RESUMEN
Annexin A11 mutations are a rare cause of amyotrophic lateral sclerosis (ALS), wherein replicated protein variants P36R, G38R, D40G and D40Y are located in a small-alpha helix within the long, disordered N-terminus. To elucidate disease mechanisms, we characterised the phenotypes induced by a genetic loss of function (LoF) and by misexpression of G38R and D40G in vivo. Loss of Annexin A11 results in a low-penetrant behavioural phenotype and aberrant axonal morphology in zebrafish homozygous knockout larvae, which is rescued by human WT Annexin A11. Both Annexin A11 knockout/down and ALS variants trigger nuclear dysfunction characterised by Lamin B2 mis-localisation. The Lamin B2 signature also presented in anterior horn, spinal cord neurons from post-mortem ALS+/-FTD patient tissue possessing G38R and D40G protein variants. These findings suggest mutant Annexin A11 acts as a dominant negative, revealing a potential early nucleopathy highlighting nuclear envelope abnormalities preceding behavioural abnormality in animal models.
RESUMEN
Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD's interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Enzima Desubiquitinante CYLD/genética , Enzima Desubiquitinante CYLD/fisiología , Demencia Frontotemporal/genética , Predisposición Genética a la Enfermedad/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Autofagosomas/metabolismo , Autofagosomas/fisiología , Axones/patología , Encéfalo/metabolismo , Proteínas de Unión al ADN , Enzima Desubiquitinante CYLD/metabolismo , Enzimas Desubicuitinizantes/metabolismo , Demencia Frontotemporal/metabolismo , Ratones , Mutación Missense/genética , FN-kappa B/antagonistas & inhibidores , Cultivo Primario de Células , TransfecciónRESUMEN
Amyotrophic lateral sclerosis (ALS) presents with focal muscle weakness due to motor neuron degeneration that becomes generalized, leading to death from respiratory failure within 3-5 years from symptom onset. Despite the heterogeneity of aetiology, TDP-43 proteinopathy is a common pathological feature that is observed in >95% of ALS and tau-negative frontotemporal dementia (FTD) cases. TDP-43 is a DNA/RNA-binding protein that in ALS and FTD translocates from being predominantly nuclear to form detergent-resistant, hyperphosphorylated aggregates in the cytoplasm of affected neurons and glia. Mutations in TARDBP account for 1-4% of all ALS cases and almost all arise in the low complexity C-terminal domain that does not affect RNA binding and processing. Here we report an ALS/FTD kindred with a novel K181E TDP-43 mutation that is located in close proximity to the RRM1 domain. To offer predictive gene testing to at-risk family members, we undertook a series of functional studies to characterize the properties of the mutation. Spectroscopy studies of the K181E protein revealed no evidence of significant misfolding. Although it is unable to bind to or splice RNA, it forms abundant aggregates in transfected cells. We extended our study to include other ALS-linked mutations adjacent to the RRM domains that also disrupt RNA binding and greatly enhance TDP-43 aggregation, forming detergent-resistant and hyperphosphorylated inclusions. Lastly, we demonstrate that K181E binds to, and sequesters, wild-type TDP-43 within nuclear and cytoplasmic inclusions. Thus, we demonstrate that TDP-43 mutations that disrupt RNA binding greatly enhance aggregation and are likely to be pathogenic as they promote wild-type TDP-43 to mislocalize and aggregate acting in a dominant-negative manner. This study highlights the importance of RNA binding to maintain TDP-43 solubility and the role of TDP-43 aggregation in disease pathogenesis.
Asunto(s)
Proteínas de Unión al ADN/genética , Mutación , Agregación Patológica de Proteínas/genética , Proteínas de Unión al ARN/genética , Médula Espinal/metabolismo , Proteinopatías TDP-43/genética , Adulto , Proteínas de Unión al ADN/metabolismo , Humanos , Masculino , Neuroglía/metabolismo , Neuronas/metabolismo , Fosforilación , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Proteínas de Unión al ARN/metabolismo , Médula Espinal/patología , Proteinopatías TDP-43/metabolismo , Proteinopatías TDP-43/patologíaRESUMEN
An intronic GGGGCC (G4C2) hexanucleotide repeat expansion inC9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 RNA can result in five different dipeptide repeat proteins (DPR: poly GA, poly GP, poly GR, poly PA, and poly PR), which aggregate into neuronal cytoplasmic and nuclear inclusions in affected patients, however their contribution to disease pathogenesis remains controversial. We show that among the DPR proteins, expression of poly GA in a cell culture model activates programmed cell death and TDP-43 cleavage in a dose-dependent manner. Dual expression of poly GA together with other DPRs revealed that poly GP and poly PA are sequestered by poly GA, whereas poly GR and poly PR are rarely co-localised with poly GA. Dual expression of poly GA and poly PA ameliorated poly GA toxicity by inhibiting poly GA aggregation both in vitro and in vivo in the chick embryonic spinal cord. Expression of alternative codon-derived DPRs in chick embryonic spinal cord confirmed in vitro data, revealing that each of the dipeptides caused toxicity, with poly GA being the most toxic. Further, in vivo expression of G4C2 repeats of varying length caused apoptotic cell death, but failed to generate DPRs. Together, these data demonstrate that C9-related toxicity can be mediated by either RNA or DPRs. Moreover, our findings provide evidence that poly GA is a key mediator of cytotoxicity and that cross-talk between DPR proteins likely modifies their pathogenic status in C9ALS/FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Proteína C9orf72/metabolismo , Células Cultivadas , Embrión de Pollo , Expansión de las Repeticiones de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dipéptidos/genética , Dipéptidos/metabolismo , Lóbulo Frontal/metabolismo , Lóbulo Frontal/fisiología , Células HEK293 , Humanos , Cuerpos de Inclusión Intranucleares/metabolismo , Neuronas/metabolismo , Agregado de ProteínasRESUMEN
OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease of motor neurons with a median survival of 2 years. Familial ALS has a younger age of onset than apparently sporadic ALS. We sought to determine whether this younger age of onset is a result of ascertainment bias or has a genetic basis. METHODS: Samples from people with ALS were sequenced for 13 ALS genes. To determine the effect of genetic variation, age of onset was compared in people with sporadic ALS carrying a pathogenic gene variant and those who do not; to determine the effect of family history, we compared those with genetic sporadic ALS and familial ALS. RESULTS: There were 941 people with a diagnosis of ALS, 100 with familial ALS. Of 841 with apparently sporadic ALS, 95 carried a pathogenic gene variant. The mean age of onset in familial ALS was 5.3 years younger than for apparently sporadic ALS (p=6.0×10-5, 95% CI 2.8 to 7.8 years). The mean age of onset of genetic sporadic ALS was 2.9 years younger than non-genetic sporadic ALS (p=0.011, 95% CI 0.7 to 5.2 years). There was no difference between the mean age of onset in genetic sporadic ALS and familial ALS (p=0.097). CONCLUSIONS: People with familial ALS have an age of onset about 5 years younger than those with apparently sporadic ALS, and we have shown that this is a result of Mendelian gene variants lowering the age of onset, rather than ascertainment bias.
Asunto(s)
Esclerosis Amiotrófica Lateral/epidemiología , Esclerosis Amiotrófica Lateral/genética , Variación Genética/genética , Adulto , Edad de Inicio , Anciano , Sesgo , Bases de Datos de Ácidos Nucleicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reino UnidoRESUMEN
Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a 'steric zipper' motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.
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Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Demencia Frontotemporal/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/química , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Distrofia Muscular de Cinturas/genética , Proteínas Mutantes/genética , Mutación/genética , Miositis por Cuerpos de Inclusión/genética , Osteítis Deformante/genética , Priones/química , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Masculino , Ratones , Datos de Secuencia Molecular , Distrofia Muscular de Cinturas/metabolismo , Distrofia Muscular de Cinturas/patología , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Miositis por Cuerpos de Inclusión/metabolismo , Miositis por Cuerpos de Inclusión/patología , Osteítis Deformante/metabolismo , Osteítis Deformante/patología , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Priones/genética , Priones/metabolismo , Estructura Terciaria de Proteína/genética , ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
A massive intronic GGGGCC hexanucleotide repeat expansion in C9ORF72 has recently been identified as the most common cause of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We have previously demonstrated that C9ORF72 mutant cases have a specific pathological profile with abundant p62-positive, TDP-43-negative cytoplasmic and intranuclear inclusions within cerebellar granular cells of the cerebellum and pyramidal cells of the hippocampus in addition to classical TDP-43 pathology. Here, we report mixed tau and TDP-43 pathology in a woman with behavioural variant FTLD who had the C9ORF72 mutation, and the p.Ala239Thr variant in MAPT (microtubule associated protein tau) gene not previously associated with tau pathology. Two of her brothers, who carried the C9ORF72 mutation, but not the MAPT variant, developed classical ALS without symptomatic cognitive changes. The dominant neuropathology in this woman with FTLD was a tauopathy with Pick's disease-like features. TDP-43 labelling was mainly confined to Pick bodies, but p62-positive, TDP-43-negative inclusions, characteristic of C9ORF72 mutations, were present in the cerebellum and hippocampus. Mixed pathology to this degree is unusual. One might speculate that the presence of the C9ORF72 mutation might influence tau deposition in what was previously thought to be a "benign" variant in MAPT in addition to the aggregation of TDP-43 and other as yet unidentified proteins decorated with ubiquitin and p62.
Asunto(s)
Proteínas de Unión al ADN/genética , Degeneración Lobar Frontotemporal/genética , Mutación/fisiología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas tau/genética , Adulto , Esclerosis Amiotrófica Lateral/genética , Autopsia , Western Blotting , Encéfalo/patología , Cromosomas Humanos Par 9/genética , ADN/genética , Expansión de las Repeticiones de ADN , Proteínas de Unión al ADN/fisiología , Femenino , Degeneración Lobar Frontotemporal/patología , Humanos , Enfermeras y Enfermeros , Conducta Obsesiva/genética , Conducta Obsesiva/psicología , Sistemas de Lectura Abierta/genética , Linaje , Fosforilación , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas tau/fisiologíaRESUMEN
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are relentlessly progressive neurodegenerative disorders with overlapping clinical, genetic and pathological features. Cytoplasmic inclusions of fused in sarcoma (FUS) are the hallmark of several forms of FTLD and ALS patients with mutations in the FUS gene. FUS is a multifunctional, predominantly nuclear, DNA and RNA binding protein. Here, we report that transgenic mice overexpressing wild-type human FUS develop an aggressive phenotype with an early onset tremor followed by progressive hind limb paralysis and death by 12 weeks in homozygous animals. Large motor neurons were lost from the spinal cord accompanied by neurophysiological evidence of denervation and focal muscle atrophy. Surviving motor neurons in the spinal cord had greatly increased cytoplasmic expression of FUS, with globular and skein-like FUS-positive and ubiquitin-negative inclusions associated with astroglial and microglial reactivity. Cytoplasmic FUS inclusions were also detected in the brain of transgenic mice without apparent neuronal loss and little astroglial or microglial activation. Hemizygous FUS overexpressing mice showed no evidence of a motor phenotype or pathology. These findings recapitulate several pathological features seen in human ALS and FTLD patients, and suggest that overexpression of wild-type FUS in vulnerable neurons may be one of the root causes of disease. Furthermore, these mice will provide a new model to study disease mechanism, and test therapies.
Asunto(s)
Envejecimiento/fisiología , Enfermedad de la Neurona Motora/genética , Enfermedad de la Neurona Motora/patología , Proteína FUS de Unión a ARN/fisiología , Animales , Western Blotting , Supervivencia Celular , Citoplasma/metabolismo , Dosificación de Gen , Regulación de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Cuerpos de Inclusión/patología , Contracción Isométrica/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/fisiología , Fibras Musculares Esqueléticas/fisiología , Parálisis/genética , Parálisis/patología , Proteína FUS de Unión a ARN/genética , Médula Espinal/patologíaRESUMEN
Glucose-6-phosphatase, an enzyme localized in the endoplasmic reticulum (ER), catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. In humans, there are three differentially expressed glucose-6-phosphatase catabolic genes (G6PC1-3). Recently, it has been shown that mutations in the G6PC3 gene result in a syndrome associating congenital neutropenia and various organ malformations. The enzymatic function of G6PC3 is dependent on G6P transport into the ER, mediated by G6P translocase (G6PT). Mutations in the gene encoding G6PT result in glycogen storage disease type-1b (GSD-1b). Interestingly, GSD-1b patients exhibit a similar neutrophil dysfunction to that observed in G6PC3-deficient patients. To better understand the causes of neutrophil dysfunction in both diseases, we have studied the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of patients with G6PC3 and G6PT syndromes. Unexpectedly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments indicated hypo-glycosylation of gp91(phox), the electron-transporting component of the NADPH oxidase, in all of these patients. Rigorous mass spectrometric glycomic profiling showed that most of the complex-type antennae which characterize the neutrophil N-glycome of healthy individuals were severely truncated in the patients' neutrophils. A comparable truncation of the core 2 antenna of the O-glycans was also observed. This aberrant neutrophil glycosylation is predicted to have profound effects on the neutrophil function and merit designation of both syndromes as a new class of congenital disorders of glycosylation.
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Glucosa-6-Fosfatasa/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Mutación/genética , Neutrófilos/fisiología , Polisacáridos/metabolismo , Adolescente , Adulto , Secuencia de Aminoácidos , Niño , Retículo Endoplásmico , Femenino , Glicómica , Glicosilación , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Neutrófilos/citología , Linaje , Polisacáridos/química , Estallido Respiratorio , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto JovenRESUMEN
Aberrantly expressed fused in sarcoma (FUS) is a hallmark of FUS-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Wildtype FUS localises to synapses and interacts with mitochondrial proteins while mutations have been shown to cause to pathological changes affecting mitochondria, synapses and the neuromuscular junction (NMJ). This indicates a crucial physiological role for FUS in regulating synaptic and mitochondrial function that is currently poorly understood. In this paper we provide evidence that mislocalised cytoplasmic FUS causes mitochondrial and synaptic changes and that FUS plays a vital role in maintaining neuronal health in vitro and in vivo. Overexpressing mutant FUS altered synaptic numbers and neuronal complexity in both primary neurons and zebrafish models. The degree to which FUS was mislocalised led to differences in the synaptic changes which was mirrored by changes in mitochondrial numbers and transport. Furthermore, we showed that FUS co-localises with the mitochondrial tethering protein Syntaphilin (SNPH), and that mutations in FUS affect this relationship. Finally, we demonstrated mutant FUS led to changes in global protein translation. This localisation between FUS and SNPH could explain the synaptic and mitochondrial defects observed leading to global protein translation defects. Importantly, our results support the 'gain-of-function' hypothesis for disease pathogenesis in FUS-related ALS.
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Esclerosis Amiotrófica Lateral/metabolismo , Proteínas Portadoras/metabolismo , Mitocondrias/metabolismo , Mutación , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Sinapsis/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Proteínas Portadoras/genética , Mitocondrias/genética , Proteínas del Tejido Nervioso/genética , Unión Neuromuscular/genética , Proteína FUS de Unión a ARN/genética , Ratas , Sinapsis/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Loss of function (LoF) mutations in Optineurin can cause recessive amyotrophic lateral sclerosis (ALS) with some heterozygous LoF mutations associated with dominant ALS. The molecular mechanisms underlying the variable inheritance pattern associated with OPTN mutations have remained elusive. We identified that affected members of a consanguineous Middle Eastern ALS kindred possessed a novel homozygous p.S174X OPTN mutation. Analysis of these primary fibroblast lines from family members identified that the p.S174X mutation reduces OPTN mRNA expression in an allele-dependent fashion by nonsense mediated decay. Western blotting correlated a reduced expression in heterozygote carriers but a complete absence of OPTN protein in the homozygous carrier. This data suggests that the p.S174X truncation mutation causes recessive ALS through LoF. However, functional analysis detected a significant increase in mitophagy markers TOM20 and COXIV, and higher rates of mitochondrial respiration and ATP levels in heterozygous carriers only. This suggests that heterozygous LoF OPTN mutations may not be causative in a Mendelian manner but may potentially behave as contributory ALS risk factors.
Asunto(s)
Alelos , Esclerosis Amiotrófica Lateral/genética , Proteínas de Ciclo Celular/genética , Genes Recesivos/genética , Estudios de Asociación Genética/métodos , Mutación con Pérdida de Función/genética , Proteínas de Transporte de Membrana/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , Anciano , Anciano de 80 o más Años , Consanguinidad , Femenino , Expresión Génica/genética , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Medio Oriente , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de RiesgoRESUMEN
Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation. Objective: To identify the genetic variants associated with juvenile ALS. Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism. Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members. Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway. Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.
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Esclerosis Amiotrófica Lateral/genética , Predisposición Genética a la Enfermedad/genética , Serina C-Palmitoiltransferasa/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Mutación , Secuenciación del Exoma , Adulto JovenRESUMEN
Evidence indicates that common variants found in genome-wide association studies increase risk of disease through gene regulation via expression Quantitative Trait Loci. Using multiple genome-wide methods, we examined if Single Nucleotide Polymorphisms increase risk of Amyotrophic Lateral Sclerosis through expression Quantitative Trait Loci, and whether expression Quantitative Trait Loci expression is consistent across people who had Amyotrophic Lateral Sclerosis and those who did not. In combining public expression Quantitative Trait Loci data with Amyotrophic Lateral Sclerosis genome-wide association studies, we used Summary-data-based Mendelian Randomization to confirm that SCFD1 was the only gene that was genome-wide significant in mediating Amyotrophic Lateral Sclerosis risk via expression Quantitative Trait Loci (Summary-data-based Mendelian Randomization beta = 0.20, standard error = 0.04, P-value = 4.29 × 10-6). Using post-mortem motor cortex, we tested whether expression Quantitative Trait Loci showed significant differences in expression between Amyotrophic Lateral Sclerosis (n = 76) and controls (n = 25), genome-wide. Of 20 757 genes analysed, the two most significant expression Quantitative Trait Loci to show differential in expression between Amyotrophic Lateral Sclerosis and controls involve two known Amyotrophic Lateral Sclerosis genes (SCFD1 and VCP). Cis-acting SCFD1 expression Quantitative Trait Loci downstream of the gene showed significant differences in expression between Amyotrophic Lateral Sclerosis and controls (top expression Quantitative Trait Loci beta = 0.34, standard error = 0.063, P-value = 4.54 × 10-7). These SCFD1 expression Quantitative Trait Loci also significantly modified Amyotrophic Lateral Sclerosis survival (number of samples = 4265, hazard ratio = 1.11, 95% confidence interval = 1.05-1.17, P-value = 2.06 × 10-4) and act as an Amyotrophic Lateral Sclerosis trans-expression Quantitative Trait Loci hotspot for a wider network of genes enriched for SCFD1 function and Amyotrophic Lateral Sclerosis pathways. Using gene-set analyses, we found the genes that correlate with this trans-expression Quantitative Trait Loci hotspot significantly increase risk of Amyotrophic Lateral Sclerosis (beta = 0.247, standard deviation = 0.017, P = 0.001) and schizophrenia (beta = 0.263, standard deviation = 0.008, P-value = 1.18 × 10-5), a disease that genetically correlates with Amyotrophic Lateral Sclerosis. In summary, SCFD1 expression Quantitative Trait Loci are a major factor in Amyotrophic Lateral Sclerosis, not only influencing disease risk but are differentially expressed in post-mortem Amyotrophic Lateral Sclerosis. SCFD1 expression Quantitative Trait Loci show distinct expression profiles in Amyotrophic Lateral Sclerosis that correlate with a wider network of genes that also confer risk of the disease and modify the disease's duration.
RESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Estudio de Asociación del Genoma Completo , Mutación , Neuronas/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Encéfalo/metabolismo , Colesterol/sangre , Progresión de la Enfermedad , Femenino , Glutamina/metabolismo , Humanos , Masculino , Análisis de la Aleatorización Mendeliana , Repeticiones de Microsatélite , Enfermedades Neurodegenerativas/genética , Sitios de Carácter Cuantitativo , RNA-Seq , Factores de RiesgoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Patients with autosomal dominant (AD), sporadic and X-linked severe congenital neutropenia (SCN) may have mutations in the elastase 2 (ELA2) or Wiskott-Aldrich syndrome (WAS) genes. Homozygous mutations in the HAX1 gene have recently been reported in autosomal recessive (AR) cases of primarily Middle-Eastern descent and the original Kostmann family. We screened 109 predominantly Caucasian SCN kindreds for mutations in these genes; 33 (30%) had 24 different ELA2 mutations, five of them novel, two kindreds (2%) had WAS mutations and four kindreds (4%) had three different HAX1 mutations, two of them novel. One HAX1 mutation (p.Ser43LeufsX11) was found in an AR Ashkenazi Jewish kindred, the other (p.Glu31LysfsX54) in two unrelated British patients with sporadic disease. Microsatellite analysis of the HAX1 locus revealed a common haplotype (maximum distance 4.1 Megabases) for the p.Glu31LysfsX54 patients, suggesting a possible ancestral founder. In functional assays, the level of spontaneous and staurosporine-induced apoptosis was increased in neutrophils from both p.Ser43LeufsX11 patients but not a p.Glu31LysfsX54 patient, suggesting the possible presence of modifying factors. The low incidence of HAX1 mutations in our study suggests that the frequency may vary between racial groups but suggests that irrespective of inheritance or racial origin, SCN patients should be screened for HAX1 mutations.
Asunto(s)
Mutación , Neutropenia/congénito , Neutropenia/genética , Proteínas/genética , Proteínas Adaptadoras Transductoras de Señales , Adolescente , Adulto , Apoptosis , Biomarcadores/análisis , Estudios de Casos y Controles , Análisis Mutacional de ADN , Femenino , Genes Dominantes , Genes Recesivos , Haplotipos , Homocigoto , Humanos , Masculino , Repeticiones de Microsatélite , Neutropenia/patología , Neutrófilos/patología , Linaje , Serina Endopeptidasas/genética , Proteína del Síndrome de Wiskott-Aldrich/genética , Adulto JovenRESUMEN
Analysis of 226 exome-sequenced UK cases of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia identified 2 individuals who harbored a P497H and P506S UBQLN2 mutation, respectively (n = 0.9%). The P506S index case presented with behavioral variant frontotemporal dementia at the age of 54 years then progressed to ALS surviving 3 years. Three sons presented with (1) slowly progressive pure spastic paraplegia with an onset at 25 years and (2) ALS with disease onset of 25 years and survival of 2 years, and (3) ALS presenting symptoms at the age of 26 years, respectively. Analysis of postmortem tissue from the index case revealed frequent neuronal cytoplasmic UBQLN2-positive inclusions in the dentate gyrus and TDP-43-positive neuronal cytoplasmic inclusions in the frontal and temporal cortex and granular cell layer of the dentate gyrus of the hippocampus. Furthermore, a comprehensive analysis of published UBQLN2 mutations demonstrated that only proline-rich domain mutations contribute to a significantly earlier age of onset in male patients (p = 0.0026).
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas de Ciclo Celular/genética , Demencia Frontotemporal/genética , Mutación/genética , Paraplejía/genética , Ubiquitinas/genética , Proteínas Adaptadoras Transductoras de Señales , Adulto , Animales , Proteínas Relacionadas con la Autofagia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Giro Dentado/metabolismo , Progresión de la Enfermedad , Femenino , Lóbulo Frontal/metabolismo , Humanos , Cuerpos de Inclusión/metabolismo , Masculino , Persona de Mediana Edad , Fenotipo , Factores Sexuales , Lóbulo Temporal/metabolismo , Ubiquitinas/metabolismoRESUMEN
To discover novel genes underlying amyotrophic lateral sclerosis (ALS), we aggregated exomes from 3,864 cases and 7,839 ancestry-matched controls. We observed a significant excess of rare protein-truncating variants among ALS cases, and these variants were concentrated in constrained genes. Through gene level analyses, we replicated known ALS genes including SOD1, NEK1 and FUS. We also observed multiple distinct protein-truncating variants in a highly constrained gene, DNAJC7. The signal in DNAJC7 exceeded genome-wide significance, and immunoblotting assays showed depletion of DNAJC7 protein in fibroblasts in a patient with ALS carrying the p.Arg156Ter variant. DNAJC7 encodes a member of the heat-shock protein family, HSP40, which, along with HSP70 proteins, facilitates protein homeostasis, including folding of newly synthesized polypeptides and clearance of degraded proteins. When these processes are not regulated, misfolding and accumulation of aberrant proteins can occur and lead to protein aggregation, which is a pathological hallmark of neurodegeneration. Our results highlight DNAJC7 as a novel gene for ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Exoma/genética , Predisposición Genética a la Enfermedad/genética , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Estudios de Casos y Controles , Femenino , Variación Genética/genética , Humanos , MasculinoRESUMEN
Mutations in TANK binding kinase 1 (TBK1) have been linked to amyotrophic lateral sclerosis. Some TBK1 variants are nonsense and are predicted to cause disease through haploinsufficiency; however, many other mutations are missense with unknown functional effects. We exome sequenced 699 familial amyotrophic lateral sclerosis patients and identified 16 TBK1 novel or extremely rare protein-changing variants. We characterized a subset of these: p.G217R, p.R357X, and p.C471Y. Here, we show that the p.R357X and p.G217R both abolish the ability of TBK1 to phosphorylate 2 of its kinase targets, IRF3 and optineurin, and to undergo phosphorylation. They both inhibit binding to optineurin and the p.G217R, within the TBK1 kinase domain, reduces homodimerization, essential for TBK1 activation and function. Finally, we show that the proportion of TBK1 that is active (phosphorylated) is reduced in 5 lymphoblastoid cell lines derived from patients harboring heterozygous missense or in-frame deletion TBK1 mutations. We conclude that missense mutations in functional domains of TBK1 impair the binding and phosphorylation of its normal targets, implicating a common loss of function mechanism, analogous to truncation mutations.