pneumonia.There were some limitations in this study. First, the patients enrolled were limited to China, which may lead to certain regional restrictions, in particular in the epidemiology of possible compromises in different regions. Global studies are needed to assess the application of the model. Second, our research was confined to early and rapid screening, with no adequate information supported on this disease progression and prognosis. Follow-up studies are necessary. Third, we screened only some critical predictors to fit the model and the overall fit of the model could be affected under the limitation of sample size and predictors.In summary, the use of this screening model to rapidly distinguish COVID-19 pneumonia patients from a large scale of suspected patients has great significance in both epidemiology and clinical therapeutics under the circumstance of continuing widespread COVID-19 infection. Unlike the methods of virus isolation, RT-PCR, or specific IgM antibody assays, this early screening model is economical, uncomplicated, and fast, and it may save some medical resources and life by reducing COVID-19 pneumonia mortalities regionally or globally.We would like to thank all the patients and staff at the multicenter hospitals involved in this study.Conceptualization: Hongyi Pan, Wei Zheng, Yining Dai.Data curation: Wei Zheng, Qingqing Wu, Qiaoqiao Yin.Formal analysis: Hongyi Pan, Wei Zheng, Qingqing Wu, Yining Dai, Nannan Sun.Funding acquisition: Yining Dai, Hongying Pan.Investigation: Suxia Bao, Tianchen Hui, Wenhao Wu, Yicheng Huang, Guobo Chen, Qiaoqiao Yin, Rong Yan, Mingshan Wang, Meijuan Chen, Jiajie Zhang, Lixia Yu, Jichan Shi, Nian Fang, Yuefei Shen, Xinsheng Xie, Chunlian Ma, Wanjun Wu, Wenhui Tu, Bin Ju, Haijun Huang, Yongxi Tong, Hongying Pan.Methodology: Suxia Bao, Hongyi Pan, Nannan Sun, Tianchen Hui, Wenhao Wu, Yicheng Huang, Guobo Chen, Qiaoqiao Yin, Rong Yan, Mingshan Wang, Meijuan Chen, Jiajie Zhang, Lixia Yu, Jichan Shi, Nian Fang, Xinsheng Xie, Chunlian Ma, Wanjun Wu, Wenhui Tu, Bin Ju, Haijun Huang, Yongxi Tong, Hongying Pan.Project administration: Xinsheng Xie, Chunlian Ma.Resources: Yicheng Huang, Guobo Chen, Lixia Yu, Yuefei Shen.Software: Suxia Bao, Hongyi Pan, Lijuan Wu, Rong Yan, Mingshan Wang, Bin Ju, Hongying Pan.Supervision: Suxia Bao, Lijuan Wu, Rong Yan, Jiajie Zhang.Validation: Lijuan Wu, Rong Yan, Nian Fang.Visualization: Mingshan Wang, Meijuan Chen, Jiajie Zhang, Jichan Shi.Writing – original draft: Suxia Bao.Writing – review & editing: Haijun Huang, Yongxi Tong, Hongying Pan.