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Anoctamins are a family of Ca2+-activated proteins that may act as ion channels and/or phospholipid scramblases with limited understanding of function and disease association. Here, we identified five de novo and two inherited missense variants in ANO4 (alias TMEM16D) as a cause of fever-sensitive developmental and epileptic or epileptic encephalopathy (DEE/EE) and generalized epilepsy with febrile seizures plus (GEFS+) or temporal lobe epilepsy. In silico modeling of the ANO4 structure predicted that all identified variants lead to destabilization of the ANO4 structure. Four variants are localized close to the Ca2+ binding sites of ANO4, suggesting impaired protein function. Variant mapping to the protein topology suggests a preliminary genotype-phenotype correlation. Moreover, the observation of a heterozygous ANO4 deletion in a healthy individual suggests a dysfunctional protein as disease mechanism rather than haploinsufficiency. To test this hypothesis, we examined mutant ANO4 functional properties in a heterologous expression system by patch-clamp recordings, immunocytochemistry, and surface expression of annexin A5 as a measure of phosphatidylserine scramblase activity. All ANO4 variants showed severe loss of ion channel function and DEE/EE associated variants presented mild loss of surface expression due to impaired plasma membrane trafficking. Increased levels of Ca2+-independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca2+-dependent scramblase activity were observed. Co-transfection with ANO4 wild-type suggested a dominant-negative effect. In summary, we expand the genetic base for both encephalopathic sporadic and inherited fever-sensitive epilepsies and link germline variants in ANO4 to a hereditary disease.
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Anoctaminas , Mutación Missense , Humanos , Anoctaminas/genética , Anoctaminas/metabolismo , Mutación Missense/genética , Masculino , Femenino , Epilepsia/genética , Niño , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de Transferencia de Fosfolípidos/metabolismo , Estudios de Asociación Genética , Linaje , Calcio/metabolismo , Genes Dominantes , Preescolar , Células HEK293 , AdolescenteRESUMEN
Zinc and RING finger 3 (ZNRF3) is a negative-feedback regulator of Wnt/ß-catenin signaling, which plays an important role in human brain development. Although somatically frequently mutated in cancer, germline variants in ZNRF3 have not been established as causative for neurodevelopmental disorders (NDDs). We identified 12 individuals with ZNRF3 variants and various phenotypes via GeneMatcher/Decipher and evaluated genotype-phenotype correlation. We performed structural modeling and representative deleterious and control variants were assessed using in vitro transcriptional reporter assays with and without Wnt-ligand Wnt3a and/or Wnt-potentiator R-spondin (RSPO). Eight individuals harbored de novo missense variants and presented with NDD. We found missense variants associated with macrocephalic NDD to cluster in the RING ligase domain. Structural modeling predicted disruption of the ubiquitin ligase function likely compromising Wnt receptor turnover. Accordingly, the functional assays showed enhanced Wnt/ß-catenin signaling for these variants in a dominant negative manner. Contrarily, an individual with microcephalic NDD harbored a missense variant in the RSPO-binding domain predicted to disrupt binding affinity to RSPO and showed attenuated Wnt/ß-catenin signaling in the same assays. Additionally, four individuals harbored de novo truncating or de novo or inherited large in-frame deletion variants with non-NDD phenotypes, including heart, adrenal, or nephrotic problems. In contrast to NDD-associated missense variants, the effects on Wnt/ß-catenin signaling were comparable between the truncating variant and the empty vector and between benign variants and the wild type. In summary, we provide evidence for mirror brain size phenotypes caused by distinct pathomechanisms in Wnt/ß-catenin signaling through protein domain-specific deleterious ZNRF3 germline missense variants.
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Mature B cells express immunoglobulin M (IgM)- and IgD-isotype B cell antigen receptors, but the importance of IgD for B cell function has been unclear. By using a cellular in vitro system and corresponding mouse models, we found that antigens with low valence activated IgM receptors but failed to trigger IgD signaling, whereas polyvalent antigens activated both receptor types. Investigations of the molecular mechanism showed that deletion of the IgD-specific hinge region rendered IgD responsive to monovalent antigen, whereas transferring the hinge to IgM resulted in responsiveness only to polyvalent antigen. Our data suggest that the increased IgD/IgM ratio on conventional B-2 cells is important for preferential immune responses to antigens in immune complexes, and that the increased IgM expression on B-1 cells is essential for B-1 cell homeostasis and function.
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Linfocitos B/inmunología , Inmunoglobulina D/inmunología , Inmunoglobulina M/inmunología , Animales , Complejo Antígeno-Anticuerpo/inmunología , Antígenos/inmunología , Sitios de Unión de Anticuerpos/inmunología , Señalización del Calcio/genética , Diferenciación Celular , Línea Celular , Exones de la Región Bisagra/genética , Homeostasis/genética , Inmunidad Humoral/genética , Inmunoglobulina D/genética , Inmunoglobulina M/genética , Ratones , Ratones Noqueados , Ingeniería de Proteínas , Eliminación de Secuencia/genéticaRESUMEN
Calcium (Ca2+) is a universal second messenger involved in synaptogenesis and cell survival; consequently, its regulation is important for neurons. ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) belongs to the family of ATP-driven calmodulin-dependent Ca2+ pumps that participate in the regulation of intracellular free Ca2+. Here, we clinically describe a cohort of 12 unrelated individuals with variants in ATP2B1 and an overlapping phenotype of mild to moderate global development delay. Additional common symptoms include autism, seizures, and distal limb abnormalities. Nine probands harbor missense variants, seven of which were in specific functional domains, and three individuals have nonsense variants. 3D structural protein modeling suggested that the variants have a destabilizing effect on the protein. We performed Ca2+ imaging after introducing all nine missense variants in transfected HEK293 cells and showed that all variants lead to a significant decrease in Ca2+ export capacity compared with the wild-type construct, thus proving their pathogenicity. Furthermore, we observed for the same variant set an incorrect intracellular localization of ATP2B1. The genetic findings and the overlapping phenotype of the probands as well as the functional analyses imply that de novo variants in ATP2B1 lead to a monogenic form of neurodevelopmental disorder.
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Discapacidad Intelectual , Malformaciones del Sistema Nervioso , Trastornos del Neurodesarrollo , Células HEK293 , Humanos , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Mutación Missense/genética , Trastornos del Neurodesarrollo/genética , Fenotipo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genéticaRESUMEN
PPFIBP1 encodes for the liprin-ß1 protein, which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster. By exome and genome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 16 individuals from 12 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy, and progressive microcephaly. Further common clinical findings included muscular hyper- and hypotonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, ventriculomegaly, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM domain region that is essential for protein-protein interaction. For further insight into the effects of PPFIBP1 loss of function, we performed automated behavioral phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model, which revealed defects in spontaneous and light-induced behavior and confirmed resistance to the acetylcholinesterase inhibitor aldicarb, suggesting a defect in the neuronal presynaptic zone. In conclusion, we establish bi-allelic loss-of-function variants in PPFIBP1 as a cause of an autosomal recessive severe neurodevelopmental disorder with early-onset epilepsy, microcephaly, and periventricular calcifications.
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Epilepsia , Microcefalia , Malformaciones del Sistema Nervioso , Trastornos del Neurodesarrollo , Acetilcolinesterasa/genética , Animales , Drosophila melanogaster/genética , Epilepsia/genética , Pérdida de Heterocigocidad , Microcefalia/genética , Trastornos del Neurodesarrollo/genética , LinajeRESUMEN
Leukodystrophies are rare genetic white matter disorders that have been regarded as mainly occurring in childhood. Recent years altered this perception, as a growing number of leukodystrophies was described to have an onset at adult ages. Still, many adult patients presenting with white matter changes remain without a specific molecular diagnosis. We describe a novel adult onset leukodystrophy in 16 patients from eight families carrying one of four different stop-gain or frameshift dominant variants in the CST3 gene. Clinical and radiological features differ markedly from the previously described Icelandic Cerebral Amyloid Angiopathy that was found in patients carrying p.Leu68Asn substitution in CST3. The clinical phenotype consists of recurrent episodes of hemiplegic migraine associated with transient unilateral focal deficits and slowly progressing motor symptoms and cognitive decline in mid-old adult ages. In addition, in some cases acute onset clinical deterioration led to a prolonged episode with reduced consciousness and even early death. Radiologically, pathognomonic changes are found at typical predilection sites involving the deep cerebral white matter sparing a periventricular and directly subcortical rim, the middle blade of corpus callosum, posterior limb of the internal capsule, middle cerebellar peduncles, cerebral peduncles, and specifically the globus pallidus. Histopathologic characterization in two autopsy cases did not reveal angiopathy, but instead micro- to macrocystic degeneration of the white matter. Astrocytes were activated at early stages and later on displayed severe degeneration and loss. In addition, despite loss of myelin, elevated numbers of partly apoptotic oligodendrocytes were observed. A structural comparison of the variants in CST3 suggests that specific truncations of Cystatin C result in an abnormal function, possibly by rendering the protein more prone to aggregation. Future studies are required to confirm the assumed effect on the protein and to determine pathophysiologic downstream events at the cellular level.
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Recently, others and we identified de novo FBXO11 (F-Box only protein 11) variants as causative for a variable neurodevelopmental disorder (NDD). We now assembled clinical and mutational information on 23 additional individuals. The phenotypic spectrum remains highly variable, with developmental delay and/or intellectual disability as the core feature and behavioral anomalies, hypotonia and various facial dysmorphism as frequent aspects. The mutational spectrum includes intragenic deletions, likely gene disrupting and missense variants distributed across the protein. To further characterize the functional consequences of FBXO11 missense variants, we analyzed their effects on protein expression and localization by overexpression of 17 different mutant constructs in HEK293 and HeLa cells. We found that the majority of missense variants resulted in subcellular mislocalization and/or reduced FBXO11 protein expression levels. For instance, variants located in the nuclear localization signal and the N-terminal F-Box domain lead to altered subcellular localization with exclusion from the nucleus or the formation of cytoplasmic aggregates and to reduced protein levels in western blot. In contrast, variants localized in the C-terminal Zn-finger UBR domain lead to an accumulation in the cytoplasm without alteration of protein levels. Together with the mutational data, our functional results suggest that most missense variants likely lead to a loss of the original FBXO11 function and thereby highlight haploinsufficiency as the most likely disease mechanism for FBXO11-associated NDDs.
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Proteínas F-Box , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Proteínas F-Box/genética , Células HEK293 , Células HeLa , Humanos , Discapacidad Intelectual/genética , Mutación Missense/genética , Trastornos del Neurodesarrollo/genética , Proteína-Arginina N-Metiltransferasas/genéticaRESUMEN
ARID1B is the most frequently mutated gene in Coffin-Siris syndrome (CSS). To date, the vast majority of causative variants reported in ARID1B are truncating, leading to nonsense-mediated mRNA decay. In the absence of experimental data, only few ARID1B amino acid substitutions have been classified as pathogenic, mainly based on clinical data and their de novo occurrence, while most others are currently interpreted as variants of unknown significance. The present study substantiates the pathogenesis of ARID1B non-truncating/NMD-escaping variants located in the SMARCA4-interacting EHD2 and DNA-binding ARID domains. Overexpression assays in cell lines revealed that the majority of EHD2 variants lead to protein misfolding and formation of cytoplasmic aggresomes surrounded by vimentin cage-like structures and co-localizing with the microtubule organisation center. ARID domain variants exhibited not only aggresomes, but also nuclear aggregates, demonstrating robust pathological effects. Protein levels were not compromised, as shown by quantitative western blot analysis. In silico structural analysis predicted the exposure of amylogenic segments in both domains due to the nearby variants, likely causing this aggregation. Genome-wide transcriptome and methylation analysis in affected individuals revealed expression and methylome patterns consistent with those of the pathogenic haploinsufficiency ARID1B alterations in CSS cases. These results further support pathogenicity and indicate two approaches for disambiguation of such variants in everyday practice. The few affected individuals harbouring EHD2 non-truncating variants described to date exhibit mild CSS clinical traits. In summary, this study paves the way for the re-evaluation of previously unclear ARID1B non-truncating variants and opens a new era in CSS genetic diagnosis.
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Proteínas de Unión al ADN , Cara , Deformidades Congénitas de la Mano , Discapacidad Intelectual , Micrognatismo , Cuello , Factores de Transcripción , Humanos , Discapacidad Intelectual/genética , Micrognatismo/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Deformidades Congénitas de la Mano/genética , Cuello/anomalías , Cara/anomalías , Anomalías Múltiples/genética , Mutación , Masculino , Agregado de ProteínasRESUMEN
Neurons form the basic anatomical and functional structure of the nervous system, and defects in neuronal differentiation or formation of neurites are associated with various psychiatric and neurodevelopmental disorders. Dynamic changes in the cytoskeleton are essential for this process, which is, inter alia, controlled by the dedicator of cytokinesis 4 (DOCK4) through the activation of RAC1. Here, we clinically describe 7 individuals (6 males and one female) with variants in DOCK4 and overlapping phenotype of mild to severe global developmental delay. Additional symptoms include coordination or gait abnormalities, microcephaly, nonspecific brain malformations, hypotonia and seizures. Four individuals carry missense variants (three of them detected de novo) and three individuals carry null variants (two of them maternally inherited). Molecular modeling of the heterozygous missense variants suggests that the majority of them affect the globular structure of DOCK4. In vitro functional expression studies in transfected Neuro-2A cells showed that all missense variants impaired neurite outgrowth. Furthermore, Dock4 knockout Neuro-2A cells also exhibited defects in promoting neurite outgrowth. Our results, including clinical, molecular and functional data, suggest that loss-of-function variants in DOCK4 probable cause a variable spectrum of a novel neurodevelopmental disorder with microcephaly.
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Proteínas Activadoras de GTPasa , Heterocigoto , Microcefalia , Mutación Missense , Trastornos del Neurodesarrollo , Humanos , Microcefalia/genética , Femenino , Masculino , Preescolar , Proteínas Activadoras de GTPasa/genética , Niño , Trastornos del Neurodesarrollo/genética , Mutación con Pérdida de Función , Animales , Discapacidades del Desarrollo/genética , Ratones , Lactante , Fenotipo , AdolescenteRESUMEN
Single nucleotide polymorphisms (SNPs) account for significant genomic variability in microbes, including the highly diverse gastric pathogen Helicobacter pylori. However, data on the effects of specific SNPs in pathogen-host interactions are scarce. Recent functional studies unravelled how a serine/leucine polymorphism in serine protease HtrA affects the formation of proteolytically active trimers and modulates cleavage of host cell-to-cell junction proteins during infection. A similar serine/leucine mutation in the carbohydrate binding domain of the adhesin BabA controls binding of ABO blood group antigens, enabling binding of either only the short Lewis b/H antigens of blood group O or also the larger antigens of blood groups A and B. Here we summarize the functional importance of these two remarkable bacterial SNPs and their effect on the outcome of pathogen-host interactions.
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Adhesinas Bacterianas , Helicobacter pylori , Leucina , Serina , Helicobacter pylori/genética , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Humanos , Serina/genética , Serina/metabolismo , Leucina/genética , Leucina/metabolismo , Polimorfismo de Nucleótido Simple/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/genética , AnimalesRESUMEN
Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of thermodynamic parameters of NEC formation of three prototypical α-, ß-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in ß- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.
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Herpesvirus Humano 3 , Membrana Nuclear , Proteínas Virales , Cristalografía por Rayos X , Herpesvirus Humano 3/química , Herpesvirus Humano 3/genética , Humanos , Membrana Nuclear/química , Membrana Nuclear/genética , Proteínas Virales/química , Proteínas Virales/genética , Liberación del VirusRESUMEN
Generalized pustular psoriasis (GPP) is a severe multi-systemic inflammatory disease characterized by neutrophilic pustulosis and triggered by pro-inflammatory IL-36 cytokines in skin. While 19%-41% of affected individuals harbor bi-allelic mutations in IL36RN, the genetic cause is not known in most cases. To identify and characterize new pathways involved in the pathogenesis of GPP, we performed whole-exome sequencing in 31 individuals with GPP and demonstrated effects of mutations in MPO encoding the neutrophilic enzyme myeloperoxidase (MPO). We discovered eight MPO mutations resulting in MPO -deficiency in neutrophils and monocytes. MPO mutations, primarily those resulting in complete MPO deficiency, cumulatively associated with GPP (p = 1.85E-08; OR = 6.47). The number of mutant MPO alleles significantly differed between 82 affected individuals and >4,900 control subjects (p = 1.04E-09); this effect was stronger when including IL36RN mutations (1.48E-13) and correlated with a younger age of onset (p = 0.0018). The activity of four proteases, previously implicated as activating enzymes of IL-36 precursors, correlated with MPO deficiency. Phorbol-myristate-acetate-induced formation of neutrophil extracellular traps (NETs) was reduced in affected cells (p = 0.015), and phagocytosis assays in MPO-deficient mice and human cells revealed altered neutrophil function and impaired clearance of neutrophils by monocytes (efferocytosis) allowing prolonged neutrophil persistence in inflammatory skin. MPO mutations contribute significantly to GPP's pathogenesis. We implicate MPO as an inflammatory modulator in humans that regulates protease activity and NET formation and modifies efferocytosis. Our findings indicate possible implications for the application of MPO inhibitors in cardiovascular diseases. MPO and affected pathways represent attractive targets for inducing resolution of inflammation in neutrophil-mediated skin diseases.
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Inflamación/genética , Interleucinas/genética , Peroxidasa/genética , Psoriasis/genética , Enfermedades de la Piel/genética , Adulto , Animales , Citocinas/genética , Trampas Extracelulares/genética , Femenino , Humanos , Inflamación/patología , Interleucina-1/genética , Interleucinas/metabolismo , Masculino , Ratones , Mutación/genética , Neutrófilos/metabolismo , Psoriasis/patología , Enfermedades Raras/enzimología , Enfermedades Raras/genética , Enfermedades Raras/patología , Piel/enzimología , Piel/patología , Enfermedades de la Piel/patologíaRESUMEN
TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.
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COVID-19 , Glicoproteína de la Espiga del Coronavirus , Animales , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Humanos , Ratones , SARS-CoV-2RESUMEN
PURPOSE: The study aimed to clinically and molecularly characterize the neurodevelopmental disorder associated with heterozygous de novo variants in CNOT9. METHODS: Individuals were clinically examined. Variants were identified using exome or genome sequencing. These variants were evaluated using in silico predictions, and their functional relevance was further assessed by molecular models and research in the literature. The variants have been classified according to the criteria of the American College of Medical Genetics. RESULTS: We report on 7 individuals carrying de novo missense variants in CNOT9, p.(Arg46Gly), p.(Pro131Leu), and p.(Arg227His), and, recurrent in 4 unrelated individuals, p.(Arg292Trp). All affected persons have developmental delay/intellectual disability, with 5 of them showing seizures. Other symptoms include muscular hypotonia, facial dysmorphism, and behavioral abnormalities. Molecular modeling predicted that the variants are damaging and would lead to reduced protein stability or impaired recognition of interaction partners. Functional analyses in previous studies showed a pathogenic effect of p.(Pro131Leu) and p.(Arg227His). CONCLUSION: We propose CNOT9 as a novel gene for neurodevelopmental disorder and epilepsy.
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Epilepsia , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Humanos , Epilepsia/genética , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Mutación Missense/genética , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Fenotipo , Convulsiones/genéticaRESUMEN
PURPOSE: LHX2 encodes the LIM homeobox 2 transcription factor (LHX2), which is highly expressed in brain and well conserved across species, but it has not been clearly linked to neurodevelopmental disorders (NDDs) to date. METHODS: Through international collaboration, we identified 19 individuals from 18 families with variable neurodevelopmental phenotypes, carrying a small chromosomal deletion, likely gene-disrupting or missense variants in LHX2. Functional consequences of missense variants were investigated in cellular systems. RESULTS: Affected individuals presented with developmental and/or behavioral abnormalities, autism spectrum disorder, variable intellectual disability, and microcephaly. We observed nucleolar accumulation for 2 missense variants located within the DNA-binding HOX domain, impaired interaction with co-factor LDB1 for another variant located in the protein-protein interaction-mediating LIM domain, and impaired transcriptional activation by luciferase assay for 4 missense variants. CONCLUSION: We implicate LHX2 haploinsufficiency by deletion and likely gene-disrupting variants as causative for a variable NDD. Our findings suggest a loss-of-function mechanism also for likely pathogenic LHX2 missense variants. Together, our observations underscore the importance of LHX2 in the nervous system and for variable neurodevelopmental phenotypes.
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Trastorno del Espectro Autista , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Humanos , Proteínas con Homeodominio LIM/genética , Trastorno del Espectro Autista/genética , Haploinsuficiencia/genética , Trastornos del Neurodesarrollo/patología , Factores de Transcripción/genética , Discapacidad Intelectual/genética , Discapacidad Intelectual/complicacionesRESUMEN
OBJECTIVE: Rare inherited missense variants in SLC32A1, the gene that encodes the vesicular gamma-aminobutyric acid (GABA) transporter, have recently been shown to cause genetic epilepsy with febrile seizures plus. We aimed to clarify if de novo missense variants in SLC32A1 can also cause epilepsy with impaired neurodevelopment. METHODS: Using exome sequencing, we identified four individuals with a developmental and epileptic encephalopathy and de novo missense variants in SLC32A1. To assess causality, we performed functional evaluation of the identified variants in a murine neuronal cell culture model. RESULTS: The main phenotype comprises moderate-to-severe intellectual disability, infantile-onset epilepsy within the first 18 months of life, and a choreiform, dystonic, or dyskinetic movement disorder. In silico modeling and functional analyses reveal that three of these variants, which are located in helices that line the putative GABA transport pathway, result in reduced quantal size, consistent with impaired filling of synaptic vesicles with GABA. The fourth variant, located in the vesicular gamma-aminobutyric acid N-terminus, does not affect quantal size, but increases presynaptic release probability, leading to more severe synaptic depression during high-frequency stimulation. Thus, variants in vesicular gamma-aminobutyric acid can impair GABAergic neurotransmission through at least two mechanisms, by affecting synaptic vesicle filling and by altering synaptic short-term plasticity. INTERPRETATION: This work establishes de novo missense variants in SLC32A1 as a novel cause of a developmental and epileptic encephalopathy. SUMMARY FOR SOCIAL MEDIA IF PUBLISHED: @platzer_k @lemke_johannes @RamiJamra @Nirgalito @GeneDx The SLC family 32 Member 1 (SLC32A1) is the only protein identified to date, that loads gamma-aminobutyric acid (GABA) and glycine into synaptic vesicles, and is therefore also known as the vesicular GABA transporter (VGAT) or vesicular inhibitory amino acid transporter (VIAAT). Rare inherited missense variants in SLC32A1, the gene that encodes VGAT/vesicular inhibitory amino acid transporter, have recently been shown to cause genetic epilepsy with febrile seizures plus. We aimed to clarify if de novo missense variants in SLC32A1 can also cause epilepsy with impaired neurodevelopment. We report on four individuals with de novo missense variants in SLC32A1 and a developmental and epileptic encephalopathy with infantile onset epilepsy. We establish causality of the variants via in silico modeling and their functional evaluation in a murine neuronal cell culture model. SLC32A1 variants represent a novel genetic etiology in neurodevelopmental disorders with epilepsy and a new GABA-related disease mechanism. ANN NEUROL 2022;92:958-973.
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Epilepsia Generalizada , Epilepsia , Convulsiones Febriles , Animales , Ratones , Epilepsia Generalizada/genética , Epilepsia/genética , Transmisión Sináptica/genética , Ácido gamma-Aminobutírico/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismoRESUMEN
BACKGROUND: SARS-CoV-2 has caused a worldwide pandemic since December 2019 and the search for pharmaceutical targets against COVID-19 remains an important challenge. Here, we studied the envelope protein E of SARS-CoV and SARS-CoV-2, a highly conserved 75-76 amino acid viroporin that is crucial for virus assembly and release. E protein channels were recombinantly expressed in HEK293 cells, a membrane-directing signal peptide ensured transfer to the plasma membrane. METHODS: Viroporin channel activity of both E proteins was investigated using patch-clamp electrophysiology in combination with a cell viability assay. We verified inhibition by classical viroporin inhibitors amantadine, rimantadine and 5-(N,N-hexamethylene)-amiloride, and tested four ivermectin derivatives. RESULTS: Classical inhibitors showed potent activity in patch-clamp recordings and viability assays. In contrast, ivermectin and milbemycin inhibited the E channel in patch-clamp recordings but displayed only moderate activity on the E protein in the cell viability assay, which is also sensitive to general cytotoxic activity of the tested compounds. Nemadectin and ivermectin aglycon were inactive. All ivermectin derivatives were cytotoxic at concentrations > 5 µM, i.e. below the level required for E protein inhibition. CONCLUSIONS: This study demonstrates direct inhibition of the SARS-CoV-2 E protein by classical viroporin inhibitors. Ivermectin and milbemycin inhibit the E protein channel but their cytotoxicity argues against clinical application.
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COVID-19 , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Humanos , Proteínas Viroporinas , SARS-CoV-2 , Supervivencia Celular , Células HEK293 , Ivermectina/farmacologíaRESUMEN
The human pathogen Helicobacter pylori is a major risk factor for gastric disease development. Serine protease HtrA is an important bacterial virulence factor that cleaves the cell junction proteins occludin, claudin-8 and E-cadherin, which causes gastric tissue damage. Using casein zymography, we discovered that HtrA trimer stability varies in clinical H. pylori strains. Subsequent sequence analyses revealed that HtrA trimer stability correlated with the presence of leucine or serine residue at position 171. The importance of these amino acids in determining trimer stability was confirmed by leucine-to-serine swapping experiments using isogenic H. pylori mutant strains as well as recombinant HtrA proteins. In addition, this sequence position displays a high sequence variability among various bacterial species, but generally exhibits a preference for hydrophilic amino acids. This natural L/S171 polymorphism in H. pylori may affect the protease activity of HtrA during infection, which could be of clinical importance and may determine gastric disease development.
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Helicobacter pylori , Humanos , Proteínas Bacterianas/metabolismo , Leucina/genética , Leucina/metabolismo , Serina Proteasas/genética , Serina Proteasas/metabolismo , Proteínas Recombinantes/genética , Mutación , Serina/genética , Serina/metabolismoRESUMEN
The Kennedy pathways catalyse the de novo synthesis of phosphatidylcholine and phosphatidylethanolamine, the most abundant components of eukaryotic cell membranes. In recent years, these pathways have moved into clinical focus because four of ten genes involved have been associated with a range of autosomal recessive rare diseases such as a neurodevelopmental disorder with muscular dystrophy (CHKB), bone abnormalities and cone-rod dystrophy (PCYT1A) and spastic paraplegia (PCYT2, SELENOI). We identified six individuals from five families with bi-allelic variants in CHKA presenting with severe global developmental delay, epilepsy, movement disorders and microcephaly. Using structural molecular modelling and functional testing of the variants in a cell-based Saccharomyces cerevisiae model, we determined that these variants reduce the enzymatic activity of CHKA and confer a significant impairment of the first enzymatic step of the Kennedy pathway. In summary, we present CHKA as a novel autosomal recessive gene for a neurodevelopmental disorder with epilepsy and microcephaly.
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Colina Quinasa , Epilepsia , Microcefalia , Malformaciones del Sistema Nervioso , Trastornos del Neurodesarrollo , Alelos , Colina Quinasa/genética , Epilepsia/genética , Humanos , Microcefalia/complicaciones , Microcefalia/genética , Malformaciones del Sistema Nervioso/genética , Trastornos del Neurodesarrollo/genéticaRESUMEN
In malignant melanoma, a highly aggressive form of skin cancer, many microRNAs are aberrantly expressed contributing to tumorigenesis and progression. Further, deregulation of microRNA processing enzymes, like the miRNA-binding protein Argonaute 2, significantly impacts microRNA function. This study characterizes a novel splice variant of Argonaut 2, AGO2-ex1/3. AGO2-ex1/3 is substantially expressed in different melanoma cell lines and patient-derived tissue samples. It is a mature mRNA, which is translated into an N-terminally truncated Argonaute 2 protein form. Molecular dynamics simulations show that the PAZ, MID, and PIWI domain largely retain their structure in AGO2-ex1/3 and that the truncation of the N-terminus leads to an increased interdomain flexibility. Expression of AGO2-ex1/3 provides a survival advantage for melanoma cells while the knockdown causes significantly reduced proliferation and increases apoptosis. RNA-sequencing revealed that in cells lacking AGO2-ex1/3 expression many miRNA target genes are deregulated, implicating a considerable role of AGO2-ex1/3 for miRNA function. This study inaugurates insights into an important role of a so far unknown splice variant of Argonaute 2 for the miRNA pathway as well as the mechanisms which drive growth and survival of melanoma cells. This knowledge provides the basis for potential new promising therapeutic targets focusing on small RNA-mediated gene regulation in melanoma.