RESUMEN
Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.
Asunto(s)
Edema Encefálico , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias , Humanos , Proteínas de la Membrana/genética , Edema Encefálico/genética , Edema Encefálico/metabolismo , Mutación/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Encéfalo/metabolismo , Astrocitos/metabolismo , Acuaporina 4/genética , Acuaporina 4/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Objective: Vanishing white matter (VWM) is a genetic brain white matter disorder caused by mutations in eIF2B. eIF2B is central in the integrated stress response (ISR), during which its activity is inhibited by various cellular stresses. VWM is a chronic progressive disease with episodes of rapid neurological deterioration provoked by stresses. VWM patients and VWM mouse models show ISR deregulation in brain, correlating with chronic disease development. ISR inhibition ameliorates the chronic disease in VWM mice. The subacute deteriorations have not been modeled yet. We hypothesized that ISR activation could worsen disease progression in mice and model the episodic neurological deterioration.Method: We chose to activate the ISR by subjecting wild-type (wt) and VWM mice to an isocaloric low protein diet. This model would allow us to investigate the contribution of ISR activation in subacute decline in VWM.Results: We found that the low protein diet did not significantly affect amino acid levels nor ISR levels in wt and VWM mouse brain. Our study serendipitously led to the discovery of increased levels of glycine, asparagine and Fgf21 mRNA in VWM mouse brain irrespective of the dietary protein content. Strikingly, the ISR was not activated by the low protein diet in the liver of VWM in contrast to wt mice, due to a modest ISR deregulation in this organ.Discussion: A model for subacute neurological deterioration in VWM was not established. Possibly, ISR deregulation in VWM results in reduced ISR responsiveness.
Asunto(s)
Leucoencefalopatías , Sustancia Blanca , Animales , Encéfalo/metabolismo , Enfermedad Crónica , Dieta con Restricción de Proteínas , Modelos Animales de Enfermedad , Factor 2B Eucariótico de Iniciación/genética , Factor 2B Eucariótico de Iniciación/metabolismo , Humanos , Leucoencefalopatías/genética , Leucoencefalopatías/metabolismo , Hígado/metabolismo , Ratones , Sustancia Blanca/metabolismoRESUMEN
Human immunodeficiency virus (HIV) uses the ESCRT (endosomal sorting complexes required for transport) protein pathway to bud from infected cells. Despite the roles of ESCRT-I and -III in HIV budding being firmly established, participation of ESCRT-II in this process has been controversial. EAP45 is a critical component of ESCRT-II. Previously, we utilised a CRISPR-Cas9 EAP45 knockout cell line to assess the involvement of ESCRT-II in HIV replication. We demonstrated that the absence of ESCRT-II impairs HIV budding. Here, we show that virus spread is also defective in physiologically relevant CRISPR/Cas9 EAP45 knockout T cells. We further show reappearance of efficient budding by re-introduction of EAP45 expression into EAP45 knockout cells. Using expression of selected mutants of EAP45, we dissect the domain requirement responsible for this function. Our data show at the steady state that rescue of budding is only observed in the context of a Gag/Pol, but not a Gag expressor, indicating that the size of cargo determines the usage of ESCRT-II. EAP45 acts through the YPXL-ALIX pathway as partial rescue is achieved in a PTAP but not a YPXL mutant virus. Our study clarifies the role of ESCRT-II in the late stages of HIV replication and reinforces the notion that ESCRT-II plays an integral part during this process as it does in sorting ubiquitinated cargos and in cytokinesis.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Infecciones por VIH/virología , VIH-1/metabolismo , Sistemas CRISPR-Cas , Línea Celular , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Linfocitos T , Ubiquitina/metabolismo , Replicación ViralRESUMEN
BACKGROUND: HIV-1 does not encode a helicase and hijacks those of the cell for efficient replication. We and others previously showed that the DEAD box helicase, DDX5, is an essential HIV dependency factor. DDX5 was recently shown to be associated with the 7SK snRNP. Cellular positive transcription elongation factor b (P-TEFb) is bound in an inactive form with HEXIM1/2 on 7SK snRNP. The Tat/P-TEFb complex is essential for efficient processivity of Pol II in HIV-1 transcription elongation and Tat competes with HEXIM1/2 for P-TEFb. We investigated the precise role of DDX5 in HIV replication using siRNA mediated knockdown and rescue with DDX5 mutants which prevent protein-protein interactions and RNA and ATP binding. RESULTS: We demonstrate a critical role for DDX5 in the Tat/HEXIM1 interaction. DDX5 acts to potentiate Tat activity and can bind both Tat and HEXIM1 suggesting it may facilitate the dissociation of HEXIM1/2 from the 7SK-snRNP complex, enhancing Tat/P-TEFb availability. We show knockdown of DDX5 in a T cell line significantly reduces HIV-1 infectivity and viral protein production. This activity is unique to DDX5 and cannot be substituted by its close paralog DDX17. Overexpression of DDX5 stimulates the Tat/LTR promoter but suppresses other cellular and viral promoters. Individual mutations of conserved ATP binding, RNA binding, helicase related or protein binding motifs within DDX5 show that the N terminal RNA binding motifs, the Walker B and the glycine doublet motifs are essential for this function. The Walker A and RNA binding motifs situated on the transactivation domain are however dispensable. CONCLUSION: DDX5 is an essential cellular factor for efficient HIV transcription elongation. It interacts with Tat and may potentiate the availability of P-TEFb through sequestering HEXIM1.
Asunto(s)
ARN Helicasas DEAD-box/genética , VIH-1/genética , Factores de Transcripción/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Regulación Viral de la Expresión Génica , Células HeLa , Humanos , Unión ProteicaRESUMEN
Cytochrome c oxidase (COX) deficiency is a frequent biochemical abnormality in mitochondrial disorders, but a large fraction of cases remains genetically undetermined. Whole-exome sequencing led to the identification of APOPT1 mutations in two Italian sisters and in a third Turkish individual presenting severe COX deficiency. All three subjects presented a distinctive brain MRI pattern characterized by cavitating leukodystrophy, predominantly in the posterior region of the cerebral hemispheres. We then found APOPT1 mutations in three additional unrelated children, selected on the basis of these particular MRI features. All identified mutations predicted the synthesis of severely damaged protein variants. The clinical features of the six subjects varied widely from acute neurometabolic decompensation in late infancy to subtle neurological signs, which appeared in adolescence; all presented a chronic, long-surviving clinical course. We showed that APOPT1 is targeted to and localized within mitochondria by an N-terminal mitochondrial targeting sequence that is eventually cleaved off from the mature protein. We then showed that APOPT1 is virtually absent in fibroblasts cultured in standard conditions, but its levels increase by inhibiting the proteasome or after oxidative challenge. Mutant fibroblasts showed reduced amount of COX holocomplex and higher levels of reactive oxygen species, which both shifted toward control values by expressing a recombinant, wild-type APOPT1 cDNA. The shRNA-mediated knockdown of APOPT1 in myoblasts and fibroblasts caused dramatic decrease in cell viability. APOPT1 mutations are responsible for infantile or childhood-onset mitochondrial disease, hallmarked by the combination of profound COX deficiency with a distinctive neuroimaging presentation.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Complejo IV de Transporte de Electrones/metabolismo , Leucoencefalopatías/genética , Leucoencefalopatías/patología , Proteínas Mitocondriales/genética , Mutación/genética , Adolescente , Adulto , Células Cultivadas , Niño , Preescolar , Deficiencia de Citocromo-c Oxidasa , Complejo IV de Transporte de Electrones/genética , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Lactante , Leucoencefalopatías/enzimología , Imagen por Resonancia Magnética , Masculino , Mitocondrias/metabolismo , Mioblastos/metabolismo , Mioblastos/patologíaRESUMEN
This study focused on the molecular characterization of patients with leukoencephalopathy associated with a specific biochemical defect of mitochondrial respiratory chain complex III, and explores the impact of a distinct magnetic resonance imaging pattern of leukoencephalopathy to detect biallelic mutations in LYRM7 in patients with biochemically unclassified leukoencephalopathy. 'Targeted resequencing' of a custom panel including genes coding for mitochondrial proteins was performed in patients with complex III deficiency without a molecular genetic diagnosis. Based on brain magnetic resonance imaging findings in these patients, we selected additional patients from a database of unclassified leukoencephalopathies who were scanned for mutations in LYRM7 by Sanger sequencing. Targeted sequencing revealed homozygous mutations in LYRM7, encoding mitochondrial LYR motif-containing protein 7, in four patients from three unrelated families who had a leukoencephalopathy and complex III deficiency. Two subjects harboured previously unreported variants predicted to be damaging, while two siblings carried an already reported pathogenic homozygous missense change. Sanger sequencing performed in the second cohort of patients revealed LYRM7 mutations in three additional patients, who were selected on the basis of the magnetic resonance imaging pattern. All patients had a consistent magnetic resonance imaging pattern of progressive signal abnormalities with multifocal small cavitations in the periventricular and deep cerebral white matter. Early motor development was delayed in half of the patients. All patients but one presented with subacute neurological deterioration in infancy or childhood, preceded by a febrile infection, and most patients had repeated episodes of subacute encephalopathy with motor regression, irritability and stupor or coma resulting in major handicap or death. LYRM7 protein was strongly reduced in available samples from patients; decreased complex III holocomplex was observed in fibroblasts from a patient carrying a splice site variant; functional studies in yeast confirmed the pathogenicity of two novel mutations. Mutations in LYRM7 were previously found in a single patient with a severe form of infantile onset encephalopathy. We provide new molecular, clinical, and neuroimaging data allowing us to characterize more accurately the molecular spectrum of LYRM7 mutations highlighting that a distinct and recognizable magnetic resonance imaging pattern is related to mutations in this gene. Inter- and intrafamilial variability exists and we observed one patient who was asymptomatic by the age of 6 years.
Asunto(s)
Leucoencefalopatía Multifocal Progresiva/diagnóstico , Leucoencefalopatía Multifocal Progresiva/genética , Imagen por Resonancia Magnética , Proteínas Mitocondriales/genética , Chaperonas Moleculares/genética , Mutación/genética , Adolescente , Secuencia de Aminoácidos , Niño , Preescolar , Femenino , Humanos , Lactante , Imagen por Resonancia Magnética/métodos , Masculino , Datos de Secuencia Molecular , Saccharomyces cerevisiaeRESUMEN
Inherited white-matter disorders are a broad class of diseases for which treatment and classification are both challenging. Indeed, nearly half of the children presenting with a leukoencephalopathy remain without a specific diagnosis. Here, we report on the application of high-throughput genome and exome sequencing to a cohort of ten individuals with a leukoencephalopathy of unknown etiology and clinically characterized by hypomyelination with brain stem and spinal cord involvement and leg spasticity (HBSL), as well as the identification of compound-heterozygous and homozygous mutations in cytoplasmic aspartyl-tRNA synthetase (DARS). These mutations cause nonsynonymous changes to seven highly conserved amino acids, five of which are unchanged between yeast and man, in the DARS C-terminal lobe adjacent to, or within, the active-site pocket. Intriguingly, HBSL bears a striking resemblance to leukoencephalopathy with brain stem and spinal cord involvement and elevated lactate (LBSL), which is caused by mutations in the mitochondria-specific DARS2, suggesting that these two diseases might share a common underlying molecular pathology. These findings add to the growing body of evidence that mutations in tRNA synthetases can cause a broad range of neurologic disorders.
Asunto(s)
Aspartato-ARNt Ligasa/genética , Leucoencefalopatías/genética , Modelos Moleculares , Espasticidad Muscular/genética , Conformación Proteica , Aspartato-ARNt Ligasa/química , Tronco Encefálico/patología , Cristalografía por Rayos X , Humanos , Pierna/patología , Leucoencefalopatías/patología , Mutación/genética , Médula Espinal/patologíaRESUMEN
OBJECTIVE: To identify the etiology of a novel, heritable encephalopathy in a small group of patients. METHODS: Magnetic resonance imaging (MRI) pattern analysis was used to select patients with the same pattern. Homozygosity mapping and whole exome sequencing (WES) were performed to find the causal gene mutations. RESULTS: Seven patients from 4 families (2 consanguineous) were identified with a similar MRI pattern characterized by T2 signal abnormalities and diffusion restriction in the posterior limb of the internal capsule, often also optic radiation, brainstem tracts, and cerebellar white matter, in combination with delayed myelination and progressive brain atrophy. Patients presented with early infantile onset encephalopathy characterized by progressive microcephaly, seizures, variable cardiac defects, and early death. Metabolic testing was unrevealing. Single nucleotide polymorphism array revealed 1 overlapping homozygous region on chromosome 20 in the consanguineous families. In all patients, WES subsequently revealed recessive predicted loss of function mutations in ITPA, encoding inosine triphosphate pyrophosphatase (ITPase). ITPase activity in patients' erythrocytes and fibroblasts was severely reduced. INTERPRETATION: Until now ITPA variants have only been associated with adverse reactions to specific drugs. This is the first report associating ITPA mutations with a human disorder. ITPase is important in purine metabolism because it removes noncanonical nucleotides from the cellular nucleotide pool. Toxicity of accumulated noncanonical nucleotides, leading to neuronal apoptosis and interference with proteins normally using adenosine triphosphate/guanosine triphosphate, probably explains the disease. This study confirms that combining MRI pattern recognition to define small, homogeneous patient groups with WES is a powerful approach for providing a fast diagnosis in patients with an unclassified genetic encephalopathy.
Asunto(s)
Encefalopatías/diagnóstico , Encefalopatías/genética , Genes Recesivos/genética , Mutación/genética , Pirofosfatasas/genética , Preescolar , Femenino , Humanos , Lactante , Masculino , Estructura Secundaria de Proteína , Pirofosfatasas/químicaRESUMEN
OBJECTIVE: Megalencephalic leukoencephalopathy with cysts (MLC) is a genetic disease characterized by infantile onset white matter edema and delayed onset neurological deterioration. Loss of MLC1 function causes MLC. MLC1 is involved in ion-water homeostasis, but its exact role is unknown. We generated Mlc1-null mice for further studies. METHODS: We investigated which brain cell types express MLC1, compared developmental expression in mice and men, and studied the consequences of loss of MLC1 in Mlc1-null mice. RESULTS: Like humans, mice expressed MLC1 only in astrocytes, especially those facing fluid-brain barriers. In mice, MLC1 expression increased until 3 weeks and then stabilized. In humans, MLC1 expression was highest in the first year, decreased, and stabilized from approximately 5 years. Mlc1-null mice had early onset megalencephaly and increased brain water content. From 3 weeks, abnormal astrocytes were present with swollen processes abutting fluid-brain barriers. From 3 months, widespread white matter vacuolization with intramyelinic edema developed. Mlc1-null astrocytes showed slowed regulatory volume decrease and reduced volume-regulated anion currents, which increased upon MLC1 re-expression. Mlc1-null astrocytes showed reduced expression of adhesion molecule GlialCAM and chloride channel ClC-2, but no substantial changes in other known MLC1-interacting proteins. INTERPRETATION: Mlc1-null mice replicate early stages of the human disease with early onset intramyelinic edema. The cellular functional defects, described for human MLC, were confirmed. The earliest change was astrocytic swelling, substantiating that in MLC the primary defect is in volume regulation by astrocytes. MLC1 expression affects expression of GlialCAM and ClC-2. Abnormal interplay between these proteins is part of the pathomechanisms of MLC.
Asunto(s)
Quistes/genética , Quistes/patología , Quistes/fisiopatología , Regulación del Desarrollo de la Expresión Génica/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/fisiopatología , Adolescente , Adulto , Factores de Edad , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Astrocitos/patología , Edema Encefálico/etiología , Cerebelo/patología , Corteza Cerebral/citología , Corteza Cerebral/patología , Niño , Preescolar , Quistes/metabolismo , Modelos Animales de Enfermedad , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Humanos , Lactante , Recién Nacido , Potenciales de la Membrana/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/genética , Equilibrio Postural/genética , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , Trastornos de la Sensación/genética , Sustancia Blanca/metabolismo , Sustancia Blanca/patología , Sustancia Blanca/ultraestructura , Adulto JovenRESUMEN
The 5' leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.
Asunto(s)
Genoma Viral/genética , VIH-1/genética , Motivos de Nucleótidos/genética , ARN Viral/genética , Replicación Viral/genética , Emparejamiento Base , Secuencia de Bases , Northern Blotting , Línea Celular Tumoral , Dimerización , Células HEK293 , VIH-1/química , VIH-1/metabolismo , Humanos , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/patología , Linfocitos T/virología , Transcripción GenéticaRESUMEN
BACKGROUND: Egress of a number of different virus species from infected cells depends on proteins of the endosomal sorting complexes required for transport (ESCRT) pathway. HIV has also hijacked this system to bud viruses outward from the cell surface. How ESCRT-I activates ESCRT-III in this process remains unclear with conflicting published evidence for the requirement of ESCRT-II which fulfils this role in other systems. We investigated the role of ESCRT-II using knockdown mediated by siRNA and shRNA, mutants which prevent ESCRT-I/ESCRT-II interaction and a CRISPR/Cas9 EAP45 knockout cell line. RESULTS: Depletion or elimination of ESCRT-II components from an HIV infected cell produces two distinct effects. The overall production of HIV-1 Gag is reduced leading to a diminished amount of intracellular virion protein. In addition depletion of ESCRT-II produces an effect similar to that seen when ESCRT-I and -III components are depleted, that of a delayed Gag p26 to p24 +p2 cleavage associated with a reduction in export of virion particles and a visible reduction in budding efficiency in virus producing cells. Mutants that interfere with ESCRT-I interacting with ESCRT-II similarly reduce virus export. The export defect is independent of the decrease in overall Gag production. Using a mutant virus which cannot use the ALIX mediated export pathway exacerbates the decrease in virus export seen when ESCRT-II is depleted. ESCRT-II knockdown does not lead to complete elimination of virus release suggesting that the late domain role of ESCRT-II is required for optimal efficiency of viral budding but that there are additional pathways that the virus can employ to facilitate this. CONCLUSION: ESCRT-II contributes to efficient HIV virion production and export by more than one pathway; both by a transcriptional or post transcriptional mechanism and also by facilitating efficient virus export from the cell through interactions with other ESCRT components.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , VIH-1/fisiología , Transporte Biológico , Sistemas CRISPR-Cas/genética , Línea Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Técnicas de Inactivación de Genes , VIH-1/genética , Humanos , Mutación , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/genética , Virión/metabolismo , Liberación del Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Central to the development of new treatments for human immunodeficiency virus 1 (HIV-1) is a more thorough understanding of the viral life cycle and the cellular cofactors upon which this depends. Targeting cellular proteins and their interaction with HIV-1 has the potential to reduce the problem of emerging viral resistance to drugs as mutational escape is more difficult. We performed a short interfering RNA (siRNA) library screen targeting 59 cellular RNA helicases, assessing the effect on both viral capsid protein production and infectious virion formation. Five RNA helicases were identified which, when knocked down, reproducibly decreased infectious particle production: DDX5, DDX10, DDX17, DDX28 and DDX52. Two of these proteins (DDX5 and DDX17) have known roles in HIV-1 replication. A further helicase (DDX10) was a positive hit from a previous genome-wide siRNA screen; however, DDX28 and DDX52 have not previously been implicated as essential cofactors for HIV-1.
Asunto(s)
VIH-1/enzimología , VIH-1/fisiología , Interacciones Huésped-Patógeno , ARN Helicasas/metabolismo , Replicación Viral , Técnicas de Silenciamiento del Gen , Pruebas Genéticas , VIH-1/genética , Humanos , ARN Helicasas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Hypomyelination with atrophy of the basal ganglia and cerebellum is a rare leukoencephalopathy that was identified using magnetic resonance imaging in 2002. In 2013, whole exome sequencing of 11 patients with the disease revealed that they all had the same de novo mutation in TUBB4A, which encodes tubulin ß-4A. We investigated the mutation spectrum in a cohort of 42 patients and the relationship between genotype and phenotype. Patients were selected on the basis of clinical and magnetic resonance imaging abnormalities that are indicative of hypomyelination with atrophy of the basal ganglia and cerebellum. Genetic testing and a clinical inventory were performed, and sequential magnetic resonance images were evaluated using a standard protocol. The heterozygous TUBB4A mutation observed in the first 11 patients was the most common (25 patients). Additionally, 13 other heterozygous mutations were identified, located in different structural domains of tubulin ß-4A. We confirmed that the mutations were de novo in all but three patients. In two of these three cases we lacked parental DNA and in one the mutation was also found in the mother, most likely due to mosaicism. Patients showed a phenotypic continuum ranging from neonatal to childhood disease onset, normal to delayed early development and slow to more rapid neurological deterioration. Neurological symptomatology consisted of extrapyramidal movement abnormalities, spasticity, ataxia, cognitive deficit and sometimes epilepsy. Three patients died and the oldest living patient was 29 years of age. The patients' magnetic resonance images showed an absent or disappearing putamen, variable cerebellar atrophy and highly variable cerebral atrophy. Apart from hypomyelination, myelin loss was evident in several cases. Three severely affected patients had similar, somewhat atypical magnetic resonance image abnormalities. The study results were strongly suggestive of a genotype-phenotype correlation. The 25 patients with the common c.745G>A mutation generally had a less rapidly progressive disease course than the 17 cases with other TUBB4A mutations. Overall, this work demonstrates that the distinctive magnetic resonance imaging pattern for hypomyelination with atrophy of the basal ganglia and cerebellum defines a homogeneous clinical phenotype of variable severity. Patients almost invariably have prominent extrapyramidal movement abnormalities, which are rarely seen in patients with hypomyelination of different origin. A dominant TUBB4A mutation is also associated with dystonia type 4, in which magnetic resonance images of the brain seem normal. It is highly likely that there is a disease continuum associated with TUBB4A mutations, of which hypomyelination with atrophy of the basal ganglia and cerebellum and dystonia type 4 are the extremes. This would indicate that extrapyramidal movement abnormalities constitute the core feature of the disease spectrum related to dominant TUBB4A mutations and that all other features are variable.
Asunto(s)
Ganglios Basales/patología , Cerebelo/patología , Leucoencefalopatías/patología , Mutación/genética , Tubulina (Proteína)/genética , Adolescente , Factores de Edad , Atrofia/patología , Niño , Preescolar , Femenino , Estudios de Asociación Genética , Humanos , Lactante , Leucoencefalopatías/genética , Imagen por Resonancia Magnética , Masculino , Adulto JovenRESUMEN
Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is a disorder caused by recessive mutations in the gene DARS2, which encodes mitochondrial aspartyl-tRNA synthetase. Recent observations indicate that the phenotypic range of the disease is much wider than initially thought. Currently, no treatment is available. The aims of our study were (i) to explore a possible genotype-phenotype correlation; and (ii) to identify potential therapeutic agents that modulate the splice site mutations in intron 2 of DARS2, present in almost all patients. A cross-sectional observational study was performed in 78 patients with two DARS2 mutations in the Amsterdam and Helsinki databases up to December 2012. Clinical information was collected via questionnaires. An inventory was made of the DARS2 mutations in these patients and those previously published. An assay was developed to assess mitochondrial aspartyl-tRNA synthetase enzyme activity in cells. Using a fluorescence reporter system we screened for drugs that modulate DARS2 splicing. Clinical information of 66 patients was obtained. The clinical severity varied from infantile onset, rapidly fatal disease to adult onset, slow and mild disease. The most common phenotype was characterized by childhood onset and slow neurological deterioration. Full wheelchair dependency was rare and usually began in adulthood. In total, 60 different DARS2 mutations were identified, 13 of which have not been reported before. Except for 4 of 42 cases published by others, all patients were compound heterozygous. Ninety-four per cent of the patients had a splice site mutation in intron 2. The groups of patients sharing the same two mutations were too small for formal assessment of genotype-phenotype correlation. However, some combinations of mutations were consistently associated with a mild phenotype. The mitochondrial aspartyl-tRNA synthetase activity was strongly reduced in patient cells. Among the compounds screened, cantharidin was identified as the most potent modulator of DARS2 splicing. In conclusion, the phenotypic spectrum of leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is wide, but most often the disease has a relatively slow and mild course. The available evidence suggests that the genotype influences the phenotype, but because of the high number of private mutations, larger numbers of patients are necessary to confirm this. The activity of mitochondrial aspartyl-tRNA synthetase is significantly reduced in patient cells. A compound screen established a 'proof of principle' that the splice site mutation can be influenced. This finding is promising for future therapeutic strategies.
Asunto(s)
Empalme Alternativo/efectos de los fármacos , Aspartato-ARNt Ligasa/deficiencia , Leucoencefalopatías/complicaciones , Leucoencefalopatías/genética , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/genética , Adolescente , Adulto , Edad de Inicio , Aspartato-ARNt Ligasa/genética , Aspartato-ARNt Ligasa/metabolismo , Cantaridina/farmacología , Niño , Preescolar , Estudios Transversales , Análisis Mutacional de ADN , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Estudios de Asociación Genética , Humanos , Lactante , Leucoencefalopatías/tratamiento farmacológico , Leucoencefalopatías/enzimología , Masculino , Persona de Mediana Edad , Enfermedades Mitocondriales/tratamiento farmacológico , Enfermedades Mitocondriales/enzimología , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
To accomplish a diagnosis in patients with a rare unclassified disorder is difficult. In this study, we used magnetic resonance imaging pattern recognition analysis to identify patients with the same novel heritable disorder. Whole-exome sequencing was performed to discover the mutated gene. We identified seven patients sharing a previously undescribed magnetic resonance imaging pattern, characterized by initial swelling with T2 hyperintensity of the basal nuclei, thalami, cerebral white matter and cortex, pons and midbrain, followed by rarefaction or cystic degeneration of the white matter and, eventually, by progressive cerebral, cerebellar and brainstem atrophy. All patients developed a severe encephalopathy with rapid deterioration of neurological functions a few weeks after birth, followed by respiratory failure and death. Lactate was elevated in body fluids and on magnetic resonance spectroscopy in most patients. Whole-exome sequencing in a single patient revealed two predicted pathogenic, heterozygous missense mutations in the SLC19A3 gene, encoding the second thiamine transporter. Additional predicted pathogenic mutations and deletions were detected by Sanger sequencing in all six other patients. Pathology of brain tissue of two patients demonstrated severe cerebral atrophy and microscopic brain lesions similar to Leigh's syndrome. Although the localization of SLC19A3 expression in brain was similar in the two investigated patients compared to age-matched control subjects, the intensity of the immunoreactivity was increased. Previously published patients with SLC19A3 mutations have a milder clinical phenotype, no laboratory evidence of mitochondrial dysfunction and more limited lesions on magnetic resonance imaging. In some, cerebral atrophy has been reported. The identification of this new, severe, lethal phenotype characterized by subtotal brain degeneration broadens the phenotypic spectrum of SLC19A3 mutations. Recognition of the associated magnetic resonance imaging pattern allows a fast diagnosis in affected infants.
Asunto(s)
Química Encefálica/genética , Exoma/genética , Enfermedad de Leigh/genética , Enfermedad de Leigh/patología , Proteínas de Transporte de Membrana/genética , Mutación/genética , Femenino , Humanos , Lactante , Enfermedad de Leigh/mortalidad , Masculino , Estudios RetrospectivosRESUMEN
The leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC) is characterized by infantile-onset macrocephaly and chronic edema of the brain white matter. With delayed onset, patients typically experience motor problems, epilepsy and slow cognitive decline. No treatment is available. Classic MLC is caused by bi-allelic recessive pathogenic variants in MLC1 or GLIALCAM (also called HEPACAM). Heterozygous dominant pathogenic variants in GLIALCAM lead to remitting MLC, where patients show a similar phenotype in early life, followed by normalization of white matter edema and no clinical regression. Rare patients with heterozygous dominant variants in GPRC5B and classic MLC were recently described. In addition, two siblings with bi-allelic recessive variants in AQP4 and remitting MLC have been identified. The last systematic overview of variants linked to MLC dates back to 2006. We provide an updated overview of published and novel variants. We report on genetic variants from 508 patients with MLC as confirmed by MRI diagnosis (258 from our database and 250 extracted from 64 published reports). We describe 151 unique MLC1 variants, 29 GLIALCAM variants, 2 GPRC5B variants and 1 AQP4 variant observed in these MLC patients. We include experiments confirming pathogenicity for some variants, discuss particularly notable variants, and provide an overview of recent scientific and clinical insight in the pathophysiology of MLC.
RESUMEN
Vanishing white matter (VWM) is a devastating autosomal recessive leukodystrophy, resulting in neurological deterioration and premature death, and without curative treatment. Pathogenic hypomorphic variants in subunits of the eukaryotic initiation factor 2B (eIF2B) cause VWM. eIF2B is required for regulating the integrated stress response (ISR), a physiological response to cellular stress. In patients' central nervous system, reduced eIF2B activity causes deregulation of the ISR. In VWM mouse models, the extent of ISR deregulation correlates with disease severity. One approach to restoring eIF2B activity is by inhibition of GSK3ß, a kinase that phosphorylates eIF2B and reduces its activity. Lithium, an inhibitor of GSK3ß, is thus expected to stimulate eIF2B activity and ameliorate VWM symptoms. The effects of lithium were tested in zebrafish and mouse VWM models. Lithium improved motor behavior in homozygous eif2b5 mutant zebrafish. In lithium-treated 2b4he2b5ho mutant mice, a paradoxical increase in some ISR transcripts was found. Furthermore, at the dosage tested, lithium induced significant polydipsia in both healthy controls and 2b4he2b5ho mutant mice and did not increase the expression of other markers of lithium efficacy. In conclusion, lithium is not a drug of choice for further development in VWM based on the limited or lack of efficacy and significant side-effect profile.
RESUMEN
BACKGROUND: Immune cell adaptor protein ADAP (adhesion and degranulation-promoting adaptor protein) mediates aspects of T-cell adhesion and proliferation. Despite this, a connection between ADAP and infection by the HIV-1 (human immunodeficiency virus-1) has not been explored. RESULTS: In this paper, we show for the first time that ADAP and its binding to SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) regulate HIV-1 infection via two distinct mechanisms and co-receptors. siRNA down-regulation of ADAP, or expression of a mutant that is defective in associating to its binding partner SLP-76 (termed M12), inhibited the propagation of HIV-1 in T-cell lines and primary human T-cells. In one step, ADAP and its binding to SLP-76 were needed for the activation of NF-κB and its transcription of the HIV-1 long terminal repeat (LTR) in cooperation with ligation of co-receptor CD28, but not LFA-1. In a second step, the ADAP-SLP-76 module cooperated with LFA-1 to regulate conjugate formation between T-cells and dendritic cells or other T-cells as well as the development of the virological synapse (VS) and viral spread between immune cells. CONCLUSIONS: These findings indicate that ADAP regulates two steps of HIV-1 infection cooperatively with two distinct receptors, and as such, serves as a new potential target in the blockade of HIV-1 infection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , VIH-1/fisiología , Linfocitos T/virología , Transcripción Genética , Replicación Viral , Adhesión Celular , Humanos , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Fosfoproteínas/metabolismoRESUMEN
In the large group of genetically undetermined infantile-onset mitochondrial encephalopathies, multiple defects of mitochondrial DNA-related respiratory-chain complexes constitute a frequent biochemical signature. In order to identify responsible genes, we used exome-next-generation sequencing in a selected cohort of patients with this biochemical signature. In an isolated patient, we found two mutant alleles for EARS2, the gene encoding mitochondrial glutamyl-tRNA synthetase. The brain magnetic resonance imaging of this patient was hallmarked by extensive symmetrical cerebral white matter abnormalities sparing the periventricular rim and symmetrical signal abnormalities of the thalami, midbrain, pons, medulla oblongata and cerebellar white matter. Proton magnetic resonance spectroscopy showed increased lactate. We matched this magnetic resonance imaging pattern with that of a cohort of 11 previously selected unrelated cases. We found mutations in the EARS2 gene in all. Subsequent detailed clinical and magnetic resonance imaging based phenotyping revealed two distinct groups: mild and severe. All 12 patients shared an infantile onset and rapidly progressive disease with severe magnetic resonance imaging abnormalities and increased lactate in body fluids and proton magnetic resonance spectroscopy. Patients in the 'mild' group partially recovered and regained milestones in the following years with striking magnetic resonance imaging improvement and declining lactate levels, whereas those of the 'severe' group were characterized by clinical stagnation, brain atrophy on magnetic resonance imaging and persistent lactate increases. This new neurological disease, early-onset leukoencephalopathy with thalamus and brainstem involvement and high lactate, is hallmarked by unique magnetic resonance imaging features, defined by a peculiar biphasic clinical course and caused by mutations in a single gene, EARS2, expanding the list of medically relevant defects of mitochondrial DNA translation.
Asunto(s)
Tronco Encefálico/patología , Glutamato-ARNt Ligasa/genética , Ácido Láctico/metabolismo , Leucoencefalopatías , Mutación/genética , Tálamo/patología , Células Cultivadas , Niño , Análisis Mutacional de ADN , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Femenino , Fibroblastos/fisiología , Humanos , Leucoencefalopatías/genética , Leucoencefalopatías/metabolismo , Leucoencefalopatías/patología , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Masculino , Proteínas Mitocondriales/genética , Consumo de Oxígeno/genética , Consumo de Oxígeno/fisiología , Protones , Piel/patologíaRESUMEN
Translation initiation on most eukaryotic mRNAs occurs via a cap-dependent scanning mechanism and its efficiency is modulated by their 5'-untranslated regions (5'-UTR). The human immunodeficiency virus type 1 (HIV-1) 5'-UTR contains a stable TAR hairpin directly at its 5'-end, which possibly masks the cap structure. In addition, the 5'-UTR is relatively long and contains several stable RNA structures that are essential for viral replication. These characteristics may interfere with ribosomal scanning and suggest that translation is initiated via internal entry of ribosomes. Literature on the HIV-1 5'-UTR-driven translation initiation mechanism is controversial. Both scanning and internal initiation have been shown to occur in various experimental systems. To gain further insight in the translation initiation process, we determined which part of the 5'-UTR is scanned. To do so, we introduced upstream AUGs at various positions across the 5'-UTR and determined the effect on expression of a downstream reporter gene that was placed under control of the gag start codon. This strategy allowed us to determine the window of ribosomal scanning on the HIV-1 5'-UTR.